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• W2078960075 abstract "Polyglutamylation is an original posttranslational modification, discovered on tubulin, consisting in side chains composed of several glutamyl units and leading to a very unusual protein structure. A monoclonal antibody directed against glutamylated tubulin (GT335) was found to react with other proteins present in HeLa cells. After immunopurification on a GT335 affinity column, two prominent proteins of ∼50 kDa were observed. They were identified by microsequencing and mass spectrometry as NAP-1 and NAP-2, two members of the nucleosome assembly protein family that are implicated in the deposition of core histone complexes onto chromatin. Strikingly, NAP-1 and NAP-2 were found to be substrates of an ATP-dependent glutamylation enzyme co-purifying on the same column. We took advantage of this property to specifically label and purify the polyglutamylated peptides. NAP-1 and NAP-2 are modified in their C-terminal domain by the addition of up to 9 and 10 glutamyl units, respectively. Two putative glutamylation sites were localized for NAP-1 at Glu-356 and Glu-357 and, for NAP-2, at Glu-347 and Glu-348. These results demonstrate for the first time that proteins other than tubulin are polyglutamylated and open new perspectives for studying NAP function. Polyglutamylation is an original posttranslational modification, discovered on tubulin, consisting in side chains composed of several glutamyl units and leading to a very unusual protein structure. A monoclonal antibody directed against glutamylated tubulin (GT335) was found to react with other proteins present in HeLa cells. After immunopurification on a GT335 affinity column, two prominent proteins of ∼50 kDa were observed. They were identified by microsequencing and mass spectrometry as NAP-1 and NAP-2, two members of the nucleosome assembly protein family that are implicated in the deposition of core histone complexes onto chromatin. Strikingly, NAP-1 and NAP-2 were found to be substrates of an ATP-dependent glutamylation enzyme co-purifying on the same column. We took advantage of this property to specifically label and purify the polyglutamylated peptides. NAP-1 and NAP-2 are modified in their C-terminal domain by the addition of up to 9 and 10 glutamyl units, respectively. Two putative glutamylation sites were localized for NAP-1 at Glu-356 and Glu-357 and, for NAP-2, at Glu-347 and Glu-348. These results demonstrate for the first time that proteins other than tubulin are polyglutamylated and open new perspectives for studying NAP function. microtubule nucleosome assembly protein monoclonal antibody polyacrylamide gel electrophoresis matrix-assisted laser desorption/ionization time of flight high performance liquid chromatography Tris-buffered saline Tubulin, the main microtubule component, is subjected to several posttranslational modifications (for a recent review, see Ref. 1.MacRae T.H. Eur. J. Biochem. 1997; 244: 265-278Crossref PubMed Scopus (262) Google Scholar). Among them, polyglutamylation and polyglycylation, which occur on both α- and β-tubulin subunits, are very unusual (2.Alexander J.E. Hunt D.F. Lee M.K. Shabanowitz J. Michel H. Berlin S.C. Macdonald T.L. Sundberg R.J. Rebhun L.I. Frankfurter A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4685-4689Crossref PubMed Scopus (221) Google Scholar, 3.Eddé B. Rossier J. Le Caer J.P. Desbruyères E. Gros F. Denoulet P. Science. 1990; 247: 83-85Crossref PubMed Scopus (425) Google Scholar, 4.Eddé B. Rossier J. Le Caer J.P. Berwald N.Y. Koulakoff A. Gros F. Denoulet P. J. Cell. Biochem. 1991; 46: 134-142Crossref PubMed Scopus (56) Google Scholar, 5.Redeker V. Melki R. Promé D. Caer J.-P.L. Rossier J. FEBS Lett. 1992; 313: 185-192Crossref PubMed Scopus (129) Google Scholar, 6.Redeker V. Levilliers N. Schmitter J.M. Le C.J. Rossier J. Adoutte A. Bré M.H. Science. 1994; 266: 1688-1691Crossref PubMed Scopus (218) Google Scholar, 7.Rüdiger M. Plessmann U. Kloppel K.D. Wehland J. Weber K. FEBS Lett. 1992; 308: 101-105Crossref PubMed Scopus (118) Google Scholar). They are formally similar to each other, consisting of the addition of a variable number of glutamyl units, in the case of polyglutamylation, or of glycyl units in the case of polyglycylation. In both cases, the first unit is linked by an amide bond between its α-amino group and the γ-carboxylic group of a glutamate residue of the polypeptide chain. Additional units are linked together by isopeptidic bonds, thus leading to the formation of a linear side chain of amino acid residues, which extends from the main polypeptidic chain and exhibits a second C terminus. In some cases, especially for polyglycylation, several glutamate residues of the polypeptide can be modified, leading to the formation of several side chains (8.Redeker V. Rossier J. Frankfurter A. Biochemistry. 1998; 37: 14838-14844Crossref PubMed Scopus (50) Google Scholar, 9.Vinh J. Langridge J.I. Bré M.H. Levilliers N. Redeker V. Loyaux D. Rossier J. Biochemistry. 1999; 38: 3133-3139Crossref PubMed Scopus (44) Google Scholar). The total number of glutamyl and glycyl units present in each subunit can reach up to ∼20 and ∼34, respectively, but is more commonly in the range of 1–6 (10.Eddé B. Rossier J. Le Caer J. Promé J.C. Desbruyères E. Gros F. Denoulet P. Biochemistry. 1992; 31: 403-410Crossref PubMed Scopus (39) Google Scholar, 11.Geimer S. Teltenkotter A. Plessmann U. Weber K. Lechtreck K.F. Cell Motil. Cytoskel. 1997; 37: 72-85Crossref PubMed Scopus (50) Google Scholar, 12.Schneider A. Plessmann U. Felleisen R. Weber K. FEBS Lett. 1998; 429: 399-402Crossref PubMed Scopus (46) Google Scholar). The sites of modification are located in the C-terminal region of α- and β-tubulin, which are known to be exposed on the surface of the MT1 lattice and to interact with various microtubule associated proteins. Polyglutamylation and polyglycylation are thought to profoundly affect the tridimensional structure of these domains and to interfere with the binding of essential effectors involved in MT function. Tubulin polyglutamylation is a very old modification, widely distributed among species, from protozoa to mammals. It is relatively abundant in centrioles, basal bodies, cilia, and flagella but is not restricted to these very particular structures. For instance, in neurons, ∼90% of α-tubulin and ≥50% of β-tubulin are polyglutamylated (13.Audebert, S., Koulakoff, A., Berwald, N. Y., Gros, F., Denoulet, P., and Eddé, B. (1994) J. Cell Sci. 2313–2322Google Scholar). Modification of the α-tubulin subunit is triggered at the beginning of neurite extension, while the β subunit is modified much later, during a phase probably related to neuronal maturation (13.Audebert, S., Koulakoff, A., Berwald, N. Y., Gros, F., Denoulet, P., and Eddé, B. (1994) J. Cell Sci. 2313–2322Google Scholar, 14.Eddé B. Denoulet P. De Néchaud B. Koulakoff A. Berwald-Netter Y. Gros F. Biol. Cell. 1989; 65: 109-117Crossref PubMed Scopus (25) Google Scholar). The modification occurs also, but at much lower levels, in proliferative non-neuronal cells, such as HeLa or 3T3 cells (15.Bobinnec Y. Moudjou M. Fouquet J.P. Desbruyères E. Eddé B. Bornens M. Cell Motil. Cytoskel. 1998; 39: 223-232Crossref PubMed Scopus (130) Google Scholar). In these cells, interphasic microtubules are poorly glutamylated while other structures like mitotic spindle MTs, and especially MTs of the centrioles, are more highly glutamylated (15.Bobinnec Y. Moudjou M. Fouquet J.P. Desbruyères E. Eddé B. Bornens M. Cell Motil. Cytoskel. 1998; 39: 223-232Crossref PubMed Scopus (130) Google Scholar). Consistent with its wide distribution, tubulin polyglutamylation is believed to be involved in multiple functions. In vitro blot overlay assays strongly suggest that the polyglutamyl side chain of tubulin can regulate differentially the binding of various microtubule-associated proteins, in a manner dependent on the length of the side chain (16.Boucher D. Larcher J.C. Gros F. Denoulet P. Biochemistry. 1994; 33: 12471-12477Crossref PubMed Scopus (168) Google Scholar, 17.Larcher J.C. Boucher D. Lazereg S. Gros F. Denoulet P. J. Biol. Chem. 1996; 271: 22117-22124Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). These results are of particular interest because binding of microtubule-associated proteins strongly influences MT dynamics and is particularly important during neuronal differentiation, where plasticity and rigidity of the axodendritic processes must be precisely regulated in relation with environmental cues. Indications that polyglutamylation is involved in other biological processes come from ex vivo experiments using a mAb raised against glutamylated tubulin (GT335). For instance, mAb GT335 was shown to strongly inhibit the motility of demembranated, re-activated spermatozoa. Interestingly, the amplitude of the beating waves was strongly reduced while the frequency was unaffected (18.Gagnon C. White D. Cosson J. Huitorel P. Eddé B. Desbruyères E. Paturle-Lafanechère L. Multigner L. Job D. Cibert C. J. Cell Sci. 1996; 109: 1545-1553Crossref PubMed Google Scholar). More recently, it was shown that introduction of mAb GT335 into HeLa cells by either microinjection or electroporation leads to the unexpected disappearance of the centrioles and the dispersion of the pericentriolar material (19.Bobinnec Y. Khodjakov A. Mir L.M. Rieder C.L. Eddé B. Bornens M. J. Cell Biol. 1998; 143: 1575-1589Crossref PubMed Scopus (302) Google Scholar). Whether polyglutamylation and polyglycylation are specific for tubulin or are common to other proteins was not yet known. Recently, we examined polyglutamylation of tubulin in HeLa cells and its variations during the cell cycle (20.Regnard, C., Desbruyères, E., Denoulet, P., and Eddé, B. (1999) J. Cell Sci. 4281–4289Google Scholar). In the course of that study, we observed a minor reactivity of mAb GT335 with proteins comigrating with tubulin on SDS-PAGE but not on two-dimensional PAGE. The possibility that this reactivity corresponded to a particularly high background seemed unlikely since other very abundant proteins were not labeled at all. In this report, we show indeed that proteins other than tubulin are subjected to polyglutamylation and identify two nucleosome assembly proteins as new targets for this modification. NAP-1 and NAP-2 are recognized by mAb GT335 in both their native and denatured states and are substrates of a polyglutamylase activity present in HeLa cells. The sites of modification have been localized in the very acidic C-terminal domains of both proteins, which bear up to 9–10 glutamyl units. Examination of other cell types and organisms indicates that polyglutamylation of NAPs appears as a general phenomenon. These results provide new insights for studying the regulation of NAP function. They furthermore suggest that polyglutamylation might be a more general posttranslational modification than previously believed. HeLa cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 2 mmglutamine, 100 IU penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified 5% CO2 atmosphere. SDS-PAGE (21.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207208) Google Scholar) was performed on 8% acrylamide, 0.11% bisacrylamide, slab gels (8 cm long) containing 0.1% (w/v) SDS (90% pure; Merck, Darmstadt, Germany). Isoelectric focusing in denaturing 9.5 m urea cylindrical gels (12 cm long) was performed as described (22.O'Farrell P.H. J. Biol. Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar), with a mixture of pH 3.5–10 and 5–8 (1:4) ampholytes (Amersham Pharmacia Biotech, Saclay, France). Second dimension was performed on 24-cm-long slab gels as described above. Exposures of radioactive gels were performed at −80 °C, typically for 2–4 weeks, using XAR-5 films (Eastman Kodak Co.) after enhancement with Amplify (Amersham Pharmacia Biotech). Electrotransfer of polyacrylamide gels was performed onto nitrocellulose (Hybond C, Amersham Pharmacia Biotech) as described (23.Towbin H. Staehelin S. Gordon H. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44922) Google Scholar). mAb GT335 was biotinylated and incubated simultaneously with streptavidin conjugated to horseradish peroxidase, as described (20.Regnard, C., Desbruyères, E., Denoulet, P., and Eddé, B. (1999) J. Cell Sci. 4281–4289Google Scholar). α- and β-tubulin were detected by DM1A (1:5000, Amersham Pharmacia Biotech) and Tub 2.1 (1:2000, Sigma), respectively. Anti-NAP-1 hybridoma supernatant (mAb 4A8; Refs. 24.Ishimi Y. Sato W. Kojima M. Sugasawa K. Hanaoka F. Yamada M. Cell Struct. Funct. 1985; 10: 373-382Crossref PubMed Scopus (22) Google Scholar and 25.Fujii-Nakata T. Ishimi Y. Okuda A. Kikuchi A. J. Biol. Chem. 1992; 267: 20980-20986Abstract Full Text PDF PubMed Google Scholar) was a gift from Dr. Y. Ishimi (Mitsubishi-Kasei Institute of Life Sciences, Tokyo, Japan) and was diluted 1:20. All steps were performed at 4 °C. HeLa cells were extracted in TBS (20 mm Tris, pH 7.2, 150 mm NaCl) containing 0.1% Triton X-100 and protease inhibitors (aprotinin, leupeptin, and 4-(2-aminoethyl)-benzenesulfonyl fluoride, each at 10 μg/ml). The high speed supernatant was obtained after centrifugation at 100,000 × g for 40 min. mAb GT335 (25 μg) was diluted in TBS and preincubated 3 h with 25 μl of a suspension of agarose beads coupled to protein G (Amersham Pharmacia Biotech) on a rotatory agitator. After washing with TBS, the beads were incubated overnight with the HeLa supernatant (400 μl, 10 mg/ml). After extensive washing, the bound proteins were eluted with 2 × 30 μl of extraction buffer containing 1 m NaCl and analyzed by SDS-PAGE and Western blotting. mAb GT335 (1 ml, 7–10 mg of Ig) was purified by affinity on a tubulin column and coupled toN-hydroxysuccinimide-activated Hi-Trap columns (1 ml, Amersham Pharmacia Biotech) according to the manufacturer's instructions. All subsequent steps were performed at 4 °C. High speed supernatant of HeLa cells, obtained as described in the previous paragraph, was diluted in TBS to a final protein concentration of 2 mg/ml and loaded on the column (50 mg/ml of gel) at a flow rate of 0.5 ml/min. After extensive washing with TBS containing 0.01% Triton X-100, bound proteins were eluted by increasing the NaCl concentration to 0.65 m. This was sufficient to break the ionic interactions involved in the antigen-antibody binding. The eluted fractions were detected by their absorbance at 280 nm, pooled, supplemented with protease inhibitors, and concentrated on an Amicon ultrafiltration device (2 ml, cut-off 10,000, Millipore). The yield of this last step was highly variable, ranging from 20% to 70%. Proteolytic fragments of proteins X and Y (see Fig. 3) were obtained according to the method of Aebersold et al. (26.Aebersold R.H. Leavitt J. Saavedra R.A. Hood L.E. Kent S.B.H. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6970-6974Crossref PubMed Scopus (630) Google Scholar). Briefly, after one-dimensional or two-dimensional PAGE, proteins were electrotransferred on nitrocellulose. After Ponceau Red staining, the bands or spots were excised from the nitrocellulose sheets, saturated with 20 mg/ml PVP40 (Sigma), and incubated overnight at 30 °C, under constant agitation in digestion buffer (Tris 50 mm, pH 8) containing 2.5 μg/ml Trypsin (sequence grade, Promega). In the experiment presented in Fig. 4, reversed phase chromatography was performed on an HPLC system (Waters) with a C18 column (Brownlee, 2.1 × 220 mm) at a flow rate of 0.2 ml/min. Solution A was 0.1% trifluoroacetic acid in water, and solution B was 0.09% trifluoroacetic acid, 70% acetonitrile in water. A gradient of 5–50% B in 50 min followed by 50–100% B in 20 min was developed. In the experiments presented in Fig. 7, a DEAE column suitable for HPLC was used (AX300, Brownlee, 4.6 × 100 mm) at a flow rate of 0.4 ml/min. Solution A was 50 mm Tris-HCl, pH 8, and solution B was 50 mm Tris-HCl, pH 8, containing 1 m NaCl. A linear gradient of 0–100% B was developed. The main glutamylated peak eluted at ∼0.75 m NaCl. In the experiment presented in Fig. 7 A, an on-line flow radioactivity detector (Flo-One, Radiomatic, Packard Instruments) was placed immediately after the UV detector. The radioactive signals were accumulated for 9-s intervals. Size exclusion chromatography was carried out to desalt the radioactive peptides eluted from the DEAE chromatography. A column (1 × 50 cm) of Trisacryl gel GF05 (IBF, France) with a separating range of 300–2500 Da was used at a flow rate of 0.1 ml/min. Radioactive peptides were followed by counting an aliquot of each fraction and eluted just before the salt peak. Before being analyzed by microsequencing and mass spectrometry, the fractions were concentrated under speed vacuum. The enzymatic assay was performed as described previously (27.Regnard C. Audebert S. Desbruyères E. Denoulet P. Eddé B. Biochemistry. 1998; 37: 8395-8404Crossref PubMed Scopus (36) Google Scholar) in 50 mm Tris-HCl, pH 9.0 (final pH: 8.7), 2 mm ATP (equilibrated to pH 7 with NaOH), 8 mmMgCl2, 2.5 mm dithiothreitol, andl-[3H]glutamate (45–55 Ci/mmol, Amersham Pharmacia Biotech). To increase the radioactive signal, the [3H]glutamate solution was concentrated 10-fold under speed vacuum and added at a final concentration of 50 μm. Incubation was performed at 30 °C, usually for 1 h. For the assay presented in Fig. 6, concentrated affinity-purified fraction was used at a final protein concentration of 0.11 mg/ml, in the presence or absence of tubulin polyglutamylase partially purified from mouse brain (fraction IV of Ref. 27.Regnard C. Audebert S. Desbruyères E. Denoulet P. Eddé B. Biochemistry. 1998; 37: 8395-8404Crossref PubMed Scopus (36) Google Scholar, at a final concentration of 0.04 mg/ml) or from HeLa cells (phosphocellulose-purified fraction of Ref. 20.Regnard, C., Desbruyères, E., Denoulet, P., and Eddé, B. (1999) J. Cell Sci. 4281–4289Google Scholar, final concentration of 0.3 mg/ml). Quantitation of the radioactivity incorporated in the different proteins was performed by scintillation counting of the protein bands after one-dimensional PAGE and electrotransfer onto nitrocellulose sheets, as described previously (20.Regnard, C., Desbruyères, E., Denoulet, P., and Eddé, B. (1999) J. Cell Sci. 4281–4289Google Scholar). A duplicate sample (10 μl) of that presented in Fig.6 A (lane 3) gave ∼1000, 500, and 800 dpm for the 52-, 48-, and 40-kDa proteins, respectively. If each of these species accounts for 10% of proteins present in the fraction, specific radioactivity could be grossly estimated to ∼10,000, 5,000, and 8,000 dpm/μg protein, respectively. For the preparative experiment presented in Fig. 7, affinity-purified fraction was assayed at a final protein concentration of 0.15 mg/ml in the presence of 50 μm l-[3H]glutamate (in the absence of any exogenous tubulin polyglutamylase). After incubation, the reaction mixture (0.8 ml) was mixed with unlabeled affinity-purified fraction (10 ml) before loading on one-dimensional PAGE. A total of 100,000 dpm were recovered in the proteolytic peptide fraction of NAP-1 plus NAP-2. Of these, 5% (5,000 dpm) were analyzed in Fig. 7 A, and the remainder was used for the preparative chromotography shown in Fig.7 B. The specific activity attained for NAPs in these conditions could be estimated from amino acid sequencing data. The aliquot of fraction A loaded on the sequencer contained 10,000 dpm and 24 pmol of NAP peptides, thus corresponding to a specific radioactivity of ∼400 dpm/pmol, that is to say that 4 × 10−3pmol of Glu/pmol of peptide were incorporated. Thus, in the conditions used for labeling the glutamylation sites in vitro, the overall level of glutamylation of native NAPs was not significantly affected. Edman sequencing was performed using a a 494-HT Procise apparatus (PE Biosystems, Foster City, CA). The reversed-phase samples were spotted on glass fiber disks treated with Biobrene (1.5 mg). The ion-exchange fractions were adsorbed onto polyvinylidene difluoride membranes and centrifuged at 500 ×g in Prospin tubes deprived of the 3-kDa ultrafiltration membrane, washed with 10% methanol, dried, and treated with 0.1 mg of Biobrene prior to loading onto the sequencer. A Hewlett-Packard MALDI-TOF G2025 equipment was utilized for reversed-phase separated peptides using α-cyano-4-hydroxycinnamic acid as matrix (Hewlett Packard). For the analysis of the ion-exchange separated peptides, mass spectra were acquired with a Voyager DE-PRO (PE-Biosystems) MALDI-TOF device, in linear mode, with dihydroxybenzoic acid as matrix (Sigma). Western blotting analysis of HeLa cell soluble extract with GT335, a mAb directed against the polyglutamylated motif of tubulin (28.Wolff A. de, N. B. Chillet D. Mazarguil H. Desbruyères E. Audebert S. Eddé B. Gros F. Denoulet P. Eur J. Cell Biol. 1992; 59: 425-432PubMed Google Scholar), revealed several reactive proteins of 48–52 kDa that fall within the region of migration of α- and β-tubulin. Two additional bands of ∼66 and ∼180 kDa were also detected after long exposure (Fig.1, lanes 2 and3). We showed previously by two-dimensional PAGE analysis that one of the reactive proteins indeed corresponds to glutamylated β-tubulin but that other ones of similar M rare more acidic than α- and β-tubulin (Ref. 20.Regnard, C., Desbruyères, E., Denoulet, P., and Eddé, B. (1999) J. Cell Sci. 4281–4289Google Scholar, see also Fig.3). Whether the reactivity observed with non-tubulin components was an artifact related to denaturation by SDS or indicated the presence of polyglutamylation in these proteins was first tested by carrying out immunoprecipitation experiments in TBS buffer. A strong enrichment of the same reactive proteins was observed in the immunoprecipitate, indicating that they are efficiently recognized by mAb GT335 under native conditions (Fig. 1, lane 6). Preparative scale purification of the immunoreactive proteins was performed by affinity chromatography on a GT335 column. SDS-PAGE analysis of the affinity-purified fraction showed the presence of prominent proteins with apparent M r of 40 and 48–52 kDa and a less represented one of ∼200 kDa (Fig.2 A, lane 3). Probing of the immunopurified fractions with mAb GT335 (Fig. 2 B) revealed a strong enrichment of the 48–52- and 66-kDa species. A faint band corresponding to the 180-kDa species was also detected. In some experiments, this band was much more intense, most likely depending on the efficiency of the transfer onto nitrocellulose. The 66- and 180-kDa species were not observed on the stained blots but could be detected after silver staining of gels run in parallel (data not shown). All the reactive proteins observed in the affinity-purified fraction were no longer detected in the flow-through, indicating that they have been efficiently retained on the column. Some of the proteins present in the affinity-purified fraction, such as the 40- and 200-kDa species, did not react with mAb GT335 on immunoblots. These proteins were probably retained on the column via their interaction with GT335-reactive proteins. However, we cannot exclude that they are recognized by the mAb only in their native form (see “Discussion”). When the affinity-purified fraction was analyzed by two-dimensional PAGE electrophoresis, two prominent spots, denoted X and Y, were observed in the 50-kDa mass range (Fig.3 A). Two other much less intense spots were also present in the same region (horizontal arrows). These four spots were all labeled by mAb GT335 proportionally to their relative abundance (Fig.3 B). An intense labeling corresponding to glutamylated β-tubulin was also observed although the protein was not detected by Coomassie Blue staining, which indicates a stronger reactivity of mAb GT335 toward glutamylated tubulin. None of the three ∼40-kDa proteins resolved in these gels (arrowheads) was detected by mAb GT335. We focused this study on the identification of the most abundant proteins in the affinity-purified fraction, proteins X and Y. Tryptic peptides were obtained after digestion of the excised two-dimensional spots and fractionated by reversed phase chromatography on a C18 column. Several well resolved peaks (Fig.4) were analyzed by automated Edman degradation and mass spectrometry (TableI). All of the peptides analyzed resulted in amino acid sequences and mass values that allowed identification of proteins X and Y as members of the nucleosome associated protein family, NAP-2 and NAP-1, respectively (29.Hu R.J. Lee M.P. Johnson L.A. Feinberg A.P. Hum. Mol. Genet. 1996; 5: 1743-1748Crossref PubMed Scopus (45) Google Scholar, 30.Simon H.U. Mills G.B. Kozlowski M. Hogg D. Branch D. Ishimi Y. Siminovitch K.A. Biochem. J. 1994; 297: 389-397Crossref PubMed Scopus (82) Google Scholar). However, two splicing variants have been described for NAP-2. Form I terminates at residue 375, while form II contains an additional C-terminal segment of 11 residues (31.Rodriguez P. Munroe D. Prawitt D. Chu L.L. Bric E. Kim J. Reid L.H. Davies C. Nakagama H. Loebbert R. Winterpacht A. Petruzzi M.J. Higgins M.J. Nowak N. Evans G. Shows T. Weissman B.E. Zabel B. Housman D.E. Pelletier J. Genomics. 1997; 44: 253-265Crossref PubMed Scopus (77) Google Scholar) Whether protein X corresponds to form I or II is not yet known.Table IAmino acid sequencing and mass spectrometry analysis of trypsinized proteins X and YPeptideaThe peptides were named after the peak number in Fig.4. X and Y indicate peptides derived from proteins X and Y, respectively.MassbFor each peptide, the first line corresponds to experimental data (partial sequences) and the second one to whole tryptic peptide sequences deduced from the known sequences of human NAP-1 or NAP-2. Dashes indicate undetermined residues.SequencebFor each peptide, the first line corresponds to experimental data (partial sequences) and the second one to whole tryptic peptide sequences deduced from the known sequences of human NAP-1 or NAP-2. Dashes indicate undetermined residues.IdentificationcThe position of each peptide in the sequence of NAP-1 or NAP-2 is indicated in parentheses.X-23901VLAALQER899VLAALQERNAP-2 (37–44)X-271338FYEEVHDLER1336FYEEVHDLERNAP-2 (84–93) or NAP-1 (95–104)X-351916LDNVPHTP-SYIETLPK1911LDNVPHTPSSYIETLPKNAP-2 (45–61)X-512499—-AEEPDPKGIPEF-FT–R2494AAATAEEPDPKGIPEFWFTIFRNAP-2 (147–168)X-574800QVPNESFFNFFNPLKAS4789QVPNESFFNFFNPLKASGDGESLEDSEFTLASDFEIGHFFRNAP-2 (283–324)Y-433989-EIINAIYEPTEEECE3915 + 71 = 3986dThe mass increment of 71 is explained by alkylation of the cysteine residue by acrylamide, as observed during protein sequencing.FEIINAIYEPTEEECEWKPDEDEISEELKEKNAP-1 (118–149)Y-514522-VSNDSFFNFFAPPEVPESGDLD4517TVSNDSFFNFFAPPEVPESGDLDDDAEAILAADFEIGHFLRNAP-1 (291–331)Calculated masses correspond to an average isotopic abundance.a The peptides were named after the peak number in Fig.4. X and Y indicate peptides derived from proteins X and Y, respectively.b For each peptide, the first line corresponds to experimental data (partial sequences) and the second one to whole tryptic peptide sequences deduced from the known sequences of human NAP-1 or NAP-2. Dashes indicate undetermined residues.c The position of each peptide in the sequence of NAP-1 or NAP-2 is indicated in parentheses.d The mass increment of 71 is explained by alkylation of the cysteine residue by acrylamide, as observed during protein sequencing. Open table in a new tab Calculated masses correspond to an average isotopic abundance. This identification was supported by immunoblotting experiments with the anti-NAP mAb 4A8 (24.Ishimi Y. Sato W. Kojima M. Sugasawa K. Hanaoka F. Yamada M. Cell Struct. Funct. 1985; 10: 373-382Crossref PubMed Scopus (22) Google Scholar, 25.Fujii-Nakata T. Ishimi Y. Okuda A. Kikuchi A. J. Biol. Chem. 1992; 267: 20980-20986Abstract Full Text PDF PubMed Google Scholar). As shown in Fig.5 A, this mAb reacted very strongly with the 48–52-kDa proteins enriched in the affinity-purified fraction. On two-dimensional gels, mAb 4A8 strongly labeled proteins X and Y, corresponding to NAP-2 and NAP-1, respectively, and faintly labeled the two minor species also detected by mAb GT335 (Fig.5 B). Although this antibody was obtained using NAP-1 as immunogen, it also recognized the spot corresponding to NAP-2. This is consistent with the suggested epitope of mAb 4A8 previously described to recognize the domain of NAP-1 from amino acids 290–305, and in particular the sequence FNF also present in NAP-2 (25.Fujii-Nakata T. Ishimi Y. Okuda A. Kikuchi A. J. Biol. Chem. 1992; 267: 20980-20986Abstract Full Text PDF PubMed Google Scholar). Immunodetection with mAb 4A8 indicated that ∼50% of the total NAPs present in the extract were retained on the GT335 column. This result suggests the existence of two subpopulations of NAPs, one being glutamylated and recognized by mAb GT335 and the other one not. It was not possible to directly identify the fractions corresponding to the glutamylated peptides in the bulk of the eluted peptides. We thus searched for a way to specifically label glutamylated peptides and allow their efficient detection. For this purpose, the affinity-purified fraction was incubated, in the presence of [3H]glutamate and MgATP, with partially purified preparations of mouse brain or HeLa cell tubulin polyglutamylase, as described previously (20.Regnard, C., Desbruyères, E., Denoulet, P., and Eddé, B. (1999) J. Cell Sci. 4281–4289Google Scholar, 27.Regnard C. Audebert S. Desbruyères E. Denoulet P. Eddé B. Biochemistry. 1998; 37: 8395-8404Crossref PubMed Scopus (36) Google Scholar). Glutamate incorporation into proteins was followed by SDS-PAGE and fluorography. Proteins of 180, 48–52, and 40 kDa were labeled similarly with either enzyme preparation (Fig.6 A, lanes 1 and 2). Surprisingly," @default.
• W2078960075 created "2016-06-24" @default.
• W2078960075 creator A5014431751 @default.
• W2078960075 creator A5035714072 @default.
• W2078960075 creator A5048959927 @default.
• W2078960075 creator A5049537801 @default.
• W2078960075 creator A5077888541 @default.
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• W2078960075 date "2000-05-01" @default.
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• W2078960075 title "Polyglutamylation of Nucleosome Assembly Proteins" @default.
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• W2078960075 cites W1959985846 @default.
• W2078960075 cites W1964716733 @default.
• W2078960075 cites W1968111328 @default.
• W2078960075 cites W1970124231 @default.
• W2078960075 cites W1972348353 @default.
• W2078960075 cites W1978661070 @default.
• W2078960075 cites W1982582935 @default.
• W2078960075 cites W1985338007 @default.
• W2078960075 cites W1986571317 @default.
• W2078960075 cites W1986676218 @default.
• W2078960075 cites W2002865057 @default.
• W2078960075 cites W2020415062 @default.
• W2078960075 cites W2024715696 @default.
• W2078960075 cites W2044835185 @default.
• W2078960075 cites W2057667659 @default.
• W2078960075 cites W2058852380 @default.
• W2078960075 cites W2069264853 @default.
• W2078960075 cites W2076799857 @default.
• W2078960075 cites W2077432173 @default.
• W2078960075 cites W2081644828 @default.
• W2078960075 cites W2086969577 @default.
• W2078960075 cites W2091352351 @default.
• W2078960075 cites W2099829773 @default.
• W2078960075 cites W2100837269 @default.
• W2078960075 cites W2101108802 @default.
• W2078960075 cites W2115685883 @default.
• W2078960075 cites W2116015519 @default.
• W2078960075 cites W2118056303 @default.
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