experimental procedures - tokushima bunri universitykp.bunri-u.ac.jp/kph02/pdf/2012 jnc; cnpase ca...

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*Department of Biochemistry, University of Oulu, Oulu, Finland

†Biocenter Oulu, University of Oulu, Oulu, Finland

‡Laboratory of Molecular and Cellular Neurosciences, Kagawa School of Pharmaceutical Sciences,

Tokushima Bunri University, Sanuki-city, Kagawa, Japan

§Institute for Biomedicine, University of Oulu, Oulu, Finland

¶Department of Pediatrics, Medical University of Vienna, Vienna, Austria

**Department of Chemistry, University of Hamburg and CSSB-HZI, DESY, Hamburg, Germany

Abstract2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) is aquantitatively major enzyme in myelin, where it localizes tothe non-compact regions and is bound to the membranesurface. Although its catalytic activity in vitro has beencharacterized, the physiological function and in vivo substrateof CNPase remain unknown. Especially the N-terminaldomain has been poorly characterized; previously, we haveshown it is involved in CNPase dimerization and RNAbinding. Here, we show that purified CNPase binds to thecalcium sensor protein calmodulin (CaM) in a calcium-dependent manner; the binding site is in the N-terminaldomain of CNPase. CaM does not affect the phosphodies-terase activity of CNPase in vitro, nor does it influence

polyadenylic acid binding. The colocalization of CNPase andCaM during Schwann cell myelination in culture wasobserved, and CaM antagonists induced the colocalizationof CNPase with microtubules in differentiated CG-4 oligoden-drocytes. An analysis of post-translational modifications ofCNPase from rat brain revealed the presence of two novelphosphorylation sites on Tyr110 and Ser169 within theN-terminal domain. The results indicate a role for theN-terminal domain of CNPase in mediating multiple molecularinteractions and provide a starting point for detailed structure-function studies on CNPase and its N-terminal domain.Keywords: 2′,3′-cyclic nucleotide 3′-phosphodiesterase, cal-modulin, interaction, myelin, protein.J. Neurochem. (2012) 123, 515–524.

Myelin is a highly specialized structure in the vertebratenervous system, formed by the differentiated plasma mem-brane of a glial cell. The myelin membrane wraps itselftightly around the axon, forming a compactly packed,multilayered proteolipid complex with a very low contentof aqueous solvent. The myelin membrane composition isunique, containing over 70% lipids, of which nearly 30% ischolesterol (Norton and Poduslo 1973). The myelin proteomeis also unusual in that it contains a handful of proteins that

Received June 15, 2012; revised manuscript received August 16, 2012;accepted August 20, 2012.Address correspondence and reprint requests to Petri Kursula,

Department of Biochemistry, University of Oulu, Oulu, Finland.E-mail: [email protected] used: BSA, bovine serum albumin; CaM, calmodulin;

CNPase, 2′,3′-cyclic nucleotide 3′-phosphodiesterase; dCG-4, differen-tiated CG-4 cells; DRG, dorsal root ganglion; DTT, dithiothreitol; ITC,isothermal titration calorimetry; MBP, myelin basic protein; PBS,phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate–poly-acrylamide gel electrophoresis; TFP, trifluoperazine dihydrochloride.

Journal of Neurochemistry © 2012 International Society for Neurochemistry, J. Neurochem. (2012) 123, 515--524 515© 2012 The Authors

JOURNAL OF NEUROCHEMISTRY | 2012 | 123 | 515–524 doi: 10.1111/jnc.12000

are often specific to myelin and present in high localconcentrations (de Monasterio-Schrader et al. 2012). Theprotein composition of myelin differs between the centraland peripheral nervous systems (CNS and PNS, respec-tively), and different regions of the myelin sheath carryspecific protein components. Despite the fact that many ofthe major myelin proteins were first described already in the1960s and early 1970s, as a result of their high concentrationin myelinated nervous tissues, little is still known about thestructure–function relationships in myelin proteins and theircomplexes. Such information will be crucial to understandingthe function of myelin proteins inside the myelin membranemultilayer.2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) is

an enzyme with a well-known catalytic activity but anunknown function, despite its initial characterization already50 years ago (Drummond et al. 1962). It is present at highlocal concentrations in the non-compact regions of myelin(Trapp et al. 1988). CNPase-deficient mice develop brainaxonal swellings and motor deficits with age (Lappe-Siefkeet al. 2003). Recent data have linked CNPase to catatonia-depression syndrome in both mice and humans (Hagemeyeret al. 2012).CNPase contains two domains, of which the

C-terminal phosphodiesterase domain has been well charac-terized. Crystal and solution structures for this catalyticdomain have been solved, and mutagenesis has highlightedresidues important for the CNPase catalytic activity (Leeet al. 2001; Kozlov et al. 2003; Sakamoto et al. 2005). Wealso recently solved the first crystal structures of the mouseCNPase phosphodiesterase domain complexed with nucleo-tide ligands (Myllykoski et al. 2012). CNPase has beensuggested to play roles in RNA metabolism within myelin;the true substrates of CNPase in vivo could be RNAmolecules, instead of the nucleotide substrates used inenzyme assays (Gravel et al. 2009), or RNA degradationproducts formed during RNA turnover (Verrier et al. 2012).However, details of CNPase interaction with other macro-molecules, including RNA, are still lacking. Our recentresults indicate that the CNPase N-terminal domain playsmajor roles in CNPase dimerization and RNA binding(Myllykoski et al. 2012). CNPase also binds cytoskeletalproteins and attaches to the plasma membrane (Braun et al.1991; De Angelis and Braun 1996; Laezza et al. 1997; Leeet al. 2005). The CNPase isoform 2 is imported intomitochondria, where it has been suggested to regulate matrixcalcium release (Lee et al. 2006; Azarashvili et al. 2009).Calmodulin (CaM) is a ubiquitous calcium sensor, capable

of interacting with and regulating hundreds of target proteins.CaM is also abundant in the nervous system, and especiallyits interactions with the myelin basic protein (MBP) havebeen characterized in detail also at the structural level(Majava et al. 2010; Bamm et al. 2011; Wang et al. 2011).Two high-throughput proteomics studies have been carried

out to find CaM targets in the brain (Berggård et al. 2006;Zhang et al. 2006). In both studies, CNPase was listed as aputative interaction partner for CaM, but the interaction hasnot been characterized using purified components, as wasdone, for example, for CRMP-2 in a similar case (Zhanget al. 2009).We have earlier prepared expression systems for different

domains of mouse CNPase, and purified several forms of it inlarge scale, to carry out detailed studies on CNPase structureand function (Myllykoski and Kursula 2010). Here, we usedfull-length CNPase and both of its domains separately to mapthe interaction site of CaM, and to study the effects of CaMon CNPase activity. In addition, the localization of CNPaseand CaM in cell culture, the effect of CaM antagonists onCNPase in cultured cells, and post-translational modifica-tions in brain CNPase were studied. The results bring aboutnovel aspects of CNPase structure and function.

Experimental procedures

Protein expression & purification

The expression and purification of different versions of murineCNPase have been previously described (Myllykoski and Kursula2010). Briefly, CNPase cDNA corresponding to residues 20-398(CNP_20-398), 25-398 (CNP_25-398), 20-420 (CNP_20-420),20-185 (CNP_20-185), and 179-398 (CNP_179-398) of CNPase(numbering according to isoform 2; the first 20 residues containthe mitochondrial targeting sequence, which was left out ofthe constructs) were cloned into the pTH27 vector (Hammarströmet al. 2006), and the recombinant proteins were expressed in E. coliand purified with Ni-affinity and size exclusion chromatography.Additional purification for the N-terminal and full-length constructswas achieved by calmodulin affinity chromatography as describedbelow, or using Blue Sepharose 6 (GE Healthcare, Uppsala,Sweden). Calmodulin was expressed in E. coli using the pETCMvector and purified as previously described (Hayashi et al. 1998;Kursula and Majava 2007). In addition, two synthetic peptidesrepresenting possible CaM-binding sites in the N-terminal domain(pep1 - KTAWRLDCAQLKEKNQWQ; pep2 - KSTLARVIVD-KYRDGTKMV) were purchased from SBS Genetech (Beijing,China); the N-termini were acetylated and the C-termini amidated.

Affinity chromatography

Concentrated CNP_20-398, CNP_20-420, CNP_20-185, andCNP_179-398 were diluted to 0.3 mg/mL with equilibration buffer(50 mM HEPES pH 7.0, 300 mM NaCl, 5% glycerol, 1 mMdithiothreitol (DTT), and 10 mM CaCl2). CNPase was applied into apre-equilibrated column containing approximately 1.2 mL of calmod-ulin agarose (Sigma-Aldrich, St. Louis, MO, USA; P4385, expected tocontain 0.5 mg CaM per mL). The column was first washed withequilibration buffer and then, with buffer containing 5 mM EGTAinstead of CaCl2. The eluted fractions were analyzed by sodiumdodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) .

Size exclusion chromatography

A Superdex 200 10/300 column was equilibrated with a buffercontaining 20 mM Bis-Tris pH 5.5, 0.2 M NaCl, 10 mM CaCl2,

Journal of Neurochemistry © 2012 International Society for Neurochemistry, J. Neurochem. (2012) 123, 515--524© 2012 The Authors

516 M. Myllykoski et al.

and 1 mM DTT. 20 nmol of CaM, CNP_20-398, and CNP_20-185were applied into the column separately, and complexes were runusing 20 nmol of CNPase mixed with 20 nmol of CaM. Elution wasmonitored by measuring absorbance at 280 nm and by SDS-PAGEanalysis of the fractions.

Affinity pull down from human brain white matter lysate

An affinity pull down experiment using CaM-agarose from ahuman white matter lysate was carried out essentially as describedbefore (Majava et al. 2010). Human brain samples were obtainedduring autopsy (Department of Pathology, Oulu Central Hospital,Finland). Permission to use human brain tissue for research wasobtained from the Finnish Medico-Legal Council (permit 102/32/200/99). Blocks of ~2 cm3 of white matter were dissected from thecerebral hemispheres of an 89-year-old male patient, with noknown neurological diseases, and stored at �20°C in 1 mMEDTA.

A block of ~1 cm3 was cut from the frozen material andhomogenized in 10 mL of CNPase solubilizing lysis buffer (Sudaand Tsukada 1980), containing 10 mM HEPES (pH 8), 1 Mammonium acetate, 100 mM NaCl, 1% Triton X-100, 10 mM CaCl2and EDTA-free Complete protease inhibitors (Roche, Penzberg,Germany). The homogenate was centrifuged for 20 min at 27 000 g,and the supernatant was collected. The supernatant was diluted 1 : 10,adjusted to contain 10 mM HEPES (pH 8), 100 mM ammoniumacetate, 100 mM NaCl, 0.2% Triton X-100, 10 mM CaCl2, andcentrifuged for 20 min at 27 000 g. 10 mL of the supernatant weremixed with 0.5 mL of CaM-agarose (Sigma, St. Louis, MO, USA)for 1 h. The unbound eluate was collected, the column was washedfive times with 1 mL of diluted lysis buffer and five times with 1 mLof elution buffer, which contained 10 mM EGTA instead of CaCl2.The fractions were analyzed with SDS-PAGE and western blottingwith a 1 : 1000 dilution of mouse monoclonal anti-CNPase (C5922,Sigma) as the primary antibody and a 1 : 100 dilution of horseradishperoxidase-conjugated goat anti-mouse IgG (32430, ThermoScientific, Waltham, MA, USA) as the secondary antibody.

Interaction assays using intrinsic tryptophan fluorescence

Tryptophan fluorescence was used as a probe for a directinteraction between purified CNPase and CaM. CaM has no Trpresidues, so the observed signal comes from the Trp residues onCNPase (5 Trp residues in full-length CNPase, of which 3 in thecatalytic domain). Measurements were done at an excitationwavelength of 295 nm, and the emission spectrum was recordedbetween 310 and 450 nm. The measurement was done in 50 mMBis-Tris pH 5.5, 200 mM NaCl, 10% glycerol, and 5 mM CaCl2.CaM was gradually added into a solution of 750 lL of 4 lMCNP_20-398 or CNP_179-398. Between additions, the solutionwas mixed for 3 min and fluorescence was measured.

Enzymatic activity assays

The activity of CNPase was measured as previously described(Sogin 1976; Myllykoski and Kursula 2010), except that Bis-Trisinstead of MES was used as the reaction buffer, because MESslightly inhibits CNPase activity (unpublished data). All measure-ments were done in triplicate. The effect of CaM was assessed byadding different amounts of CaM from 3 fmol to 1 nmol into thereaction containing 1 pmol of CNP_20-398.

Polyadenylic acid-binding assays

Poly(A)-binding assays were carried out as previously described(Myllykoski et al. 2012). 10 mg of polyadenylic acid (Pharmacia,Uppsala, Sweden) were covalently coupled to 1 mL of CNBr-activated sepharose (GE Healthcare), according to the manufac-turer’s instructions. 50 lL of the coupled poly(A) sepharose wereequilibrated with binding buffer (10 mM Tris-HCl at pH 7.5,50 mM NaCl, 2.5 mM MgCl2, and 1 mM DTT). 500 lL ofCNP_20-398 at 0.5 mg/mL, with and without 0.5 mg/mL CaMand 10 mM CaCl2, was mixed with poly(A) sepharose for 2 h at 4°C with rotation in microcentrifuge tubes. After mixing, the matrixwas pelleted by centrifuging at 6600 g for 30 s, and thesupernatant was removed. The matrix was washed five times with500 lL of binding buffer and diluted to 100 lL. SDS-PAGEloading buffer was added, and 15 lL of the matrix were examinedwith SDS-PAGE.

Surface plasmon resonance

Surface plasmon resonance was used to analyze the CNPase–CaMinteraction, with the Biacore T100 instrument (GE Healthcare).CaM was immobilized onto a CM5 chip as previously described(Majava et al. 2008), and the binding of CNP_20-398 andCNP_179-398 was analyzed. A channel with no immobilizedCaM was used as a control, and the data were analyzed using theBiaEvaluation software (GE Healthcare).

Isothermal titration calorimetry

Isothermal titration calorimetry (ITC) was carried out essentially asdescribed (Zhang et al. 2009). CNP_25-398, CaM, and the twoCNPase peptides were dialyzed into the assay buffer (20 mMHEPES (pH 7.5), 100 mM NaCl, 10 mM CaCl2). ITC experimentswere done on a Microcal VP-ITC (Microcal, Northampton, MA,USA) instrument at 30°C. For the CaM-CNPase titration, CNP_25-398 was in the cell at 19 lM and CaM in the syringe at 190 lM.For the CaM-peptide titrations, the CaM concentration in thecell was 16 lM and the peptide concentration in the syringe200–300 lM. The data were analyzed with Microcal Origin.

Molecular modeling

The sequence similarity of the N-terminal domain of CNPase toother proteins is low. To find homologs and build a model, thePhyre2 server (Kelley and Sternberg 2009) at http://www.sbg.bio.ic.ac.uk/phyre2 was used. The best hit (sequence identity of 22% over133 residues, model confidence 99.9%) was the crystal structure ofT4 polynucleotide kinase [(Zhu et al. 2007), PDB entry 2IA5], andthe homology model built by Phyre based on this template wasdirectly used in further analyses. Secondary structure predictions ofCNPase matched well those observed in the template, and theP-loop, the only highly conserved feature of the N-terminal domain,is located in the same position. Several other hits to structures fromthe same fold family were obtained from the Phyre2 server,highlighting the reliability of the result. The CaM-binding site of theN-terminal domain of CNPase was predicted using the CaM targetdatabase (Yap et al. 2000).

Analysis of CaM antagonists in GC-4 cell cultures

CG-4 cells, a rat oligodendrocyte progenitor cell line, were culturedas described previously (Louis et al. 1992). The CG-4 cells were

© 2012 The AuthorsJournal of Neurochemistry © 2012 International Society for Neurochemistry, J. Neurochem. (2012) 123, 515--524

CNPase binds calmodulin 517

routinely expanded in Dulbecco’s Modified Eagle’s Medium(D-MEM; Invitrogen, Carlsbad, CA, USA), supplemented withthe N2 supplements (Invitrogen), 10 ng/mL biotin (Sigma) and10 ng/mL human platelet-derived growth factor-AA (PDGF-AA,Sigma). The cells were passed every 5 days on polyornithine(10 lg/mL) and fibronectin (5 lg/mL)-coated coverslips at aseeding density of ~10–50 cells/mm2. To differentiate the CG-4cells, the medium was exchanged to serum-free chemically definedD-MEM (CDM), supplemented with 10 lg/mL insulin, 0.5 lg/mLtransferrin, 100 lg/mL bovine serum albumin (BSA) , 60 ng/mLprogesterone, 16 lg/mL putrescine, 40 ng/mL sodium selenite,60 ng/mL N-acetyl-L-cysteine, 5 lM forskolin, 40 ng/mL L-thyrox-ine (T3), 30 ng/mL 3,3′,5-triiodothyronine (T4), and 5 ng/mLneurotrophin-3 (NT-3). The culture was continued for 7 days.

Differentiated CG-4 (dCG-4) cells were treated with the calmod-ulin inhibitors W-7 HCl (W-7) (5 lM, Wako) and trifluoperazinedihydrochloride (TFP) (2.5 lM, Sigma) overnight. To determine theexpression and distribution of CNPase and tubulin in the culturedcells, double immunofluorescence staining for CNPase and tubulinwas performed as previously described (Watanabe et al. 2006).The dCG-4 cells were fixed in 4% paraformaldehyde in 0.1 Mphosphate-buffered saline (PBS, pH 7.4) for 15 min at 20°C. Thefixed cells were permeabilized with 0.5% Triton X-100 for 15 minbefore intracellular staining and were treated with 10% normal goatserum (Vector Laboratories, Burlingame, CA, USA) in PBS for30 min to block non-specific binding. Following washing, the cellswere incubated with the primary CNPase mAb (1 : 25, Sigma)overnight at 4°C. After washing, the cells were incubated with goatanti-mouse IgG Alexa Fluor® 488 (1 : 1000) for 1 h at 20°C. Fordouble immunolabeling, the same procedures were repeatedlyperformed. After overnight incubation with the 2nd primaryantibody, a-tubulin pAb (1 : 1500, Abcam, Cambridge, UK,15246), at 4°C, the cells were incubated with goat anti-rabbit IgGAlexa Fluor® 594 (1 : 1000) for 1 h at 20°C. The stained cells weremounted using Vectashield® mounting medium (Vector Laborato-ries, Burlingame, CA, USA). Fluorescence microscopy was per-formed with an Olympus confocal laser-scanning FV1000 systemwith an inverted microscope (IX81; Olympus, Tokyo, Japan).

Localization of CNPase and CaM in myelinating Schwann cells

Mice (C57BL/6J strain) employed for the establishment of thedorsal root ganglion (DRG) explant cultures were acquired from theLaboratory Animal Centre, University of Oulu, and the AnimalWelfare Committee has approved their use and the protocolsemployed (permit number 026/10).

DRG explant cultures, containing endogenous Schwann cells,were prepared according to a modified protocol (Fex Svenningsenet al. 2003). Cultures were maintained under a 5% CO2 atmo-sphere, at 37°C. DRG isolation was performed as described(Päiväläinen et al. 2008). Briefly, the DRGs were removed fromthe spinal cords of 13.5-day-old mouse embryos and placed in L15medium. The pooled DRGs were incubated with 2 lg/mL dispasein 2 mL of L15 medium at 37°C for 45 min, washed three timeswith L15 medium containing 10% HS, and seeded in a drop ofDRG growth medium (Päiväläinen et al. 2008) onto 3D-Matrigel-coated 13-mm glass cover slips on four well cluster plates, with 1–2DRGs on each cover slip. The next day, 250 lL of DRG growthmedium were added into each well, and the explants were grown inDRG growth medium for 12 days, renewing the medium every

2–3 days, to allow the endogenous Schwann cells to proliferate andpopulate the axons. Myelination was then started by the addition ofmyelination medium to the cultures (Päiväläinen et al. 2008).Myelination was allowed to proceed for 10 days or 3 weeks,renewing the myelination medium every 3 days.

The DRG explant cultures were fixed for immunocytochemistryafter 12 days in DRG growth medium (= 0 days in myelinationmedium), and after 10 and 21 days in myelination medium. Thecocultures were rinsed 3 times with PBS, fixed with 4% parafor-maldehyde in PBS for 10 min at 20°C, and then, rinsed three timeswith PBS. Before the staining, the cells were permeabilized for6 min with ice-cold methanol. The cultures were blocked with 5%goat serum in PBS at 4°C overnight. The primary antibodies (rabbitpolyclonal anti-CNPase (Santa Cruz Biotechnology, Santa Cruz,CA, USA; dilution 1 : 75) and mouse monoclonal anti-CaM (LabVision, Kalamazoo, MI, USA; dilution 1 : 75) were diluted in 1%BSA in PBS, and incubation on the fixed cultures was carried out at20°C for 80 min. The cultures were then washed three times for5 min with PBS, incubated for 80 min with the secondaryantibodies (goat anti-mouse Alexa Fluor 488 (Molecular Probes,Eugene, OR, USA), dilution 1 : 150 and goat anti-rabbit AlexaFluor 546 (Molecular Probes), dilution 1 : 150 in 1% BSA in PBS)at 20°C, washed twice with PBS for 5 min and again overnight at 4°C in PBS. After rinsing twice with sterile H2O, the cultures weremounted with Immu-Mount on objective slides.

The immunolabeled cultures were examined with an OLYMPUSFluoView-1000 laser-scanning confocal microscope equipped withargon and HeNe1 lasers, and using a UPLSAPO 60x O (NA: 1.35)objective, or a UPLSAPO 100x O (NA:1.40) oil immersionobjective. Pictures were taken using Z-stack (11–13 slices), andan optical slice thickness of 0.25–0.31 lm.

Identification of post-translational modifications

Most myelin proteomics experiments were carried out exactly asdescribed before (Baer et al. 2009; Chen et al. 2010; Majava et al.2010), including the preparation of myelin protein extracts,electrophoresis, in-gel digestion, and peptide analysis. Details aregiven in the supporting information.

Results and discussion

In two earlier studies (Berggård et al. 2006; Zhang et al.2006), focused on the identification of CaM-binding proteinsin the brain, CNPase was for the first time identified as apotential CaM target, but follow-up studies have not beenperformed to date. Our aim was to validate the directmolecular CNPase–CaM interaction and study its potentialfunctional significance.

CNPase binds directly to CaM in a calcium-dependent manner

CNPase binding to calmodulin, the domain localization ofthe binding, and the dependence of the binding on calciumwere studied by affinity chromatography on calmodulin-coupled agarose (Fig. 1a). CNP_20-398 and the N-terminaldomain bound to CaM, while the C-terminal domain did not.Both bound fragments were eluted from the matrix, whencalcium was removed by EGTA. The result shows that the



CNPase N-terminal domain harbors a calcium-dependentbinding site for CaM. Interestingly, the inclusion of the22-residue C-terminal tail in the CNP_20-420 constructabolishes CaM binding in this assay. This tail is known to

mediate the binding of CNPase to the membrane, and it isrequired for CNPase–tubulin interactions (De Angelis andBraun 1994; Lee et al. 2005). The structural basis for thiseffect is currently unknown, as the available crystal struc-tures are missing the tail region. It is known, however, thatthe tail must be located close to both the N-terminal domainand the active site, and it may, hence, block the CaM-bindingsite (see below).As a further test of interaction, CaM affinity pulldown was

performed from a human white matter lysate (Fig. 1b).Using western blotting, a band corresponding to nativeCNPase was detected in the EGTA eluate. Previously, asimilar experiment indicated that MBP is the major CaM-binding protein in a human white matter lysate (Majava et al.2010), and indeed, we could again detect MBP in theaffinity-purified fractions. Size exclusion chromatography ofCNPase with CaM indicated the presence of a new species,corresponding to a complex, with both the CNP_20-398 andthe N-terminal domain (Fig. 1c).Surface plasmon resonance (Fig. 2a–c) was used to obtain

an estimate for the CNPase–CaM affinity. Full-lengthCNPase bound to covalently immobilized CaM with anapparent Kd of 100 nM, while no interaction was seenbetween CaM and the phosphodiesterase domain (Fig. 2a).Binding in solution was also detected using ITC (Figure S1)and covalent cross-linking (unpublished data). Using intrin-sic tryptophan fluorescence spectroscopy, binding betweenCNPase and CaM was also detected (Fig. 2d). A change wasseen in the wavelength of the spectral maximum as a functionof CaM concentration. Taken together, our results indicatethat CNPase binds to CaM in a calcium-dependent manner,and that the interaction requires the presence of theN-terminal domain.

The presence of CaM does not affect the activity of CNPase

The enzymatic activity of CNPase was measured in thepresence and absence of CaM. To get rid of possiblecontaminating nucleotides, CNPase was additionally purifiedwith Blue Sepharose. No changes in CNPase activity in thepresence of CaM were detected (Fig. 3a). This indicates thatCaM binding does not directly regulate CNPase activityin vitro. However, the activity assay uses 2′,3′-cyclic NADP+

as the substrate, while the physiological substrate is likely to bean RNA molecule. Earlier, upon characterization of CNPaseactivity in bovine adrenal medulla, CaM was also found tohave no effect on the activity in vitro toward 2′,3′-cyclic AMP(Tirrell and Coffee 1986). Considering the assumption that thephysiological substrate of CNPase could be RNA, it cannot beruled out, based on this assay, that CaM might indirectlyregulate CNPase activity by influencing substrate binding.

Polyadenylic acid binding by CNPase

CNPase has previously been shown to bind RNA (Gravelet al. 2009; Myllykoski et al. 2012). We measured binding

(a)

(b)

(c)

Fig. 1 Interaction between 2′,3′-cyclic nucleotide 3′-phosphodiester-

ase (CNPase) and CaM in vitro. (a) Affinity chromatography ofCNPase on CaM-sepharose. CNPase was passed through CaM-agarose in the presence of calcium. The column was first washed with

a calcium-containing buffer and then, eluted with an EGTA-containingbuffer. The fractions were analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using Coomassiestaining. 1 - input sample; 2 - flow-through; 3 - combined calcium

washes; 4–6 - EGTA eluates. (b) CaM affinity pull down from whitematter lysate. The brain lysate was mixed with CaM-agarose andapplied to a column. The column was washed first with a CaCl2-

containing buffer and then, with an EGTA-containing buffer. Top:western blot with anti-CNPase; bottom: Coomassie staining. 1 - inputsample; 2 - unbound sample; 3–8 - calcium washes; 9–13 - EGTA

washes. CNPase is indicated with an asterisk and MBP with a double;in the EGTA elutions, a lot of myelin basic protein (MBP) is present,which gives a cross-reaction with the antibody. (c) Size exclusionchromatography indicates that the peaks of CNP_20-398 and

CNP_20-185 are shifted when the proteins are mixed with CaM.



of purified full-length CNPase to sepharose-immobilizedpolyadenylic acid in the presence and absence of CaM. CaMhad no significant effects toward the binding of RNA, whenusing polyadenylic acid binding as the model system(Fig. 3b). In an earlier report, it was suggested theN-terminal domain, containing the P-loop, would be a majorsite for binding nucleoside triphosphates, especially GTP(Stingo et al. 2007). It is, thus, possible that CaM may affectnucleotide binding by CNPase.

CNPase and CaM in differentiated CG-4 cells

To study the interaction of CNPase with CaM in dCG-4 cells,we performed immunocytochemical studies with CaM inhib-itors (W7 and TFP) using anti-CNPase and anti-tubulinantibodies (Fig. 4). CNPase and tubulin were stronglyexpressed in the processes and cell bodies of dCG-4 cells.CNPase was expressed at the tips of processes, such as growthcones, while tubulin was absent. Treatment of dCG-4 with10 lM W7 or 5 lM TFP for 24 h caused morphologicalchanges, including the disappearance of processes, a roundshape, and cell death. Hence, lower doses of these CaMantagonists were used (Fig. 4). The dCG-4 cells were supple-mented with 5 lMW7 or 2.5 lM TFP for 24 h. CNPase wasnot colocalized with tubulin in non-treated dCG-4 cells. On theother hand, following exposure to W7 and TFP, CNPasecolocalized with tubulin in the cytoplasm of dCG-4. Theseresults suggest that the interaction of CNPase with tubulincould be directly or indirectly regulated by CaM.

(a) (b)

(c) (d)

Fig. 2 Quantitative analysis of 2′,3′-cyclic

nucleotide 3′-phosphodiesterase (CNPase)-CaM binding with surface plasmonresonance and fluorescence spectroscopy.(a) Binding ofCNPase to immobilizedCaM in

surface plasmon resonance. CNP_20-398binds to CaM strongly (black), while nointeraction is detected with the catalytic

domain (CNP_179-398; red). (b) Injectionsof a concentration series of CNP_20-398over immobilized CaM. The kinetic analysis

indicatesKd�0.1 lM. (c) Abinding plot of theresponse at the end of the injection versusCNPase concentration. (d) Tryptophanfluorescence analysis of CaM binding to

CNPase. The catalytic domain shows nochange in the spectrum (black, no CaM; red,CaM). CNP_20-398 has a shift of the

fluorescence peak with CaM (blue, no CaM;green, CaM).

(a)

(b)

Fig. 3 Analysis of the effects of CaM on 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) activity and RNA binding. (a) Activityassay. CNPase activity was determined with the coupled enzyme

assay (Sogin 1976) in the presence of a range of CaM concentrations.Absorption at 340 nm from the produced NADPH is plotted againsttime for the first 100 s of the reaction. (b) RNA binding. CNP_20-398

was mixed with agarose-coupled polyadenylic acid with and withoutCaM. 1 - input sample; 2 - unbound fraction; 3–6 - washes; 7 - sampleof poly-A matrix after washes. CNPase is indicated with a singleasterisk and CaM with a double.



Localization of CNPase and CaM in myelinatingSchwann cells

Double immunofluorescence microscopy analysis of CNPaseand CaM expression in mouse DRG explant cultures revealstheir partial colocalization during myelination (Fig. 5). Thesimultaneous presence of both CNPase and CaM in the samecellular compartments supports the possibility of adirect physical interaction between these two proteins. The

apparent colocalization was stronger during early stages ofmyelination.

The putative CaM- binding site of the CNPase N-terminal

domain

To get a more detailed view of the N-terminal domain, ahomology model was built (Fig. 6a). The predicted CaM-binding segment (Yap et al. 2000) is in the immediate

Fig. 4 The effects of CaM antagonists on 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) localization in dCG-4 cells. The panels

are as follows: top left – merge, top right – CNPase, bottom left –tubulin, bottom right – DIC. 50-lm scale bars are shown in the bottom

left panels in white. 157, 118, and 132 cells were examined for theuntreated, trifluoperazine dihydrochloride (TFP)-treated, and W7-

treated experiments, respectively, and representative results areshown.

Fig. 5 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) andCaM expression during myelination. Confocal immunofluorescencemicroscopy reveals the partial colocalization (orange–yellow) of

CNPase (red) and CaM (green) in mouse dorsal root ganglion(DRG) explant cultures after 0, 10, and 21 days in conditions

permissive for myelination. Colocalization is most evident in immaturestructures (inset at 0 days). CaM appears to be absent, or very weaklyexpressed, in compacted sheaths (yellow arrows in insets at 10 and

21 days). Scale bars: 100 lm. The insets in the merged images aredigitally magnified from the areas outlined with dotted lines.



vicinity of the P-loop, on the protein surface. CaM bindingcould, thus, regulate the binding of CNPase to the ligands ofits N-terminal domain. We also noted another predictedamphipathic helix in the N-terminal domain (Fig. 6a).Interestingly, in an ITC experiment, the latter gave a strongsignal indicative of binding (Figure S1).

Identification of two novel phosphorylation sites in

brain CNPase

A mass spectrometric approach (Figure S2, Table S1) wastaken to identify novel post-translational modifications in ratbrain CNPase. Two previously unreported phosphorylation

sites were detected and confirmed by phosphatase treatment(Figure S3). Tyr100 is predicted to lie in the middle of along helix, on the surface of the N-terminal domain. Ser169is located some 10 residues before the beginning of the C-terminal phosphodiesterase domain, in a region predicted tobe helical and connecting the two domains. In thehomology model of the N-terminal domain, both residuesare solvent-accessible, and their phosphorylation couldaffect the interactions of CNPase with its ligands (Fig. 6a).

Implications of CaM binding for CNPase function

The function of CNPase can be divided into three distinct, butprobably connected, aspects. First, the enzymatic phosphodi-esterase activity of CNPase converts 2′,3′-cyclic nucleotidesinto 2′-nucleotides (Drummond et al. 1962). The biologicalrole of this activity, and –more specifically – the high amountof such activity within the developing myelin sheath, haspuzzled researchers through decades. CNPase activity and its2′,3′-cyclic substrates were found to modulate the mitochon-drial permeability transition pore, which releases matrixcontents with apoptotic consequences after elevated matrixcalcium levels (Azarashvili et al. 2009). Calmodulin, on theother hand, has been shown to regulate the uptake of calciuminto the matrix (Moreau et al. 2006). Recently, 2′, 3′-cyclicnucleotides were detected in the brain of CNPase knock-outmice (Verrier et al. 2012). The catalytic activity of CNPasewas also linked as a possible player in the 2′,3′-cyclic AMP-adenosine pathway (Verrier et al. 2012); free 2′,3′-cyclicAMP is a side product of mRNA degradation (Thompsonet al. 1994) coupled to cell injury (Jackson et al. 2009;Jackson 2011). Our assays, however, revealed no directeffects on CNPase catalytic activity by CaM.Another recently discovered function for CNPase is the

binding of RNA molecules (Gravel et al. 2009). In ourearlier study, we showed that the N-terminal domain isrequired for efficient RNA binding in vitro (Myllykoski et al.2012). In this study, we did not detect any difference inpolyadenylic acid binding with and without CaM, indicatingthat the role of the CaM interaction is unlikely to be related toRNA ligand binding.A third property of CNPase is its affinity toward the cellular

cytoskeleton, including both actin and tubulin (De Angelisand Braun 1996; Laezza et al. 1997). CNPase is copurifiedwith cytoskeletal elements in the detergent-resistant fraction(Kim and Pfeiffer 1999), and its C-terminus, mediatinglinkage to the lipid membrane, is essential for interactions withtubulin (Lee et al. 2005). The cytoskeleton plays a leadingrole during myelination, and dynamic polymerization anddepolymerization of microfilaments and microtubules arenecessary for a functional outcome (Bauer et al. 2009). Theinflux of Ca2+ and depolymerization of microtubules havebeen reported as responses for antibodies against cell surfacegalactocerebroside in oligodendrocyte cultures (Dyer andBenjamins 1990). Also, as a response to axonal stimulation in

(a)

(b)

Fig. 6 Models for 2′,3′-cyclic nucleotide 3′-phosphodiesterase(CNPase) structure and function. (a) Homology model for the CNPaseN-terminal domain. The predicted CaM-binding site by the CaM targetdatabase is indicated in yellow, a second potentially amphipathic helix

in pink, and the ATP-binding P-loop in red. An ADP molecule hasbeen added to denote the ATP-binding site. The two phosphorylatedresidues are also shown. The locations of the C- and N-termini are

indicated; the catalytic domain will follow directly after the C-terminus.(b) Schematic view of the current data on CNPase function at themolecular level. CNPase is comprised of two folded domains (green/

red), and a C-terminal membrane anchor (orange). As the N- and C-termini of the catalytic domain are next to each other in the structure(Myllykoski et al. 2012), the C-terminal tail formed by the last 22

residues must lie close to the domain interface, as depicted. Althoughthe catalytic domain performs the phosphodiesterase reaction (blue),our current and earlier data indicate a role for the N-terminal domainin dimerization, RNA binding, and CaM binding. CNPase also

interacts with the actin and tubulin cytoskeleton, and it is likely thatat least the C-terminus of CNPase is involved in these interactions.



frog sciatic nerve, Ca2+ influx into Schwann cell cytoplasmhas been detected (Lev-Ram and Ellisman 1995). CNPase hasbeen shown to promote microtubule polymerization andreorganization (Lee et al. 2005), and it can be speculated thatCaM binding might modulate this effect, depending on thevariation of intracellular [Ca2+].Both CNPase and the myelin basic protein (MBP) interact

with membrane surfaces, cytoskeletal proteins, and calmod-ulin. The binding to cytoskeletal components and calmodulincreates an interesting analogy between CNPase and MBP.MBP also is likely to be involved in the regulation of CNPaselocalization outside the compacted regions of myelin (Aggar-wal et al. 2011). MBP and CNPase could be involved in aregulatory network involving myelin membrane surfaces, themyelinating cell cytoskeleton, CaM, and other factors.A schematic view of the current data on the functional

domains of CNPase is shown in Fig. 6b. Although bindingpartners for the N-terminal domain and structural propertiesand catalytic activity for the C-terminal domain have beenstudied, it still remains unclear, how all these properties areinterconnected. Our results warrant further studies on thecomplex interactions of CNPase with other molecules.

Concluding remarks

We have shown that CNPase directly interacts with CaM in acalcium-dependent manner. Using in vitro functional assays,we were unable to detect an effect of CaM on CNPaseenzymatic activity or RNA binding. It is possible that theinteraction plays a role in an as-of-yet unidentified function ofCNPase; such functions could be related to CNPase localiza-tion or its interactions with the cytoskeleton.When the structureand function of the CNPase N-terminal domain, as well asthose of the full-length protein, have been better elucidated,CNPase function in vivo will also become more clear.

Acknowledgements

We thank Dr. Young-Hwa Song for supervising the fluorescencetitrations. This study has been financially supported by the Academyof Finland (Finland), the Sigrid Jusélius Foundation (Finland),the Magnus Ehrnrooth Foundation (Finland), the Department ofBiochemistry, University of Oulu (Finland), and the Researchand Science Foundation of the City of Hamburg (Germany). Theauthors declare no conflicts of interest.

Supporting information

Additional supporting information may be found in the onlineversion of this article:

Appendix S1. Supplemental Methods.Figure S1. Analysis of the CNPase–CaM interaction using ITC.Figure S2. Separation of MPEs using BN/SDS/SDS-PAGE

(3DE).

Figure S3. MS/MS spectra of phosphorylated peptides fromCN37_Rat identification with verification by phosphatase treatment.

Table S1. Peptide list with sequence coverage from spotsidentified as CN37_RAT on BN/SDS/SDS-PAGE.

As a service to our authors and readers, this journal providessupporting information supplied by the authors. Such materials arepeer-reviewed and may be re-organized for online delivery, but arenot copy-edited or typeset. Technical support issues arising fromsupporting information (other than missing files) should beaddressed to the authors.

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Supplemental information Myllykoski et al.

Supplemental methods

Preparation of myelin membrane substrates and myelin protein extracts (MPEs)

Myelin was purified by 2 rounds of discontinuous density gradient centrifugation and osmotic

disintegration. The entire brains of young Sprague–Dawley rats were homogenized mechanically in

ice-cold 0.32 M sucrose using a mechanical blender (Ultra-Turrax T18 basic, IKA Works). (Sucrose

was dissolved in sterile 2.5 mM Tris/HCl, pH 7.0, to form 0.25, 0.32, and 0.88 M solutions.) The

homogenate in 0.32 M sucrose solution was diluted with Tris/HCl to form a solution with a final

molality of 0.25, which was then pelleted in an ultracentrifuge (55,000 g, 4°C, 15 minutes). The

pellet was resuspended in 0.88 M sucrose solution and overlaid with 0.25 M sucrose. After an

ultracentrifugation step (100,000 g, 4°C, 1 hour), the material at the interface was collected and

washed in 30 ml of distilled H2O (55,000 g, 4°C, 10 minutes). The pellet was resuspended in

distilled H2O and incubated for 60 minutes on ice for osmotic disintegration. After centrifugation

(55,000 g, 4°C, 10 minutes), the flotation step was repeated. The material at the interface was

collected and washed twice in distilled H2O (55,000 g, 4°C, 10 minutes), and the pellet was stored

at –80°C until isolation of the myelin protein.

To prepare MPE, the pellets were resuspended in 1% N-octyl β-D-glucopyranoside, 0.2 M sodium

phosphate pH 6.8, 0.1 M Na2SO4, and 1 mM ethylenediaminetetraacetic acid and incubated at 23°C

for 2 hours. Following ultracentrifugation (100,000 g, 18°C, 30 minutes), the supernatant was

collected and stored at –20°C until further use.

One dimensional electrophoresis (1DE): Blue native – polyacrylamide gel electrophoresis (BN-

PAGE)

MPEs were dialyzed against a buffer containing 750 mM 6-aminocaproic acid, 50 mM Bis-Tris, 5%

(w/v) glycerol, 0.5 mM EDTA, 0.1 % Triton X 100, pH 7.0 using Microcon® YM-3 (Bedford, MA,

USA). The sample volume of 60 µL (protein content 1 µg/µL) was added to 10 µL of G250 solution

[5 % (w/v) Coomassie G250 in 10 mM 6-aminocaproic acid] and loaded onto the gel. BN-PAGE

was performed in a PROTEAN II xi Cell (BioRad, Germany) using a 4 % stacking and a 5 – 13 %

separating gel. The gel buffer contained 250 mM 6-aminocaproic acid, 25 mM Bis-Tris, pH 7.0; the

cathode buffer 50 mM Tricine, 15 mM Bis-Tris, 0.05 % (w/v) Coomassie G250, pH 7.0; and the

anode buffer 50 mM Bis-Tris, pH 7.0. For electrophoresis, the voltage was set to 70 V for 2 h, and

was increased to 250 V (10 mA/gel) until the dye front reached the bottom of the gel. BN-PAGE

gels were cut into small pieces of approximately 1-3 cm depending on the intensity of protein bands

for the BN/SDS/SDS-PAGE (2). High molecular mass markers were obtained from Invitrogen

(Carlsbad, CA, USA).

Three dimensional electrophoresis (3DE): BN/SDS/SDS-PAGE

1-3 cm gel pieces from BN-PAGE were soaked for 2 h in a solution of 1% (w/v) SDS and 1% (v/v)

2-mercaptoethanol. Gel pieces were then rinsed twice with SDS-PAGE electrophoresis buffer (25

mM Tris–HCl, 192 mM glycine and 0.1% (w/v) SDS; pH 8.3), then the gel pieces were placed onto

the wells. 2DE-SDS-PAGE was performed in PROTEAN II xi Cell using a 4 % stacking and a 6–

13 % separating gel for BN/SDS-PAGE (2DE). Electrophoresis was carried out at 25°C with an

initial current of 70 V (during the first hour). Then, the voltage was set to 100 V for the next 12 h

(overnight), and increased to 200 V until the bromophenol blue marker moved 17 cm from the top

of separation gel.

2DE gels were cut again into lanes and gel strips from each lane were soaked for 20 min in a

solution of 1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol. Gel strips were then rinsed twice with

SDS-PAGE electrophoresis buffer (25 mM Tris–HCl, 192 mM glycine and 0.1% (w/v) SDS; pH

8.3), and were placed onto the wells of another gel (3DE). SDS-PAGE was performed in

PROTEAN II xi Cell using a 4% stacking and a 7.5–17% separating gel. Electrophoresis was

carried out at 25°C with an initial current of 70 V (during the first hour). Then, the voltage was set

to 100 V for the next 12 h (overnight), and increased to 200 V until the dye front reached the bottom

of the gel. Colloidal Coomassie Blue staining was used for visualization.

In-gel digestion

Spots of interest were excised into small pieces to increase surface and put into a 0.6 mL tube. They

were initially washed with 50 mM ammonium bicarbonate and then two times with 50% 50 mM

ammonium bicarbonate/50% acetonitrile for 30 min with occasional vortexing. The washing

solution was discarded at the end of each step. 100 µL of 100% acetonitrile was added to each tube

to cover the gel piece completely and incubated for at least 5 min. The gel pieces were dried

completely in a Speedvac Concentrator 5301 (Eppendorf, Germany). Reducing cysteines was

carried out with a 10 mM dithiothreitol solution in 0.1 M ammonium bicarbonate pH 8.6 for 60 min

at 56°C. The same volume of a 55 mM solution of iodoacetamide in 0.1 M ammonium bicarbonate

buffer pH 8.6 was added and incubated in darkness for 45 min at 25°C to alkylate cysteine residues.

The reduction/alkylation solutions were replaced by 50 mM ammonium bicarbonate buffer for 10

min. Gel pieces were washed and dried in acetonitrile followed by Speedvac concentration.

The dried gel pieces were re-swollen with 12.5 ng/µL trypsin (Promega, Germany) solution

buffered in 25 mM ammonium bicarbonate. Gel pieces were incubated for 16 h (overnight) at 37°C.

Supernatants were transferred to new 0.6 mL tubes, and gel pieces were extracted again with 50 µL

of 0.5% formic acid/20% acetonitrile for 15 min in a sonication bath. This step was performed two

times. Samples in extraction buffer were pooled in a 0.6 mL tube and evaporated in a Speedvac. The

volume was reduced to approximately 10 µL and then 10 µL HPLC grade water (Sigma, Germany)

was added for nano-LC-ESI-(CID/ETD)-MS/MS analysis via high capacity ion trap (HCT; Bruker,

Germany).

Peptide analysis by Nano-LC-ESI-(CID/ETD)-MS/MS HCT

For CNPase identification including phosphorylation, peptides were separated by biocompatible

Ultimate 3000 nano-LC system (Dionex, Sunnyvale, CA, USA) equipped with a PepMap100 C-18

trap column (300 µm id × 5 mm long cartridge, from Dionex) and PepMap100 C-18 analytic

column (75 µm id × 150 mm long, from Dionex). The gradient consisted of (A) 0.1% formic acid in

water, (B) 0.08% formic acid in ACN: 4–30% B from 0 to 105 min, 80% B from 105 to 110 min

and 4% B from 110 to 125 min. The flow rate was 300 nL/min from 0 to 12 min, 75 nL/min from

12 to 105 min, 300 nL/min from 105 to 125 min. An HCT ultra-PTM discovery system (Bruker

Daltonics, Bremen, Germany) was used to record peptide spectra over the mass range of m/z 350–

1500, and MS/MS spectra in information-dependent data acquisition over the mass range of m/z

100–2800. Repeatedly, MS spectra were recorded followed by four data-dependent collision

induced dissociation (CID) MS/MS spectra and four electron transfer dissociation (ETD). MS/MS

spectra were generated with parameters set as follows: The voltage between ion spray tip and spray

shield was set to 1550 V. Drying nitrogen gas was heated to 170°C and the flow rate was 10 L/min.

The collision energy was set automatically according to the mass and charge state of the peptides

chosen for fragmentation. Doubly or triply charged peptides were chosen for MS/MS experiments

due to their good fragmentation characteristics. MS/MS spectra were interpreted by the MASCOT

software (mascot.dll 1.6b21; Matrix Science, London, UK). Searches were done by using the

MS/MS spectra were interpreted and peak lists were generated by DataAnalysis 4.0 (Bruker

Daltonics) with parameters set to an absolute threshold limit 5000 and the relative threshold 0.1%.

Searches were performed by using the MASCOT v2.2 (Matrix Science, London, UK) against

UniProtKB/Swiss-prot database (Rattus; 524,420 sequence entries, 11-Jan-2011) for protein

identification. MASCOT searching parameters were set as follows: trypsin as used with two

maximum missing cleavage sites, species limited to Rattus, a mass tolerance of 0.2 Da for peptide

tolerance, 0.2 Da for MS/MS tolerance, ions score cut-off lower than 15, fixed modification of

carbamidomethyl(C) and variable modification of oxidation (M), acetylation (K), deamidation (N,

Q), methylation (D, E) and phosphorylation (S, T, Y). Positive protein identifications were based on

significant MOWSE scores. After protein identification, an error-tolerant search with parameter set

was performed to detect unspecific cleavage and unassigned modifications. For the verification of

phosphorylation sites, phosphatase in-gel digestion was carried out and the mass shift correction

was used for the interpretation [1].

References

[1] Chen, W. Q., Graf, C., Zimmel, D., Rovina, P., et al., Ceramide kinase profiling by mass

spectrometry reveals a conserved phosphorylation pattern downstream of the catalytic site. J

Proteome Res 2010, 9, 420-429.

Supplemental results

Supplemental Figure 1. Analysis of the CNPase-CaM interaction using ITC. Titration curves are

shown for CNP_25-298 and the two CNPase peptides. CNP_25-398 titrated with CaM, open

circles; CaM titrated with pep1 – red; CaM titrated with pep2 – green. All titrations were done at

+30°C. Estimated Kd values for CNP_25-398 and pep1 were 27 and 23 µM, respectively.

Supplemental figure 2. Separation of MPEs using BN/SDS/SDS-PAGE (3DE)

(A) 60 µg of MPEs were separated by 1D BN-PAGE using a 5%-13% separating gel. The BN gel was then

dissected into 10 pieces. (B) These gel pieces were equilibrated in SDS sample buffer and applied on a 6%-

13% SDS gel in the second dimension. (C) Individual lanes resulting from this second dimension gel were

then applied to a 7.5%-17% SDS gel (BN/SDS/SDS 3DE). Colloidal Coomassie Blue staining was used for

protein visualization. Spots identified as CN37_RAT with sequence coverage are indicated by arrows. All

matched peptides on CN37_Rat are given in Supplementary Table 1.

Supplemental Figure 3. MS/MS spectra of phosphorylated peptides from CN37_Rat

identification with verification by phosphatase treatment

MS/MS spectra of phosphorylated peptides from CNPase identification were shown comparing with

verified spectra by treatment of phosphatase from spot 4 (A; Y110) and spot 7 (B; S169). Protein spots from

BN/SDS/SDS-PAGE [3DE] were treated by phosphatase and subsequently digested by trypsin. Peptides

were extracted and analyzed by nano-LC-ESI-MS/MS (high-capacity ion trap). MS/MS spectra were

interpreted and peak lists were generated by DataAnalysis 3.4. Searches were done by using the MASCOT

2.2 against latest UniProtKB database for protein identification and PTM search. Asterisk marks from

figures indicate amino acids which contain phosphorylation. All identified peptide list of CN37_Rat are

given in Supplemental Table 1.

a SwissProt accession number (Acc. Nr.) referred in website (http://www.uniprot.org/uniprot)

b Charge state of the spectrum

c Ion score for the match between peptide and spectrum

d Observed mass in Dalton

e Calculated (theoretical) mass to charge ratio in Dalton

f Error (delta value) between measured and theoretical mass in Dalton

http://www.uniprot.org/uniprot

Supplementary table 1. Peptide list with sequence coverage from spots identified as CN37_RAT on BN/SDS/SDS-PAGE

Spot 1 (sequence coverage: 29.5%)Residue Nr. Observed Mr (expt) Mr (Calc) Delta Ions score Sequence Post-translational modifications16 - 27 680.414 1358.812 1358.725 0.087 17 K.IFFRKMSSSGAK.D -57 - 68 572.909 1143.803 1143.648 0.155 19 R.GLPGSGKSTLAR.L -80 - 87 442.714 883.413 883.411 0.021 21 K.MVSADAYK.I -94 - 101 494.782 987.551 987.419 0.132 23 R.ADFSEEYK.R -103 - 112 578.307 1154.600 1154.507 0.093 20 R.LDEDLAGYCR.R -204 - 216 481.573 1441.698 1441.720 -0.022 27 K.AGQVFLEELGNHK.A Deamidated (N214)224 - 232 515.200 1028.385 1028.493 -0.108 38 R.HFISGDEPK.E -283 - 293 588.350 1174.686 1174.696 -0.011 44 K.LSISALFVTPK.T -356 - 368 657.771 1313.528 1313.621 -0.093 42 K.GGSQGEEVGELPR.G -356 - 368 658.281 1314.546 1314.605 -0.059 50 K.GGSQGEEVGELPR.G Deamidated (Q359)391 - 399 510.293 1018.570 1018.512 0.058 28 K.AIFTGYYGK.G -402 - 420 655.673 1963.996 1964.057 -0.061 16 K.PVPVHGSRKGGAMQICTII.- -

Spot 2 (sequence coverage: 21.7%)Residue Nr. Observed Mr (expt) Mr (Calc) Delta Ions score Sequence Post-translational modifications80 - 87 441.769 883.524 883.411 0.113 27 K.MVSADAYK.I -94 - 101 494.664 987.313 987.419 -0.105 31 R.ADFSEEYK.R -103 - 112 594.284 1186.553 1186.497 0.056 29 R.LDEDLAGYCR.R Dioxidation (R112)224 - 232 515.234 1028.454 1028.493 -0.039 30 R.HFISGDEPK.E -235 - 243 521.239 1040.464 1040.554 -0.090 28 K.LDLVSYFGK.R -261 - 274 768.772 1535.530 1535.722 -0.192 78 K.ATGAEEYAQQDVVR.R -283 - 293 588.348 1174.682 1174.696 -0.014 39 K.LSISALFVTPK.T -356 - 368 657.792 1313.570 1313.621 -0.051 45 K.GGSQGEEVGELPR.G -391 - 399 510.205 1018.395 1018.512 -0.117 21 K.AIFTGYYGK.G -

Spot 3 (sequence coverage: 16.9%)

Residue Nr. Observed Mr (expt) Mr (Calc) Delta Ions score Sequence Post-translational modifications74 - 93 742.072 2223.195 2223.111 0.084 25 K.YHNGTKMVSADAYKIIPGSR.A Oxidation (M80)80 - 87 442.718 883.422 883.411 0.011 32 K.MVSADAYK.I -94 - 101 494.766 987.518 987.419 0.099 30 R.ADFSEEYK.R -103 - 112 578.355 1154.695 1154.507 0.188 27 R.LDEDLAGYCR.R -283 - 293 588.363 1174.711 1174.696 0.015 35 K.LSISALFVTPK.T -355 - 368 658.289 1314.561 1314.628 -0.067 20 K.GGSQGEEVGELPR.G -

Spot 4 (sequence coverage: 14.0%)Residue Nr. Observed Mr (expt) Mr (Calc) Delta Ions score Sequence Post-translational modifications80 - 87 442.728 883.441 883.411 0.030 31 K.MVSADAYK.I -94 - 101 494.705 987.396 987.419 -0.022 29 R.ADFSEEYK.R -103 - 112 627.161 1252.306 1252.491 -0.186 23 R.LDEDLAGYCR.R Phosphorylation (Y110)224 - 232 515.268 1028.520 1028.493 0.027 19 R.HFISGDEPK.E -283 - 293 588.390 1174.764 1174.696 0.068 31 K.LSISALFVTPK.T -356 - 368 657.755 1313.495 1313.621 -0.126 33 K.GGSQGEEVGELPR.G -

Spot 4 + Phosphatase (sequence coverage: 16.7%)Residue Nr. Observed Mr (expt) Mr (Calc) Delta Ions score Sequence Post-translational modifications74 - 93 737.088 2208.242 2208.123 0.119 21 K.YHNGTKMVSADAYKIIPGSR.A -80 - 87 442.809 883.603 883.411 0.192 17 K.MVSADAYK.I -94 - 101 494.777 987.539 987.419 0.120 25 R.ADFSEEYK.R -103 - 112 578.320 1154.624 1154.507 0.117 33 R.LDEDLAGYCR.R -283 - 293 588.411 1174.806 1174.696 0.110 26 K.LSISALFVTPK.T -356 - 368 657.780 1313.545 1313.621 -0.076 18 K.GGSQGEEVGELPR.G -


Residue Nr. Observed Mr (expt) Mr (Calc) Delta Ions score Sequence Post-translational modifications63 - 79 930.051 1858.086 1858.053 0.033 17 K.STLARLIVEKYHNGTK.M Methylation (K73 and K79)117 - 127 437.936 1310.783 1310.670 0.113 33 R.VLVLDDTNHER.E -283 - 293 588.394 1174.773 1174.696 0.077 32 K.LSISALFVTPK.T -356 - 368 657.822 1313.629 1313.621 0.008 44 K.GGSQGEEVGELPR.G -379 - 390 732.972 1463.929 1463.811 0.118 29 R.WMLSLAKKMEVK.A Acetylation (K385)


Residue Nr. Observed Mr (expt) Mr (Calc) Delta Ions score Sequence Post-translational modifications80 - 87 451.283 900.550 900.412 0.137 29 K.MVSADAYK.I Oxidation (M80)94 - 101 494.777 987.540 987.419 0.121 24 R.ADFSEEYK.R -103 - 112 578.251 1154.497 1154.507 -0.020 29 R.LDEDLAGYCR.R -117 - 127 437.579 1309.714 1309.663 0.051 25 R.VLVLDDTNHER.E -276 - 282 400.692 799.370 799.423 -0.053 27 R.SYGKAFK.L -283 - 293 588.401 1174.786 1174.696 0.090 52 K.LSISALFVTPK.T -356 - 368 657.907 1313.799 1313.621 0.178 23 K.GGSQGEEVGELPR.G -


Residue Nr. Observed Mr (expt) Mr (Calc) Delta Ions score Sequence Post-translational modifications80 - 87 442.713 883.412 883.411 0.001 25 K.MVSADAYK.I -94 - 101 494.705 987.396 987.419 -0.023 26 R.ADFSEEYK.R -103 - 112 578.167 1154.319 1154.507 -0.188 26 R.LDEDLAGYCR.R -117 - 127 437.567 1309.680 1309.663 0.018 27 R.VLVLDDTNHER.E -117 - 127 655.853 1309.690 1309.663 0.028 42 R.VLVLDDTNHER.E -164 - 177 865.543 1729.070 1728.941 0.130 28 K.NQWQLSLDDLKKLK.P Phosphorylation (S169)283 - 293 588.345 1174.675 1174.696 -0.022 36 K.LSISALFVTPK.T -356 - 368 657.857 1313.699 1313.621 0.078 26 K.GGSQGEEVGELPR.G -

Spot 7 + Phosphatase (sequence coverage: 19.0%)

Residue Nr. Observed Mr (expt) Mr (Calc) Delta Ions score Sequence Post-translational modifications80 - 87 442.795 883.575 883.411 0.164 22 K.MVSADAYK.I -103 - 112 578.164 1154.312 1154.507 -0.195 19 R.LDEDLAGYCR.R -117 - 127 655.889 1309.763 1309.663 0.100 27 R.VLVLDDTNHER.E -164 - 177 577.361 1729.059 1728.964 0.094 35 K.NQWQLSLDDLKKLK.P -164 - 177 865.564 1729.121 1728.941 0.171 19 K.NQWQLSLDDLKKLK.P Deamidated (Q167)204 - 216 481.600 1441.775 1441.743 0.032 20 K.AGQVFLEELGNHK.A -283 - 293 588.390 1174.764 1174.696 0.068 31 K.LSISALFVTPK.T -356 - 368 657.799 1313.582 1313.621 -0.039 20 K.GGSQGEEVGELPR.G -

experimental procedures - tokushima bunri universitykp.bunri-u.ac.jp/kph02/pdf/2012 jnc; cnpase ca...

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