insights into the pathogenesis of dominant retinitis...

O R I G I N A L A R T I C L E

Insights into the pathogenesis of dominant retinitis

pigmentosa associated with a D477G mutation

in RPE65Elliot H Choi1 Susie Suh1 Christopher L Sander1Christian J Ortiz Hernandez12 Elizabeth R Bulman13 Nimesh Khadka1Zhiqian Dong14 Wuxian Shi5 Krzysztof Palczewski16 andPhilip D Kiser1361Department of Pharmacology School of Medicine Case Western Reserve University Cleveland OH 44106USA 2University of Puerto Rico at Humacao Humacao PR USA 3Research Service Louis Stokes Cleveland VAMedical Center Cleveland OH 44106 USA 4Polgenix Inc Cleveland OH 44106 USA 5National SynchrotronLight Source-II Brookhaven National Laboratory Upton NY 11973 USA and 6Cleveland Center for Membraneand Structural Biology Case Western Reserve University Cleveland OH 44106 USA

To whom correspondence should be addressed at Department of Pharmacology School of Medicine Case Western Reserve University 10900 Euclid AveCleveland OH 44106-4965 USA Tel thorn1 2163684631 Fax thorn1 2163681300 Email kxp65caseedu (KP) Department of Pharmacology School of MedicineCase Western Reserve University 10900 Euclid Ave Cleveland OH 44106-4965 USA Telthorn1 2163680040 Fax thorn1 2163681300 Email pdk7caseedu (PDK)

AbstractRPE65 is the essential transndashcis isomerase of the classical retinoid (visual) cycle Mutations in RPE65 give rise to severe retinaldystrophies most of which are associated with loss of protein function and recessive inheritance The only known exceptionis a c1430GgtA (D477G) mutation that gives rise to dominant retinitis pigmentosa with delayed onset and choroidal andmacular involvement Position 477 is distant from functionally critical regions of RPE65 Hence the mechanism of D477Gpathogenicity remains unclear although protein misfolding and aggregation mechanisms have been suggested Wecharacterized a D477G knock-in mouse model which exhibited mild age-dependent changes in retinal structure and functionImmunoblot analysis of protein extracts from the eyes of these knock-in mice demonstrated the presence of ubiquitinatedRPE65 and reduced RPE65 expression We observed an accumulation of retinyl esters in the knock-in mice as well as a delayin rhodopsin regeneration kinetics and diminished electroretinography responses indicative of RPE65 functional impairmentinduced by the D477G mutation in vivo However a cell line expressing D477G RPE65 revealed protein expression levelscellular localization and retinoid isomerase activity comparable to cells expressing wild-type protein Structural analysis ofan RPE65 chimera suggested that the D477G mutation does not perturb protein folding or tertiary structure Instead themutation generates an aggregation-prone surface that could induce cellular toxicity through abnormal complex formation assuggested by crystal packing analysis These results indicate that a toxic gain-of-function induced by the D477G RPE65substitution may play a role in the pathogenesis of this form of dominant retinitis pigmentosa

Received February 28 2018 Revised April 2 2018 Accepted April 6 2018

VC The Author(s) 2018 Published by Oxford University Press All rights reserved For permissions please email journalspermissionsoupcom

2225

Human Molecular Genetics 2018 Vol 27 No 13 2225ndash2243

doi 101093hmgddy128Advance Access Publication Date 12 April 2018Original Article

Downloaded from httpsacademicoupcomhmgarticle-abstract271322254969374by Case Western Reserve University useron 25 June 2018

IntroductionThe retinoid (visual) cycle is an enzymatic pathway that regen-erates visual chromophore in vertebrates This metabolic cycletakes place mainly in the retinal pigment epithelium (RPE) asingle layer of cells adjacent to the photoreceptor outer seg-ments Continuous regeneration of visual chromophore is criti-cal for detection of light and defects in the enzymes involved inthe retinoid cycle often culminate in retinal dystrophy One ofthe indispensable enzymes of the visual cycle is an RPE-specificprotein known as RPE65 which is the retinoid isomerase thatcatalyzes cleavage and isomerization of all-trans-retinyl estersto form 11-cis-retinol (1ndash3)

RPE65 is a 61 kDa protein bound to the endoplasmic reticu-lum (ER) membranes of the RPE that is highly conserved amongvertebrates and belongs to the carotenoid cleavage oxygenase(CCO) enzyme family (4ndash7) To date over 100 mutations in RPE65have been implicated in Leber congenital amaurosis (LCA) andretinitis pigmentosa (RP) (8) and almost all of these are inher-ited in a recessive manner However an autosomal dominantform of RPE65-associated RP with choroidal involvement hasalso been identified (910) This novel form of RP arises from amissense mutation in exon 13 of RPE65 (c1430GgtA) thatchanges the amino acid residue encoded at position 477 fromAsp to Gly (D477G)

Carriers of the D477G mutation exhibit varying pheno-types including disease severity and age of disease onsetThey also display areas of retinal atrophy ranging from thechoroid to the photoreceptor layer (910) Unlike typical RPpatients who present with progressive visual field loss fromthe mid to far periphery most carriers of the D477G mutationinitially present with a central visual defect (10) Moreoverchoroidal atrophy and RPE hypertrophy are frequently ob-served and distinctive features associated with this mutation(910)

Previous studies of the RPE65 D477G mutation have sug-gested that the pathological phenotype is attributable to struc-tural destabilization of RPE65 due to increased folding freeenergy (9) and delayed chromophore regeneration resultingfrom dominant-negative suppression of RPE65 retinoid isomer-ase activity (11) However visual cycle activity in heterozygousD477G knock-in (KI) mice can replenish sufficient 11-cis-retinal-dehyde to support relatively normal retinal function (11) Thedisease-associated phenotypes in homozygous KI mice havenot been reported to date so the precise classification of thispoint mutation remains unclear Understanding the pathologi-cal consequences of this mutation is likely to aid in the develop-ment of treatment strategies for this and potentially otherRPE65-associated dominant forms of RP For exampledominant-negative or haploinsufficiency mechanisms wouldimply that RPE65 gene therapy may be of value to D477G sub-jects (12) whereas gain-of-function mechanisms would likelynot be responsive to this intervention

In this study we assessed the phenotype of anindependently-generated D477G mouse line in both the hetero-zygous and homozygous states using a combination of high-resolution imaging histology electroretinography (ERG) andretinoid profile analyses We further studied the consequencesof this mutation at the molecular level with in vitro isomeriza-tion assays RNA sequencing immunoblotting of ubiquitinatedRPE65 and X-ray crystallographic analyses These comprehen-sive studies provide novel insights into the pathogenesis of theD477G mutation

ResultsThe D477G mutation does not affect expressionsubcellular localization and isomerization activityin vitro

To evaluate the intracellular localization and enzymatic func-tion of RPE65 carrying the D477G mutation we used retroviraltransduction to generate two NIH3T3 cell lines that stablyexpress either LRAT that generates substrate for the isomeraseand human RPE65 (hRPE65) or LRAT and the hRPE65 D477G mu-tant Immunoblotting analysis of the cell lines showed compa-rable expression levels of hRPE65 and the D477G mutant(Fig 1A) which differs from R91W G244V and Y368H substitu-tions that cause decreased stability of RPE65 in vitro (1314)Next we examined whether the D477G mutant was properly lo-calized within NIH3T3 cells Immunofluorescence confocal mi-croscopy revealed that hRPE65 co-localized with the ER markercalnexin consistent with previous reports (4515) (Fig 1B) Thislocalization was unchanged for D477G RPE65 suggesting themutation does not affect protein trafficking or membrane asso-ciation in this heterologous expression system (Fig 1B) Ourresults cannot definitively exclude the possibility that protein ismisfolded and retained in the ER However it would be expectedthat such a protein would be quickly degraded Moreover previ-ous work has shown that aggregates of mutant RPE65 includingL22P T101I and L408P point mutants exhibited punctate-like in-clusion bodies whereas wild-type (WT) RPE65 exhibited a ho-mogenous ER distribution (16) Next we assessed whether theD477G mutation changes RPE65 isomerization activity by incu-bating the NIH3T3 cell lines stably expressing LRAT and theD477G mutant or WT RPE65 protein with 10 mM all-trans-retinolfor 16 h Analysis of retinoids extracted from these cellsrevealed comparable production of 11-cis-retinol between thetwo cell lines (Fig 1CndashE) These findings suggest that the D477Gmutation does not affect ER localization and isomerization ac-tivity of RPE65 in cell culture assays

mRNA from D477G RPE65 and ubiquitination of RPE65induced by the D477G mutation

We generated D477G RPE65 KI mice by replacing the endoge-nous RPE65 gene with a targeting vector including exons 8ndash14along with the D477G mutation (Fig 2A) A neomycin (Neo) cas-sette flanked by Flp-recombinase target (FRT) sites was used forclonal selection and later excised by FLP recombinase expressedin the embryonic stem cells The embryonic stem cells carryingthe Neo deleted targeting vector were microinjected into C57Bl6 blastocysts The final KI allele contained the D477G mutationwith one 85-bp FRT site remaining between exons 13 and 14 Aprimer set flanking the FRT site was used to distinguish the WTallele and mutant allele WT mice had a 495-bp PCR productwhereas homozygous mice had a 580-bp PCR product heterozy-gous mice had both PCR products (Fig 2B) Additional sequenc-ing of cDNA from the heterozygous mice showed transcriptionof both WT and D477G mRNA (Fig 2C)

Next we examined whether the D477G mutation affectedthe transcriptional level of RPE65 using real-time PCR RPE65mRNA levels were comparable between WT heterozygous andhomozygous KI mice (Supplementary Material Fig S1) consis-tent with prior data (11) To evaluate the ratio of transcribed WTand D477G mRNA we collected 95 RPE65 cDNA clones fromthe RPE layer of heterozygous mice and sequenced them

2226 | Human Molecular Genetics 2018 Vol 27 No 13


Importantly the number of D477G clones was 375-fold lessthan that of WT clones (Table 1) We note that small differencesin the mRNA secondary structure arising from the single nucle-otide change could potentially bias the outcome of RT-PCR forthe mutant transcript

Next we examined whether the D477G mutation affects trans-lation or susceptibility of the protein to degradationImmunoblotting of RPE lysates revealed that levels of RPE65 ex-pression were decreased in heterozygous and to a greater extentin homozygous KI mice (Fig 3A) Previous studies have showndegradation of RPE65 mutants via the ubiquitin-proteasome path-way in vitro and in vivo (17ndash19) To examine possible ubiquitinationof RPE65 induced by the D477G substitution we immunoprecipi-tated ubiquitinated proteins and then probed for the presence ofRPE65 by immunoblotting Lysates from WT mice did not exhibitubiquitinated RPE65 whereas lysates from heterozygous and ho-mozygous KI mice showed two distinct bands of increased molec-ular weight (Fig 3B) These findings suggest that the D477Gmutation facilitated mono- and di-ubiquitination of RPE65 in vivo

Changes in retinal morphology associated with theD477G mutation

To assess the consequences of the D477G mutation on retinalmorphology we compared WT heterozygous and homozygousKI mice at different ages ranging from 3 to 9 months First weused scanning laser ophthalmoscopy (SLO) with an excitationwavelength of 490 nm and an emission filter of 500ndash700 nm tovisualize the retinal fundus Autofluorescent spots were pre-sent in 9-month-old heterozygous and 6-month-old homozy-gous KI mice (Fig 4) No such spots were observed inage-matched WT mice This may represent inflammatoryresponses in the retina through activation of Muller cells mac-rophages or microglia cells which were observed in several ani-mal models displaying retinal degeneration (20ndash23) Next weperformed optical coherence tomography (OCT) imaging to vi-sualize the retina in cross-section allowing us to determinewhether the D477G mutation leads to retinal degenerationA subtle reduction in outer nuclear layer (ONL) thickness was

RPE65

β-actin

10 20 25 3015

10 20 25 3015

5

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30

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25

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6

Absorb

ance

280 300 320 340 360

11

- cis

-re

tin

ol

(pm

ol1

06

ce

lls)

Absorb

ance a

t 325 n

mA

bsorb

ance a

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m

Time (min)

Time (min)

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G7

74

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318 328

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D477G

calnexin

calnexin

RPE65

RPE65

merged

merged

D

E

Figure 1 Analysis of NIH3T3 cells expressing LRAT and WT hRPE65 or D477G hRPE65 (A) Immunoblot of NIH3T3-LRAT-WT hRPE65 and NIH3T3-LRAT-D477G hRPE65

cells Sample loading was normalized based on the signal for b-actin (B) Subcellular localization of hRPE65 and D477G hRPE65 The merged images show the co-locali-

zation of hRPE65 or D477G RPE65 with the ER marker (calnexin) (C) HPLC analysis of retinoids extracted from NIH3T3 cells expressing LRAT and WT hRPE65 or D477G

hRPE65 Chromatography peaks were identified based on elution time and their characteristic UVvis absorbance spectra Peaks lsquoarsquo correspond to retinyl esters peaks

lsquobrsquo correspond to 11-cis-retinol and peaks lsquocrsquo correspond to 13-cis-retinol (D) UVvis spectra of peaks lsquobrsquo (green) and lsquocrsquo (red) confirming their identities as 11-cis-retinol

and 13-cis-retinol respectively by the characteristic spectral shapes and maximal absorbance wavelengths (E) Quantification of the HPLC results presented in (C)

nfrac143 Data are shown as means 6 SEM

2227Human Molecular Genetics 2018 Vol 27 No 13 |


observed in 6-month-old heterozygous and homozygous KImice and this thinning became more prominent in 9-month-old heterozygous and homozygous KI mice compared with age-matched WT mice (Fig 5A and B) Taken together these resultsindicate the D477G mutation is associated with a slow degener-ation that becomes more evident with aging This finding isconsistent with the phenotype observed in human patientsharboring the same mutation

Reduction in scotopic ERG responses associated with theD477G mutation

A previous study of the D477G mutation reported that rod ERGresponses in patients harboring the mutation were impaired

even in individuals with mild structural abnormalities (9) Todetermine whether D477G affects the functional integrity ofmouse retina and the rod photoreceptors in particular we per-formed dark-adapted (scotopic) ERG recordings in both hetero-zygous and homozygous KI mice at 3 6 and 9 months of ageERG analysis in mice did not reveal a significant difference in a-and b-wave amplitudes between WT heterozygous and homo-zygous KI mice up to 6-months of age However at 9-months ofage scotopic a- and b-wave responses in both heterozygous andhomozygous KI mice declined compared with the correspond-ing responses in WT mice (Fig 6A and B and SupplementaryMaterial Fig S2A) Similar results were obtained for photopic a-and b-wave amplitudes (Fig 6C and D and SupplementaryMaterial Fig S2B) This result is consistent with the progressivethinning of the ONL in heterozygous and homozygous KI miceobserved by OCT imaging and the later age of onset in humanswith the D477G mutation compared with those with RPE65-associated recessive RP

Impact of the D477G mutation on the mouseretinoid cycle

Animal models carrying P25L R91W and L450M mutations inRPE65 have demonstrated delayed visual chromophore

WT

WT

WT

KI

KIK

I

WT RPE65D477G RPE65

Neo

Neo

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475 476 477 478 479

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Codon

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A

B C

Genotyping whole KI locus

D G

F1

R1

D G

Ex7 8 9 10 11 12 13 14

WT RPE65

Targeting

Vector

Targeted

RPE65

Final

KI RPE65

Figure 2 Targeting strategy used to generate D477G KI mice and molecular characterization (A) D477G KI mice were generated by homologous recombination with an

113 kb targeting vector containing the FRT-flanked Neo cassette between exons 13 and 14 Codon 477 in exon 13 was changed from GAT to GGT This change results in

a D to G amino acid substitution The FRT-flanked Neo cassette was deleted by FRT-FLP recombination to generate D477G RPE65 KI mice The mouse retained an 85-bp

FRT site in the intron between exons 13 and 14 (B) PCR primers flanking the FRT site as shown in the bottom scheme of (A) were used for genotyping The WT allele

and D477G KI allele produced 495- and 580-bp PCR products respectively (C) Sequencing chromatograms of two RPE65 cDNA clones from the RPE of a 3-month-

old heterozygous KI mouse confirmed the presence of both WT and mutant mRNAs F forward R reverse KI knock-in WT wild-type

Table 1 cDNA ratio of D477G RPE65 to WT RPE65 revealed a decreasein mRNAs transcribed from RPE65 (c1430GgtA)

Mouse ID No of WT clones No of D477G clones

1 41 72 34 13Total 75 20



regeneration (182425) Therefore we examined whether theD477G mutation perturbs the visual cycle We analyzed the reti-noid composition in dark-adapted WT heterozygous and ho-mozygous KI mice at different ages ranging from 1 to 3 monthsThe amount of 11-cis-retinal in heterozygous and homozygousKI mice did not differ from WT mice whereas the amount ofretinyl esters in heterozygous and homozygous KI mice washigher than that in WT mice (Fig 7AndashC) This elevated level ofretinyl esters could be due to a feedback response to aberrantmutant protein RPE65 could also play a role in retinyl ester mo-bilization from lipid droplet stores (retinosomes) (2627) outsideof its role in cleaving and isomerizing retinyl esters for visualchromophore production as recently suggested (28)

Next we examined the amount of 11-cis-retinal in WT het-erozygous and homozygous KI mice dark adapted for 4 8 12and 20 h following a 10 min bleach Compared with WT micehomozygous KI mice had significantly lower amounts of 11-cis-retinal after 4 and 8 h of dark adaptation however the amountsbecame comparable with those of WT mice after 20 h of darkadaptation (Fig 7D) Heterozygous KI mice had significantlylower amounts of 11-cis-retinal after 4 h of dark adaptation butlevels became comparable with WT mice at 8 h of dark adapta-tion This result was further supported by the elevated quantityof retinyl esters found in homozygous (and at some time pointsheterozygous) KI mice (Fig 7E) Additionally we assessed the re-generation of 11-cis-retinal with the shorter dark-adaption pe-riod WT heterozygous and homozygous KI mice were exposedto a 10 min bleach followed by dark adaptation for 10 min As

Ub Pulldown

A B

WT

WT

WT

KI

KIK

I

150

10075

50

37

20

RPE65

β-actin

WT

WT

WT

KI

KIK

I

Figure 3 Decreased RPE65 protein level and ubiquitination of RPE65 in D477G KI

mice (A) RPE lysates from wild-type (WTWT) heterozygous (WTKI) and homo-

zygous (KIKI) mice were subjected to immunoblotting using an in-house gener-

ated RPE65 antibody Equal protein sample loading was verified by b-actin

immunoblotting Note the slight reduction in RPE65 protein levels in the WTKI

sample and more substantial reduction in the KIKI sample Lower molecular

weight bands recognized by the RPE65 antibody were present in the WTKI and

KIKI samples but not WTWT samples suggesting an elevated susceptibility to

proteolysis induced by the D477G substitution (B) RPE lysates from WTWT

WTKI and KIKI mice were subjected to immunoprecipitation with a ubiquitin

antibody followed by immunoblotting with RPE65 antibody Ubiquitinated RPE65

was present in WTKI and to a greater extent KIKI samples whereas in the WT

WT sample it was scarcely detectable This finding suggests that proteolysis

could play a role in the reduced RPE65 levels observed in the KI mice

6-month-old3-month-old 9-month-old

WTWT

WTKI

KIKI

Figure 4 Fundus autofluorescent spots in D477G KI mice Representative SLO fundus images from wild-type (WTWT) heterozygous (WTKI) and homozygous (KIKI)

mice at 3 6 and 9 months of age Autofluorescent spots were first noted in 9-month-old heterozygous and 6-month-old homozygous KI mice fundus images whereas

such spots were scarcely visible in the WTWT fundus



expected levels of 11-cis-retinal were lower in heterozygousand homozygous mice compared to WT (Fig 7F) This resultwas further supported by the elevated levels of retinyl estersfound in heterozygous and homozygous KI mice (Fig 7G) Takentogether this retinoid profile suggests the 11-cis-retinal regener-ation rate was reduced in both heterozygous and homozygousKI mice

Analysis of the D477 structural environment

To better understand the pathogenicity of the D477G substitu-tion we analyzed the structure of the region of the RPE65 se-quence near this position Asp is conserved at position 477 inRPE65 from primates to sea lamprey consistent with an impor-tant role for this residue (Fig 8A) The ExAC database revealedno missense mutations at position 477 although a synonymoussubstitution was documented (29) Interestingly glycine isfound at a position equivalent to position 477 of human RPE65in some CCO enzymes including human BCO1 and BCO2 as wellas Synechocystis ACO indicating that Gly is tolerated for proteinstructure and function in the presence of a suitable surroundingsequence (Fig 8A) The D477-containing loop and nearby resi-dues in the surrounding bstrands are relatively well conservedbetween RPE65 and ACO compared with the protein chains as awhole which exhibited only 25 sequence identity

D477 occupies the nthorn 2 position of a type-1 b-turn elementfound within a loop connecting strands 32 and 33 of theb-propeller fold (30) Our inspection of known RPE65 crystalstructures revealed a consistent secondary structure for thisprotein region with some rigid body variation in its overall posi-tion This loop is located distant from the active site does notcontribute to the membrane binding surface of the enzyme andis not involved in formation of the conserved dimeric interfaceor any other proteinndashprotein interaction preserved acrossknown crystal structures of RPE65 (Fig 8B) (3132) Hence a criti-cal structural or functional role for D477 is not obvious from itsposition within the context of the three-dimensional structureHowever bturns are known to exert pivotal roles in proteinfolding (33) and in proteinndashprotein interactions (34) Thus the

D477G substitution could perturb the normal structure of thisbturn causing pathogenic protein misfolding as discussed pre-viously (911) For example a glycyl residue in the nthorn 2 positionof the bturn could promote switching to a type-II conforma-tion (34) thereby promoting the above-mentioned effectsIndeed inspection of PDB entries containing the ldquoHPGArdquo se-quence within a bturn motif revealed that most are in a type-IIconformation (35) bturns with an ldquoHPGArdquo sequence wereobserved only in non-metazoan species and are less commonthan the turns comprised of ldquoHPDArdquo and ldquoRPGGrdquo sequencesfound in RPE65 and ACO respectively

Design and expression of a chimeric ACO proteincontaining the D477 loop

To date it has not proven feasible to crystallize recombinantlyproduced RPE65 hampering direct examination of amino acidsubstitution effects on RPE65 structure To circumvent this diffi-culty we exploited the high structural similarity between RPE65and ACO near position 477 (Fig 8C) to design a chimeric proteinin which residues 474ndash485 of RPE65 were replaced in the equiva-lent position of the ACO sequence (Fig 8A) We also swappedfour other residues in the ACO sequence to their RPE65 counter-parts to maintain important chemical interactions and avoidsteric clashes (Fig 8A) We expressed this hybrid protein referredto herein as ldquoACORPE65ndash477-looprdquo and its corresponding D477Gmutant in E coli using the procedure previously described for WTACO (36) The two hybrid proteins were expressed in the solublefraction at levels comparable with WT ACO (Fig 9A left) andcould also be purified by the same protocol with a similar yield asACO (Fig 9A right and Fig 9B) All three enzymes also possessedcomparable catalytic activities (Fig 9C and SupplementaryMaterial Fig S3) Given that the D477 loop is structurally isolated(Fig 8B) we expected that sequence differences outside of thisloop would have minimal impact on the potential influence ofthe D477G substitution on protein folding in either RPE65 or ACORPE65ndash477-loop Based on this assumption these results indicatethat the D477G substitution does not have a marked effect onprotein folding which is consistent with the in vitro data we

B

AWTWT

WTKI

KIKI

3-month-old 6-month-old 9-month-old

ON

L T

hic

kness (

microm

)

0

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70

WTWT WTKI KIKI

57 microm

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50 microm

ON

L T

hic

kness (

microm

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ON

L T

hic

kness (

microm

)

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70

WTWT WTKI KIKI WTWT WTKI KIKI

Figure 5 Retinal degeneration in D477G KI mice over time (A) Representative OCT images of retinae crossing the optic nerve head from wild-type (WTWT) heterozy-

gous (WTKI) and homozygous (KIKI) mice at 9 months of age Heterozygous and homozygous retinae displayed decreased thickness of the ONL (indicated by the

brackets) compared with age-matched wild-type retina (B) ONL layer thickness in wild-type (WTWT) heterozygous (WTKI) and homozygous (KIKI) mice at 3 6 and 9

months of age as measured by OCT Both heterozygous and homozygous mice underwent mild progressive photoreceptor degeneration nfrac144ndash5 for the WTWT group

nfrac145 for the WTKI group nfrac145 for the KIKI group indicates Plt001 Data are shown as mean 6 SEM



A

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-10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 250

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3-month-old

3-month-old

3-month-old

6-month-old

6-month-old

6-month-old

9-month-old

9-month-old

9-month-old

WTWTWTKIKIKI

WTWTWTKIKIKI

WTWTWTKIKIKI

WTWTWTKIKIKI

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)

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top

ic b

-wa

ve

am

plit

ud

e (

microV

)

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top

ic b

-wa

ve

am

plit

ud

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microV

)

Light intensity [log (cdsmsup2)] Light intensity [log (cdsmsup2)] Light intensity [log (cdsmsup2)]

Sco

top

ic a

-wa

ve

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plit

ud

e (

microV

)

Light intensity [log (cdsmsup2)]

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plit

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e (

microV

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)

Light intensity [log (cdsmsup2)] Light intensity [log (cdsmsup2)]

Ph

oto

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a-w

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microV

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ave

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microV

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ave

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microV

)



Ph

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b-w

ave

am

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microV

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ave

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microV

)

Ph

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b-w

ave

am

plit

ud

e (

microV

)

Figure 6 ERG responses of wild-type (WTWT) heterozygous (WTKI) and homozygous (KIKI) mice at 3 6 and 9 months of age (A) To compare the retinal function be-

tween WT and KI mice the scotopic a- and b-wave amplitudes were plotted as a function of light stimulus intensity (37 to 23 log cdsm2) The a-wave amplitude

which reflects the initial response of photoreceptors to a light stimulus did not show significant difference between 3-month-old and 6-month-old WTWT WTKI

and KIKI mice However at 9 months of age both WTKI and KIKI mice displayed significant reduction in a-wave amplitudes compared with WTWT mice (Plt001)

suggesting that the mutation may cause a delayed onset of pathological phenotype (B) Likewise the amplitudes of the scotopic b-wave which reflects the response of

downstream retinal neurons to photoreceptor stimulation also began to decline in 9-month-old WTKI and KIKI mice compared with WTWT mice (Plt005) (C) To

specifically examine the cone function the photopic a- and b-wave amplitudes were plotted as a function of light stimulus intensity (07 to 23 log cdsm2) Similar to

the results of scotopic ERG the photopic a-wave amplitude began to decline in 9-month-old WTKI and KIKI mice (Plt005) (D) The photopic b-wave amplitude was

also reduced in 9-month-old WTKI and KIKI mice (Plt 005) Different sets of animals were used for ERG recordings at each time point nfrac144 for the WTWT group

nfrac144 for the WTKI group nfrac144 for the KIKI group Data are shown as mean 6 SEM



presented on RPE65 D477G expression and activity in mamma-lian cells (Fig 1A and E)

Crystallization and structural determination of the ACORPE65 D477 chimera

To directly examine the structural preservation of the D477 loopin RPE65 and the ACORPE65 chimera we crystallized ACO

RPE65ndash477 loop under the same conditions used for WT ACO (36)The crystals diffracted X-rays to 25 A resolution and were iso-morphous to the previously reported orthorhombic crystal formof ACO with four monomers in the asymmetric unit (Table 2) (37)Inspection of the initial electron density maps revealed cleardifference density for the substituted residues Following refine-ment (Table 2) we found the loop conformation to be nearlyidentical to that of RPE65 with an overall root-mean-square

A B

D E

C

F G

0

100

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300

400

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11-c

is-r

etin

al (

pm

ole

ye)

0

100

200

300

400

500

600

11-c

is-r

etin

al (

pm

ole

ye)

0

50

100

150

200

250

300

Re

tinyl

est

ers

(p

mo

leye

)

0

50

100

150

200

250

300

Re

tinyl

est

ers

(p

mo

leye

)

1-month-old 3-month-old


WTWT WTKI KIKIWTWT WTKI KIKI


0

200

400

600

800

Re

tin

yl e

ste

rs (

pm

ole

ye

)

Time after bleaching (h)0 5 10 15 20


0

100

200

300

400

500

11

-cis

-re

tin

al (p

mo

le

ye

)

WTWT

WTKI

KIKI

a

b

a

b

a

b

0 10 20 30 40Time (min)

0 10 20 30 40Time (min)

0 10 20 30 40Time (min)

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

WTWT WTKI KIKI0

10

20

30

40

50

60

11

-cis

-re

tin

al (p

mo

le

ye

)

WTWT WTKI KIKI0

200

400

600

800

1000

1200

Re

tin

yl e

ste

rs (

pm

ole

ye

)

WTWTWTKIKIKI

WTWTWTKIKIKI

Figure 7 Retinoid profile of D477G KI mice and analysis of 11-cis-retinal regeneration after bleaching (A) HPLC chromatograms showing the retinoid composition of

eye extracts from fully dark-adapted wild-type (WTWT) heterozygous (WTKI) and homozygous (KIKI) mice Peaks in the chromatograms were identified based on

elution time and characteristic UVvis absorbance spectra Peaks lsquoarsquo correspond to retinyl esters and peaks lsquobrsquo correspond to 11-cis-retinal (B) Amount of retinyl esters

in fully dark-adapted wild-type heterozygous and homozygous mice nfrac146 for 1-month-old WTWT group nfrac148 for 1-month-old WTKI group nfrac144 for 1-month-old

KIKI group nfrac144 for 3-month-old WTWT group nfrac144 for 3-month-old WTKI group and nfrac143 for 3-month-old KIKI group indicates Plt 005 Data are shown as

mean 6 SEM (C) Amount of 11-cis-retinal in fully dark-adapted wild-type heterozygous and homozygous mice nfrac146 for 1-month-old WTWT group nfrac148 for 1-

month-old WTKI group nfrac144 for 1-month-old KIKI group nfrac144 for 3-month-old WTWT group nfrac144 for 3-month-old WTKI group and nfrac143 for 3-month-old KIKI

group Data are shown as mean 6 SEM Differences between groups all had P-valuesgt005 (D) Regeneration of 11-cis-retinal following a bleach (5000 lux light for 10

min) in 3-month-old wild-type heterozygous and homozygous mice after 0 4 8 and 20 h of dark adaptation nfrac143ndash4 for each time point The recovery of the chromo-

phore was significantly delayed in heterozygous mice until 4 h of dark adaptation (Plt 005) and homozygous mice until 8 h of dark adaptation (Plt 0001) Data are

shown as mean 6 SEM Significance between groups was determined with one-way ANOVA and the Bonferroni post-test Curve fitting was performed with the follow-

ing equation in SigmaPlot [exponential rise to the max yfrac14a(1 ebx)] (E) Changes of retinyl esters following a bleach (5000 lux light for 10 min) in 3-month-old wild-

type heterozygous and homozygous mice after 0 4 8 and 20 h of dark adaptation nfrac143ndash4 for each time point Data are shown as mean 6 SEM (F) Regeneration of 11-

cis-retinal following a bleach (5000 lux light for 10 min) in 9-month-old wild-type heterozygous and homozygous mice after 10 min of dark adaptation nfrac14 3 for the

WTWT group nfrac14 5 for the WTKI group and nfrac143 for the KIKI group Data are shown as mean 6 SEM (G) Levels of retinyl esters following a bleach (5000 lux light for

10 min) in 9-month-old wild-type heterozygous and homozygous mice after 10 min of dark adaptation nfrac143 for the WTWT group nfrac145 for the WTKI group and nfrac143

for the KIKI group Data are shown as mean 6 SEM



TT

P GI LT LW P GI LT LW P GI LT LW P GI LR TW P GI IK SF P GI LS SW P GF LN KW P GI LQ AW

343434343434762442 P GI LQ PD

TT

TT TT

------ 472 F P ED G V D VVL SH DALE ------ 472 F P ED G V D VVL SH DALE ------ 472 F P ED G V D VVL SQ DALE ------ 472 F P ED G V D VVL SH DALE ------ 472 F P ED G V D ILM QT DGVD ------ 472 F P ED G I D VLL AR DAQD ------ 519 F P ED G V G VIL PA GTNE ------ 460 F P ED G V D VIL PA GAKD ------ 429 F P ED G V D WLL PR GGVA

TT TT

------ 505 L A KD N ------ 505 L A KD N ------ 505 L A KD N ------ 505 L A KD N ------ 504 L A KD N ------ 506 L A KD N ------ 551 L A KN D ------ 492 L A KS D ------ 460 L A QD D

TT

HumanCowMouseChickenZebrafishSea lampreyHuman BCO2Human BCO1Synechocystis ACO

Membrane-binding surface

Dimerization

interface

Asp477

Phe472

Asp477

Leu487

Phe429

Gly434

Leu444

A

B C RPE65 ACO

Figure 8 Structural analysis of the D477 loop region in RPE65 and its homologs (A) Alignment of RPE65 orthologs human BCO enzymes and Synechocystis ACO amino

acid sequences Positions swapped to generate the ACORPE65 chimera are marked with black triangles The purple star marks position 477 in RPE65 Numbers in front

of the aligned sequences denote the beginning sequence position Secondary structure elements determined by X-ray crystallography for RPE65 and ACO are displayed

on the top and bottom of the aligned sequences respectively (B) Location of the Asp477 loop within the three-dimensional structure of RPE65 (C) leftmdashStick represen-

tation of the RPE65 loop region containing Asp477 RightmdashStick representation of the corresponding loop in ACO Note the close structural similarity between the two

proteins as well as the difference in b-turn conformation marked by an asterisk

A

250

150

10075

50

37

25

20

ACOACO

RPE65

ACO

RPE65

D477G

ACOACO

RPE65

ACO

RPE65

D477G

Soluble Purified B C

Volume (mL)

20 40 60 80

Absorb

ance a

t 280 n

m

ACOACORPE65ACORPE65

D477G

Time (min)

0 1 2 3

Pro

du

ct

En

zym

e (microm

olmicrom

ol)

0

20

40

60 ACORPE65

D477G

ACOACORPE

Figure 9 Expression purification and enzymatic activity of ACORPE65 chimeras (A) SDS-PAGE of soluble fractions obtained from lysates of E coli expressing the indi-

cated constructs (left) as well as the corresponding final purified protein samples (right) Proteins were visualized by Coomassie Blue staining (B) Gel filtration elution

profiles of the indicated proteins The horizontal bracket indicates the protein peak corresponding to monomeric protein (C) Apocarotenoid oxygenase activity of the

three purified proteins Data are shown as mean 6 SD from three separate experiments each performed in triplicate



difference (RMSD) of 056 A for all atoms in the loop based on aglobal superposition of the two structures (Fig 10A) These resultsvalidated our use of ACORPE65-D477 loop to infer the structuralconsequences of the RPE65 D477G mutation

Crystallization and structural determination of the ACORPE65 D477G mutant

Next we crystallized the D477G chimera variant again underthe same conditions used for WT ACO Strikingly this singleamino acid change resulted in the formation of trigonal andmonoclinic crystals unprecedented amongst prior ACO andRPE65 crystallographic studies These crystals grew eitheralone or as polymorphs with both forms present in a singledrop (Supplementary Material Fig S4) whereas the original or-thorhombic crystals were not observed for the point mutantThe trigonal crystals diffracted X-rays anisotropically to 31 Aresolution and belonged to the rare space group P312 with twomolecules in the asymmetric unit The monoclinic crystals dif-fracted X-rays more isotropically to a resolution of 26 A and

belonged to space group C2 also with two molecules in theasymmetric unit (Table 2) Refined structures of these two crys-tal forms revealed the expected absence of an aspartic acid sidechain at position 434 (equivalent to position 477 in RPE65)The overall structure of the D477G loop region was like that ofthe D477 chimera (main chain RMSD of 048ndash086 A dependingon the specific chain) with a few notable differences (Fig 10Band C) Focusing on the monoclinic crystal form for which theelectron density support was strongest the D477G-containingbturn was found in both a type-I (chain A) and a type-II (chainB) conformation This variability is not unexpected given theconflicting tendencies of the His residue in position ldquonrdquo to favora type-I conformation through hydrogen bonding with the am-ide nitrogen of residue 434 and the glycyl residue at the nthorn 2 po-sition allowing the flipped orientation of the 433ndash434 peptidebond characteristic of a type-II turn Additionally Leu436 wasdisplaced by 05ndash14 A depending on the subunit and the sidechains for Glu437 and Glu440 adopted different rotameric con-formations compared with the ACORPE65-D477 loop The latteralterations are likely due to a difference in crystal packing asdiscussed below In both crystal forms the electron density

Table 2 X-ray diffraction data collection and structure refinement statistics

Data collection and processing

Crystal ACORPE65 chimera ACORPE65 chimeraD477Ga trigonal crystal form

ACORPE65 chimeraD477G monoclinic crystal form

X-ray source NSLS-II 17-ID-2 APS 24-IDENSLS-II 17-ID-1 NSLS-II 17-ID-1Wavelength (A) 0979357 0979180918401 0918401Space group P212121 P312 C2Unit cell lengths (A) a frac14 11935 b frac14 12566 c frac14 20441 a frac14 12652 c frac14 159767 a frac14 19143 b frac14 5617 c frac14 13691Unit cell angles () b frac14 13383Resolution (A)b 50mdash25 (265 25) 50mdash315 (323mdash315) 50mdash260 (276mdash260)Unique reflections 106654 (16940) 25537 (4090) 31744 (5023)Multiplicity 87 (86) 186 (180) 31 (30)Completeness () 998 (994) 999 (100) 967 (96)ltIrIgt 95 (099) 160 (12) 116 (093)RmergeI () 196 (1752) 125 (2916) 66 (998)CC12 () 996 (525) 100 (673) 998 (489)Wilson B factor (A2) 62 121 76RefinementResolution (A) 4884ndash25 4965ndash315 4176 (26)No reflectionsc 101 462 (5100) 24 267 (1269) 30 156 (1588)RworkRfree () 181217 232266 220260No atoms 15 813 7541 7546Protein 15 082 7536 7491Iron 4 2 2Water 725 3 53ltB-factorgt (A2) 62 168 79Protein 62 168 79Iron 53 177 80Water 56 106 58RMS deviationsBond lengths (A) 0012 0006 0007Bond angles () 153 0968 111Ramachandran plot ( favoredoutliers)4 9790 9530 9690Molprobity score () 100 100 100PDB accession code 6C7K 6C7O 6C7P

aData from two isomorphous crystals were merged to produce the final datasetbValues in parentheses are for the highest resolution shell of datacValues in parentheses are the number of reflections used for cross-validation



corresponding to the D477 loop and surrounding residues waswell resolved indicating that the D477G substitution does notstructurally destabilize this loop region

The D477G substitution causes a gain of proteinndashproteininteraction potential for the 477 loop

Molecular packing in the D477G crystals differed in a strikingway from that of the original ACORPE65-D477-loop proteinWhereas the D477 loop was not involved in significant proteinndashprotein contacts in the former crystal form (SupplementaryMaterial Fig S5) contacts involving this loop were integrally in-volved in the crystal packing of both new crystal forms (Fig 11)In the trigonal crystals the D477G loop regions of the twomonomers within the asymmetric unit engage in two non-equivalent proteinndashprotein interactions with contact surfaceareas of 477 and 411 A2 involving predominantly van der Waalsinteractions with a few H-bonds and salt bridges (Fig 11Aand B) The two interactions occur at roughly the same site ofthe ACORPE65-D477G molecule but are rotated by 180 with re-spect to each other and involve two distinct sets of residues Inthe monoclinic crystals the D477G loop regions of the two mol-ecules in the asymmetric unit engage in two non-identical butsimilar proteinndashprotein interactions with contact surface areasof 415 and 389 A2 (Fig 11C and D) The two contact points are re-lated by a slight rotation and involve similar sets of residuesforming distinct combinations of van der Waals and polarionicinteractions In both crystal forms these predominantly hydro-phobic interactions involving the D477G loop are among themajor contact points responsible for crystal formation In allfour cases multiple van der Waals interactions formed betweenthe G434 a carbon and interacting residues indicate that the ab-sence of the Asp side chain at position 434 crucially enabled for-mation of the proteinndashprotein contacts The D477G substitutionthus triggered a gain of proteinndashprotein interaction potential byallowing the 477 loop to form contacts with diverse molecularsurfaces

DiscussionUntil recently RPE65-associated RP and LCA were only knownto occur as recessive diseases with complete penetrance andgenerally early onset typically progressing to legal blindnesswithin the first or second decade of human life (38) The discov-ery of a phenotypically distinct dominant form of RPE65-associated RP linked to the D477G mutation with a differenttime course of disease and incomplete penetrance was thussurprising when it was reported (9) The D477G mutation isqualitatively different from other pathogenic RPE65 mutationsreported to date Recessive RP-associated mutations are typi-cally located in critical active site regions or at the interfaces ofthe beta propeller blades which directly affect the catalytic ac-tivity folding trajectory or tertiary structure stability of the en-zyme (13) By contrast D477 is located in a solvent-exposed looplocated away from functionally critical regions of the protein(Fig 8B) It is also striking that closely related RPE65 familymembers encode a Gly residue at a position in their proteinchain equivalent to residue 477 in RPE65 (Fig 8A) Hence thepathogenic mechanism of this missense mutation hasremained mysterious although it was previously proposed thatthe D477G substitution could result in protein misfolding andoraggregation (910)

In this report we provide evidence that the D477G substitu-tion does not induce a protein-folding defect similarly sparingany significant perturbation of the three-dimensional struc-ture and catalytic activity of the protein Instead we findthat it changes the physiochemical properties of theD477-containing loop to produce an aggregation-prone surfacecapable of engaging in abnormal proteinndashprotein interactionsThis change in interaction potential was directly observed athigh resolution with an ACORPE65 chimeric protein contain-ing the D477 loop and surrounding residues of RPE65The change of position 477 from Asp to Gly in this protein withall other experimental variables held constant enabled the477 loop to engage in diverse proteinndashprotein interactions asobserved by crystal packing analysis in two distinct crystal

ACORPE65

A B C

vs RPE65D477G conf 1

vs ACORPE65D477G conf 2

vs ACORPE65

Trp45

(37)

Lys463

(508)

Asn461

(506)

Ser431

(474)

Val442

(485)

Asp434

(477)

Figure 10 Structure of the D477 loop region in ACORPE65 chimera proteins (A) Structure of the D477 loop region in ACORPE65 chimera (blue to green gradient) com-

pared with bovine RPE65 (gray) Auxiliary substituted residues in ACORPE65 chimera and the corresponding residues in bovine RPE65 are colored light orange and

gray respectively The Leu residue substituted at position 44 is omitted for clarity The D477 position is marked by an asterisk Top residue labeling refers to the ACO

RPE65 sequence whereas the numbers on bottom (in gray) refer to the native RPE65 sequence Note the close structural correspondence between the two proteins in

this region (B) Comparison of the same loop region of ACORPE65 D477G chimera monoclinic crystal form with the ACORPE65 D477 chimera (shown in gray) (C) Same

as panel B but showing the conformation of the second molecule located in the asymmetric unit of the monoclinic crystal form Note the two distinct orientations of

the b-turn marked with asterisks with (B) showing a type II turn and (C) showing a type I turn



forms Among the possible interactions that could be alteredin vivo we observed that D477G RPE65 is subject to recognitionby ubiquitin ligases likely due to the increased hydrophobicityof the 477 loop imparted by loss of the negatively charged Aspside chain (Supplementary Material Fig S6) This interpreta-tion is supported by previous studies demonstrating that hy-drophobic surfaces with the potential to cause insolubility oraggregation are recognized by the Ubc6Ubc7-containing ubiq-uitination complex (39) Ubiquitination of RPE65 likely targetsit for proteasomal degradation as reported previously for re-cessive RP-associated RPE65 mutations in vitro (17) A similarphenomenon was also observed in R91W RPE65 KI mice wheremutant RPE65 was polyubiquitinated and subsequently de-graded by the 26S proteasome (19) We observed that the de-gree of ubiquitination and degeneration was more extensive inhomozygous KI mice consistent with the retinoid profile insuch mice Therefore two copies of the mutant allele in homo-zygous KI mice exacerbated their phenotypes which could beinterpreted as the D477G allele being semi-dominant (40)Besides the protein-associated effect we also observed thatthe c1430GgtA point mutation reduces mRNA levels relative toWT RPE65 mRNA suggesting a defect in RNA stability or proc-essing (Fig 12) It remains possible that the reduction in D477GmRNA is a result of the remaining loxP site in the mutant

allele Nevertheless total RPE65 mRNA levels were comparablebetween the three genotypes as observed previously (11) sug-gesting that reduced processing or stability of D477G mRNAcan be compensated for by transcriptional upregulation

The reduced levels of RPE65 we and others have observed inD477G KI mice raise the possibility of haploinsufficiency beinginvolved in the disease pathogenesis Although a few studieshave suggested the possibility of retinal disease in carriers of re-cessive RP-associated RPE65 alleles (1641) others specificallylooking at the potential presence of retinal disease in RPE65-null mutant carriers have consistently concluded that heterozy-gous loss of RPE65 function (pure haploinsufficiency) does notadversely affect visual function or retinal structure in a clini-cally meaningful way (42ndash44) Likewise P25L and R91W KI micewhich are models of mild and severe RP respectively do not ex-hibit abnormal visual phenotypes in marked contrast to the re-spective homozygous KI mice Heterozygous RPE65 KO mouseretinal structure and function are also indistinguishable fromWT mice (4) Thus haploinsufficiency appears not to underliethe retinal structural and functional defects associated with theD477G mutation

The potential pathogenic mechanisms mentioned in thepreceding text help explain the phenotypes we and others (11)have observed in RPE65 D477G KI mice These mice exhibit an

A B

C D

Figure 11 Involvement of the D477 loop in crystal packing of ACORPE65 chimera D477G mutant (A) Molecular packing observed in the trigonal crystal form revealing

an interaction of the D477G loop and surrounding residues (all shown as van der Waals spheres) with the second molecule in the asymmetric unit (contact I) This sec-

ond molecule in turn also interacts with a symmetry-related molecule through the region surrounding the D477G loop (contact II) (B) Details of the interactions formed

between the D477G loop and surrounding residues with the interaction partner Dashed lines indicate van der Waals contacts (width proportional to number of atomic

contacts) solid blue lines indicate hydrogen bonds and solid red lines indicate salt bridges (C) Molecular packing of molecules in the monoclinic crystal form mediated

by the D477G loop and surrounding residues Contact III involves the two molecules in the asymmetric unit whereas contact IV involves a symmetry-related molecule

These two contacts are related by a slight rotation (D) Interactions formed between the D477G loop and residues from interacting partners in the monoclinic crystal

form In all four unique contacts the introduction of an Asp side chain at position 434 would result in steric clashes which explains why these two crystal forms are

only observed for the D477G mutant



overall visual cycle insufficiency manifested by ERG suppres-sion in older mice a delay in visual chromophore regenerationand the accumulation of retinyl esters in both heterozygous andhomozygous mice These phenotypes can be attributed to re-duced levels of functional RPE65 in KI mice likely due to a re-duction in RPE65 protein levels resulting from its ubiquinationand proteolytic degradation (Fig 12) as well as a potentialdominant-negative effect exerted by the D477G mutant onvisual chromophore production which could suppress visualcycle activity to a pathogenic level (11) Given that RPE65 islikely a dimeric protein in vivo (32) the dominant-negativeeffect could be explained by heterodimer formation betweenWT and D477G resulting in potential mislocalization andordegradation of the dimeric complex thus reducing WT proteinfunction (Fig 12)

Besides effects on visual cycle function we also observedpathological autofluorescent bodies in the retinas of heterozy-gous and homozygous KI mice by SLO imaging Although thesestructures cannot be definitively identified by SLO prior studieson animal disease models including Palm (20) E150K opsin(22) and ABCA4Rdh8 (23) mice indicate that autofluores-cent bodies reflect the infiltration of phagocytic cells into theretina In addition to this abnormality we also observed a milddecrement in photoreceptor number in mice of both genotypesrelative to WT controls by OCT imaging This loss of photore-ceptors could originate either from visual chromophore

deficiency as described previously for RPE65-associated reces-sive RP or from a generalized RPE dysfunction that perturbs theability of the RPE to support photoreceptor health (45) The lattermechanism which implicates a direct toxic effect of D477GRPE65 on the RPE is suggested by RPE hypertrophy and choroi-dal atrophy observed in humans carrying the RPE65 D477G sub-stitution (910) We note that the mild loss of photoreceptors inthese KI mice cannot fully account for the observed decrease inERG responses which again points to visual cycle insufficiencyin these animals

Although gain-of-function mutations affecting protein fold-ing and aggregation are most frequently associated with the for-mation of intracellular fibrils and amyloid as in Alzheimerdisease and systemic amyloidosis (46) such mutations can alsoexert subtler effects on cellular homeostasis not involving for-mation of beta-amyloid or other beta-sheet rich aggregates(4748) Recent studies on proteinndashchaperone interactions havesuggested that potentially aggregation-prone regions of a pro-tein are frequently protected by so-called ldquogatekeeperrdquo residuespresent within the peptide sequence (4849) These typically hy-drophilic residues (Arg Lys Glu and Asp) interrupt regions ofhydrophobicity and may also increase surface entropy at other-wise aggregation-prone sites of a protein Our data suggest thatD477 could serve such a role in RPE65 as the Gly replacementallows formation of predominantly hydrophobic contacts thatwould be prohibited by the negatively charged Asp side chain

Figure 12 Schematic diagram showing a proposed pathogenic mechanism of RP resulting from the D477G RPE65 mutation In the RPE of WTWT mice RPE65 is

localized in the ER membrane following translation and contributes to the visual cycle by isomerizing all-trans-retinyl esters to form 11-cis-retinol In RPE of WTKI

mice mRNA from the D477G RPE65 allele is significantly reduced The hydrophobic neomorphic surface of D477G RPE65 could produce aggregates in the cytoplasm and

impair the ER localization of RPE65 Due to the possibility of dimerization between D477G and WT RPE65 the mutation could also result in mislocalization of WT RPE65

resulting in a dominant-negative effect as described previously for D477G KI mice The aggregates are ubiquitinated and degraded by proteasomes



Loss of a surface-exposed gatekeeper residue leading to theaggregation of properly folded protein molecules as we suggesthere for D477G RPE65 has previously been documented for thesickle-cell anemia-associated E6V substitution in hemoglobin(known as hemoglobin S) which predisposes the protein to ab-normal oligomerization (50) This mutation does not affect thesecondary tertiary or quaternary structures of hemoglobin atnormal oxygen concentrations (51) However during hypoxiathe protein can form abnormal homo-oligomers that generatecharacteristic sickling of red blood cells Individuals carryingone abnormal allele only exhibit symptoms of sickle-cell ane-mia in the presence of stressors such as severe hydration orhigh-intensity physical activity (52ndash54) Considering the rela-tively mild phenotype in the D477G KI mouse model and the in-complete penetrance of D477G-associated disease in humans(910) the pathogenic mechanism of D477G conceivablyrequires extra factors such as environmental stressors and age-related physiological changes as triggers

The R36S mutation in cD crystallin also exhibits abnormalproteinndashprotein interactions enabled by loss of a gatekeeper res-idue that leads to dominant ophthalmic disease in spite of pre-served native tertiary structure of the mutant protein (55) TheR36S missense mutation results in the formation of a novel pro-teinndashprotein interaction surface that allows crystallin to crystal-lize in the lens Notably the structure of the mutant proteinwas determined from an actual crystal extracted from the af-fected patientrsquos lens which showed that the site of mutationallowed formation of new predominantly hydrophobic proteinndashprotein interactions (55) Other crystallin mutants have alsobeen shown to exhibit abnormal aggregation behavior but withpreserved tertiary structures (56) While we do not present datathat suggests the D477G promotes abnormal RPE65 homo-oligomerization the same concept of abnormal hydrophobicinteractions could be applicable to RPE65 heterotypic interac-tions within the cell Indeed the fact that RPE65 is relativelyabundant within the RPE suggests that it could be present atsufficient levels to generate substantial cellular stress throughbinding to cellular components

The D477G RPE65 KI mouse that we report here recapitulatessome but not all features of the human disease As mentionedabove additional stressors required for the full phenotypic ex-pression of the disease that are experienced by humans but notmice in a well-controlled laboratory environment could under-lie the differences in disease severity Lack of a macula inmouse retina might be another key factor contributing to thephenotypic discrepancy between humans and mice Severalpatients with the D477G mutation developed a dystrophy of themacula which is the area of highest cone density (910) In addi-tion mouse RPE conceivably could tolerate the existence of mu-tant RPE65 and clear protein aggregation products moreeffectively than human RPE Therefore RPE cells or 3-D retinalorganoids differentiated from human-induced pluripotent stemcells carrying the D477G RPE65 mutation could be alternativetools for future studies on the pathogenesis and treatment ofthis form of RP

Materials and MethodsAnimals

D477G KI mice were generated by the InGenious TargetingLaboratory (Ronkonkoma NY) C57BL6J WT mice (JacksonLaboratory Bar Harbor ME) were used for mating with D477G KImice Mice were housed at the animal resource center at Case

Western Reserve University School of Medicine in a 12-h light(lt10 lux)12-h dark cyclic environment with ad libitum access tofood (LabDiet 5053) and water All animal procedures andexperiments were approved by the Institutional Animal Careand Use Committee of Case Western Reserve University con-formed to the recommendations of the American VeterinaryMedical Association Panel on Euthanasia and were conductedin accordance with the Association for Research in Vision andOphthalmology Statement for the Use of Animals inOphthalmic and Visual Research

Generation of D477G KI mice

D477G KI mice were generated as follows a 113 kb region usedto construct the targeting vector was first subcloned from a pos-itively identified C57BL6 BAC clone The construct wasdesigned as follows the 50 homology arm extended 89 kb 50 tothe KI mutation the 30 homology arm extended 22 kb 30 to theFRT flanked Neo cassette the Neo cassette was inserted be-tween exons 13 and 14 exon 13 with a point mutation was engi-neered by three-step PCR mutagenesis to introduce AgtGmutations resulting in Asp to Gly substitutions This BAC con-struct was subcloned into a 245 kb backbone vector (pSP72Promega Madison WI) containing an ampicillin selection cas-sette for transformation of the construct before electroporationThe targeting vector was linearized by NotI and then electropo-rated into embryonic stem cells (129SvEV X C57BL6 FLP hybrid)expressing FLP recombinase After selection with G418 antibi-otic positive clones were expanded for PCR analysis (FN1 for-ward 50-TATCCGTACGTTCGTGGGATTGTC-30 and A3 reverse50-AGAACTTCTCGAGGACTCACCAGG-30) and southern blottinganalysis to identify recombinant ES clones Validated cloneswere then microinjected into C57BL6 blastocysts and theresulting chimeric mice with a high percentage of agouti coatcolor were mated with C57BL6 mice (InGenious TargetingLaboratory) to generate germline Neo-deleted mice Thesefounders were then screened for deletion of the Neo Cassette byPCR analysis (SC1 forward 50-GCATACGGACTTGGGTTGAATCAC-30 and NDEL2 reverse 50-ACTATGCTGCAGAAGGTGTTTTACTGTGG-30) of their genomic DNA obtained from tail biop-sies The Rd8 mutation in Crb1 was absent in the animals usedfor this study (57)

Mutagenesis and stable transduction of NIH3T3cell lines

An NIH3T3 cell line stably expressing LRAT was generated by apreviously published protocol (58) The retroviral expressionvectors were generated by introducing EcoRI and NotI restrictionsites at the ends of the hRPE65 coding sequence by PCR withthe following primers forward 50-TAGGAATTCATGTCTATCCAGGTTGAG-30 reverse 50-TAAGCGGCCGCTCAAGATTTTTTGAACAG-30 The modified hRPE65 cDNA was inserted into a pMX-IGretroviral vector provided by Dr T Kitamura from theUniversity of Tokyo (59) An internal ribosomal entry site (IRES)and enhanced green fluorescence protein (EGFP) wereplaced downstream of the hRPE65 coding region allowingco-expression of hRPE65 and EGFP from the same mRNA ThehRPE65 (D477G) mutation was introduced by PCR amplificationof the entire plasmid by using Phusion High Fidelity polymerase(New England Biolabs Ipswich MA)

NIH3T3-LRAT-hRPE65 and NIH3T3-LRAT-hRPE65 (D477G)stable cell lines were generated by transduction of NIH3T3-



LRAT cells with retrovirus collected from Phoenix-Eco cellstransfected with the pMXs-IG containing hRPE65 or hRPE65(D477G) according to a previously published protocol (58)Transduced cells were sorted by a FACSAria cell sorter (BDBiosciences San Jose CA) to selectively collect transduced cellsand ensure comparable EGFP expression between NIH3T3-LRAT-hRPE65 and NIH3T3-LRAT-hRPE65 (D477G)

Cells were maintained in growth medium (GM) composed ofDulbeccorsquos modified Eaglersquos medium (Gibco Gaithersburg MD)pH 72 with 4 mM L-glutamine 4500 mgl glucose and 110 mgl sodium pyruvate supplemented with 10 (vv) heat-inactivated fetal bovine serum 100 unitsml penicillin and 100unitsml streptomycin After reaching confluence cells werewashed twice with PBS trypsinized passaged and then main-tained at 37C in 5 CO2

Immunoblotting of RPE65

Cultured cells were lysed with 200 ll of RIPA buffer (CellSignaling Technology Danvers MA) with protease inhibitors(Sigma-Aldrich St Louis MO) and sonicated for 5 s to shear theDNA Enucleated eyes from WT heterozygous KI and homozy-gous KI mice were dissected and lysed according to a previouslypublished protocol (60) Protein concentrations were deter-mined with a BCA Assay kit (Bio-Rad Hercules CA) followingthe manufacturerrsquos instructions Protein samples were mixedwith NuPAGE LDS sample buffer and NuPAGE reducing agentand then separated using NuPAGE 4ndash12 BisndashTris gels(Invitrogen Carlsbad CA) and transferred to polyvinylidenedifluoride (PVDF) membranes Membranes were blocked with5 (wv) non-fat milk or 5 (wv) BSA and then incubated witheither anti-RPE65 monoclonal antibody (1 1000) (61) anti-b-ac-tin antibody (1 1000) (Cell Signaling Technology) or anti-GAPDHantibody (Abcam Cambridge UK) overnight at 4C After wash-ing with PBS containing 01 (vv) Tween 20 membranes wereincubated with peroxidase-linked anti-mouse or anti-rabbit IgG(1 10 000) (Jackson ImmunoResearch Laboratories West GrovePA) for 1 h at room temperature Protein bands were visualizedafter exposure to SuperSignal West Pico Chemiluminescentsubstrate (ThermoFisher Scientific Waltham MA)

Immunoprecipitation

Immunoprecipitation was performed with anti-ubiquitin anti-body (Abcam) coupled to magnetic protein G Dynabeads(Invitrogen) Total RPE cell extracts isolated from WT heterozy-gous KI or homozygous KI mice (two eyes per genotype) preparedin RIPA buffer were incubated with anti-ubiquitin antibody con-jugated magnetic beads (15 mg beads and 12 mg antibody per re-action) for 20 min at room temperature under constant mildagitation The supernatant was then removed and kept for furtheranalysis and the beads were washed three times in PBS contain-ing 01 (vv) Tween-20 by gentle pipetting and subsequent re-moval of wash buffer Proteins pulled down by the beads wereeluted by incubation for 20 min in a room temperatureThermomixer (Eppendorf Hauppauge NY) at 300 rpm in 20 mlElution Buffer (NuPAGE LDS sample buffer and NuPAGE samplereducing agent mixed per manufacturerrsquos instructions) The ly-sate flow through and eluate were separated on a NuPAGE 4ndash12BisndashTris gel (Invitrogen) Immunoblotting was performed as de-scribed in the preceding section

Immunocytochemistry

Cells were fixed with 4 (wv) paraformaldehyde (ThermoFisherScientific) for 10 min and permeabilized with PBST (PBS contain-ing 01 Triton X-100) for 10 min Then cells were blocked inPBST containing 5 goat serum at room temperature Cellswere immunostained with anti-RPE65 monoclonal antibody (11000) and anti-Calnexin polyclonal antibody (1 200) (Abcam)followed by the corresponding Alexa Fluor 647 anti-mouse IgG(1 1000) (ThermoFisher Scientific) Alexa Fluor 594 anti-rabbitIgG (1 1000) (ThermoFisher Scientific) and DAPI Samples thenwere mounted in ProLong Gold antifade reagent (Invitrogen)and imaged with a Leica TCS SP8 confocal microscope

Retinoid isomerization activity assay

NIH3T3-LRAT-hRPE65 or NIH3T3-LRAT-hRPE65 (D477G) cellswere plated in six-well culture plates at 08 106 cells per wellin GM The isomerization reaction was initiated 16 h later byreplacing GM with fresh GM containing 10 mM all-trans-retinoldelivered in N N-dimethylformamide Cells shielded from lightwere maintained at 37C in 5 CO2 After 16 h cells and me-dium were collected mixed with an equal volume of 4 M KOH inmethanol and incubated at 52C for 25 h to hydrolyze retinoidesters Next an equal volume of hexane was added and themixture was vigorously shaken Following a 15 min centrifuga-tion at 4000 rpm to facilitate phase separation the organicphase was collected dried in vacuo and dissolved in 250 ml ofhexane Extracted retinoids were separated on a normal phaseHPLC column (Sil 5 mm 46 250 mm Agilent TechnologiesSanta Clara CA) equilibrated with 10 ethyl acetate in hexaneat an isocratic flow rate of 14 mlmin 11-cis-retinol wasdetected by its characteristic retention time and absorptionspectrum and quantified by correlating peak areas to a standardcurve

Real-time PCR analysis

Total RNA from the RPE layer of WT heterozygous KI and homo-zygous KI mice was isolated according to a previously publishedmethod (62) cDNA was synthesized with a high capacity RNA-to-cDNA kit (Applied Biosystems Foster City CA) following themanufacturerrsquos instructions The same amount of total RNA wasprocessed without reverse transcriptase as a negative controlReal-time PCR was performed using iQTM SYBR Green Supermix(Bio-rad) with the following primers forward 50-TATGAAGACAATGGATTTCTGATTGTGGATCTCTGTTGCT-30rsquo reverse 50-ATGTTCAGGATCTTTTGAACAGTCCATGGAAGGTCACAGG-30 Gapdhwas used as a housekeeping gene for normalization Triplicatereal-time PCR reactions were performed for each animal

RPE65 cDNA fragment cloning

Total RNA from the RPE layer of WT heterozygous KI and ho-mozygous KI mice was isolated according to a previously pub-lished method (62) cDNA was synthesized with a high capacityRNA-to-cDNA kit (Applied Biosystems Foster City CA) followingthe manufacturerrsquos instructions The same amount of totalRNA was processed without reverse transcriptase as a negativecontrol PCR was performed using Phusion High-FidelityPCR Master Mix with the following primers forward50-TATGAAGACAATGGATTTCTGATTGTGGATCTCTGTTGCT-30



reverse 50-ATGTTCAGGATCTTTTGAACAGTCCATGGAAGGTCACAGG-30 The 655-bp predicted PCR products were not generatedby negative controls involving either non-reverse-transcribedtotal RNA or water as templates After purification with aQIAquick PCR purification kit PCR products were ligated intopCR 4Blunt-TOPO (Invitrogen) and transformed into One ShotTop10 Chemically Competent E coli (Invitrogen) Sequencingwas used to determine the presence of WT RPE65 and D477GRPE65 fragments (examples of sequence chromatograms fromheterozygous KI animals are shown in Fig 2C) To examine theratio of WT RPE65 and D477G RPE65 mRNA 95 cDNA clonesfrom two heterozygous KI mice were randomly selected andsequenced

SLO and OCT imaging

Mice were anesthetized by intraperitoneal injection of a cocktailconsisting of 20 mgml ketamine and 175 mgml xylazine inphosphate-buffered saline at a dose of 01ndash013 ml per 25 g bodyweight Pupils were dilated with 1 tropicamide (Henry ScheinMelville NY) before imaging Heidelberg Retinal Angiograph II(Heidelberg Engineering Franklin MA) was used for whole fundusimaging of retinas in vivo Ultra-high resolution spectral domainOCT (Bioptigen Morrisville NC) was employed for imaging of reti-nas in vivo Five frames of OCT images were acquired in the B-mode and then used to construct final averaged images For quan-titative measurements the thickness of the ONL was measured050 mm away from the optic nerve head in the superior inferiortemporal and nasal retina These values were then averaged

ERG

Prior to recording mice were dark adapted for 24 h Under asafety light mice were anesthetized by intraperitoneal injectionof a cocktail consisting of 20 mgml ketamine and 175 mgmlxylazine in phosphate-buffered saline at a dose of 01ndash013 mlper 25 g body weight Pupils were dilated with 1 tropicamide(Henry Schein) and then 25 hypromellose was applied tokeep the corneas hydrated Contact lens electrodes were placedonto the corneas and reference and ground electrodes were po-sitioned subdermally between the ears and on the tail respec-tively All ERGs were recorded with the universal testing andelectrophysiological system UTAS E-3000 (LKC TechnologiesGaithersburg MD)

For single-flash scotopic ERG recording the duration ofwhite-light flash stimuli (from 20 ms to 1 ms) was adjusted toprovide a range of illumination intensities from 37 to 23 logcdsm2 For each intensity 3 to 20 recordings were made at suf-ficient intervals between flash stimuli (from 3 to 90 s) to allowrecovery from any photobleaching effects The photopic ERGrecordings were performed after bleaching at 14 log cdsm2 for10 minutes For photopic ERG the cone response was measuredat four different light intensities (07 to 23 log cdsm2) in thepresence of rod-desensitizing white-light background

HPLC retinoid profiling

Eyes were enucleated from dark-adapted WT heterozygous KIand homozygous KI mice and then homogenized in 10 mM so-dium phosphate buffer pH 80 containing 50 methanol (vv)and 100 mM hydroxylamine After 15 min of incubation at roomtemperature 2 ml of 3 M sodium chloride was added The result-ing sample was extracted twice with 3 ml of ethyl acetate Then

the combined organic phase was dried in vacuo and reconsti-tuted in 400 ll of hexane Extracted retinoids (100 ml) were sepa-rated on a normal phase HPLC column (Sil 5 mm 46 250 mmAgilent Technologies) equilibrated with a stepwise gradient of06 ethyl acetate in hexane at an isocratic flow rate of 14 mlmin for 17 min and 10 ethyl acetate in hexane at an isocraticflow rate of 14 mlmin for 25 min Retinoids were detected bymonitoring their absorbance at 325 nm and quantified based oncorrelations of their peak areas

ACORPE65 chimera construct design

A chimeric ACORPE65 coding sequence was synthesized byreplacing the nucleotides encoding residues 44ndash45 431ndash442 461and 463 of Synechocystis ACO (GI 81671293) with those encodingresidues 36ndash37 474ndash485 506 and 508 of bovine RPE65 (GI27806125) (Genscript Piscataway NJ) The open reading framewas subcloned into the pET3a expression vector The D434Gpoint mutant (corresponding to D477G in RPE65 and referred toas such in the manuscript) was generated by the QuikChangesite-directed mutagenesis protocol (Stratagene San Diego CA)Coding regions of all expression plasmids were verified bysequencing

ACORPE65 chimera expression purificationand crystallization

ACORPE65 chimera and its D477G variant were expressed puri-fied and crystallized as described previously for ACO (36) withthe following modifications Protein expression was not chemi-cally induced but instead relied on basal T7 promoter activityFollowing purification proteins were diluted into a buffer con-sisting of 20 mM HEPES-NaOH pH 7 (Sigma-Aldrich) and 002vv Triton X-100 (Anatrace Maumee OH) to a final concentra-tion of 10 mgml Crystals were formed by the hanging-drop va-por diffusion method after mixing 2 ll drops of protein solutionwith 2 ll of a crystallant consisting of 01 M BisndashTris propane pH6 (Sigma-Aldrich) and 21 wv sodium polyacrylate 2100(Molecular Dimensions Maumee OH) After 2ndash3 weeks ofgrowth crystals were cryoprotected in crystallant supple-mented with 15 vv ethylene glycol flash cooled in liquid ni-trogen and then stored in nitrogen vapor prior to X-rayexposure

Diffraction data collection structure solutionand refinement

Diffraction data were collected at the NSLS II AMX and FMXbeamlines or the APS NECAT IDE beamline Data reduction wascarried out with XDS (63) The ACORPE65 chimera structurewhich was isomorphous to previous ACO structures (3637) wassolved by direct refinement in REFMAC (64) using the coordi-nates deposited under PDB accession code 4OU9 as a startingmodel Amino acid replacements were performed with COOT(65) The ACORPE65 chimera coordinates were used as a searchmodel to solve the structures of the two D477G crystal forms bymolecular replacement in the program Phaser (66) Structuralmodels were iteratively improved by manual real space refine-ment in COOT and reciprocal space refinement in REFMACSuch models were periodically checked for geometric sound-ness and fit to the electron density maps with MOLPROBITY (67)and the wwPDB validation server (68)



Structure analysis

CCO amino acid sequence alignments were carried out withClustal Omega (69) and were annotated with Espript (70)Structural motifs involving specific amino acid sequences wereidentified and analyzed with PDBeMotif using the MSDmotif al-gorithm (35) Secondary structures and contact surfaces for theACORPE65 chimera crystal structures were analyzed withPDBsum (30) and PISA (71) Electrostatics calculations were car-ried out with APBS (72) Figures displaying three-dimensionalstructures were rendered using PyMOL (Schrodinger LLC)

ACORPE65 chimera activity assays

Enzymatic activity of purified ACO and ACORPE65 proteinswere assayed spectrophotometrically by monitoring formationof the all-trans-retinal product at 340 nm using all-trans-b-apo-80-carotenol as the reaction substrate and assay conditionsidentical to those previously described (SupplementaryMaterial Fig S3) (36)

Statistics

Data representing the means 6 SEMs (unless otherwise indi-cated) are presented for results of at least three independentexperiments Significance between groups was determinedeither with the unpaired Studentrsquos t-test or one-way ANOVAwith the Bonferroni post-test for comparisons of more than twogroups Sigma Plot 110 (Systat Software) was used to performthe statistical analyses

Supplementary MaterialSupplementary Material is available at HMG online

Acknowledgements

We thank Dr Leslie T Webster Jr (CWRU) for valuable com-ments on the manuscript We thank Drs Jianye Zhang LukasHofmann and Marcin Golczak (CWRU) for sharing their expertiserelated to retinoid analysis and the RPE65 isomerization assay

Conflict of Interest statement None declared

FundingThis research was supported in part by grants from the NationalInstitutes of Health (EY009339 to KP and PDK T32GM007250to EHC and SS T32EY007157 to CLS EY027283 and EY024864to KP and core grants P30EY011373 and P30EY025585) and theDepartment of Veterans Affairs (IK2BX002683 to PDK) Thiswork is based in part upon research conducted at the APSNortheastern Collaborative Access Team beamlines supportedby grants GM103403 RR029205 and DE-AC02ndash06CH11357 Datafor this study were measured at beamlines 17-ID-1 and 17-ID-2of the National Synchrotron Light Source-II which are sup-ported by NIH grant GM111244 and the DOE Office of Biologicaland Environmental Research KP1605010 The NSLS-II is sup-ported in part by the DOE Office of Science Office of BasicEnergy Sciences Program under contract number DE-SC0012704(KC0401040) The Case Center for Synchrotron Biosciences issupported by NIH grant EB009998 KP is the John H HordProfessor of Pharmacology

References1 Jin M Li S Moghrabi WN Sun H and Travis GH (2005)

Rpe65 is the retinoid isomerase in bovine retinal pigmentepithelium Cell 122 449ndash459

2 Moiseyev G Chen Y Takahashi Y Wu BX and Ma JX(2005) RPE65 is the isomerohydrolase in the retinoid visualcycle Proc Natl Acad Sci USA 102 12413ndash12418

3 Redmond TM Poliakov E Yu S Tsai JY Lu Z andGentleman S (2005) Mutation of key residues of RPE65 abol-ishes its enzymatic role as isomerohydrolase in the visualcycle Proc Natl Acad Sci USA 102 13658ndash13663

4 Redmond TM Yu S Lee E Bok D Hamasaki D ChenN Goletz P Ma JX Crouch RK and Pfeifer K (1998)Rpe65 is necessary for production of 11-cis-vitamin A in theretinal visual cycle Nat Genet 20 344ndash351

5 Nicoletti A Wong DJ Kawase K Gibson LH Yang-FengTL Richards JE and Thompson DA (1995) Molecularcharacterization of the human gene encoding an abundant61 kDa protein specific to the retinal pigment epitheliumHum Mol Genet 4 641ndash649

6 Schwartz SH Tan BC Gage DA Zeevaart JA andMcCarty DR (1997) Specific oxidative cleavage of carote-noids by VP14 of maize Science 276 1872ndash1874

7 Hamel CP Tsilou E Pfeffer BA Hooks JJ Detrick B andRedmond TM (1993) Molecular cloning and expression ofRPE65 a novel retinal pigment epithelium-specific micro-somal protein that is post-transcriptionally regulatedin vitro J Biol Chem 268 15751ndash15757

8 Chacon-Camacho OF and Zenteno JC (2015) Review andupdate on the molecular basis of Leber congenital amauro-sis World J Clin Cases 3 112ndash124

9 Bowne SJ Humphries MM Sullivan LS Kenna PFTam LC Kiang AS Campbell M Weinstock GMKoboldt DC and Ding L (2011) A dominant mutation inRPE65 identified by whole-exome sequencing causes retini-tis pigmentosa with choroidal involvement Eur J HumGenet 19 1074ndash1081

10 Hull S Mukherjee R Holder GE Moore AT and WebsterAR (2016) The clinical features of retinal disease due to adominant mutation in RPE65 Mol Vis 22 626ndash635

11 Shin Y Moiseyev G Chakraborty D and Ma JX (2017) Adominant mutation in Rpe65 D477G delays dark adaptationand disturbs the visual cycle in the mutant knock-in miceAm J Pathol 187 517ndash527

12 Wilson JH and Wensel TG (2003) The nature of dominantmutations of rhodopsin and implications for gene therapyMol Neurobiol 28 149ndash158

13 Bereta G Kiser PD Golczak M Sun W Heon ESaperstein DA and Palczewski K (2008) Impact of retinaldisease-associated RPE65 mutations on retinoid isomeriza-tion Biochemistry 47 9856ndash9865

14 Takahashi Y Chen Y Moiseyev G and Ma JX (2006)Two point mutations of RPE65 from patients with retinaldystrophies decrease the stability of RPE65 protein andabolish its isomerohydrolase activity J Biol Chem 28121820ndash21826

15 Bavik CO Busch C and Eriksson U (1992)Characterization of a plasma retinol-binding protein mem-brane receptor expressed in the retinal pigment epitheliumJ Biol Chem 267 23035ndash23042

16 Li S Izumi T Hu J Jin HH Siddiqui AA Jacobson SGBok D and Jin M (2014) Rescue of enzymatic function for



disease-associated RPE65 proteins containing various mis-sense mutations in non-active sites J Biol Chem 28918943ndash18956

17 Li SH Izumi T Hu J Jin HH Siddiqui AAA JacobsonSG Bok D and Jin MH (2014) Rescue of enzymatic func-tion for disease-associated RPE65 proteins containing vari-ous missense mutations in non-active sites J Biol Chem289 18943ndash18956

18 Li S Hu J Jin RJ Aiyar A Jacobson SG Bok D and JinM (2015) Temperature-sensitive retinoid isomerase activityof RPE65 mutants associated with Leber congenital amauro-sis J Biochem 158 115ndash125

19 Li S Samardzija M Yang Z Grimm C and Jin M (2016)Pharmacological amelioration of cone survival and vision ina mouse model for Leber congenital amaurosis J Neurosci36 5808ndash5819

20 Maeda A Okano K Park PS Lem J Crouch RK MaedaT and Palczewski K (2010) Palmitoylation stabilizes unli-ganded rod opsin Proc Natl Acad Sci USA 107 8428ndash8433

21 Mustafi D Maeda T Kohno H Nadeau JH andPalczewski K (2012) Inflammatory priming predisposesmice to age-related retinal degeneration J Clin Invest 1222989ndash3001

22 Zhang N Kolesnikov AV Jastrzebska B Mustafi DSawada O Maeda T Genoud C Engel A Kefalov VJ andPalczewski K (2013) Autosomal recessive retinitis pigmen-tosa E150K opsin mice exhibit photoreceptor disorganiza-tion J Clin Invest 123 121ndash137

23 Chen Y Palczewska G Masuho I Gao S Jin H Dong ZGieser L Brooks MJ Kiser PD Kern TS et al (2016)Synergistically acting agonists and antagonists of Gprotein-coupled receptors prevent photoreceptor cell degen-eration Sci Signal 9 ra74

24 Samardzija M von Lintig J Tanimoto N Oberhauser VThiersch M Reme CE Seeliger M Grimm C and WenzelA (2007) R91W mutation in Rpe65 leads to milderearly-onset retinal dystrophy due to the generation of lowlevels of 11-cis-retinal Hum Mol Genet 17 281ndash292

25 Redmond TM Weber CH Poliakov E Yu S andGentleman S (2007) Effect of LeuMet variation at residue450 on isomerase activity and protein expression of RPE65and its modulation by variation at other residues Mol Vis13 1813ndash1821

26 Imanishi Y Batten ML Piston DW Baehr W andPalczewski K (2004) Noninvasive two-photon imagingreveals retinyl ester storage structures in the eye J Cell Biol164 373ndash383

27 Imanishi Y Gerke V and Palczewski K (2004)Retinosomes new insights into intracellular managing ofhydrophobic substances in lipid bodies J Cell Biol 166447ndash453

28 Sheridan C Boyer NP Crouch RK and Koutalos Y(2017) RPE65 and the accumulation of retinyl esters inmouse retinal pigment epithelium Photochem Photobiol93 844ndash848

29 Lek M Karczewski KJ Minikel EV Samocha KEBanks E Fennell T OrsquoDonnell-Luria AH Ware JSHill AJ Cummings BB et al (2016) Analysis ofprotein-coding genetic variation in 60 706 humansNature 536 285ndash291

30 Laskowski RA Jabłonska J Pravda L Varekova RS andThornton JM (2018) PDBsum structural summaries of PDBentries Protein Sci 27 129ndash134

31 Kiser PD Golczak M Lodowski DT Chance MR andPalczewski K (2009) Crystal structure of native RPE65 theretinoid isomerase of the visual cycle Proc Natl Acad SciUSA 106 17325ndash17330

32 Kiser PD Farquhar ER Shi W Sui X Chance MR andPalczewski K (2012) Structure of RPE65 isomerase in a li-pidic matrix reveals roles for phospholipids and iron in ca-talysis Proc Natl Acad Sci USA 109 E2747ndashE2756

33 Richardson JS (1981) The anatomy and taxonomy of pro-tein structure Adv Protein Chem 34 167ndash339

34 Hutchinson EG and Thornton JM (1994) A revised set ofpotentials for beta-turn formation in proteins Protein Sci 32207ndash2216

35 Golovin A and Henrick K (2008) MSDmotif exploring pro-tein sites and motifs BMC Bioinform 9 312

36 Sui X Kiser PD Che T Carey PR Golczak M Shi Wvon Lintig J and Palczewski K (2014) Analysis of carotenoidisomerase activity in a prototypical carotenoid cleavage en-zyme apocarotenoid oxygenase (ACO) J Biol Chem 28912286ndash12299

37 Kloer DP Ruch S Al-Babili S Beyer P and Schulz GE(2005) The structure of a retinal-forming carotenoid oxygen-ase Science 308 267ndash269

38 Cideciyan AV (2010) Leber congenital amaurosis due toRPE65 mutations and its treatment with gene therapy ProgRetin Eye Res 29 398ndash427

39 Laney JD and Hochstrasser M (1999) Substrate targeting inthe ubiquitin system Cell 97 427ndash430

40 Wilkie AO (1994) The molecular basis of genetic domi-nance J Med Genet 31 89ndash98

41 Felius J Thompson DA Khan NW Bingham ELJamison JA Kemp JA and Sieving PA (2002) Clinicalcourse and visual function in a family with mutations in theRPE65 gene Arch Ophthalmol 120 55ndash61

42 Poehner WJ Fossarello M Rapoport AL Aleman TSCideciyan AV Jacobson SG Wright AF Danciger Mand Farber DB (2000) A homozygous deletion in RPE65 in asmall Sardinian family with autosomal recessive retinaldystrophy Mol Vis 6 192ndash198

43 Yzer S van den Born LI Schuil J Kroes HY vanGenderen MM Boonstra FN van den Helm B BrunnerHG Koenekoop RK and Cremers FP (2003) A Tyr368HisRPE65 founder mutation is associated with variable expres-sion and progression of early onset retinal dystrophy in 10families of a genetically isolated population J Med Genet40 709ndash713

44 Morimura H Fishman GA Grover SA Fulton ABBerson EL and Dryja TP (1998) Mutations in the RPE65gene in patients with autosomal recessive retinitis pigmen-tosa or Leber congenital amaurosis Proc Natl Acad SciUSA 95 3088ndash3093

45 Strauss O (2005) The retinal pigment epithelium in visualfunction Physiol Rev 85 845ndash881

46 Aguzzi A and OrsquoConnor T (2010) Protein aggregation dis-eases pathogenicity and therapeutic perspectives Nat RevDrug Discov 9 237ndash248

47 Rousseau F Schymkowitz J and Serrano L (2006) Proteinaggregation and amyloidosis confusion of the kinds CurrOpin Struct Biol 16 118ndash126

48 De Baets G Van Doorn L Rousseau F and Schymkowitz J(2015) Increased aggregation is more frequently associatedto human disease-associated mutations than to neutralpolymorphisms PLoS Comput Biol 11 e1004374



49 Reumers J Maurer-Stroh S Schymkowitz J and RousseauF (2009) Protein sequences encode safeguards against aggre-gation Hum Mutat 30 431ndash437

50 Monplaisir N Merault G Poyart C Rhoda MD CraescuC Vidaud M Galacteros F Blouquit Y and Rosa J (1986)Hemoglobin-S antilles - a variant with lower solubility thanhemoglobin-S and producing sickle-cell disease in heterozy-gotes Proc Natl Acad Sci USA 83 9363ndash9367

51 Harrington DJ Adachi K and Royer WE (1997) The highresolution crystal structure of deoxyhemoglobin S J MolBiol 272 398ndash407

52 Tripette J Loko G Samb A Gogh BD Sewade E SeckD Hue O Romana M Diop S Diaw M et al (2010) Effectsof hydration and dehydration on blood rheology in sicklecell trait carriers during exercise Am J Physiol Heart CircPhysiol 299 H908ndashH914

53 Kark JA Posey DM Schumacher HR and Ruehle CJ(1987) Sickle-cell trait as a risk factor for sudden death inphysical training N Engl J Med 317 781ndash787

54 Anzalone ML Green VS Buja M Sanchez LAHarrykissoon RI and Eichner ER (2010) Sickle cell trait andfatal rhabdomyolysis in football training a case study MedSci Sports Exerc 42 3ndash7

55 Kmoch S Brynda J Asfaw B Bezouska K Novak PRezacova P Ondrova L Filipec M Sedlacek J and EllederM (2000) Link between a novel human gammaD-crystallinallele and a unique cataract phenotype explained by proteincrystallography Hum Mol Genet 9 1779ndash1786

56 Pande A Pande J Asherie N Lomakin A Ogun O KingJ and Benedek GB (2001) Crystal cataracts human geneticcataract caused by protein crystallization Proc Natl AcadSci USA 98 6116ndash6120

57 Mattapallil MJ Wawrousek EF Chan CC Zhao HRoychoudhury J Ferguson TA and Caspi RR (2012) TheRd8 mutation of the Crb1 gene is present in vendor lines ofC57BL6N mice and embryonic stem cells and confoundsocular induced mutant phenotypes Invest Ophthalmol VisSci 53 2921ndash2927

58 Chelstowska S Widjaja-Adhi MAK Silvaroli JA andGolczak M (2017) Impact of LCA-associated E14L LRAT mu-tation on protein stability and retinoid homeostasisBiochemistry 56 4489ndash4499

59 Kitamura T Koshino Y Shibata F Oki T Nakajima HNosaka T and Kumagai H (2003) Retrovirus-mediated genetransfer and expression cloning powerful tools in functionalgenomics Exp Hematol 31 1007ndash1014

60 Wei H Xun Z Granado H Wu A and Handa JT (2016)An easy rapid method to isolate RPE cell protein from themouse eye Exp Eye Res 145 450ndash455

61 Golczak M Kiser PD Lodowski DT Maeda A andPalczewski K (2010) Importance of membrane structural in-tegrity for RPE65 retinoid isomerization activity J BiolChem 285 9667ndash9682

62 Xin-Zhao Wang C Zhang K Aredo B Lu H and Ufret-Vincenty RL (2012) Novel method for the rapid isolation ofRPE cells specifically for RNA extraction and analysis ExpEye Res 102 1ndash9

63 Kabsch W (2010) XDS Acta Crystallogr D 66 125ndash13264 Murshudov GN Skubak P Lebedev AA Pannu NS

Steiner RA Nicholls RA Winn MD Long F and VaginAA (2011) REFMAC5 for the refinement of macromolecularcrystal structures Acta Crystallogr D 67 355ndash367

65 Emsley P Lohkamp B Scott WG and Cowtan K (2010)Features and development of Coot Acta Crystallogr D 66486ndash501

66 McCoy AJ Grosse-Kunstleve RW Adams PD WinnMD Storoni LC and Read RJ (2007) Phaser crystallo-graphic software J Appl Crystallogr 40 658ndash674

67 Chen VB Arendall WB 3rd Headd JJ Keedy DAImmormino RM Kapral GJ Murray LW Richardson JSand Richardson DC (2010) MolProbity all-atom structurevalidation for macromolecular crystallography ActaCrystallogr D 66 12ndash21

68 Read RJ Adams PD Arendall WB 3rd Brunger ATEmsley P Joosten RP Kleywegt GJ Krissinel EBLutteke T Otwinowski Z et al (2011) A new generation ofcrystallographic validation tools for the protein data bankStructure 19 1395ndash1412

69 Sievers F Wilm A Dineen D Gibson TJ Karplus K LiW Lopez R McWilliam H Remmert M Soding J et al(2014) Fast scalable generation of high-quality proteinmultiple sequence alignments using Clustal Omega MolSyst Biol 7 539

70 Robert X and Gouet P (2014) Deciphering key features inprotein structures with the new ENDscript server NucleicAcids Res 42 W320ndashW324

71 Krissinel E and Henrick K (2007) Inference of macromolecu-lar assemblies from crystalline state J Mol Biol 372 774ndash797

72 Baker NA Sept D Joseph S Holst MJ and McCammonJA (2001) Electrostatics of nanosystems application tomicrotubules and the ribosome Proc Natl Acad Sci USA98 10037ndash10041



ddy128-TF1

ddy128-TF2

ddy128-TF3

IntroductionThe retinoid (visual) cycle is an enzymatic pathway that regen-erates visual chromophore in vertebrates This metabolic cycletakes place mainly in the retinal pigment epithelium (RPE) asingle layer of cells adjacent to the photoreceptor outer seg-ments Continuous regeneration of visual chromophore is criti-cal for detection of light and defects in the enzymes involved inthe retinoid cycle often culminate in retinal dystrophy One ofthe indispensable enzymes of the visual cycle is an RPE-specificprotein known as RPE65 which is the retinoid isomerase thatcatalyzes cleavage and isomerization of all-trans-retinyl estersto form 11-cis-retinol (1ndash3)

RPE65 is a 61 kDa protein bound to the endoplasmic reticu-lum (ER) membranes of the RPE that is highly conserved amongvertebrates and belongs to the carotenoid cleavage oxygenase(CCO) enzyme family (4ndash7) To date over 100 mutations in RPE65have been implicated in Leber congenital amaurosis (LCA) andretinitis pigmentosa (RP) (8) and almost all of these are inher-ited in a recessive manner However an autosomal dominantform of RPE65-associated RP with choroidal involvement hasalso been identified (910) This novel form of RP arises from amissense mutation in exon 13 of RPE65 (c1430GgtA) thatchanges the amino acid residue encoded at position 477 fromAsp to Gly (D477G)

Carriers of the D477G mutation exhibit varying pheno-types including disease severity and age of disease onsetThey also display areas of retinal atrophy ranging from thechoroid to the photoreceptor layer (910) Unlike typical RPpatients who present with progressive visual field loss fromthe mid to far periphery most carriers of the D477G mutationinitially present with a central visual defect (10) Moreoverchoroidal atrophy and RPE hypertrophy are frequently ob-served and distinctive features associated with this mutation(910)

Previous studies of the RPE65 D477G mutation have sug-gested that the pathological phenotype is attributable to struc-tural destabilization of RPE65 due to increased folding freeenergy (9) and delayed chromophore regeneration resultingfrom dominant-negative suppression of RPE65 retinoid isomer-ase activity (11) However visual cycle activity in heterozygousD477G knock-in (KI) mice can replenish sufficient 11-cis-retinal-dehyde to support relatively normal retinal function (11) Thedisease-associated phenotypes in homozygous KI mice havenot been reported to date so the precise classification of thispoint mutation remains unclear Understanding the pathologi-cal consequences of this mutation is likely to aid in the develop-ment of treatment strategies for this and potentially otherRPE65-associated dominant forms of RP For exampledominant-negative or haploinsufficiency mechanisms wouldimply that RPE65 gene therapy may be of value to D477G sub-jects (12) whereas gain-of-function mechanisms would likelynot be responsive to this intervention

In this study we assessed the phenotype of anindependently-generated D477G mouse line in both the hetero-zygous and homozygous states using a combination of high-resolution imaging histology electroretinography (ERG) andretinoid profile analyses We further studied the consequencesof this mutation at the molecular level with in vitro isomeriza-tion assays RNA sequencing immunoblotting of ubiquitinatedRPE65 and X-ray crystallographic analyses These comprehen-sive studies provide novel insights into the pathogenesis of theD477G mutation

ResultsThe D477G mutation does not affect expressionsubcellular localization and isomerization activityin vitro

To evaluate the intracellular localization and enzymatic func-tion of RPE65 carrying the D477G mutation we used retroviraltransduction to generate two NIH3T3 cell lines that stablyexpress either LRAT that generates substrate for the isomeraseand human RPE65 (hRPE65) or LRAT and the hRPE65 D477G mu-tant Immunoblotting analysis of the cell lines showed compa-rable expression levels of hRPE65 and the D477G mutant(Fig 1A) which differs from R91W G244V and Y368H substitu-tions that cause decreased stability of RPE65 in vitro (1314)Next we examined whether the D477G mutant was properly lo-calized within NIH3T3 cells Immunofluorescence confocal mi-croscopy revealed that hRPE65 co-localized with the ER markercalnexin consistent with previous reports (4515) (Fig 1B) Thislocalization was unchanged for D477G RPE65 suggesting themutation does not affect protein trafficking or membrane asso-ciation in this heterologous expression system (Fig 1B) Ourresults cannot definitively exclude the possibility that protein ismisfolded and retained in the ER However it would be expectedthat such a protein would be quickly degraded Moreover previ-ous work has shown that aggregates of mutant RPE65 includingL22P T101I and L408P point mutants exhibited punctate-like in-clusion bodies whereas wild-type (WT) RPE65 exhibited a ho-mogenous ER distribution (16) Next we assessed whether theD477G mutation changes RPE65 isomerization activity by incu-bating the NIH3T3 cell lines stably expressing LRAT and theD477G mutant or WT RPE65 protein with 10 mM all-trans-retinolfor 16 h Analysis of retinoids extracted from these cellsrevealed comparable production of 11-cis-retinol between thetwo cell lines (Fig 1CndashE) These findings suggest that the D477Gmutation does not affect ER localization and isomerization ac-tivity of RPE65 in cell culture assays

mRNA from D477G RPE65 and ubiquitination of RPE65induced by the D477G mutation

We generated D477G RPE65 KI mice by replacing the endoge-nous RPE65 gene with a targeting vector including exons 8ndash14along with the D477G mutation (Fig 2A) A neomycin (Neo) cas-sette flanked by Flp-recombinase target (FRT) sites was used forclonal selection and later excised by FLP recombinase expressedin the embryonic stem cells The embryonic stem cells carryingthe Neo deleted targeting vector were microinjected into C57Bl6 blastocysts The final KI allele contained the D477G mutationwith one 85-bp FRT site remaining between exons 13 and 14 Aprimer set flanking the FRT site was used to distinguish the WTallele and mutant allele WT mice had a 495-bp PCR productwhereas homozygous mice had a 580-bp PCR product heterozy-gous mice had both PCR products (Fig 2B) Additional sequenc-ing of cDNA from the heterozygous mice showed transcriptionof both WT and D477G mRNA (Fig 2C)

Next we examined whether the D477G mutation affectedthe transcriptional level of RPE65 using real-time PCR RPE65mRNA levels were comparable between WT heterozygous andhomozygous KI mice (Supplementary Material Fig S1) consis-tent with prior data (11) To evaluate the ratio of transcribed WTand D477G mRNA we collected 95 RPE65 cDNA clones fromthe RPE layer of heterozygous mice and sequenced them







RPE65

β-actin

10 20 25 3015

10 20 25 3015

5

10

15

20

25

30

0

5

10

15

20

25

30

0

0

2

4

6

Absorb

ance

280 300 320 340 360

11

- cis

-re

tin

ol

(pm

ol1

06

ce

lls)

Absorb

ance a

t 325 n

mA

bsorb

ance a

t 325 n

m

Time (min)

Time (min)

WT D477G

WT

G7

74

D

0

20

40

60

80

100

120

WT D477G

318 328

Wavelength (nm)

ns

A B

C

a

b

c

b

c

a

WT

D477G

calnexin

calnexin

RPE65

RPE65

merged

merged

D

E
















WT

WT

WT

KI

KIK

I

WT RPE65D477G RPE65

Neo

Neo


475 476 477 478 479


Codon

WTD477G

A

B C


D G

F1

R1

D G

Ex7 8 9 10 11 12 13 14

WT RPE65

Targeting

Vector

Targeted

RPE65

Final

KI RPE65









1 41 72 34 13Total 75 20





Ub Pulldown

A B

WT

WT

WT

KI

KIK

I

150

10075

50

37

20

RPE65

β-actin

WT

WT

WT

KI

KIK

I
















WTWT

WTKI

KIKI













B

AWTWT

WTKI

KIKI


ON

L T

hic

kness (

microm

)

0

10

20

30

40

50

60

70

WTWT WTKI KIKI

57 microm

52 microm

50 microm

ON

L T

hic

kness (

microm

)

0

10

20

30

40

50

60

70

ON

L T

hic

kness (

microm

)

0

10

20

30

40

50

60

70









A

B

C

D

0

200

400

600

800

1000

1200

1400

1600

0

200

400

600

800

1000

-4 -3 -2 -1 0 1 2 3

0

200

400

600

800

1000

0

200

400

600

800

1000

-4 -3 -2 -1 0 1 2 3 -4 -3 -2 -1 0 1 2 3

0

200

400

600

800

1000

1200

1400

1600

0

200

400

600

800

1000

1200

1400

1600

-10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 250

20

30

10

40

50

0

20

30

10

40

50

0

20

30

10

40

50

-10 -05 00 05 10 15 20 25

-4 -3 -2 -1 0 1 2 3-4 -3 -2 -1 0 1 2 3-4 -3 -2 -1 0 1 2 3

-10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 250

50

100

150

200

250

300

350

0

50

100

150

200

250

300

350

0

50

100

150

200

250

300

350


3-month-old

3-month-old

3-month-old

6-month-old

6-month-old

6-month-old

9-month-old

9-month-old

9-month-old

WTWTWTKIKIKI

WTWTWTKIKIKI

WTWTWTKIKIKI

WTWTWTKIKIKI

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)


Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)


Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)


Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)



Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

















A B

D E

C

F G

0

100

200

300

400

500

600

11-c

is-r

etin

al (

pm

ole

ye)

0

100

200

300

400

500

600

11-c

is-r

etin

al (

pm

ole

ye)

0

50

100

150

200

250

300

Re

tinyl

est

ers

(p

mo

leye

)

0

50

100

150

200

250

300

Re

tinyl

est

ers

(p

mo

leye

)





0

200

400

600

800

Re

tin

yl e

ste

rs (

pm

ole

ye

)



0

100

200

300

400

500

11

-cis

-re

tin

al (p

mo

le

ye

)

WTWT

WTKI

KIKI

a

b

a

b

a

b

0 10 20 30 40Time (min)

0 10 20 30 40Time (min)

0 10 20 30 40Time (min)

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

WTWT WTKI KIKI0

10

20

30

40

50

60

11

-cis

-re

tin

al (p

mo

le

ye

)

WTWT WTKI KIKI0

200

400

600

800

1000

1200

Re

tin

yl e

ste

rs (

pm

ole

ye

)

WTWTWTKIKIKI

WTWTWTKIKIKI




















TT


343434343434762442 P GI LQ PD

TT

TT TT


TT TT


TT



Dimerization

interface

Asp477

Phe472

Asp477

Leu487

Phe429

Gly434

Leu444

A

B C RPE65 ACO







A

250

150

10075

50

37

25

20

ACOACO

RPE65

ACO

RPE65

D477G

ACOACO

RPE65

ACO

RPE65

D477G


Volume (mL)

20 40 60 80

Absorb

ance a

t 280 n

m

ACOACORPE65ACORPE65

D477G

Time (min)

0 1 2 3

Pro

du

ct

En

zym

e (microm

olmicrom

ol)

0

20

40

60 ACORPE65

D477G

ACOACORPE
























ACORPE65

A B C



vs ACORPE65

Trp45

(37)

Lys463

(508)

Asn461

(506)

Ser431

(474)

Val442

(485)

Asp434

(477)














A B

C D








































Immunoprecipitation


Immunocytochemistry











SLO and OCT imaging


ERG














Structure analysis




Statistics



Acknowledgements




















































































ddy128-TF1

ddy128-TF2

ddy128-TF3





RPE65

β-actin

10 20 25 3015

10 20 25 3015

5

10

15

20

25

30

0

5

10

15

20

25

30

0

0

2

4

6

Absorb

ance

280 300 320 340 360

11

- cis

-re

tin

ol

(pm

ol1

06

ce

lls)

Absorb

ance a

t 325 n

mA

bsorb

ance a

t 325 n

m

Time (min)

Time (min)

WT D477G

WT

G7

74

D

0

20

40

60

80

100

120

WT D477G

318 328

Wavelength (nm)

ns

A B

C

a

b

c

b

c

a

WT

D477G

calnexin

calnexin

RPE65

RPE65

merged

merged

D

E
















WT

WT

WT

KI

KIK

I

WT RPE65D477G RPE65

Neo

Neo


475 476 477 478 479


Codon

WTD477G

A

B C


D G

F1

R1

D G

Ex7 8 9 10 11 12 13 14

WT RPE65

Targeting

Vector

Targeted

RPE65

Final

KI RPE65









1 41 72 34 13Total 75 20





Ub Pulldown

A B

WT

WT

WT

KI

KIK

I

150

10075

50

37

20

RPE65

β-actin

WT

WT

WT

KI

KIK

I
















WTWT

WTKI

KIKI













B

AWTWT

WTKI

KIKI


ON

L T

hic

kness (

microm

)

0

10

20

30

40

50

60

70

WTWT WTKI KIKI

57 microm

52 microm

50 microm

ON

L T

hic

kness (

microm

)

0

10

20

30

40

50

60

70

ON

L T

hic

kness (

microm

)

0

10

20

30

40

50

60

70









A

B

C

D

0

200

400

600

800

1000

1200

1400

1600

0

200

400

600

800

1000

-4 -3 -2 -1 0 1 2 3

0

200

400

600

800

1000

0

200

400

600

800

1000

-4 -3 -2 -1 0 1 2 3 -4 -3 -2 -1 0 1 2 3

0

200

400

600

800

1000

1200

1400

1600

0

200

400

600

800

1000

1200

1400

1600

-10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 250

20

30

10

40

50

0

20

30

10

40

50

0

20

30

10

40

50

-10 -05 00 05 10 15 20 25

-4 -3 -2 -1 0 1 2 3-4 -3 -2 -1 0 1 2 3-4 -3 -2 -1 0 1 2 3

-10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 250

50

100

150

200

250

300

350

0

50

100

150

200

250

300

350

0

50

100

150

200

250

300

350


3-month-old

3-month-old

3-month-old

6-month-old

6-month-old

6-month-old

9-month-old

9-month-old

9-month-old

WTWTWTKIKIKI

WTWTWTKIKIKI

WTWTWTKIKIKI

WTWTWTKIKIKI

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)


Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)


Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)


Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)



Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

















A B

D E

C

F G

0

100

200

300

400

500

600

11-c

is-r

etin

al (

pm

ole

ye)

0

100

200

300

400

500

600

11-c

is-r

etin

al (

pm

ole

ye)

0

50

100

150

200

250

300

Re

tinyl

est

ers

(p

mo

leye

)

0

50

100

150

200

250

300

Re

tinyl

est

ers

(p

mo

leye

)





0

200

400

600

800

Re

tin

yl e

ste

rs (

pm

ole

ye

)



0

100

200

300

400

500

11

-cis

-re

tin

al (p

mo

le

ye

)

WTWT

WTKI

KIKI

a

b

a

b

a

b

0 10 20 30 40Time (min)

0 10 20 30 40Time (min)

0 10 20 30 40Time (min)

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

WTWT WTKI KIKI0

10

20

30

40

50

60

11

-cis

-re

tin

al (p

mo

le

ye

)

WTWT WTKI KIKI0

200

400

600

800

1000

1200

Re

tin

yl e

ste

rs (

pm

ole

ye

)

WTWTWTKIKIKI

WTWTWTKIKIKI




















TT


343434343434762442 P GI LQ PD

TT

TT TT


TT TT


TT



Dimerization

interface

Asp477

Phe472

Asp477

Leu487

Phe429

Gly434

Leu444

A

B C RPE65 ACO







A

250

150

10075

50

37

25

20

ACOACO

RPE65

ACO

RPE65

D477G

ACOACO

RPE65

ACO

RPE65

D477G


Volume (mL)

20 40 60 80

Absorb

ance a

t 280 n

m

ACOACORPE65ACORPE65

D477G

Time (min)

0 1 2 3

Pro

du

ct

En

zym

e (microm

olmicrom

ol)

0

20

40

60 ACORPE65

D477G

ACOACORPE
























ACORPE65

A B C



vs ACORPE65

Trp45

(37)

Lys463

(508)

Asn461

(506)

Ser431

(474)

Val442

(485)

Asp434

(477)














A B

C D








































Immunoprecipitation


Immunocytochemistry











SLO and OCT imaging


ERG














Structure analysis




Statistics



Acknowledgements




















































































ddy128-TF1

ddy128-TF2

ddy128-TF3







WT

WT

WT

KI

KIK

I

WT RPE65D477G RPE65

Neo

Neo


475 476 477 478 479


Codon

WTD477G

A

B C


D G

F1

R1

D G

Ex7 8 9 10 11 12 13 14

WT RPE65

Targeting

Vector

Targeted

RPE65

Final

KI RPE65









1 41 72 34 13Total 75 20





Ub Pulldown

A B

WT

WT

WT

KI

KIK

I

150

10075

50

37

20

RPE65

β-actin

WT

WT

WT

KI

KIK

I
















WTWT

WTKI

KIKI













B

AWTWT

WTKI

KIKI


ON

L T

hic

kness (

microm

)

0

10

20

30

40

50

60

70

WTWT WTKI KIKI

57 microm

52 microm

50 microm

ON

L T

hic

kness (

microm

)

0

10

20

30

40

50

60

70

ON

L T

hic

kness (

microm

)

0

10

20

30

40

50

60

70









A

B

C

D

0

200

400

600

800

1000

1200

1400

1600

0

200

400

600

800

1000

-4 -3 -2 -1 0 1 2 3

0

200

400

600

800

1000

0

200

400

600

800

1000

-4 -3 -2 -1 0 1 2 3 -4 -3 -2 -1 0 1 2 3

0

200

400

600

800

1000

1200

1400

1600

0

200

400

600

800

1000

1200

1400

1600

-10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 250

20

30

10

40

50

0

20

30

10

40

50

0

20

30

10

40

50

-10 -05 00 05 10 15 20 25

-4 -3 -2 -1 0 1 2 3-4 -3 -2 -1 0 1 2 3-4 -3 -2 -1 0 1 2 3

-10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 250

50

100

150

200

250

300

350

0

50

100

150

200

250

300

350

0

50

100

150

200

250

300

350


3-month-old

3-month-old

3-month-old

6-month-old

6-month-old

6-month-old

9-month-old

9-month-old

9-month-old

WTWTWTKIKIKI

WTWTWTKIKIKI

WTWTWTKIKIKI

WTWTWTKIKIKI

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)


Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)


Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)


Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)



Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

















A B

D E

C

F G

0

100

200

300

400

500

600

11-c

is-r

etin

al (

pm

ole

ye)

0

100

200

300

400

500

600

11-c

is-r

etin

al (

pm

ole

ye)

0

50

100

150

200

250

300

Re

tinyl

est

ers

(p

mo

leye

)

0

50

100

150

200

250

300

Re

tinyl

est

ers

(p

mo

leye

)





0

200

400

600

800

Re

tin

yl e

ste

rs (

pm

ole

ye

)



0

100

200

300

400

500

11

-cis

-re

tin

al (p

mo

le

ye

)

WTWT

WTKI

KIKI

a

b

a

b

a

b

0 10 20 30 40Time (min)

0 10 20 30 40Time (min)

0 10 20 30 40Time (min)

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

WTWT WTKI KIKI0

10

20

30

40

50

60

11

-cis

-re

tin

al (p

mo

le

ye

)

WTWT WTKI KIKI0

200

400

600

800

1000

1200

Re

tin

yl e

ste

rs (

pm

ole

ye

)

WTWTWTKIKIKI

WTWTWTKIKIKI




















TT


343434343434762442 P GI LQ PD

TT

TT TT


TT TT


TT



Dimerization

interface

Asp477

Phe472

Asp477

Leu487

Phe429

Gly434

Leu444

A

B C RPE65 ACO







A

250

150

10075

50

37

25

20

ACOACO

RPE65

ACO

RPE65

D477G

ACOACO

RPE65

ACO

RPE65

D477G


Volume (mL)

20 40 60 80

Absorb

ance a

t 280 n

m

ACOACORPE65ACORPE65

D477G

Time (min)

0 1 2 3

Pro

du

ct

En

zym

e (microm

olmicrom

ol)

0

20

40

60 ACORPE65

D477G

ACOACORPE
























ACORPE65

A B C



vs ACORPE65

Trp45

(37)

Lys463

(508)

Asn461

(506)

Ser431

(474)

Val442

(485)

Asp434

(477)














A B

C D








































Immunoprecipitation


Immunocytochemistry











SLO and OCT imaging


ERG














Structure analysis




Statistics



Acknowledgements




















































































ddy128-TF1

ddy128-TF2

ddy128-TF3



Ub Pulldown

A B

WT

WT

WT

KI

KIK

I

150

10075

50

37

20

RPE65

β-actin

WT

WT

WT

KI

KIK

I
















WTWT

WTKI

KIKI













B

AWTWT

WTKI

KIKI


ON

L T

hic

kness (

microm

)

0

10

20

30

40

50

60

70

WTWT WTKI KIKI

57 microm

52 microm

50 microm

ON

L T

hic

kness (

microm

)

0

10

20

30

40

50

60

70

ON

L T

hic

kness (

microm

)

0

10

20

30

40

50

60

70









A

B

C

D

0

200

400

600

800

1000

1200

1400

1600

0

200

400

600

800

1000

-4 -3 -2 -1 0 1 2 3

0

200

400

600

800

1000

0

200

400

600

800

1000

-4 -3 -2 -1 0 1 2 3 -4 -3 -2 -1 0 1 2 3

0

200

400

600

800

1000

1200

1400

1600

0

200

400

600

800

1000

1200

1400

1600

-10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 250

20

30

10

40

50

0

20

30

10

40

50

0

20

30

10

40

50

-10 -05 00 05 10 15 20 25

-4 -3 -2 -1 0 1 2 3-4 -3 -2 -1 0 1 2 3-4 -3 -2 -1 0 1 2 3

-10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 250

50

100

150

200

250

300

350

0

50

100

150

200

250

300

350

0

50

100

150

200

250

300

350


3-month-old

3-month-old

3-month-old

6-month-old

6-month-old

6-month-old

9-month-old

9-month-old

9-month-old

WTWTWTKIKIKI

WTWTWTKIKIKI

WTWTWTKIKIKI

WTWTWTKIKIKI

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)


Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)


Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)


Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)



Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

















A B

D E

C

F G

0

100

200

300

400

500

600

11-c

is-r

etin

al (

pm

ole

ye)

0

100

200

300

400

500

600

11-c

is-r

etin

al (

pm

ole

ye)

0

50

100

150

200

250

300

Re

tinyl

est

ers

(p

mo

leye

)

0

50

100

150

200

250

300

Re

tinyl

est

ers

(p

mo

leye

)





0

200

400

600

800

Re

tin

yl e

ste

rs (

pm

ole

ye

)



0

100

200

300

400

500

11

-cis

-re

tin

al (p

mo

le

ye

)

WTWT

WTKI

KIKI

a

b

a

b

a

b

0 10 20 30 40Time (min)

0 10 20 30 40Time (min)

0 10 20 30 40Time (min)

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

WTWT WTKI KIKI0

10

20

30

40

50

60

11

-cis

-re

tin

al (p

mo

le

ye

)

WTWT WTKI KIKI0

200

400

600

800

1000

1200

Re

tin

yl e

ste

rs (

pm

ole

ye

)

WTWTWTKIKIKI

WTWTWTKIKIKI




















TT


343434343434762442 P GI LQ PD

TT

TT TT


TT TT


TT



Dimerization

interface

Asp477

Phe472

Asp477

Leu487

Phe429

Gly434

Leu444

A

B C RPE65 ACO







A

250

150

10075

50

37

25

20

ACOACO

RPE65

ACO

RPE65

D477G

ACOACO

RPE65

ACO

RPE65

D477G


Volume (mL)

20 40 60 80

Absorb

ance a

t 280 n

m

ACOACORPE65ACORPE65

D477G

Time (min)

0 1 2 3

Pro

du

ct

En

zym

e (microm

olmicrom

ol)

0

20

40

60 ACORPE65

D477G

ACOACORPE
























ACORPE65

A B C



vs ACORPE65

Trp45

(37)

Lys463

(508)

Asn461

(506)

Ser431

(474)

Val442

(485)

Asp434

(477)














A B

C D








































Immunoprecipitation


Immunocytochemistry











SLO and OCT imaging


ERG














Structure analysis




Statistics



Acknowledgements




















































































ddy128-TF1

ddy128-TF2

ddy128-TF3








B

AWTWT

WTKI

KIKI


ON

L T

hic

kness (

microm

)

0

10

20

30

40

50

60

70

WTWT WTKI KIKI

57 microm

52 microm

50 microm

ON

L T

hic

kness (

microm

)

0

10

20

30

40

50

60

70

ON

L T

hic

kness (

microm

)

0

10

20

30

40

50

60

70









A

B

C

D

0

200

400

600

800

1000

1200

1400

1600

0

200

400

600

800

1000

-4 -3 -2 -1 0 1 2 3

0

200

400

600

800

1000

0

200

400

600

800

1000

-4 -3 -2 -1 0 1 2 3 -4 -3 -2 -1 0 1 2 3

0

200

400

600

800

1000

1200

1400

1600

0

200

400

600

800

1000

1200

1400

1600

-10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 250

20

30

10

40

50

0

20

30

10

40

50

0

20

30

10

40

50

-10 -05 00 05 10 15 20 25

-4 -3 -2 -1 0 1 2 3-4 -3 -2 -1 0 1 2 3-4 -3 -2 -1 0 1 2 3

-10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 250

50

100

150

200

250

300

350

0

50

100

150

200

250

300

350

0

50

100

150

200

250

300

350


3-month-old

3-month-old

3-month-old

6-month-old

6-month-old

6-month-old

9-month-old

9-month-old

9-month-old

WTWTWTKIKIKI

WTWTWTKIKIKI

WTWTWTKIKIKI

WTWTWTKIKIKI

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)


Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)


Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)


Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)



Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

















A B

D E

C

F G

0

100

200

300

400

500

600

11-c

is-r

etin

al (

pm

ole

ye)

0

100

200

300

400

500

600

11-c

is-r

etin

al (

pm

ole

ye)

0

50

100

150

200

250

300

Re

tinyl

est

ers

(p

mo

leye

)

0

50

100

150

200

250

300

Re

tinyl

est

ers

(p

mo

leye

)





0

200

400

600

800

Re

tin

yl e

ste

rs (

pm

ole

ye

)



0

100

200

300

400

500

11

-cis

-re

tin

al (p

mo

le

ye

)

WTWT

WTKI

KIKI

a

b

a

b

a

b

0 10 20 30 40Time (min)

0 10 20 30 40Time (min)

0 10 20 30 40Time (min)

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

WTWT WTKI KIKI0

10

20

30

40

50

60

11

-cis

-re

tin

al (p

mo

le

ye

)

WTWT WTKI KIKI0

200

400

600

800

1000

1200

Re

tin

yl e

ste

rs (

pm

ole

ye

)

WTWTWTKIKIKI

WTWTWTKIKIKI




















TT


343434343434762442 P GI LQ PD

TT

TT TT


TT TT


TT



Dimerization

interface

Asp477

Phe472

Asp477

Leu487

Phe429

Gly434

Leu444

A

B C RPE65 ACO







A

250

150

10075

50

37

25

20

ACOACO

RPE65

ACO

RPE65

D477G

ACOACO

RPE65

ACO

RPE65

D477G


Volume (mL)

20 40 60 80

Absorb

ance a

t 280 n

m

ACOACORPE65ACORPE65

D477G

Time (min)

0 1 2 3

Pro

du

ct

En

zym

e (microm

olmicrom

ol)

0

20

40

60 ACORPE65

D477G

ACOACORPE
























ACORPE65

A B C



vs ACORPE65

Trp45

(37)

Lys463

(508)

Asn461

(506)

Ser431

(474)

Val442

(485)

Asp434

(477)














A B

C D








































Immunoprecipitation


Immunocytochemistry











SLO and OCT imaging


ERG














Structure analysis




Statistics



Acknowledgements




















































































ddy128-TF1

ddy128-TF2

ddy128-TF3

A

B

C

D

0

200

400

600

800

1000

1200

1400

1600

0

200

400

600

800

1000

-4 -3 -2 -1 0 1 2 3

0

200

400

600

800

1000

0

200

400

600

800

1000

-4 -3 -2 -1 0 1 2 3 -4 -3 -2 -1 0 1 2 3

0

200

400

600

800

1000

1200

1400

1600

0

200

400

600

800

1000

1200

1400

1600

-10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 250

20

30

10

40

50

0

20

30

10

40

50

0

20

30

10

40

50

-10 -05 00 05 10 15 20 25

-4 -3 -2 -1 0 1 2 3-4 -3 -2 -1 0 1 2 3-4 -3 -2 -1 0 1 2 3

-10 -05 00 05 10 15 20 25 -10 -05 00 05 10 15 20 250

50

100

150

200

250

300

350

0

50

100

150

200

250

300

350

0

50

100

150

200

250

300

350


3-month-old

3-month-old

3-month-old

6-month-old

6-month-old

6-month-old

9-month-old

9-month-old

9-month-old

WTWTWTKIKIKI

WTWTWTKIKIKI

WTWTWTKIKIKI

WTWTWTKIKIKI

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic b

-wa

ve

am

plit

ud

e (

microV

)


Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)


Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)

Sco

top

ic a

-wa

ve

am

plit

ud

e (

microV

)


Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

a-w

ave

am

plit

ud

e (

microV

)



Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

Ph

oto

pic

b-w

ave

am

plit

ud

e (

microV

)

















A B

D E

C

F G

0

100

200

300

400

500

600

11-c

is-r

etin

al (

pm

ole

ye)

0

100

200

300

400

500

600

11-c

is-r

etin

al (

pm

ole

ye)

0

50

100

150

200

250

300

Re

tinyl

est

ers

(p

mo

leye

)

0

50

100

150

200

250

300

Re

tinyl

est

ers

(p

mo

leye

)





0

200

400

600

800

Re

tin

yl e

ste

rs (

pm

ole

ye

)



0

100

200

300

400

500

11

-cis

-re

tin

al (p

mo

le

ye

)

WTWT

WTKI

KIKI

a

b

a

b

a

b

0 10 20 30 40Time (min)

0 10 20 30 40Time (min)

0 10 20 30 40Time (min)

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

WTWT WTKI KIKI0

10

20

30

40

50

60

11

-cis

-re

tin

al (p

mo

le

ye

)

WTWT WTKI KIKI0

200

400

600

800

1000

1200

Re

tin

yl e

ste

rs (

pm

ole

ye

)

WTWTWTKIKIKI

WTWTWTKIKIKI




















TT


343434343434762442 P GI LQ PD

TT

TT TT


TT TT


TT



Dimerization

interface

Asp477

Phe472

Asp477

Leu487

Phe429

Gly434

Leu444

A

B C RPE65 ACO







A

250

150

10075

50

37

25

20

ACOACO

RPE65

ACO

RPE65

D477G

ACOACO

RPE65

ACO

RPE65

D477G


Volume (mL)

20 40 60 80

Absorb

ance a

t 280 n

m

ACOACORPE65ACORPE65

D477G

Time (min)

0 1 2 3

Pro

du

ct

En

zym

e (microm

olmicrom

ol)

0

20

40

60 ACORPE65

D477G

ACOACORPE
























ACORPE65

A B C



vs ACORPE65

Trp45

(37)

Lys463

(508)

Asn461

(506)

Ser431

(474)

Val442

(485)

Asp434

(477)














A B

C D








































Immunoprecipitation


Immunocytochemistry











SLO and OCT imaging


ERG














Structure analysis




Statistics



Acknowledgements




















































































ddy128-TF1

ddy128-TF2

ddy128-TF3





A B

D E

C

F G

0

100

200

300

400

500

600

11-c

is-r

etin

al (

pm

ole

ye)

0

100

200

300

400

500

600

11-c

is-r

etin

al (

pm

ole

ye)

0

50

100

150

200

250

300

Re

tinyl

est

ers

(p

mo

leye

)

0

50

100

150

200

250

300

Re

tinyl

est

ers

(p

mo

leye

)





0

200

400

600

800

Re

tin

yl e

ste

rs (

pm

ole

ye

)



0

100

200

300

400

500

11

-cis

-re

tin

al (p

mo

le

ye

)

WTWT

WTKI

KIKI

a

b

a

b

a

b

0 10 20 30 40Time (min)

0 10 20 30 40Time (min)

0 10 20 30 40Time (min)

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

0

5

10

15

20

25

Ab

sorb

an

ce a

t 3

25

nm

WTWT WTKI KIKI0

10

20

30

40

50

60

11

-cis

-re

tin

al (p

mo

le

ye

)

WTWT WTKI KIKI0

200

400

600

800

1000

1200

Re

tin

yl e

ste

rs (

pm

ole

ye

)

WTWTWTKIKIKI

WTWTWTKIKIKI




















TT


343434343434762442 P GI LQ PD

TT

TT TT


TT TT


TT



Dimerization

interface

Asp477

Phe472

Asp477

Leu487

Phe429

Gly434

Leu444

A

B C RPE65 ACO







A

250

150

10075

50

37

25

20

ACOACO

RPE65

ACO

RPE65

D477G

ACOACO

RPE65

ACO

RPE65

D477G


Volume (mL)

20 40 60 80

Absorb

ance a

t 280 n

m

ACOACORPE65ACORPE65

D477G

Time (min)

0 1 2 3

Pro

du

ct

En

zym

e (microm

olmicrom

ol)

0

20

40

60 ACORPE65

D477G

ACOACORPE
























ACORPE65

A B C



vs ACORPE65

Trp45

(37)

Lys463

(508)

Asn461

(506)

Ser431

(474)

Val442

(485)

Asp434

(477)














A B

C D








































Immunoprecipitation


Immunocytochemistry











SLO and OCT imaging


ERG














Structure analysis




Statistics



Acknowledgements




















































































ddy128-TF1

ddy128-TF2

ddy128-TF3

TT


343434343434762442 P GI LQ PD

TT

TT TT


TT TT


TT



Dimerization

interface

Asp477

Phe472

Asp477

Leu487

Phe429

Gly434

Leu444

A

B C RPE65 ACO







A

250

150

10075

50

37

25

20

ACOACO

RPE65

ACO

RPE65

D477G

ACOACO

RPE65

ACO

RPE65

D477G


Volume (mL)

20 40 60 80

Absorb

ance a

t 280 n

m

ACOACORPE65ACORPE65

D477G

Time (min)

0 1 2 3

Pro

du

ct

En

zym

e (microm

olmicrom

ol)

0

20

40

60 ACORPE65

D477G

ACOACORPE
























ACORPE65

A B C



vs ACORPE65

Trp45

(37)

Lys463

(508)

Asn461

(506)

Ser431

(474)

Val442

(485)

Asp434

(477)














A B

C D








































Immunoprecipitation


Immunocytochemistry











SLO and OCT imaging


ERG














Structure analysis




Statistics



Acknowledgements




















































































ddy128-TF1

ddy128-TF2

ddy128-TF3


















ACORPE65

A B C



vs ACORPE65

Trp45

(37)

Lys463

(508)

Asn461

(506)

Ser431

(474)

Val442

(485)

Asp434

(477)














A B

C D








































Immunoprecipitation


Immunocytochemistry











SLO and OCT imaging


ERG














Structure analysis




Statistics



Acknowledgements




















































































ddy128-TF1

ddy128-TF2

ddy128-TF3





A B

C D








































Immunoprecipitation


Immunocytochemistry











SLO and OCT imaging


ERG














Structure analysis




Statistics



Acknowledgements




















































































ddy128-TF1

ddy128-TF2

ddy128-TF3





























Immunoprecipitation


Immunocytochemistry











SLO and OCT imaging


ERG














Structure analysis




Statistics



Acknowledgements




















































































ddy128-TF1

ddy128-TF2

ddy128-TF3


SLO and OCT imaging


ERG














Structure analysis




Statistics



Acknowledgements




















































































ddy128-TF1

ddy128-TF2

ddy128-TF3

Structure analysis




Statistics



Acknowledgements




















































































ddy128-TF1

ddy128-TF2

ddy128-TF3






























































ddy128-TF1

ddy128-TF2

ddy128-TF3

insights into the pathogenesis of dominant retinitis...

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