Phosphorylation and O-Glycosylation Sites of Human Chromogranin A (CGA79–439) from Urine of Patients with Carcinoid Tumors
1998; Elsevier BV; Volume: 273; Issue: 51 Linguagem: Inglês
10.1074/jbc.273.51.34087
ISSN1083-351X
AutoresPatrice Gadroy, Mats Stridsberg, Calliope Capon, Jean‐Claude Michalski, Jean‐Marc Strub, Alain Van Dorsselaer, Dominique Aunis, Marie‐Hélène Metz‐Boutigue,
Tópico(s)Neuroendocrine Tumor Research Advances
ResumoBecause of their water-soluble properties, chromogranins (CGs) and chromogranin-derived fragments are released together with catecholamines from adrenal chromaffin cells during stress situations and can be detected in the blood by radiochemical and enzyme assays. It is well known that chromogranins can serve as immunocytochemical markers for neuroendocrine tissues and as a diagnostic tool for neuroendocrine tumors. In 1993, large CGA-derived fragments have been shown to be excreted into the urine in patients with carcinoid tumors and the present study deals with the characterization of the post-translational modifications (phosphorylation and O-glycosylation) located along the largest natural CGA-derived fragment CGA79–439. Using mild proteolysis of peptidic material, high performance liquid chromatography, sequencing, and mass spectrometry analysis, six post-translational modifications were detected along the C-terminal CGA-derived fragment CGA79–439. Three O-linked glycosylation sites were located in the core of the protein on Thr163, Thr165, and Thr233, consisting in di-, tri-, and tetrasaccharides. Three phosphorylation sites were located in the middle and C-terminal domain, on serine residues Ser200, Ser252, and Ser315. These modified sites were compared with sequences of others species and discussed in relation with the post-translational modifications that we have reported previously for bovine CGA. Because of their water-soluble properties, chromogranins (CGs) and chromogranin-derived fragments are released together with catecholamines from adrenal chromaffin cells during stress situations and can be detected in the blood by radiochemical and enzyme assays. It is well known that chromogranins can serve as immunocytochemical markers for neuroendocrine tissues and as a diagnostic tool for neuroendocrine tumors. In 1993, large CGA-derived fragments have been shown to be excreted into the urine in patients with carcinoid tumors and the present study deals with the characterization of the post-translational modifications (phosphorylation and O-glycosylation) located along the largest natural CGA-derived fragment CGA79–439. Using mild proteolysis of peptidic material, high performance liquid chromatography, sequencing, and mass spectrometry analysis, six post-translational modifications were detected along the C-terminal CGA-derived fragment CGA79–439. Three O-linked glycosylation sites were located in the core of the protein on Thr163, Thr165, and Thr233, consisting in di-, tri-, and tetrasaccharides. Three phosphorylation sites were located in the middle and C-terminal domain, on serine residues Ser200, Ser252, and Ser315. These modified sites were compared with sequences of others species and discussed in relation with the post-translational modifications that we have reported previously for bovine CGA. chromogranins/secretogranins chromogranin A chromogranin B chromogranin C liquid chromatography/mass spectrometry matrix-assisted laser desorption ionization time-of-flight high performance liquid chromatography. Chromogranins/secretogranins (CGs/Sgs)1 constitute a family of acidic secretory glycoproteins widely expressed in a large number of endocrine and neuroendocrine cells and in neurons (1Simon J.P. Aunis D. Biochem. J. 1989; 262: 1-13Crossref PubMed Scopus (242) Google Scholar, 2Helle K.B. Neurochem. Int. 1990; 17: 165-175Crossref PubMed Scopus (58) Google Scholar, 3Huttner W.B. Gerdes H.H. Rosa P. Trends Biochem. Sci. 1991; 16: 27-30Abstract Full Text PDF PubMed Scopus (415) Google Scholar, 4Winkler H. Fischer-Colbrie R. Neuroscience. 1992; 49: 497-528Crossref PubMed Scopus (611) Google Scholar). Chromogranin A (CGA), the major member (40% of total soluble granule proteins) of this family, has been studied extensively. At the subcellular level, chromogranins are exclusively found in the soluble core of hormone and neurotransmitter storage vesicles and are released during exocytosis. Chromogranins have been proposed to play multiple roles in the secretory process. An intracellular function as a "helper" protein in the packaging of peptides, hormones, and neuropeptides by virtue of their ability to aggregate in the low pH and high calcium environment of the trans-Golgi network and as modulators of the processing of these components has been suggested (3Huttner W.B. Gerdes H.H. Rosa P. Trends Biochem. Sci. 1991; 16: 27-30Abstract Full Text PDF PubMed Scopus (415) Google Scholar). Extracellularly, different members of the chromogranin family are now considered as precursor proteins, which are actively processed into peptides within the secretory granules (see Refs. 1Simon J.P. Aunis D. Biochem. J. 1989; 262: 1-13Crossref PubMed Scopus (242) Google Scholar and 5Dillen L. Miserez B. Claeys M. Aunis D. De Potter W. Neurochem. Int. 1993; 22: 315-352Crossref PubMed Scopus (73) Google Scholar for reviews). Previously, we reported a detailed study of the intracellular and extracellular processing of CGA and CGB/SgI (6Metz-Boutigue M.H. Garcia-Sablone P. Hogue-Angeletti R. Aunis D. Eur. J. Biochem. 1993; 217: 247-257Crossref PubMed Scopus (184) Google Scholar, 7Strub J.M. Garcia-Sablone P. Lonning K. Taupenot L. Hubert P. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. Eur. J. Biochem. 1995; 229: 356-368Crossref PubMed Scopus (113) Google Scholar) and a preliminary analysis of the post-translational proteolysis of CGC/SgII (8Soszynski D. Metz-Boutigue M.H. Aunis D. Bader M.F. J. Neuroendocrinol. 1993; 5: 655-662Crossref PubMed Scopus (15) Google Scholar) in bovine chromaffin granules. The proteolytic processing of CGA is a topic of growing interest, as biological activities have been attributed to peptides located along the sequence of CGA. For example, in the N-terminal domain, a peptide corresponding to the sequence 1–113 has been shown to inhibit hormone secretion in the bovine parathyroid gland (9Drees B.M. Rouse J. Johnson J. Hamilton J.W. Endocrinology. 1991; 129: 3381-3387Crossref PubMed Scopus (98) Google Scholar); a homologous peptide, β-granin, corresponding to the sequence 1–115 has been isolated from rat pancreas, but its function has not yet been defined (10Hutton J.C. Davidson H.W. Peshavaria M. Biochem. J. 1987; 244: 457-464Crossref PubMed Scopus (94) Google Scholar). Vasostatins are peptides containing the N-terminal sequence (1–76/113) (11Helle K.B. Marley P.D. Hogue-Angeletti R. Galindo E. Aunis D. Small D.H. Livett B.G. J. Neuroendocrinol. 1993; 5: 413-420Crossref PubMed Scopus (55) Google Scholar) that exhibit vasoinhibitory activity of isolated human blood vessels (12Aardal S. Helle K.B. Regul. Pept. 1992; 41: 9-18Crossref PubMed Scopus (181) Google Scholar, 13Aardal S. Helle K.B. Elsayed S. Reed R.K. Serck-Hanssen G. J. Neuroendocrinol. 1993; 5: 405-412Crossref PubMed Scopus (173) Google Scholar). As early as 1988, it was established that CGA is the precursor of a peptide that inhibits the secretory activity on chromaffin cells (14Simon J.P. Bader M.F. Aunis D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1712-1716Crossref PubMed Scopus (186) Google Scholar), and recently, catestatin, a novel CGA fragment (344–364), was characterized as a noncompetitive nicotinic cholinergic antagonist (15Mahata S.K. O'Connor D.T. Mahata M. Yoo S.H. Taupenot L. Wu H. Gill B.M. Parmer R.J. J. Clin. Invest. 1997; 100: 1623-1633Crossref PubMed Scopus (334) Google Scholar). In addition, pancreastatin (248–293) is a peptide with multiple properties, since it negatively modulates insulin secretion from endocrine pancreatic islets (16Tatemoto K. Efendic S. Mutt V. Makk G. Feistner G.J. Barchas J.D. Nature. 1986; 324: 476-478Crossref PubMed Scopus (616) Google Scholar, 17Efendic S. Tatemoto K. Mutt V. Quan C. Chang D. Ostenson C.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 4: 7257-7260Crossref Scopus (154) Google Scholar), amylase release from exocrine pancreas (18Ishizuka J. Asada I. Poston G. Lluis F. Tatemoto K. Greeley G. Thompson J. Pancreas. 1989; 4: 277-281Crossref PubMed Scopus (41) Google Scholar), and acid secretion from parietal cells (19Lewis J.J. Goldenring J.R. Asher V.A. Modlin I.M. Biochem. Biophys. Res. Commun. 1989; 163: 667-673Crossref PubMed Scopus (31) Google Scholar). Parastatin (347–419) is another CGA-derived peptide located in the C-terminal domain of CGA that inhibits parathyroid cell secretion (20Fasciotto B.H. Trauss C.A. Greeley G.H. Cohn D.V. Endocrinology. 1993; 133: 461-466Crossref PubMed Scopus (0) Google Scholar). In addition to the autocrine or paracrine role in hormone secretion of these CGA-derived peptides, we have shown recently that numerous peptides present as water-soluble components of bovine chromaffin granules and released during secretion display antibacterial activity (7Strub J.M. Garcia-Sablone P. Lonning K. Taupenot L. Hubert P. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. Eur. J. Biochem. 1995; 229: 356-368Crossref PubMed Scopus (113) Google Scholar, 21Strub J.M. Hubert P. Nullans G. Aunis D. Metz-Boutigue M.H. FEBS Lett. 1996; 379: 273-278Crossref PubMed Scopus (34) Google Scholar, 22Strub J.M. Goumon Y. Lugardon K. Capon C. Lopez M. Moniatte M. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. J. Biol. Chem. 1996; 271: 28533-28540Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 23Goumon Y. Strub J.M. Moniatte M. Nullans G. Poteur L. Hubert P. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. Eur. J. Biochem. 1996; 235: 516-525Crossref PubMed Scopus (80) Google Scholar, 24Metz-Boutigue M.H. Strub J.M. Goumon Y. Lugardon K. Aunis D. Cell. Mol. Neurobiol. 1998; 18: 249-266Crossref PubMed Scopus (79) Google Scholar). Human CGA is a single polypeptide chain of 439 residues, with an apparent molecular mass of 70 kDa as estimated by SDS-polyacrylamide gel electrophoresis gel and a pI of 4.7–5.2. The amino acid sequence of human CGA (25Helman L. Ahn T. Levine M. Allison A. Cohen P. Cohn D. Israel M. J. Biol. Chem. 1988; 263: 11559-11563Abstract Full Text PDF PubMed Google Scholar, 26Konecki D. Benedum U. Gerdes H. Huttner W. J. Biol. Chem. 1987; 262: 17026-17030Abstract Full Text PDF PubMed Google Scholar) indicates a real molecular mass of 48 kDa for the unmodified form of this protein. The difference between the apparent (70 kDa) and theoretical molecular mass (48 kDa) probably results from post-translational modifications (i.e. glycosylation, phosphorylation) (27Kiang W.L. Krusius T. Finne J. Margolis R.U. Margolis R.K. J. Biol. Chem. 1982; 257: 1651-1659Abstract Full Text PDF PubMed Google Scholar, 28Settleman J. Fonseca R. Nolan J. Hogue-Angeletti R. J. Biol. Chem. 1985; 260: 1645-1651Abstract Full Text PDF PubMed Google Scholar) and the abundance of acidic residues (25%), which cause a slower migration during electrophoresis in the presence of sodium dodecyl sulfate (see Ref. 1Simon J.P. Aunis D. Biochem. J. 1989; 262: 1-13Crossref PubMed Scopus (242) Google Scholar, for review). In 1997, using mild proteolysis, peptide separation, microsequencing, and mass analysis techniques, seven post-translational modification sites were detected in bovine CGA (29Strub J.M. Sorokine O. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. J. Biol. Chem. 1997; 272: 11928-11936Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Two glycosylation sites, each consisting of the trisaccharide NeuAcα2–3Galβ1–3GalNAcα1-O-linked to Ser186 and Thr231. The former residue is present in the antibacterial peptide named chromacin (22Strub J.M. Goumon Y. Lugardon K. Capon C. Lopez M. Moniatte M. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. J. Biol. Chem. 1996; 271: 28533-28540Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Five phosphorylation sites were located on serine residues at positions Ser81, Ser307, Ser372, Ser376, and on Tyr173, this latter residue being the N-terminal amino acid of chromacin. Furthermore, studying the new antibacterial bovine CGA-derived peptides G- and PG-chromacin (CGA173–194), we demonstrated that the two post-translational modifications (Tyr173 and Ser186) are both necessary for the antibacterial activity of chromacin. Since 1989, it is well known that chromogranins can serve as immunocytochemical markers for neuroendocrine tissues and as a diagnostic tool for neuroendocrine tumors (3Huttner W.B. Gerdes H.H. Rosa P. Trends Biochem. Sci. 1991; 16: 27-30Abstract Full Text PDF PubMed Scopus (415) Google Scholar, 4Winkler H. Fischer-Colbrie R. Neuroscience. 1992; 49: 497-528Crossref PubMed Scopus (611) Google Scholar, 30Wiedenmann B. Huttner W.B. Virchows Archiv. B Cell Pathol. 1989; 58: 95-121Crossref Scopus (296) Google Scholar, 31Deftos L.J. Endocr. Rev. 1991; 12: 181-187Crossref PubMed Scopus (239) Google Scholar, 32Degorce F. Goumon Y. Jacquemart L. Vidaud C. Bellanger L. Pons-Anicet D. Seguin P. Metz-Boutigue M.H. Aunis D. Br. J. Cancer. 1998; (in press)Google Scholar). Because of their water-soluble properties, chromogranins and chromogranin-derived fragments are released together with catecholamines from adrenal chromaffin cells during stress situations and can be detected in the blood by radioimmunoassay techniques and enzyme assays (33O'Connor D.T. Deftos L.J. N. Engl. J. Med. 1986; 314: 1145-1151Crossref PubMed Scopus (419) Google Scholar, 34Eiden L.E. Iacangelo A. Hsu C.M. Hotchkiss A.J. Bader M.F. Aunis D. J. Neurochem. 1987; 49: 65-74Crossref PubMed Scopus (35) Google Scholar, 35Eriksson B. Arnberg H. Oberg K. Hellman U. Lundqvist G. Wernsted C. Wilander E. Acta Oncol. 1989; 28: 325-329Crossref PubMed Scopus (75) Google Scholar, 36O'Connor D.T. Takiyyuddin M.A. Cervenka J.H. Parmer R.J. Barbosa J.A. Chang Y.M. Hsiao R.J. Acta Histochem. 1990; 38: 27-33Google Scholar, 37Mouland A.J. Hendy G.N. Endocrinology. 1991; 128: 441-449Crossref PubMed Scopus (25) Google Scholar, 38Stridsberg M. Hellman U. Wilander E. Lundqvist G. Hellsing K. Oberg K. J. Endocrinol. 1993; 139: 329-337Crossref PubMed Scopus (70) Google Scholar). Previously, we have shown that large fragments of CGA are excreted into the urine in some patients with carcinoid tumors (38Stridsberg M. Hellman U. Wilander E. Lundqvist G. Hellsing K. Oberg K. J. Endocrinol. 1993; 139: 329-337Crossref PubMed Scopus (70) Google Scholar). The present paper deals with the determination of the phosphorylation and carbohydrate binding sites of a large natural C-terminal CGA-derived fragment, CGA79–439, present into the urine of these patients. The strategy consists in characterizing the primary structure of modified phosphorylated and O-glycosylated peptides, which were isolated after proteolytic cleavage of CGA79–439 with endoproteinase Lys-C. Then, using successively separation by reverse phase HPLC, enzymatic modification of phosphorylated peptides, and complete analysis by sequencing and mass spectrometry (liquid chromatography/mass spectrometry and matrix-assisted laser desorption ionization time-of-flight), a detailed study was carried out. These post-translational modifications were located along the polypeptidic chain, compared with sequences of others species and discussed in relation with biological activity of natural CGA-derived fragments. Urine was collected during a 24-h period from a patient with a histologically verified carcinoid tumor and multiple liver metastasis. The sample was collected, after informed consent, when the patient was on a clinical trial at the Endocrine Unit of Uppsala University Hospital. The study was also approved by the local Ethical Committee. The urine sample was first filtered through a 0.22-μm membrane and then concentrated about 100 times in a dialysis tube (Spectra/Por; cutoff value, 6–8 kDa). CGA-derived fragments were isolated in a one-step separation on an anion exchange column (Mono Q, FPLC; Amersham Pharmacia Biotech) using a linear gradient of 0.2 mammonium acetate buffer at pH 6.0 to 1.0 m ammonium acetate buffer at pH 6.0 containing 1.0 m sodium chloride. Fractions containing CGA-derived fragments were concentrated on a Minicon concentrator (Amicon) and stored in −70 °C before further analysis. CGA79–439 (2.5 nmol) was digested for 18 h at 37 °C with endoproteinase Lys-C at a protein to enzyme weight ratio of 1000:1 in 100 mm Tris-HCl, pH 8.3. Generated peptides were then separated by HPLC, using the SMART system (Amersham Pharmacia Biotech), on a Macherey Nagel 300–5C18 column (4 × 250 mm; particle size 5 μm and pore size 100 Å). Absorbance was monitored at 215 nm, and the solvent system consisted of 0.1% trifluoroacetic acid in water (solvent A) and 0.09% trifluoroacetic acid/acetonitrile (solvent B). Material was eluted at a flow rate of 550 μl/min using, successively, a gradient of 0–30% solvent B in solvent A over 46 min, followed by a gradient of 30–50% over 13 min, and achieved by a gradient 50–100% over 10 min. Each peak fraction was automatically collected by the SMART system and concentrated by evaporation, but not to dryness. CGA209/210–245 mixture (100 pmol) was digested for 18 h at 37 °C with endoproteinase Glu-C at a protein to enzyme weight ratio of 100:1 in 100 mm Tris-HCl, pH 8.3. Released fragments were then separated by HPLC, using the SMART system (Amersham Pharmacia Biotech) on a μRPC-C2/C18 (2.1 × 100 mm; particle size 3 μm and pore size 120 Å). Absorbance was monitored at 215 nm, and the solvent system consisted of 0.1% trifluoroacetic acid in water (solvent A) and 0.085% trifluoroacetic acid, 60% acetonitrile, 39.915% water (solvent B). Elution was performed at a flow rate of 200 μl/min using successively, a gradient 0–65% solvent B in 60 min, followed by a gradient 65–100% over 10 min. The sequence of purified CGA-derived peptides was determined in our laboratory by automatic Edman degradation on an Applied Biosystems 473 A microsequencer. Samples (10–20 pmol) were loaded onto polybrene-treated and precycled glass fiber filters (6Metz-Boutigue M.H. Garcia-Sablone P. Hogue-Angeletti R. Aunis D. Eur. J. Biochem. 1993; 217: 247-257Crossref PubMed Scopus (184) Google Scholar). Carbohydrate analysis was performed using gas chromatography with a silicone OV 101 capillary column (0.32 mm × 25 m). Samples were analyzed after methanolysis (0.5 mhydrochloric acid-methanol for 24 h at 80 °C),N-reacetylation, and trimethylsilylation (39Kamerling J.P. Gerwig G.J. Vliegenthart J.F.G. Clamp J.R. Biochem. J. 1975; 151: 491-495Crossref PubMed Scopus (315) Google Scholar, 40Montreuil J. Bouquelet S. Debray H. Fournet B. Spik G. Strecker G. Chaplin M.F. Kennedy J.F. Carbohydrate Analysis, A Practical Approach. IRL Press, Oxford1986: 143-204Google Scholar). Dot slots were performed using Bio-Dot SF microfiltration apparatus, and 1 nmol of glycoprotein was slot-blotted onto nitrocellulose membrane. Fetuin was used as control. Desialylation of one-half of the dots was performed by treatment with 50 milliunits/ml sialidase from Clostridium perfringens in 0.9% NaCl, 0.1% CaCl2, 50 mm citrate buffer, pH 6.0, for 16 h at 37 °C prior incubation with the digoxigenin-labeled lectins. The different wells were incubated with each lectin dissolved in Tris-buffered saline: Maackia amurensis agglutinin (MAA-dig, 5 μg/ml), Arachis hypogaea agglutinin (PNA-dig, 2 μg/ml), and Sambucus nigra agglutinin (SNA-dig, 2 μg/ml). Then, the nitrocellulose membrane was incubated for 1 h in Tris-buffered saline with anti-digoxigenin alkaline phosphatase-labeled Fab fragments (1 μg/ml). After washing, labeled glycoproteins were revealed by 4-nitro blue tetrazolium chloride 5-bromo-4-chloro-3-indolyl phosphate staining. To isolate and characterize glycopeptides, we have performed LC/MS analysis of CGA-derived peptides obtained after endoproteinase Lys-C digestion of CGA. Then, CGA (500 pmol) was digested for 2 h at 37 °C with endoproteinase Lys-C at a protein-to-proteinase weight ratio of 1000:1 in 100 mm Tris-HCl, pH 8.3. Then, peptides were separated with an HPLC system (Applied Biosystems 140 A Solvent Delivery System) equipped with a UV detector (UV Waters Detector 386) on a Narrowbore Macherey Nagel Nucleosil 300–5C18 column (2 × 150 mm). Absorbance was monitored at 214 nm, and the solvent system consisted of 0.1% trifluoroacetic acid/water (solvent A) and 0.1% trifluoroacetic acid/acetonitrile (solvent B). Material was eluted at a flow rate of 250 μl/min using a gradient of 0–80% solvent B in solvent A over 80 min. A major part of the eluent (90%) was analyzed by UV detection and an aliquot (10%) was measured by LC-MS. The mass spectrometer was calibrated under conditions using a mixture of polyethylene glycols (average masses 400 and 2000 Da). Spectra were scanned overm/z 320–1800 for 6 s, and the total ion current was recorded. The mass spectrometry analysis was carried out on a Brucker BIFLEXTMmatrix-assisted laser time-of-flight mass spectrometer equipped with the ScoutTM high resolution optics with X-Y multisample probe, a gridless reflector, and the HIMASTM linear detector. This instrument has a maximum accelerating potential of 30 kV and may be operated either in the linear or reflector mode. Ionization was accomplished with a 337-nm beam from a nitrogen laser with a repetition rate of 3 Hz. The output signal from the detector was digitized at a sampling rate of 250 MHz in linear mode and 500 MHz in reflector mode using a 1-GHz digital oscilloscope (Lecroy model). The instrument control and data processing were accomplished with software supplied by Brucker using a Sun Sparc workstation. These studies were realized according to the procedure previously described (22Strub J.M. Goumon Y. Lugardon K. Capon C. Lopez M. Moniatte M. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. J. Biol. Chem. 1996; 271: 28533-28540Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Sequence alignment of bovine CGA sequences with corresponding fragments of CGA from different species was performed using the Cameleon sequence alignment program using default parameters (41.Cameleon Sequence Analysis, Program V3.1, Oxford Molecular Ltd., Oxford, UK.Google Scholar). Chromogranin sequences were retrieved from the Swiss-Prot data base. In this study, we have isolated a major fragment of human CGA corresponding predominantly to the domain CGA116–439 and a minor larger CGA-derived fragment CGA79–439, which are both excreted in urine of patients with carcinoid tumors (38Stridsberg M. Hellman U. Wilander E. Lundqvist G. Hellsing K. Oberg K. J. Endocrinol. 1993; 139: 329-337Crossref PubMed Scopus (70) Google Scholar). To determine phosphorylation and O-glycosylation sites included within tumoral CGA, the large C-terminal fragment was digested by endoproteinase Lys-C and the generated fragments were separated by HPLC on a reverse phase C18 column (Fig.1 A). The different peaks of the chromatogram were directly submitted to automatic Edman degradation and mass spectra analysis to detect post-translational modifications. It is important to note that all the sequences determined in this study are in accordance with the primary structure proposed by Koneckiet al. (26Konecki D. Benedum U. Gerdes H. Huttner W. J. Biol. Chem. 1987; 262: 17026-17030Abstract Full Text PDF PubMed Google Scholar); in contrast, they diverge on 15 points (Fig.2) from the primary structure reported by Helman et al. (25Helman L. Ahn T. Levine M. Allison A. Cohen P. Cohn D. Israel M. J. Biol. Chem. 1988; 263: 11559-11563Abstract Full Text PDF PubMed Google Scholar).Figure 2Amino acid sequence for human chromogranin A (CGA). Sequence corresponding to the large CGA-derived fragment 79–439 is underlined. The sequence shown is that of Koneckiet al. (26Konecki D. Benedum U. Gerdes H. Huttner W. J. Biol. Chem. 1987; 262: 17026-17030Abstract Full Text PDF PubMed Google Scholar) as confirmed by our sequence data.Arrows point on previously determined cleavage sites (38Stridsberg M. Hellman U. Wilander E. Lundqvist G. Hellsing K. Oberg K. J. Endocrinol. 1993; 139: 329-337Crossref PubMed Scopus (70) Google Scholar).View Large Image Figure ViewerDownload (PPT) Carbohydrate analysis was performed using gas chromatography (22Strub J.M. Goumon Y. Lugardon K. Capon C. Lopez M. Moniatte M. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. J. Biol. Chem. 1996; 271: 28533-28540Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), and the carbohydrate content was evaluated to 5% (m/m, carbohydrate/protein): NeuAc, Gal, and GalNAc were detected in a molar ratio 1.2: 1.7: 1, suggesting short glycans with different structures. Fragments resulting from endoproteinase Lys-C digestion of CGA79–439 were analyzed by LC-MS to detect areas containing O-glycosylated peptides (Fig. 1 B). The HPLC chromatogram (a), the single ion recording of specific ions characteristic of glycosylation sites (b), and the total ionic current of the chromatogram (c) are indicated. In b, the presence of glycans was recovered in peaks of regions I–IV (lineated in Fig. 1 A), and each area was analyzed to characterize the O-glycosylated sites. Area I included peaks corresponding to free saccharides, since areas II–IV contained glycopeptides. Sequencing of material included in the two major peaks (30Wiedenmann B. Huttner W.B. Virchows Archiv. B Cell Pathol. 1989; 58: 95-121Crossref Scopus (296) Google Scholar, 31Deftos L.J. Endocr. Rev. 1991; 12: 181-187Crossref PubMed Scopus (239) Google Scholar) eluting in region II (Fig. 1 A) indicates the presence of peptides with N-terminal end located in position 145 and 124, respectively (Table I). MALDI-TOF MS analysis (negative mode) of peptide included in peak 31 reveals an experimental molecular mass of 2216 Da corresponding to the oxidized form of 124–144 (oxidation of Met140; calculated molecular mass 2200 Da). MALDI-TOF MS analysis of material included in peak 30 (Fig. 3 A) shows four different major molecular species with respective molecular masses of 3682, 3974, 4048, and 4264 Da (Table I), indicating the presence of several different glycans. By comparison with the expected molecular mass of CGA145–175 (3321 Da), the two molecular masses of 3682 and 3974 Da might be attributed to the O-glycosylated peptide CGA145–175 with the disaccharide Galβ1–3GalNAcα1, corresponding to the antigens T described previously as characteristic of human adenocarcinoids (42Springer G.F. Science. 1984; 224: 1198-1206Crossref PubMed Scopus (937) Google Scholar), and the trisaccharide NeuAcα2–3Galβ1–3GalNAcα1 reported previously for bovine CGA (22Strub J.M. Goumon Y. Lugardon K. Capon C. Lopez M. Moniatte M. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. J. Biol. Chem. 1996; 271: 28533-28540Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 29Strub J.M. Sorokine O. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. J. Biol. Chem. 1997; 272: 11928-11936Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), respectively (Fig. 3 A). In addition, the two other molecular masses of 4048 and 4264 Da are likely to correspond, respectively, to the O-glycosylated peptide CGA145–175 with two disaccharides Galβ1–3GalNAcα1 and the tetrasaccharide with an additional NeuAc linked in 2–6 on GalNAc (Fig. 3 A) as reported previously for fetuin (43Spiro R.G. Bhoyroo V.D. J. Biol. Chem. 1974; 249: 5704-5717Abstract Full Text PDF PubMed Google Scholar). To obtain confirmation of the structure of these glycans, material included in peak 30 was slot-blotted onto nitrocellulose sheet and immunodetected with a panel of three lectins (MAA, SNA, and PNA), the specificity of which was reported previously (22Strub J.M. Goumon Y. Lugardon K. Capon C. Lopez M. Moniatte M. Van Dorsselaer A. Aunis D. Metz-Boutigue M.H. J. Biol. Chem. 1996; 271: 28533-28540Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Experimental data have shown unambiguously the simultaneous presence of the following linkages: Galβ1–3GalNAcα1-O (PNA), NeuAcα2–3Galβ (MAA), and NeuAcα2–6GalNAc. Thus, our data revealed the presence of fourO-glycosylated moities on the peptide CGA145–175, including complete forms with a tri- or a tetrasaccharide and two truncated glycans corresponding to disaccharides (Fig. 3 A).Table IStructural characterization of human CGA-derived glyco- and phosphopeptides, generated after endoproteinase Lys-C digestion of human excreted CGA79–439Peak numberN-terminal sequenceExperimental molecular massLocationCalculated molecular massDaDa30AEGNNQAPGEEEEEEEEATNT3682145–17533213974145–1754048145–1754264145–17531SGEATDGARPQALPEPMQESK2216124–144220037EIRKGESRSEALAVDGAGKPGA3317246–2773240SGELEQEEERLSK1611304–316153450EEEEEEEEEAEAGEEAVPEEEGP4345210–24539834636210–2454928210–24563GLSAEPGWQAKREEEEEEEEEA5497198–24552646019198–2456212198–245Each fragment was sequenced, submitted to MALDI-TOF MS analysis (see "Experimental Procedures"), and located by comparison with the full CGA sequence (26Konecki D. Benedum U. Gerdes H. Huttner W. J. Biol. Chem. 1987; 262: 17026-17030Abstract Full Text PDF PubMed Google Scholar). Open table in a new tab Each fragment was sequenced, submitted to MALDI-TOF MS analysis (see "Experimental Procedures"), and located by comparison with the full CGA sequence (26Konecki D. Benedum U. Gerdes H. Huttner W. J. Biol. Chem. 1987; 262: 17026-17030Abstract Full Text PDF PubMed Google Scholar). Primary structure of CGA145–175 (Fig. 3 A) includes four potential O-glycosylated residues corresponding to Thr163, Thr165, Ser170, and Ser173. The sequence in the vicinity of the serine residues Ser170 and Ser173 (PPAS170LPS173QKYPGP) fits with the sequence patterns described by Wilson forO-glycosylation sites and characterized by high proline, serine, and threonine content (44Wilson I.B.H. Gavel Y. Von Heijne G. Biochem. J. 1991; 275: 529-534Crossref PubMed Scopus (241) Google Scholar). In addition, the presence of clusters of several closely spaced glycosyla
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