functional analyses of human apolipoprotein cii by site ... · abbreviations used: apocii,...

Running title: Apolipoprotein CII mutants

1

Functional analyses of human apolipoprotein CII by site-directed

mutagenesis. Identification of residues important for activation of

lipoprotein lipase*

Yan Shen§, #, Aivar Lookene§, ¶, Solveig Nilsson§ and Gunilla Olivecrona§, **

From the §Department of Medical Biosciences, Umeå University, SE-90 187 Umeå,

Sweden and ¶ National Institute of Chemical Physics and Biophysics, Tallinn, Estonia

*This work was supported by the Swedish Medical Research Council (727 and 12203),

by the Swedish Royal Academy of Sciences, by the Estonian Science Foundation (4925)

and by Donation Funds at the Medical Faculty, Umeå University

#Present address: Cardiovascular Research Center, Cardiology Division, Massachusetts

General Hospital, Harvard Medical School, Boston, Massachusetts, USA

Additional key words: protein interaction, lipid interaction, kinetics, liposomes, mutants,

lipase activation

Abbreviations used: ApoCII, apolipoprotein CII; ApoAI, apolipoprotein AI; CD, circular

dichroism; GdmCl, guanidinium chloride; IPTG, isopropyl-β-D-thiogalactoside; LCAT,

lecithin:cholesterol acyl transferase; LPL, lipoprotein lipase; PCR, polymerase chain

reaction

**To whom correspondence should be addressed: Prof. Gunilla Olivecrona

Tel. +46-90-7867762, Fax. +46-90-7867840, E-mail.

[email protected]

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on November 21, 2001 as Manuscript M105421200 by guest on June 8, 2018

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SUMMARY

Apolipoprotein CII (apoCII) activates lipoprotein lipase (LPL). Seven residues, located on

one face of a model α-helix spanning residues 59-75, are fully conserved in apoCII from

ten different animal species. We have mutated these residues one by one. Substitution of

Ala59 by glycine, or Thr62 and Gly65 by alanine did not change the activation, indicating

that these residues are outside the LPL-binding site. Replacement of Tyr63, Ile66, Asp69

or Gln70 by alanine lowered the affinity for LPL and the catalytic activity of the

LPL/apoCII complex. For each residue several additional replacements were made. Most

mutants retained some activating ability, but replacement of Tyr63 by phenylalanine or

tryptophan and Gln70 by glutamate caused almost complete loss of activity. All mutants

bound to liposomes with similar affinity as wild-type apoCII, and they also bound with

similar affinity to LPL in the absence of hydrolyzable lipids. However, the inactive

mutants did not compete with wild-type apoCII in the activation assay. Therefore we

conclude that the productive apoCII/LPL interaction may be dependent on substrate

molecules. In summary, our data demonstrate that residues 63, 66, 69 and 70 are of

special importance for the function of apoCII, but no single amino acid residue is

absolutely crucial.

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INTRODUCTION

Human apolipoprotein CII (apoCII) is a 79-residue protein synthesized in the intestine

and liver and secreted as a surface component of chylomicrons, very low density

lipoproteins and high-density lipoproteins (1-3). ApoCII plays an important role in plasma

lipid metabolism as an activator of lipoprotein lipase (LPL) (1,4), clearly illustrated by the

severe hypertriglyceridemia that is associated with genetic deficiency of apoCII (5). A

number of natural mutations affecting the apoCII gene have been identified and

characterized (6). Most mutations reported so far result in either very low levels of

plasma apoCII or major structural changes in the protein, e.g. prematurely truncated

forms. A Trp26Arg mutation was recently reported to lead to apoCII deficiency, probably

because of a disturbed lipid interaction of the activator (7). This is the only example of a

single point mutation in apoCII leading to loss of function, but the interpretation is not

straightforward since the protein was not detectable in plasma.

The structure of full-length human apoCII in complex with SDS-micelles was recently

reported based on studies by NMR (8). The protein contains three regions with helical

conformation spanning residues 16-36, 50-56 and 63-77. The lipase-activating region of

apoCII has previously been localized to the C-terminal one third of the sequence, from

about residue 56, while the N-terminal two thirds of the sequence is involved in lipid

binding (9-11). In most studies it has been found that apoCII causes an increase in the

catalytic efficiency of LPL, and not an enhanced lipid affinity of the enzyme (10, 12). In

spite of a long history of studies on the effects of apoCII, the detailed mechanism for the

activation remains unknown. It has been proposed that apoCII binds to LPL and induces

conformational changes in the lipase, or changes its orientation at the lipid-water

interface. As a result of these changes, LPL may become more efficient in binding of

individual substrate molecules or in release of lipolysis products (fatty acids) (13). It has

also been proposed that apoCII, as an amphipathic molecule, may influence the structural

organization of lipids such that substrate molecules become more available to the active

site of LPL (8, 12). However, synthetic peptides corresponding to the C-terminal part,


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which does not bind to lipid, are able to activate LPL in vitro (14). The strongest evidence

for activation of LPL via formation a complex with apoCII come from data showing that

LPL and apoCII bind to each other even in the absence of a lipid/water interface (15).

In the present study we have focused on the region comprising residues 56-79 in human

apoCII. Compared to the corresponding region in apoCII from 9 other animal species,

there are only 7 fully conserved residues: Ala59, Thr62, Tyr63, Gly65, Ile66, Asp69 and

Gln70 (16). In the rest of the apoCII molecule there are only two other fully conserved

residues, Leu15 and Ala33, but both are outside the activating region. We have

systematically evaluated the functional role of each of the seven conserved residues by

using site-directed mutagenesis, followed by analyses of the secondary structure by

circular dichroism (CD) measurements and analyses of activating ability. We have also

directly studied the interaction of the mutants with LPL using energy transfer

measurements. The results suggest that the LPL-binding site in apoCII consists of mainly

4 residues located on one face of the third α-helix located closest to the C-terminus.

EXPERIMENTAL PROCEDURES

Materials

Restriction endonucleases were from Amersham Phamacia Biotech. T4 ligase was from

Boehringer Mannheim. Kanamycin was from Duchefa, Haarle, The Netherlands.

Isopropyl-β-D-thiogalactoside (IPTG), dansyl chloride and egg yolk phosphatidylcholine

were from Sigma, St. Louis, MO. Low-melting agarose and polyvinylidene difluoride

filters PVDF (0.2 µm) were from BioRad, Hercules, CA. JET sorb Gel extraction kit was

from Genomed. The expression vector pET-29a, S-protein agarose and BL21(DE3)

competent cells were from Novagen, Inc., Madison, WI. Epicurian Coli BL21-

codonPlus(DE3)-RIL competent Cells and QuikChangeTM Site-Directed Mutagenesis Kit

were from Stratagene, La Jolla, CA. Penta His-tag antibody and Ni2+-NTA agarose were

purchased from QIAGEN GmbH, Hilden, Germany. Factor Xa from bovine plasma was

from Biolads, Beverly, MA. Serva Blue G was from Serva, GmbH, Heidelberg, Germany.


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Transfer Membrane was from Pall Corporation, Ann Arbor, MI. Dialysis tubes were from

spectrum Medical Industries, Houston, TX. Low molecular weight marker was from

Amersham, Buckinghamshire, UK. For the analyses of LPL activity an emulsion with a

composition corresponding to Intralipid 10 % (soy bean triacylglycerols emulsified in

egg yolk phospholipids), but containing in addition a trace amount of 3H-oleic acid-

labeled trioleoylglycerol (kindly prepared by KABI-Fresenius, Uppsala, Sweden) was

used. BCA protein Assay was from Pierce, Rockford, IL. LPL was purified from bovine

milk as described (17). The stock solutions contained about 0.5 mg LPL/ml 10 mM

Bistris, pH 6.5, 1M NaCl.1,2-Di-O-hexadecyl-sn-Glycero-3-phosphatidylcholine was

from Larodan Fine Chemicals AB, Malmö, Sweden. Oligonucleotides were synthesized

by Pharmacia Biotech, Uppsala, Sweden. An antiserum against human apoCII, purified

from plasma and coupled as hapten to keyhole limpet hemocyanin, was produced in a

rabbit by a standard immunization procedure (18). Computer-assisted analyses were

accomplished with the programs Lasergene DNA Star Inc., Madison, WI and Wisconsin

sequence analysis package, Genetics Computer Group (GCG). For secondary structure

prediction of the mutants the programs at the NPS@(dNetwork Protein Sequence

analysis) web sites were used (http://pbil.icbp.fr/cgibin/npsa_automat.

pl?page=/NPSA7npsa_seccons.html). Binding and activation curves were fitted using the

program Fig. P (BIOSOFT, Cambridge, UK).

Plasmid constructions

Two plasmids were used for expression of recombinant human apoCII: pET29a-hapoCII

and pET-histag-hapoCII.

cDNA for human apolipoprotein CII in the pUC18 vector (hapoCII, a kind gift from Dr

Steven Humphries, London) was connected to an EcoRI linker coding for the cleavage

site for Factor Xa (coding sequence for factor Xa cleavage: Ile-Glu-Gly-Arg-) attached to

the first codon of the mature apoCII cDNA sequence. The whole DNA fragment was

cloned into the EcoRI/XhoI site of the vector pET29a(+) to form the expression plasmid

pET29a-hapoCII. The recombinant was verified by EcoRI/XhoI cleavage and DNA


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sequencing. The plasmid was transformed into E. coli BL21(DE3) to express the fusion

protein S-tag-“factor Xa cleavage site”-hapoCII (117 amino acid residues in total). For

production of pET-histag-hapoCII, the pET29a-hapoCII plasmid was cleaved with NdeI

and EcoRI to delete the sequence encoding the S-tag and the thrombin cleavage site. The

complementary oligonucleotides 5’ TATGCACCATCATCATCATCATG 3’ and 5’

AATTCATGATGATGATGATGGTGCA 3’, coding for an N-terminal 6xHis-tag, were

ligated to the cleaved pET29a vector. The new construct resulted in a vector with the T7

RNA polymerase promoter and the lac operator followed by sequences coding for 6xHis-

tag, Factor Xa cleavage site and human apoCII.

Site-directed mutagenesis of human apoCII

Mutant 63A was generated by overlap extension by using the polymerase chain reaction

(PCR) and four different primers (Table 1). All other mutants were generated using

QuikchangeTM Site- Directed Mutagenesis Kit. The basic procedure for generation of

each mutant utilizes a template vector with apoCII cDNA (pET29a-hapoCII or pET-

histag-hapoCII) and two synthetic oligonucleotide primers containing the desired

mutation (Table 1). The oligonucleotide primers, each complementary to opposite strands

of the vector, were extended by Pfu Turbo DNA polymerase. All mutants were sequenced

using ABI 337 DNA sequencer, Dye Terminator Cycle Sequencing Ready Reaction Kit,

Perkin Elmer.

Expression and purification of wild-type and mutated human apoCII proteins

pET29-hapoCII and its mutants (A59G, T62A, Y63A, G65A, I66A, D69A and Q70A)

were introduced into BL21(DE3) competent cell. Expression, purification and cleavage of

the fusion proteins by factor Xa were made as described (Martinez et al., manuscript in

preparation), but in LB medium.

pET-histag-hapoCII ant its mutants (Y63A, Y63T, Y63L, Y63F, Y63W, I66A, I66L,

I66V, I66F, D69A, D69S, D69N, D69E, Q70A, Q70A, Q70N, Q70E, L72A, S73A and

V74A) were transformed into Epicuran Coli BL21-CodonPlus(DE3)-RIL competent


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cell. The bacteria were grown at 37°C in Terrific Broth containing kanamycin (50 µg/ml)

to an OD600nm of 1.0. At this point the temperature was lowered to 16°C and one hour

later expression of human apoCII and its variants was induced with 0.5 mM IPTG. On the

next day, the bacteria were harvested by centrifugation and purified by Ni2+-NTA agarose

as described (19). Briefly, buffers containing 8 M urea, 0.1 M NaH2PO4, 0.01 M Tris-Cl

were used to produce a stepwise gradient from pH 8.0 to pH 4.5. The fusion proteins that

eluted at pH 7.2 were dialyzed for 24 hours at 4°C against ammonium bicarbonate and

were then lyophilized.

High performance liquid chromatography of wild-type and mutated His-tagged

human apoCII proteins

High performance ion exchange was performed on an ÄKTA purifier 900 (Amersham-

Pharmacia) using a Mini Q PE 4.6/50 column. The fusion proteins eluted between pH 6.8

and pH 4.5 from Ni2+-NTA agarose were dialyzed against 6 M urea, 0.01 M Tris pH 8.2

and were filtered through a 0.2 µm filter (Gelman Sciences, 600 S, Wagner Rd, Ann

Arbor, MI 48103) before they were applied to the column at a pressure of 5 MPa. Proteins

were then eluted by a linear gradient of Tris-Cl (pH 8.2) from 0.01 to 0.15 M. Remaining

proteins were washed out by 1M NaCl and/or 6 M guanidinium chloride (GdmCl).

Protein concentrations in all the fractions were measured by the BCA assay. Fractions

were combined and the proteins were analyzed by electrophoresis on SDS-Tricine gels

and by N-terminal amino acid sequence analyses as previously described (16). Two forms

of apoCII were detected by matrix-assisted laser desorption/ionization time-of-flight mass

spectrometry (MALDI-TOF MS) on a homemade instrument at National Institute of

Chemical Physics and Biophysics, Tallinn, Estonia.

Proteins analyzed by Tricine SDS-PAGE and Western blotting

The expressed proteins were separated on Tricine -SDS-PAGE and were stained by

0.025% Serva-blue G in 10% acetic acid (19). The separated proteins were transferred

onto a nitrocellulose filter. The filter was put in TBST (10 mM Tris-Cl, pH 8.0, 0.5 M

NaCl and 0.05% Tween-20) containing 5% bovine serum albumin and 3% gelatin for 30


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minutes at 42°C. Human apoCII and mutants were detected by incubation with the rabbit

anti-human apoCII serum (dilution 1:100). After incubation overnight the filter was

washed 3 x 10 min in TBST. Then the filter was incubated with goat anti-rabbit IgG

(conjugated to alkaline phosphatase) diluted 1:3000 in TBST. Finally the filter was

washed 3 x 10 min as before. Bound antibodies were detected by addition of substrate to

alkaline phosphatase and the reaction was stopped by rinsing the filter in H2O. For

blotting with the penta His-tag antibody, the nitrocellulose filter was handled as

recommended by the manufacturer.

Circular dichroism

Possible global changes of the secondary structure of some of the mutated apoCIIs were

investigated by measurements of far UV spectra at 25 °C on a CD6 spectrodichrograph

(Jobin-Yvon Instruments SA, Longjumeau, France) in a 0.5 mm path-length cell. For this

the protein concentrations were determined by absorbancy at 280 nm.

Lipase assays

For measurement of LPL activity we used an assay mixture of 200 µl containing 2 mg of

triacylglycerols from the lipid emulsion. The medium contained 0.1 M NaCl, 0.1 M Tris-

Cl, 20 µg heparin, and 12 mg of bovine serum albumin. The pH was 8.5. To study the

activation, wild-type apoCII and its mutants were dissolved in 5 M urea, 10 mM Tris-Cl

(pH 8.2). The protein concentration was in each case determined by the BCA protein

assay. Five µl of the stock solution of apoCII, or the same volume of dilutions in 5 M

urea, 10 mM Tris (pH 8.2), was added to the incubations. The reactions were started by

addition of LPL and were stopped after incubation for 15 min at 25 º C by addition of

organic solvents for extraction of the labeled free fatty acids (19). The lipase activity is

expressed in U/mg of LPL, where 1 U corresponds to release of 1 µmol fatty acid per

min. For studies of competition between the wild-type apoCII and mutants for activation

of LPL the experiments were carried out as described, except for the presence of 2.5 µM

Q70E, Y63F or Y63W in the incubation mixture together with wild-type apoCII.


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Determination of kinetic parameters from activation curves

Quinn et al. have shown that the activation of LPL by apoCII can be described by a

random mechanism (20). Based on this mechanism, the velocity of the reaction (v) at

substrate saturation is expressed as

++⋅⋅= ⋅

d

dcat

KC

KC�Lk v

Where L corresponds to LPL and C corresponds to apo CII.

This equation was used for determination of Kd and β. Kd characterizes the apparent

affinity of apoCII for LPL, and β indicates how much more active the LPL/apoCII

complex was compared to LPL alone (fold activation). When β is equal to 1, apoCII does

not activate LPL. The kinetic constants were determined by using the program Fig. P.

Preparation of liposomes

Two different phospholipids were used in this study. One was 1,2-di-O-hexadecyl-sn-

glycero-3-phosphatidylcholine, which cannot be hydrolyzed by LPL. This ether lipid was

used for studies of the apoCII-LPL interaction. The other was egg yolk

phosphatidylcholine, used for studies of the interaction between apoCII and lipids. For

preparation of liposomes the lipids were first dissolved in chloroform and dried under

nitrogen. The dry lipid was then dispersed in 5 mM Tris-Cl, 0.15 M NaCl, 0.02% NaN3,

pH 7.4 and sonicated without break in ice-cold buffer by an MSE Soniprep 150 equipped

with a 5 mm probe. Egg yolk phosphatidylcholine was sonicated for 30 minutes and 1,2-

di-O-hexadecyl-sn-glycero-3-phosphatidylcholine was sonicated for 1 hr. The liposomes

were kept for 2-3 hours at 37 °C before they were centrifuged for 10 min at 3000 rpm at 4

°C. The supernatant was collected and dialyzed against 0.1 M Tris-Cl (pH 8.1). Any

possible large aggregates were removed by filtration through 0.2 µM filter. The

concentrations of phospholipids in the final preparations were measured using an

enzymatic kit from Wako Chemical GmbH, Germany.


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Determination of equilibrium dissociation constants by dansylation of apoCII

The wild-type apoCII and the mutants (His-tagged, not cleaved) were dansylated

according to the procedure described in a previous study (19). The relative increase of

fluorescence at 490 nm on excitation at 280 nm was measured after each addition of

bovine LPL to the solution of dansylated apoCII in 0.1 M Tris-Cl (pH 8.1) with 10 µl 1,2-

di-O-hexadecyl-sn-glycero-3-phosphatidylcholine liposomes (final concentration 10 µg

/ml). For the measurements a Spex FluoroMax-2 fluorometer or a Shimadzu

spectofluorophotometer model RF500 were used. The fluorescence background of apoCII

was subtracted from the experimental values. Binding of wild-type apoCII to LPL was

also studied in the absence of liposomes.

Binding of apoCII to liposomes

Binding of apoCII to liposomes was measured by using the change of fluorescence of the

single tryptophan at residue 26 as described in our previous study (19). The increase of

fluorescence was attributed to binding of apoCII to the liposomes.

RESULTS

Expression and purification of apoCII and mutants

Comparison of the amino acid sequences of apoCII from the 10 different animal species

shows that there are seven fully conserved residues in the C-terminal region (Fig. 1A). All

were located on the same side of a model α-helix spanning residue 59 to 75 (Fig. 1B). To

study the role of the conserved residues for the function of apoCII we first made

substitutions with alanine using the codons GCC, GCA and GCT. Ala59 was instead

substituted with glycine. The seven mutants and the wild-type apoCII were expressed

using the pET29a-hapoCII vector, generating S-tagged apoCII proteins that were purified

on S-protein agarose and cleaved by factor Xa (Fig. 2).


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In order to identify critical chemical groups or side chains, more mutants were generated

at residues 63, 66, 69 and 70. These mutants were expressed using the pET-histag-

hapoCII vector and purified on Ni2+-NTA-agarose. When the pH of the elution buffer was

decreased to 6.8 or lower, a protein with an apparent molecular weight of 26 kDa was

eluted in addition to apoCII (Fig. 3). For separation of this protein from apoCII, ion-

exchange chromatography on a Mini Q column was used (Fig. 4). The contaminating

protein bound stronger than apoCII to the column and was eluted by 1 M NaCl and/or 6M

GdmCl (Fig. 4, inset). N-terminal sequence analyses of the contaminant gave the

sequence KVAKDLVVS, which was found to correspond to an E. Coli protein consisting

of a domain homologous to FK506-binding proteins (21). This protein is rich in

potentially metal-binding amino acids, such as histidine, cysteine, and acidic amino acids,

explaining why it bound very strongly to the Ni2+-NTA agarose. ApoCII was separated

into two forms by the ion-exchange column (Fig. 4, inset). N-terminal amino acid

sequence analysis showed that they both had the same sequence, MHHHHH. Mass

spectrometry revealed that there was a 260 dalton difference between the two forms. This

corresponded to a lack of the last two glutamate residues at the C-terminal end (the

theoretical difference is 258.2 dalton). The expressed wild-type apoCII contained more

than 75% of the short form. Since the two forms of apoCII showed similar ability to

activate LPL (data not shown), we used a mixture of the two forms of each expressed

mutant for the following experiments. In another set of experiments we compared in

detail the ability of His-tagged apoCII with apoCII purified from human plasma with

regard to actvation of LPL. From these data we know that the His-tag at the N-terminus

does not interfere with the activation (Shen Y, Lookene A, Vija H, Olivecrona G, data

unpublished). Therefore we decided to use the His-tagged recombinant proteins without

further cleavage for the present functional studies.

Activation of LPL by apoCII mutants

Substitution of Ala59 by glycine and residues Thr62, Gly65 by alanine did not have much

effect on the activating ability of apoCII (Fig. 5). In contrast, the mutants with alanine

substitutions at position 63, 66, 69 and 70 showed a defective function (Fig. 5). For


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quantitative comparisons, the kinetic parameters Kd and β were determined from the

activation curves. Under our experimental conditions the parameters for wild-type apoCII

were Kd = 0.29 ± 0.07 µM and β = 6.2 ± 0.1. Corresponding data for all mutants are

shown in Table 2.

To investigate whether the markedly reduced activation ability of some of the mutants

was due to a major change of secondary structure, mutants Y63A, I66A, D69A and Q70A

were analyzed by CD measurements in the presence of liposomes of phosphatidylcholine

(Fig. 6). No major differences were found compared to the spectrum for wild-type apoCII.

All mutants retained the typical, largely helical, structure represented by double minima at

208 and 222 nm.

In addition to the alanine substitutions mentioned above, Tyr63 was replaced by threonine

(Y63T), leucine (Y63L), phenylalanine (Y63F) and tryptophan (Y63W) to investigate the

importance of the hydroxyl group and/or the aromatic ring (Fig. 7A). The Y63F and

Y63W mutants showed very low, though detectable, activation. Since the activation

curves remained linear even at the maximally achievable apoCII concentrations, it was

not possible to calculate Kd and β for these mutants. To elucidate whether the poor

activation was caused by low affinity of apoCII for LPL, or by low activity of the mutant

apoCII/LPL complexes, the mutants Y63W and Y63F were mixed with wild-type apoCII

in the same assay (Fig. 8). The activity of wild-type apoCII was not affected even at a 20-

fold excess of the inactive mutants. Therefore we concluded that the low activation by

mutants Y63F and Y63W was mainly due to a reduced affinity for LPL. In contrast, the

Y63T and the Y63L mutants were more efficient that Y63A (Fig. 7A). The Y63T bound

to LPL with Kd = 1.73 µM and enhanced the catalytic activity four-fold (β= 4.0, Table 2).

Corresponding values for the Y63L mutant were 3.14 µM and 4.2.

Substitution of Ile66 by leucine (I66L), alanine (I66A) or phenylalanine (I66F) caused

significant changes in the activation properties of apoCII (Fig. 7B). The activation curve

for I66L remained close to linear up to 3 µM. Therefore the Kd and β values for this


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mutant were determined with large deviation (Table 2). Also I66A exhibited low affinity

Kd=3.27 µM, and the maximal activation was about 3-fold (β= 2.9). I66V was the most

active among the mutants at position 66. It appeared to bind to LPL even more tightly

than wild-type apoCII (Kd = 0.11 µM). However, I66V could not activate LPL to the level

reached with wild-type apoCII (β = 3.9). At high concentrations (>3 µM) there was even

a tendency for inhibition of LPL by this mutant (Fig. 7B).

Substitution of Asp69 by hydrophilic residues such as serine (D69S), glutamate (D69E)

and asparagine (D69N) did not lead to prominent changes in the activating properties

(Fig. 7C). Effects were mainly seen on the Kd values, which were increased

approximately 6-fold compared to wild-type apoCII. Mutants D69S and D69E had similar

β values as wild-type apoCII, but for D69N it was somewhat lower (β = 4.9). The

weakest binding affinity was seen with the D69A mutant (Kd=3.40 µM).

Three mutants were generated at position Gln70: Q70A, Q70N and Q70E (Fig, 7D). The

most active one was Q70N with Kd = 2.07 µM and β=3.0. Both Q70A and Q70N showed

lowered binding affinity to LPL and decreased maximal activation (Table 2). In contrast

to these mutants, the Q70E mutant was completely inactive and could not compete with

the wild-type apoCII in the assay (Fig. 8). Thus, a negative charge at position 70 appeared

to block binding of apoCII to LPL.

In addition to substitutions at the fully conserved residues, three mutants were generated

at residue Leu72, Ser73 and Val74 by alanine replacement. These mutants bound to LPL

with an apparent affinity comparable to that of wild-type apoCII and they activated the

enzyme to the same extent as the wild-type apoCII did (data not shown) Thus, the

structures of these three residues did not appear to be essential for the function of apoCII.

Interaction between LPL and apoCII in the absence of hydrolyzable lipids

The interaction of apoCII mutants with LPL was studied in the presence of liposomes of a

non-hydrolysable lipid (1, 2-di-O-hexadecyl-sn-glycero-3-phosphatidylcholine). In these


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experiments the apoCII mutants were dansylated, and their association to LPL was

measured by monitoring fluorescence energy transfer from LPL tryptophans to the dansyl

groups of the modified apoCII molecules. Dissociation constants (Kd’) were determined

for the apoCII/LPL complexes. All studied apoCII mutants appeared to bind to LPL with

an affinity comparable to that of wild-type apoCII. The values for the dissociation

constants were 0.3-0.8 µM (Fig. 9). For wild-type apoCII this value was similar in the

absence of liposomes (data not shown). Thus, in the absence of hydrolysable lipids the

dissociation constants (Kd’) did not vary as much as they did in the presence of the

radiolabeled emulsion of triacylglycerols, where the Kd varied��

2). Figure 9 demonstrates that there was no correlation between the Kd and Kd’ values.

Interaction of apoCII with lipids

To elucidate whether the poor activation of LPL by some mutants was caused by a

reduced interaction with lipids, the mutants were titrated with liposomes of

phosphatidylcholine. Formation of lipsome/apoCII complexes was measured by

monitoring fluorescence of the single tryptophan (Trp26) of apoCII on excitation at 280

nm. We had previously shown that on association of wild-type apoCII with liposomes, the

tryptophan fluorescence increased and shifted the emission maximum from 345nm to 320

nm (19). In the present study, we detected similar changes in the fluorescence for all of

the studied apoCII mutants. Consequently, even the inactive mutants were able to interact

with lipids. Apparent dissociation constants (Klip) calculated from the titration curves,

ranged between 101-420 µM (Table 2).

DISCUSSION

The starting point for the present study was our previous observation that there are only

seven fully conserved residues in the C-terminal one third of the apoCII molecule, the

part to which the ability to activate LPL had previously been localized (16). This implied

that the detailed structure of this region, limited to residues 59-70, was important and had

therefore not changed during evolution. We generated a set of single-residue substitution


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mutants of human apoCII, mainly at the seven fully conserved sites. ApoCII was

expressed either as a fusion protein with the S-tag or the 6xHis-tag at the N-terminal end.

The expressed apoCII appeared in two forms, one of which was due to truncation of the

last two amino acid residues at the C-terminus (Glu78-Glu79). Although the short form in

some cases constituted about 75% of the total apoCII protein, we did not further separate

the individual forms since both were shown to activate LPL. Based on sequence

comparisons of apoCII from different animal species we had previously concluded that

the negatively charged residues (Glu78-Glu79) in the C-terminal end of human apoCII are

not important for the activation (16). This was further supported by the present findings

with the recombinant, truncated forms.

Effects of mutations

There was little or no effect of some of the presently investigated mutations on the

secondary structure of apoCII. We can, however, not absolutely exclude conformational

effects for some of the other mutants, which were not produced in sufficient amounts for

CD measurements. However, theoretical structure prediction using several available

computer programs did not indicate major changes for any of the mutants studied here.

To investigate if the mutations affected formation of the apoCII/ LPL complex, the

catalytic power of the complex, or both, we derived two kinetic parameters; the apparent

dissociation constant Kd and the activation factor β. The aim was to test the hypothesis

that the LPL-interaction domain of apoCII contains two regions: one contributing mainly

to activation, while the other is mainly responsible for binding of apoCII to LPL (22). For

most of our mutants, however, both parameters were affected, indicating that

discrimination between these functional parts of apoCII was not possible.

Tyr63 appeared to be the most sensitive residue. Both Kd and β were reduced for all

studied mutants. Thus, Tyr63 is probably involved both in the binding to LPL and in the

activation. Substitution by Phe, Trp, Ala or Leu reduced the activation factor more than

substitution by Thr. This indicated that the hydroxyl group could be involved in formation


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of a hydrogen bond between residue 63 and LPL during activation, but the difference

between Y63T and the wild-type was still very large. The importance of Tyr63 was

previously demonstrated by substitution with Trp and Gly in a synthetic apoCII peptide

spanning residues 44-79. This caused reduced but not lost ability to activate LPL (23).

The mutations of Ile66 suggested that at this position both shape and hydrophobicity of

the side chain are essential properties. I66A and I66F were much less efficient than I66L

and I66V, indicating that a hydrophobic aliphatic side-chain is preferred. I66V

demonstrated even stronger apparent binding affinity to LPL than wild-type apoCII. The

maximal activation level for I66V was, however, somewhat lower than that of the wild-

type apoCII. Thus, even minor structural changes at position 66 led to marked changes in

the activation. It appears as if the sterical fit of Ile66 into a narrow hydrophobic pocket in

LPL is important for full activation.

For residue Asp69, mutants D69S, D69E and D69N did not show very dramatic changes

in the activating ability, suggesting that apoCII may tolerate some variation in hydrophilic

residues at this position. The main effect was an increase in the Kd values. The D69S

mutant was practically as efficient as the D69E mutant. Thus, a negative charge at this

position did not seem to play a crucial role. In contrast to hydrophilic residues,

substitution with alanine caused a more pronounced decrease in apparent binding affinity.

One of the most dramatic effects on activation was obtained by substitution of Gln70 by

glutamate, indicating that the corresponding binding region in LPL is probably negatively

charged. Also the Q70N mutant was rather inefficient, demonstrating that the additional

carbon in the side chain of the glutamine residue is probably important for the interaction

with LPL. Substitution with alanine decreased both the binding affinity (Kd) and

activation ability (β). Thus, not only the amide group in the side chain of residue 70, but

also the length of the carbon side chain was important for the function.

LPL interactions


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To further characterize the properties of our mutants, we studied the apoCII-LPL

interaction in the absence of hydrolyzable lipids. In the system with ether phospholipids

the mutants did not show significant differences with regard to LPL affinity. These results

are in conflict with the apparent LPL affinities from the activation experiments (Kd),

where several of the mutants showed little or no activation. The competition experiments

showed that the low-activity mutants could not compete with wild-type apoCII for

binding to LPL, even at a 20-fold higher concentration. Thus, the apoCII/LPL complex

observed in the absence of hydrolyzable lipid may not be the true functional complex that

can hydrolyze lipids at optimal rate. One explanation could be that the lipid substrate is

needed for induction of the right conformation of either LPL or apoCII, and that some of

the mutations affected this interaction. It is, unfortunately, not possible to study the

interaction by fluorescence with a hydrolysable lipid. The substrate would almost

immediately be consumed by the high amounts of lipase needed for the experiments.

Recently it was reported that the synthetic apoCII fragment 39-62 activates LPL to the

same extent as full-length apoCII (24). These data cannot be understood from our present

and previous experiences. In our study, this region is intact in all affected mutants. Still

their ability to activate LPL was severely reduced or absent. Therefore, the proposed

region cannot be crucial for activation, at least not as part of native apoCII. It is possible

that the peptide, by non-specific effects, causes an increase in LPL activity. From

previous experiments the activation seems to be specific and similar effects have not been

found by other peptides. We cannot exclude that the LPL-binding site is extended towards

the N-terminal end to also involve residues 59 and 62. These residues were only

substituted by glycine and alanine, respectively, with little change in the activation.

Considering the evolutionary conservation of these residues, and their location preceding

the third α-helix (8), it is possible that other substitutions would have had effects.

Lipid interactions

The investigation of the apoCII-liposome interaction showed that all mutants had lipid-

binding affinities comparable to that of wild-type apoCII. Thus, the loss or reduction in


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the activation ability of some of the mutants was not due to reduced ability to interact

with lipid/water interfaces. This is consistent with the previous observations that the N-

terminal domain of apoCII is the part mainly responsible for binding to lipids (9-11). The

mutations could, however, still affect details in the steric arrangement of apoCII at the

lipid/water interface ways that were not detectable by the methods used here to measure

lipid interaction.

ApoCII-binding site

It is still not clear where the apoCII-binding site is located on LPL. Studies with lipase

chimeras of LPL and hepatic lipase suggest that apoCII binds to the N-terminal folding

region of LPL (25, 26). It was also suggested that apoCII interacts simultaneously with

regions located in the N- and C-terminal domains of opposing subunits of the LPL dimer

(27). The crystal structure of the related pancreatic lipase shows that its activator,

colipase, binds to the loop covering active site (28). It is, however, well known that

apoCII and colipase differ both in structure and functions. Recently the binding site of

apolipoprotein AI (apoAI) for lecithin:cholesterol acyl transferases (LCAT) was reported

by Roosbeek et al. (29). In apoAI three strictly conserved arginine residues point out from

the same side of an amphipathic α-helix, which on the other side interacts with lipids.

The corresponding binding site in LCAT for its activator has not yet been defined. It is

clear, however, that this interaction has a different nature than that between apoCII and

LPL, which does not appear to involve charged residues. Thus additional studies are

required to identify the apoCII-binding site on LPL.

Conclusions

Our results suggest that matching between Ile66 and LPL is important for full activation.

Introduction of other hydrophobic side chains like those of Phe, Ala, Leu and Val led to

decreased activation. The other clear result is that a negative charge cannot be tolerated in

position 70. From our data we conclude that residues 66, 69 and 70 contribute, together

with Tyr63, to the LPL-binding site of apoCII. The lack of change of the activation ability

by substitution of residues 72, 73 and 74 by alanine indicated that these residues are


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outside the LPL-interaction site, even though residue 73 is situated on the same side of

the α-helix as residues 69 and 70 (8). Taken together, multiple residues (Tyr63, Ile66,

Asp69, Gln70) are involved in formation of the LPL-binding site of apoCII, but no single

amino acid was absolutely crucial for the activation.

Acknowledgements

We thank Dr. Per-Ingvar Ohlsson for the N-terminal amino acid sequence analyses and

Dr. Juhan Subbi for the analyses by mass spectrometry.


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REFERENCES 1. Bier, D. M., and Havel, R. J. (1970) J. Lipid Res. 11, 565-570 2. Havel, R. J., Kane, J. P., and Kashyap, M. L. (1973) J. Clin. Invest. 52, 32-38 3. Applebaum-Bowden, D., Goldberg, A. P., Hazzard, W. R., Sherrard, D. J., Brunzell, J. D., Huttunen, J. K., Nikkilä, E. A., and Ehnholm, C. (1979) Metabolism 28, 917-924 4. LaRosa, J. C., Levy, R. I., Herbert, P., Lux, S. E., and Fredrickson, D. S. (1970) Biochem. Biophys. Res. Commun. 41, 57-62 5. Breckenridge, W. C., Little, J. A., Steiner, G., Chow, A., and Poapst, M. (1978) N. Engl. J. Med. 298, 1265-1273 6. Santamarina-Fojo, S. (1992) Curr. Opin. Lipidol. 3, 186-195 7. Kuniyoshi, A., Okamoto, Y., Tamagawa, T., Matsuyama, Y., and Fuku, H. (1999) Intern. Med. 38, 140-144 8. MacRaild, C. A., Hatters, D. M., Howelett, G. J., and Gooley, P. R. (2001) Biochemistry 40, 5414-5421 9. Sparrow, J. T., and Gotto, A. M. Jr. (1980) Ann. N. Y. Acad. Sci. 348, 187-208 10. Vainio, P., Virtanen, J. A., Kinnunen, P. K., Gotto, A. M. J., Sparrow, J. T., Pattus, F., Bougis, P., and Verger, R. (1983) J. Biol Chem. 258, 5477-5482 11. MacPhee, C.E., Howlett, G. J., and Sawyer W. H. (1999) Anal. Biochem. 275, 22-29. 12. Olivecrona, G. and Beisiegel, U. (1997) Arterioscler. Thromb. Vasc. Biol. 17, 1545-1549 13. Bengtsson, G. and Olivecrona, T. (1982) FEBS Lett. 147, 183-187 14. Voyta, J. C., Vainio, P., Kinnunen, P. K. J., Gotto.A.M.Jr., Sparrow, J. T., and Smith, L. C. (1983) J. Biol. Chem. 258, 2934-2939 15. Clarke, A. R. and Holbrook, J. J. (1985) Biochim. Biophys. Acta 827, 358-368 16. Shen, Y., Lindberg, A., and Olivecrona, G. (2000) Gene 254, 189-198 17. Bengtsson-Olivecrona, G. and Olivecrona, T. (1991) Methods Enzymol. 197, 345-356 18. Harlow, E. and Lane, D. (1988) Antibodies, A Laboratory Manual, Cold Spring


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Harbor Laboratory, Cold Spring Harbor, NY 19. Andersson, Y., Lookene, A., Shen, Y., Nilsson, S., Thelander, L., and Olivecrona, G. (1997) J. Lipid Res. 38, 2111-2124 20. Quinn, D., Shirai, K., and Jackson, R. L. (1983) Prog. Lipid Res. 22, 35-78 21. Wülfing, C., Lombardero, J., and Plückthun, A. (1994) J. Biol Chem 269, 2895-2901 22. Cheng, Q., Blackett, P. R., Jackson, K. W., McConathy, W. J., and Wang, C.-S. (1990) Biochem. J. 269, 403-407 23. Smith, L. C., Voyta, J. C., Catapano, A. L., Kinnunen, P. K. J., Gotto, A. M. Jr. and Sparrow, J. T. (1980) Ann. N. Y. Acad. Sci. 348, 213-221 24. MacPhee, C. E., Hatters, D. M., Sawyer, W. H., and Howelett, G. J. (2000) Biochemistry 39, 3433-3440 25. Wong, H., Davis, R. C., Nikazy, J., Seebart, K. E., and Schotz, M. C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11290-11294 26. Dichek, H. L., Parrott, C., Ronan, R., Brunzell, J. D., Brewer, H. B.,Jr., and Santamarina-Fojo, S. (1993) J. Lipid Res. 34, 1393-1401 27. Hill, J. S., Yang, D., Nikazy, J., Curtiss, L. K., Sparrow, J. T., and Wong, H. (1998) J. Biol. Chem. 273, 30979-30984 28. van Tilbeurgh, H., Sarda, L., Verger, R., and Cambillau, C. (1992) Nature 359, 159-162 29. Roosbeek, S., Vanloo, B., Duverger, N., Caster, H., Breyne, J., De Beun, I., Vandekerckhove, J., Shoulders, C., and Rosseneu, M. (2001) J. Lipid Res. 42, 31-40


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FIGURE LEGENDS

Fig. 1. Panel A. Schematic view of functional domains of human apoCII. The seven

fully conserved residues in the activating region (16) are shaded and numbered.

Panel B. Helical wheel model of residues 59-75 in human apoCII. The seven fully

conserved residues are shaded.

Fig. 2. Analysis of S-tagged apoCII on Tricine-SDS-PAGE

The wild-type (WT) and mutant apoCII proteins were purified on S-protein agarose and

repurified by passage over the same column after cleavage by factor Xa. Panel A shows a

gel stained by Serva Blue G. Panel B shows a Western blot of the same fractions as in

panel A using rabbit anti-human apoCII serum.

Fig. 3. Analysis of His-tagged wild-type apoCII on Tricine-SDS-PAGE

His-tagged, wild-type apoCII was eluted from the Ni2+-NTA-agarose column by a pH

gradient from pH 8.0 to pH 4.5 in 8M urea, 0.1 M Tris and 0.01 M NaH2PO4. Panel A

shows a gel stained with Serva blue G. Panel B shows Western blot of the same fractions

as in panel A using the penta-His tag antibody.

Fig. 4. Purification of His-tagged wild-type apoCII by ion-exchange

chromatography and analysis on Tricine-SDS-PAGE

The fraction eluted from Ni2+-NTA-agarose column at pH 6.8 (see Fig. 3) was applied on

a Mini-Q column. The chromatogram shows the separation of the two forms of apoCII.

The inset shows the analysis of the fractions on Tricine-SDS-PAGE. Lanes: Mw - low

molecular weight markers; a.s.- applied sample; b.t.- break through (material that was not

bound to Mini-Q); fractions that were run on gel are shown by numbers; concentrations of

NaCl and GdmCl used for elution were 1 M and 6 M, respectively.

Fig. 5. Activation of lipoprotein lipase by recombinant wild-type apoCII and

mutants produced as fusion proteins with the S-tag. Purified and cleaved wild-type


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apoCII and mutants (shown in Figure 2) were incubated with LPL (7.85 ng) in a total

volume of 200 µl assay mixture. Each data point is the mean of triple determinations.

Fig. 6. Circular dichroism spectra of wild-type apoCII and alanine replacement

mutants of apoCII in the presence of liposomes of egg yolk phosphatidyl choline

The concentration of phospholipids was 240 µM and the concentration of apoCII was in

all cases 4.7 µM. The experiments were performed in 0.1 M phosphate buffer, pH 7.4, at

25oC All spectra are means of three scans. WT, ⋅ ⋅ D69A, ⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅⋅

Y63A, I66A, ⋅ ⋅ Q70A.

Fig. 7. Stimulation of lipoprotein lipase by His-tagged apoCII mutants at residues

63, 66, 69 and 70. Conditions for the incubations were the same as in Figure 5. Each data

point is the mean of triple determinations. Panel A shows stimulation of LPL by apoCII

and mutants at residue 63; panel B shows stimulation of LPL by mutants at residue 66;

panel C shows stimulation of LPL by mutants at residue 69; panel D shows stimulation of

LPL by mutants at residue 70. In all panels the fitted curve for wild-type apoCII is shown

by a dotted line.

Fig. 8. Competitive assay using wild-type apoCII and mutants with low activity

The incubations were carried out as described in figure 6 except for the presence of 2.5

µM of the mutants Q70E, Y63F or Y63W in the incubation mixture together with the

indicated amounts of wild-type apoCII. Filled circle, wild-type alone; open circle, in the

presence of Y63W; filled square, in the presence of Y63F; open square, in the presence of

Q70E.

Fig. 9. Lack of correlation between Kd and Kd’ values. The determination of the Kd and

Kd’ is described under Experimental procedures. Kd originates from activation curves,

while Kd’ was determined from studies of the direct interaction between apoCII and LPL

using dansylated apoCII.


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Table 1. Primers used for mutagenesis. The first four primers were used for generation

of mutant Y63A by overlap extension by PCR. The other primers were used together with

QuickchangeTMSite-Directed Mutagenesis Kit.

Y63A1a 5’ GGCTGATATCGGATCCGA 3’

Y63A1b 5’ GCTCATGGCTGCTGTGCTTTTGCT 3’

Y63A2

5’GCACAGCAGCCATGAGCACTGCTACAGGCATTTTTACTGACCAAGTT 3’

T7 terminator primer 5’ CGATCAATAACGAGTCGCC 3’

A59G1 5’ GCAAAAGCACAGCAGGCATGAGCACTTACACAGG 3’

A59G2 5’ CCTGTGTAAGTGCTCATGCCTGCTGTGCTTTTGC 3’

T62A1 5’ GCAGCCATGAGCGCTTACACAGGCATTTTTACTGACC 3’

T62A2 5’ GGTCAGTAAAAATGCCTGTGTAAGCGCTCATGGCTGC 3’

Y63F1 5’ CACAGCAGCCATGAGCACTTTTACAGGCATTTTTACTGACC 3’

Y63F2 5’ GGTCAGTAAAAATGCCTGTAAAAGTGCTCATGGCTGCTGTG 3’

Y63L1 5’ CACAGCAGCCATGCGCACTCTCACAGGCATTTTTACTGACC 3’

Y63L2 5’ GGTCAGTAAAAATGCCTGTGAGAGTGCTCATGGCTGCTGTG 3’

Y63T1 5’ CACAGCAGCCATGAGCACTACCACAGGCATTTTTACTGACC 3’

Y63T2 5’ GGTCAGTAAAAATGCCTGTGGTAGTGCTCATGGCTGCTGTG 3’

Y63W1 5’ CACAGCAGCCATGAGCACTTGGACAGGCATTTTTACTGACC 3’

Y63W2 5’ GGTCAGTAAAAATGCCTGTCCAAGTGCTCATGGCTGCTGTG 3’

G65A1 5’ GCAGCCATGAGCACTTACACAGCGATTTTTACTGACCAAG 3’

G65A2 5’ CTTGGTCAGTAAAAATCGCTGTGTAAGTGCTCATGGCTGC 3’

I66A1 5’ GCCATGAGCACTTACTCAGGCGCTTTTACTGACCAAGTTC 3’

I66A2 5’ GAACTTGGTCAGTAAAAGCGCCTGTGTAAGTGCTCATGGC 3’

I66V1 5’ GCCATGAGCACTTACTCAGGCGTGTTTACTGACCAAGTTC 3’

I66V2 5’ GAACTTGGTCAGTAAACACGCCTGTGTAAGTGCTCATGGC 3’

I66L1 5’ GCCATGAGCACTTACACAGGCCTCTTTACTGACCAAGTTC 3’

I66L2 5’ GAACTTGGTCAGTAAAGAGGCCTGTGTAAGTGCTCATGGC 3’


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I66F1 5’ GCCATGAGCACTTACACAGGCTTTTTTACTGACCAAGTTC 3’

I66F2 5’ GAACTTGGTCAGTAAAAAAGCCTGTGTAAGTGCTCATGGC 3’

D69A1 5’ CACAGGCATTTTTACTGCCCAAGTTCTTTCTGTGC 3’

D69A2 5’ GCACAGAAAGAACTTGGGCAGTAAAAATGCCTGTG 3’

D69E1 5’ CTTACACAGGCATTTTTACTGAGCAAGTTCTTTCTGTGCTG 3’

D69E2 5’ CAGCACAGAAAGAACTTGCTCAGTAAAAATGCCTGTGTAAG 3’

D69S1 5’ CTTACACAGGCATTTTTACTAGCCAAGTTCTTTCTGTGCTGAAG 3’

D69S2 5’ CTTCAGCACAGAAAGAACTTGGCTAGTAAAAATGCCTGTGTAAG 3’

D69N1 5’ CTTACACAGGCATTTTTACTAACCAAGTTCTTTCTGTGCTG 3’

D69N2 5’ CAGCACAGAAAGAACTTGGTTAGTAAAAATGCCTGTGTAAG 3’

Y70A1 5’ CACAGGCATTTTTACTGACGCAGTTCTTTCTGTGCTGAAGG 3’

Y70A2 5’ CCTTCAGCACAGAAAGAACTGCGTCAGTAAAAATGCCTGTG 3’

Q70N1 5’ CACAGGCATTTTTACTGACAACGTTCTTTCTGTGCTGAAGG 3’

Q70N2 5’ CCTTCAGCACAGAAAGAACGTTGTCAGTAAAAATGCCTGTG 3’

Q70E1 5’ CACAGGCATTTTTACTGACGAGGTTCTTTCTGTGCTGAAGG 3’

Q70E2 5’ CCTTCAGCACAGAAAGAACCTCGTCAGTAAAAATGCCTGTG 3’

L72A1 5’ GCATTTTTACTGACCAAGTTGCTTCTGTGCTGAAGGGAGAG 3’

L72A2 5’ CTCTCCCTTCAGCACAGAAGCAACTTGGTCAGTAAAAATGC 3’

S73A1 5’ CTGACCAAGTTCTTGCTGTGCTGAAGGGAGAG 3’

S73A2 5’ CTCTCCCTTCAGCACAGCAAGAACTTGGTCAG 3’

V74A1 5’ GACCAAGTTCTTTCTGCGCTGAAGGGAGAGGAG 3’

V74A2 5’ CTCCTCTCCCTTCAGCGCAGAAAGAACTTGGTC 3’


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Table 2. Kinetic parameters for the interaction of apoCII mutants with LPL and

with liposomes. Kd and β were determined from the activation curves. Klip is the

dissociation constant for the apoCII/liposome complex determined by Trp fluorescence.

Apo CII variant

Kd (µM) β Klip(µM)

WT 0.29±0.07 6.2±0.1 164±43

Y63T 1.73±0.09 4.0±1.0 n.d.

Y63A 2.30±0.32 2.5±0.3 157±27

Y63L 3.14±0.93 4.2±1.6 n.d.

Y63F almost no activation n.d.

Y63W almost no activation n.d.

I66V 0.11±0.04 3.9±0.5 420±153

I66L 4.60±2.25 7.7±2.7 270±51

I66A 3.27±1.56 2.9±1.1 254±54

I66F 0.99±0.44 1.5±0.3 370±44

D69S 1.51±0.56 6.3±1.0 101±33

D69E 0.81±0.15 6.3±0.9 n.d.

D69N 1.49±0.61 4.9±0.6 n.d.

D69A 3.40±1.62 5.8±1.9 n.d.

Q70A 1.36±0.62 3.8±0.7 170±38

Q70N 2.07±1.05 3.0±0.5 n.d.

Q70E no activation 112±41

n.d.: not determined


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LIPID-BINDING LPL ACTIVATION / BINDING

1 50 56 79

Human sequence

Residue number 59 62 63 65 66 69 70

STAAMSTYTGIFTDQVLSVLKGEE

N - terminal domain C - terminal domain

Fig. 1A


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A59

I66V74

F67

M60

V71

T64

L75

T68S61

L72

G65

D69

T62

S73

Y63Q70

Fig. 1B


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A

WT A59G T62A Y63A G65A I66A D69A Q70A

6.5 kDa

14 kDa

B

6.5 kDa

14 kDa

Fig.2


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Mw 8.0 7.2 6.8 6.3 5.9 5.9 4.5 4.5

30 kDa21.5 kDa

14 kDa 6.5 kDa

A

B

Mw 8.0 7.2 6.8 6.3 5.9 5.9 4.5 4.5

14 kDa

6.5 kDa

30 kDa

pH

pH

Fig. 3


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24 26 28 30 32 34 36 38 40 42

0

50

100

150

200

250

300

1

2

1

2

3

A

B

Fraction number

Pro

tein

co

nce

ntr

atio

n (

g/m

l)

Mw a..s. b.t. fr.28 fr.33 fr.35 NaCl GdmClFig. 4


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0 0.5 1.0 1.5 2.0 2.5 3

Apo CII (µ M)

0

50

100

150

200

250

300

LP

L a

cti

vit

y (

U/m

g)

WT

A59G

T62A

Y63A

G65A

I66A

D69A

Q70A

Fig. 5


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0 1 2 3 4 5 60

50

100

150

200

250

300

350

400L

PL

act

ivit

y (U

/mg

)

0 1 2 3 4 5 60

50

100

150

200

250

300

350

400

0 1 2 3 4 5 6

Apo CII ( M)

0

50

100

150

200

250

300

350

400

LP

L a

ctiv

ity

(U/m

g)

0 1 2 3 4 5 6

Apo CII ( M)

0

50

100

150

200

250

300

350

400

WT

WTWT

WT

Q70A

Q70N

Q70E

D69SD69E

D69ND69A

I66VI66L

I66A

I66F

Y63T

Y63AY63L

Y63WY63F

A B

C D

Fig. 7


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0 1 2 3 4

Apo CII ( µM)

0

50

100

150

200

250

300

LP

L a

ctiv

ity

(U/m

g)

Fig. 8 by guest on June 8, 2018http://w

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Yan Shen, Aivar Lookene, Solveig Nilsson and Gunilla Olivecronamutagenesis.Identification of residues important for activation of lipoprotein lipase

Functional analyses of human apolipoprotein CII by site-directed

published online November 21, 2001J. Biol. Chem.

10.1074/jbc.M105421200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

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http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M105421200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2001/11/21/jbc.M105421200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2001/11/21/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2001/11/21/jbc.M105421200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

functional analyses of human apolipoprotein cii by site ... · abbreviations used: apocii,...

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