Identification of Disulfide Bonds among the Nine Core 2 N-Acetylglucosaminyltransferase-M Cysteines Conserved in the Mucin β6-N-Acetylglucosaminyltransferase Family
2004; Elsevier BV; Volume: 279; Issue: 37 Linguagem: Inglês
10.1074/jbc.m401046200
ISSN1083-351X
AutoresJaswant Singh, Gausal A. Khan, Leo Kinarsky, Helen Cheng, Jason A. Wilken, Kyung H. Choi, Elliott Bedows, Simon Sherman, Pi‐Wan Cheng,
Tópico(s)Galectins and Cancer Biology
ResumoBovine core 2 β1,6-N-acetylglucosaminyltransferase-M (bC2GnT-M) catalyzes the formation of all mucin β1,6-N-acetylglucosaminides, including core 2, core 4, and blood group I structures. These structures expand the complexity of mucin carbohydrate structure and thus the functional potential of mucins. The four known mucin β1,6-N-acetylglucosaminyltransferases contain nine conserved cysteines. We determined the disulfide bond assignments of these cysteines in [35S]cysteine-labeled bC2GnT-M isolated from the serum-free conditioned medium of Chinese hamster ovary cells stably transfected with a pSecTag plasmid. This plasmid contains bC2GnT-M cDNA devoid of the 5′-sequence coding the cytoplasmic tail and transmembrane domain. The C18 reversed phase high performance liquid chromatographic profile of the tryptic peptides of reduced-alkylated 35S-labeled C2GnT-M was established using microsequencing. Each cystine pair was identified by rechromatography of the C8 high performance liquid chromatographic radiolabeled tryptic peptides of alkylated bC2GnT-M on C18 column. Among the conserved cysteines in bC2GnT-M, the second (Cys113) was a free thiol, whereas the other eight cysteines formed four disulfide bridges, which included the first (Cys73) and sixth (Cys230), third (Cys164) and seventh (Cys384), fourth (Cys185) and fifth (Cys212), and eighth (Cys393) and ninth (Cys425) cysteine residues. This pattern of disulfide bond formation differs from that of mouse C2GnT-L, which may contribute to the difference in substrate specificity between these two enzymes. Molecular modeling using disulfide bond assignments and the fold recognition/threading method to search the Protein Data Bank found a match with aspartate aminotransferase structure. This structure is different from the two major protein folds proposed for glycosyltransferases. Bovine core 2 β1,6-N-acetylglucosaminyltransferase-M (bC2GnT-M) catalyzes the formation of all mucin β1,6-N-acetylglucosaminides, including core 2, core 4, and blood group I structures. These structures expand the complexity of mucin carbohydrate structure and thus the functional potential of mucins. The four known mucin β1,6-N-acetylglucosaminyltransferases contain nine conserved cysteines. We determined the disulfide bond assignments of these cysteines in [35S]cysteine-labeled bC2GnT-M isolated from the serum-free conditioned medium of Chinese hamster ovary cells stably transfected with a pSecTag plasmid. This plasmid contains bC2GnT-M cDNA devoid of the 5′-sequence coding the cytoplasmic tail and transmembrane domain. The C18 reversed phase high performance liquid chromatographic profile of the tryptic peptides of reduced-alkylated 35S-labeled C2GnT-M was established using microsequencing. Each cystine pair was identified by rechromatography of the C8 high performance liquid chromatographic radiolabeled tryptic peptides of alkylated bC2GnT-M on C18 column. Among the conserved cysteines in bC2GnT-M, the second (Cys113) was a free thiol, whereas the other eight cysteines formed four disulfide bridges, which included the first (Cys73) and sixth (Cys230), third (Cys164) and seventh (Cys384), fourth (Cys185) and fifth (Cys212), and eighth (Cys393) and ninth (Cys425) cysteine residues. This pattern of disulfide bond formation differs from that of mouse C2GnT-L, which may contribute to the difference in substrate specificity between these two enzymes. Molecular modeling using disulfide bond assignments and the fold recognition/threading method to search the Protein Data Bank found a match with aspartate aminotransferase structure. This structure is different from the two major protein folds proposed for glycosyltransferases. There are two types of mucins (1Moniaux P. Escande F. Porchet N. Aubert J.-P. Batra S.K. Front. Biosci. 2001; 6: 1192-1206Crossref PubMed Google Scholar, 2Strous G.J. Dekker J. Crit. Rev. Biochem. Mol. Biol. 1992; 27: 57-92Crossref PubMed Scopus (773) Google Scholar): secreted and membrane-bound. MUC2, MUC5AC, and MUC5B are representatives of secreted mucins, whereas MUC1, MUC4, leukosialin, and P-selectin glycoprotein ligand-1 are examples of membrane-bound mucins (3Fukuda M. Biochim. Biophys. Acta. 2002; 1573: 394-405Crossref PubMed Scopus (115) Google Scholar, 4Tsuboi S. Fukuda M. BioEssays. 2001; 23: 46-53Crossref PubMed Google Scholar). Secreted mucins are produced by epithelial mucus cells and play important roles in the rheological and bacteria-binding properties of the mucus covering the epithelial tissues (5Lamblin G. Lhermitte Klein A. Houdret N. Scharfman A. Ramphal R. Roussel P. Am. Rev. Respir. Dis. 1991; 144: S19-S24Crossref PubMed Google Scholar, 6Ramphal R. Carnoy C. Fierre S. Michelski J. Houdret N. Lamblin G. Strecker G. Roussel P. Infect. Immun. 1991; 59: 700-704Crossref PubMed Google Scholar). Membrane-bound mucins are found at the cell surface throughout the body (3Fukuda M. Biochim. Biophys. Acta. 2002; 1573: 394-405Crossref PubMed Scopus (115) Google Scholar, 4Tsuboi S. Fukuda M. BioEssays. 2001; 23: 46-53Crossref PubMed Google Scholar). They can modulate immune functions, such as maturation of B cells and trafficking of leukocytes during inflammatory response (3Fukuda M. Biochim. Biophys. Acta. 2002; 1573: 394-405Crossref PubMed Scopus (115) Google Scholar, 4Tsuboi S. Fukuda M. BioEssays. 2001; 23: 46-53Crossref PubMed Google Scholar). The biological properties of both secreted and membrane-bound mucins are attributed to the structurally heterogeneous carbohydrates covalently bound to the peptide backbones. The highly heterogeneous mucin-type carbohydrate is characterized by the α-anomeric linkage between N-acetylgalactosamine and serine/threonine in the peptide backbone. Following the formation of this linkage, mucin carbohydrate is assembled by the sequential addition of one sugar at a time as catalyzed by various glycosyltransferases (7Van den Steen P. Rudd P.M. Dwek R.A. Opdenakker G. Crit. Rev. Biochem. Mol. Biol. 1998; 33: 151-208Crossref PubMed Scopus (624) Google Scholar, 8Brockhausen I. Kuhns W. Glycoproteins and Human Diseases. R. G. Landes Co., Austin, TX1997: 5-48Google Scholar). This template-independent synthetic process is responsible for the heterogeneity of mucin carbohydrate. The branching enzymes, which catalyze the synthesis of β1,6-N-acetylglucosaminides, are unique among the glycosyltransferases involved in the synthesis of mucin glycans (9Beum P.V. Cheng P.-W. Adv. Exp. Med. Biol. 2001; 491: 279-312Crossref PubMed Scopus (18) Google Scholar). The mucin β1,6-N-acetylglucosaminides formed by these enzymes constitute the initiation sites from which additional carbohydrate can be added, thus extending the complexity of mucin carbohydrate structure and increasing the functional potential of mucins. The mucin branching enzymes that have been reported to date include C2GnT-1(or L) (10Bierhuizen M.F. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Crossref PubMed Scopus (279) Google Scholar, 11Li C.-M. Adler K.B. Cheng P.-W. Am. J. Respir. Cell Mol. Biol. 1998; 18: 343-352Crossref PubMed Scopus (15) Google Scholar, 12Yen T.-Y. Macher B.A. Bryson S. Chang X. Tvaroska I. Tse R. Takeshita S. Lew A.M. Datti A. J. Biol. Chem. 2003; 278: 45864-45881Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar), C2GnT-2(or M) (13Ropp P.A. Little M.R. Cheng P.-W. J. Biol. Chem. 1991; 266: 23863-23871Abstract Full Text PDF PubMed Google Scholar, 14Yeh J.-C. Ong E. Fukuda M. J. Biol. Chem. 1999; 274: 3215-3221Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 15Schwientek T. Nomoto M. Levery S.B. Merkx G. van Kessel A.G. Bennett E.P. Hollingworth M.A. Clausen H. J. Biol. Chem. 1999; 274: 4504-4512Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 16Vanderplasschen A. Markine-Goriynoff N. Lomonte P. Suzuki M. Hiraoka N. Yeh J.C. Bureau F. Willems L. Thiry E. Fukuda M. Pastoret P.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5756-5761Crossref PubMed Scopus (47) Google Scholar, 17Choi K.H. Osorio F.A. Cheng P.-W. Am. J. Respir. Cell Mol. Biol. 2004; 30: 710-719Crossref PubMed Scopus (10) Google Scholar), C2GnT-3 (18Schwientek T. Yeh J.-C. Levery S.B. Keck B. Merkx G. van Kessel A.G. Fukuda M. Clausen H. J. Biol. Chem. 2000; 275: 11106-11113Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), and IGnT (19Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar). The core 2 structure, Galβ1-3(GlcNAcβ1-6)GalNAcα-Ser/Thr, can be synthesized by C2GnT-L, C2GnT-M, and C2GnT-3 (9Beum P.V. Cheng P.-W. Adv. Exp. Med. Biol. 2001; 491: 279-312Crossref PubMed Scopus (18) Google Scholar). Core 4 structure, GlcNAcβ1-3(GlcNAcβ1-6)GalNAcα-Ser/Thr, can be formed by C2GnT-M, whereas blood group I structure (9Beum P.V. Cheng P.-W. Adv. Exp. Med. Biol. 2001; 491: 279-312Crossref PubMed Scopus (18) Google Scholar), GlcNAc β1-3(GlcNAcβ1-6)Galβ-R, can be generated by C2GnT-M and IGnT (9Beum P.V. Cheng P.-W. Adv. Exp. Med. Biol. 2001; 491: 279-312Crossref PubMed Scopus (18) Google Scholar, 19Bierhuizen M.F.A. Mattei M.-G. Fukuda M. Genes Dev. 1993; 7: 468-478Crossref PubMed Scopus (138) Google Scholar). The reactions catalyzed by the enzyme activities exhibited by these glycosyltransferases are shown in Scheme 1. These β1,6GlcNAc transferase (β6GnT) 1The abbreviations used are: β6GnT, β1,6GlcNAc transferase; HPLC, high performance liquid chromatography; RP, reversed phase; CHO, Chinese hamster ovary; aa, amino acid(s); GT, glycosyltransferase. isozymes differ by their nucleotide and amino acid sequences, tissue distribution, and the carbohydrate structures they are able to form (9Beum P.V. Cheng P.-W. Adv. Exp. Med. Biol. 2001; 491: 279-312Crossref PubMed Scopus (18) Google Scholar). Despite these differences, all β6GnTs contain nine conserved cysteines (9Beum P.V. Cheng P.-W. Adv. Exp. Med. Biol. 2001; 491: 279-312Crossref PubMed Scopus (18) Google Scholar, 12Yen T.-Y. Macher B.A. Bryson S. Chang X. Tvaroska I. Tse R. Takeshita S. Lew A.M. Datti A. J. Biol. Chem. 2003; 278: 45864-45881Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). In an effort to elucidate the structural determinants that distinguish the difference in substrate specificity among members of this gene family, we characterized the disulfide linkages formed among these nine conserved cysteines in bC2GnT-M. To facilitate the effort, we generated a secreted form of the recombinant bC2GnT-M by removing the N-terminal region that contains the cytoplasmic tail and transmembrane domain and cloned the cDNA into pSecTag2B, which contains Ig κ-chain leader sequence at the N terminus and Myc epitope and polyhistidine tag at the C-terminal end. By microsequencing of the [35S]cysteine-containing tryptic peptides separated by reversed phase high performance liquid chromatography (RP-HPLC), we identified four cystine pairs between first and sixth, third and seventh, fourth and fifth, and eighth and ninth cysteine residues. The second cysteine was not conjugated. This pattern of disulfide bond distribution is different from that of mouse C2GnT-L recently reported (12Yen T.-Y. Macher B.A. Bryson S. Chang X. Tvaroska I. Tse R. Takeshita S. Lew A.M. Datti A. J. Biol. Chem. 2003; 278: 45864-45881Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The results indicate that the conservation of nine cysteines does not lead to the formation of same disulfide bonds between different isozymes, suggesting that other factors such as secondary structures may play a crucial role in determining the formation of disulfide bonds and substrate specificity. Molecular modeling using distribution of disulfide bonds and fold recognition/threading method to search the Protein Data Bank showed a match with the crystal structure of aspartate aminotransferase (20Malashkevich V.N. Strokopytov B.V. Borisov V.V. Dauter Z. Wilson K.S. Torchinsky Y.M. J. Mol. Biol. 1995; 247: 111-124Crossref PubMed Scopus (83) Google Scholar, 21Malashkevich V.N. Jager J. Ziak M. Sauder U. Gehring H. Christen P. Jansonius J.N. Biochemistry. 1995; 34: 405-414Crossref PubMed Scopus (26) Google Scholar). This template permits proper spatial arrangement of the cysteines involved in the formation of the four cystine pairs determined for bC2GnT-M. The structure is different from either the glycosyltransferase B-fold structure proposed for mouse C2GnT-L (12Yen T.-Y. Macher B.A. Bryson S. Chang X. Tvaroska I. Tse R. Takeshita S. Lew A.M. Datti A. J. Biol. Chem. 2003; 278: 45864-45881Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 22Coutinho P.M. Deleury E. Davies G.J. Henrissat B. J. Mol. Biol. 2003; 328: 307-317Crossref PubMed Scopus (931) Google Scholar) or glycosyltransferase A-fold, the major protein fold proposed for glycosyltransferases (22Coutinho P.M. Deleury E. Davies G.J. Henrissat B. J. Mol. Biol. 2003; 328: 307-317Crossref PubMed Scopus (931) Google Scholar, 23Franco O.L. Rigden D.J. Glycobiology. 2003; 13: 707-712Crossref PubMed Scopus (29) Google Scholar). Materials—Iodoacetamide and diphenylcarbamyl chloride-treated trypsin (T-10005) were purchased from Sigma. Dithiothreitol, ninhydrin, phenyl isothiocyanate, anhydrous trifluoroacetic acid, benzene, n-butyl acetate, ethyl acetate, and pyridine used for Edman degradation were obtained from Pierce. Methanol and acetonitrile for high pressure liquid chromatography was obtained from Water Associates (Milford, MA). [35S]Cysteine and [35S]methionine with specific activity of 1,075 and 540 Ci/mmol, respectively, were purchased from ICN (Costa Mesa, CA). The enzymatic deglycosylation kit was purchased from Prozyme, Inc. (San Leandro, CA). Cys-Arg was provided by Dr. Sam Sanderson at the University of Nebraska Medical Center (Omaha, NE). Cell Culture—Wild-type Chinese hamster ovary (CHO) cells were grown in Ham's F-12 medium plus 10% fetal bovine serum at 37 °C under 5% CO2 and a water-saturated environment. The CHO cells, stably transfected with pSecTag2B (Invitrogen) containing bC2GnT-M catalytic domain, were cultured in CHO III A medium (Invitrogen) supplemented with 10 μm hypoxanthine, 1.6 μm thymidine, 2 mm l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 1% fetal bovine serum (Medium A). To prepare the recombinant C2GnT-M secreted into the conditioned medium, the recombinant CHO cells were switched to serum-free CHO III A medium containing the supplements. Cloning of pSecTag bC2GnT-M and Generation of Stable Clones in CHO Cells—The bC2GnT-M cDNA coding the catalytic domain devoid of 47 amino acids at the N terminus containing the cytoplasmic tail and the transmembrane domain of the open reading frame was cloned from pCDNA6 containing the full-length bC2GnT-M cDNA (17Choi K.H. Osorio F.A. Cheng P.-W. Am. J. Respir. Cell Mol. Biol. 2004; 30: 710-719Crossref PubMed Scopus (10) Google Scholar) by PCR using 5′ and 3′ primers containing EcoRI and KpnI restriction sites, respectively. The PCR product was cloned into a Pichia vector (pPIC6αC) (Invitrogen) first and then transferred via EcoRI and NotI sites to pSecTag 2B vector, which contains Ig κ-chain at the N terminus and a Myc and a His6 tag at the C terminus. After confirmation by sequencing and then enzyme activity assay of the recombinant protein following a transient transfection of CHO cells by published methods (24Cheng P.-W. Hum. Gene Ther. 1996; 7: 175-282Crossref Scopus (170) Google Scholar), the pSecTag2B-bC2GnT-M was used to generate stable clones in CHO cells as described next. After they were cultured in Ham's F-12 medium plus 10% fetal bovine serum to 70% confluence, the CHO cells were transfected under serum-free conditions with pSecTag2B-bC2GnT-M delivered with Lipofectin supplemented with insulin as previously described (24Cheng P.-W. Hum. Gene Ther. 1996; 7: 175-282Crossref Scopus (170) Google Scholar). Two days later, cells were split 1:4 and cultured in Ham's F-12 medium plus 10% fetal bovine serum and 300 μg/ml Zeocin (Invitrogen). After 10 days, 24 clones were picked and characterized for C2GnT activity. The clone that expressed highest C2GnT activity after four passages was used for the current study. Assay of C2GnT-L, C4GnT-M, and IGnT Activities of Recombinant bC2GnT-M—The recombinant bC2GnT-M generated by the CHO cells stably transfected with pSecTag bC2GnT-M was assayed for C2GnT-L, C4GnT-M, and IGnT activities in the cells and conditioned medium as previously described (17Choi K.H. Osorio F.A. Cheng P.-W. Am. J. Respir. Cell Mol. Biol. 2004; 30: 710-719Crossref PubMed Scopus (10) Google Scholar). The conditioned medium was first concentrated 10-fold at 4 °C by centricon filtration with a 30-kDa molecular weight cut-off membrane (Millipore Corp.). Metabolic Labeling of bC2GnT-M—The [35S]cysteine (or [35S]methionine)-labeled bC2GnT-M was prepared from CHO cells stably transfected with pSecTag2B-bC2GnT-M as follows. The CHO cells that had grown in T-75 flasks to 90% confluence in Medium A (see “Cell Culture”) were switched to 12 ml of serum-free Medium A containing 2 mm sodium butyrate and cultured for 6-7 h. Then the cells were exposed for 1 h to Dulbecco's modified Eagle's medium (catalog no. 21013-024; Invitrogen) supplemented with 2 mm l-glutamine, 1 mm sodium pyruvate, 15 μg/ml methionine (for preparing [35S]cysteine-labeled bC2GnT-M) (25Bedows E. Huth J.R. Ruddon R.W. J. Biol. Chem. 1992; 267: 8880-8886Abstract Full Text PDF PubMed Google Scholar) or 24 μg/ml L-cysteine-HCl (for preparing [35S]methionine-labeled bC2GnT-M), and 2 mm butyrate. Following the addition of 63 μl of [35S]cysteine-HCl (11 mCi/ml at 1,075 Ci/mmol) or 22 μl of [35S]methionine (10 mCi/ml at 540 Ci/mmol) (ICN) to each T-75 flask and incubated for 1-2 h, the medium was replaced with serum-free Medium A containing 2 mm butyrate. After the cells were cultured for 24-48 h, the conditioned medium was harvested, centrifuged at 1,000 × g for 5 min to remove cell debris, and used for purification. Purification of 35S-labeled bC2GnT-M—The 35S-labeled bC2GnT-M was purified from the combined supernatant in a two-step process. First, the medium was concentrated at 4 °C from 180 to 10 ml using an Amicon YM 30 centricon (Amicon Bioseparations Centriprep; Millipore) in a centrifuge (Jouan model MR 22i) at 1,500 × g for 30 min. Two ml of nickel-nitrilotriacetic acid metal affinity resin (Qiagen), which has a 5-10-mg protein-binding capacity/ml of resin, was added to the concentrated medium. After a gentle shaking at 4 °C overnight, the resin was packed in a column. Following successive rinsing of the packed resin with the supernatant twice, 10 ml of 10 mm imidazole (pH 8.0), 10 ml of 20 mm imidazole (pH 8.0), and 10 ml of 20 mm imidazole (pH 6.2), the protein was eluted with 300 mm imidazole buffer (pH 8.0) (26Le Grice S.F.J. Ruddon R.W. Eur. J. Biochem. 1992; 187: 307-314Crossref Scopus (300) Google Scholar) and collected in 1.5 ml/fraction. Polyacrylamide Gel Electrophoresis and Western Blot Analysis—The purity of the recombinant bC2GnT-M purified by a nickel-nitrilotriacetic acid column was analyzed by SDS-10% PAGE under reduced conditions followed by Coomassie Blue stain or Western blotting using anti-Myc antibody (1:500) (Invitrogen). The anti-Myc antibody-treated membrane was further treated with horseradish peroxidase-conjugated secondary antibody (1:1000) (Invitrogen) and developed with ECL (Amersham Biosciences). Alkylation and Reduction-Alkylation of Recombinant bC2GnT-M—To prepare bC2GnT-M with free cysteines alkylated, 20-25 μg of recombinant protein in 1.5 ml of elution buffer in a silanized tube was treated with 10 mm iodoacetamide in the dark at 37 °C for 30 min (27Huth J.R. Mountjoy K. Perini F. Ruddon R.W. J. Biol. Chem. 1992; 267: 8870-8879Abstract Full Text PDF PubMed Google Scholar). To prepare bC2GnT-M with all cysteines alkylated, the same amount of the recombinant bC2GnT-M was treated first with 15 mm dithiothreitol under argon gas at 37 °C for 2 h and then 10 mm iodoacetamide for 30 min. Trypsin Digestion of bC2GnT-M—Both alkylated and reduced-alkylated bC2GnT-M (90 μg in 1.5 ml of elution buffer adjusted to pH 8.0 with 1 m Tris-HCl buffer in silanized polypropylene tubes) were digested for 16 h with 50 μg of diphenylcarbamyl chloride-treated trypsin in 50-150 mm Tris-HCl (pH 8) containing 5 mm CaCl2 (25Bedows E. Huth J.R. Ruddon R.W. J. Biol. Chem. 1992; 267: 8880-8886Abstract Full Text PDF PubMed Google Scholar, 27Huth J.R. Mountjoy K. Perini F. Ruddon R.W. J. Biol. Chem. 1992; 267: 8870-8879Abstract Full Text PDF PubMed Google Scholar). Digestions continued for 4 h after the addition of another 50 μg of trypsin (67 μg/ml final concentration), which was maintained at pH 8.0 with 1 m Tris-HCl, pH 8.0. Samples were then centrifuged (1,500 × g), and the supernatant was kept at 4 °C prior to HPLC separation of the tryptic peptides. Tryptic Mapping Strategy—The tryptic mapping strategy consisted of three steps (25Bedows E. Huth J.R. Ruddon R.W. J. Biol. Chem. 1992; 267: 8880-8886Abstract Full Text PDF PubMed Google Scholar, 26Le Grice S.F.J. Ruddon R.W. Eur. J. Biochem. 1992; 187: 307-314Crossref Scopus (300) Google Scholar, 27Huth J.R. Mountjoy K. Perini F. Ruddon R.W. J. Biol. Chem. 1992; 267: 8870-8879Abstract Full Text PDF PubMed Google Scholar). First, the [35S]cysteine-containing bC2GnT-M was fully reduced and alkylated and then digested with trypsin. The tryptic peptides were separated by C18 RP-HPLC. Those HPLC fractions containing cysteine were identified by virtue of their radiolabel and pooled, and the attendant peptides were identified by Edman degradation. In the second step, the [35S]cysteine-labeled bC2GnT-M was digested with trypsin without prior reduction and alkylation. The tryptic digests were subjected to chromatography on C8 RP-HPLC. In the third step, each [35S]cysteine-containing peak from C8 chromatographic profile was reduced, alkylated, and rechromatographed by C18 RP-HPLC. The cysteines involved in cystine pairing were identified by comparing the profile with that obtained in step one. In this study, [35S]methionine labeling was also employed to identify the peptides that contain both cysteine and methionine. RP-HPLC Separation of Tryptic Peptides from Reduced-Alkylated bC2GnT-M Labeled with [35S]Cysteine or [35S]Methionine—C18 (0.46 × 25 cm) (Vydac; 300 Å, 5 μm) column was used for establishing the profile of fully reduced and alkylated tryptic peptides first. It was then used for identification of the cysteines involved in cystine pairing. Tryptic peptides prepared from reduced-alkylated bC2GnT-M labeled with [35S]cysteine or [35S]methionine were injected onto a C18 column equilibrated with 0.1% trifluoroacetic acid (buffer A) at 42 °C. The column was eluted isocratically at 1 ml/min for 3 min with buffer A followed by an acetonitrile gradient at 0.32%/min for 100 min, 4.2%/min for 15 min and then re-equilibrated with buffer A. One-minute fractions were collected in silanized polypropylene tubes containing 4.5 μg of myoglobin/tube as carrier (25Bedows E. Huth J.R. Ruddon R.W. J. Biol. Chem. 1992; 267: 8880-8886Abstract Full Text PDF PubMed Google Scholar, 27Huth J.R. Mountjoy K. Perini F. Ruddon R.W. J. Biol. Chem. 1992; 267: 8870-8879Abstract Full Text PDF PubMed Google Scholar). The fractions were monitored by liquid scintillation counting. The fractions containing 35S label were concentrated by Speed-Vac and stored at -20 °C before amino acid sequencing. RP-HPLC Separation of Tryptic Peptides from Alkylated bC2GnT-M Labeled with [35S]Cysteine or [35S]Methionine—A C8 reversed phase column (0.46 × 25 cm) (Vydac; 300 Å, 5 μm) was used to isolate glycopeptides linked via disulfide bonds. Tryptic peptides of alkylated bC2GnT-M labeled with [35S]cysteine or [35S]methionine were injected onto a C8 column equilibrated with buffer A at 42 °C. The column was eluted isocratically at 1 ml/min for 3 min with buffer A followed by acetonitrile gradients (0.8%/min for 30 min, 0.23%/min for 70 min, 4.2%/min for 15 min) and then re-equilibrated with buffer A. The 35S-containing fractions collected and analyzed as described above were concentrated by Speed-Vac, reconstituted in 1.5 ml of 150 mm Tris-HCl (pH 8.4) containing 20 mm dithiothreitol under argon gas, and incubated at 37 °C for 3-5 h. Then free thiols were alkylated with 15 mm iodoacetamide under subdued light (26Le Grice S.F.J. Ruddon R.W. Eur. J. Biochem. 1992; 187: 307-314Crossref Scopus (300) Google Scholar, 27Huth J.R. Mountjoy K. Perini F. Ruddon R.W. J. Biol. Chem. 1992; 267: 8870-8879Abstract Full Text PDF PubMed Google Scholar). The carboxymethylated [35S]cysteine (or methionine)-containing peptides were then separated by C18 RP-HPLC as described above. Amino Acid Sequencing—[35S]Cysteine- or [35S]methionine-labeled tryptic peptides were concentrated to less than 50 μl using a Speed-Vac concentrator (Savant). Each sample was loaded onto a Polybrene-coated, trifluoroacetic acid-treated cartridge filter (Applied Biosystems) and sequenced using a pulse liquid protein sequencer (Applied Biosystems model 477A). After each cycle of Edman degradation, the released amino acid derivatives were collected and analyzed by liquid scintillation counting to determine the position(s) of radiolabeled cysteine or methionine in each peptide (28Carlson R.B. Bahl O.P. Swaminathan N. J. Biol. Chem. 1973; 248: 6810-6827Abstract Full Text PDF PubMed Google Scholar). Amino acid sequencing was performed in the protein sequencing facility at the University of Nebraska Medical Center (Omaha, NE). Bio-Gel P-4 Column Chromatography—A Bio-Gel P-4 (200-400-mesh) column (1 × 50 cm) was employed to separate the two cysteine-containing peptides, which co-eluted at peak a (see Fig. 2) of the C18 RP-HPLC chromatogram of the tryptic peptides prepared from reduced-alkylated bC2GnT-M. The column was eluted with water at 1 ml/min and collected at 1 ml/fraction. Fractions were analyzed by liquid scintillation counting to localize the [35S]cysteine in these two [35S]cysteine-containing peptides. Deglycosylation of bC2GnT-M—The purified bC2GnT-M was treated under denaturing conditions at 37 °C for 3 h with 1) buffer alone, 2) N-glycanase, 3) sialidase A plus O-glycanase, and 4) N-glycanase and sialidase A plus O-glycanase according to the protocol provided (Prozyme, Inc.). The samples were then analyzed by SDS-PAGE and stained with Coomassie Blue. The fetuin provided by the supplier was treated under same conditions to validate the reagents and the protocol. Fold Recognition and Molecular Modeling of bC2GnT-M—Due to the lack of appropriate templates (with sequence similarity greater than 30%) for homology modeling, the “inverse folding” approach (29Godzik A. Skolnick J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12098-12102Crossref PubMed Scopus (126) Google Scholar) was used to determine a set of known three-dimensional protein structures, which were compatible with our sequence of interest. The Matchmaker module of SYBYL 6.8 software package (TRIPOS, Inc., St. Louis, MO) was utilized to find crystal structures from the RCSB Protein Data Bank (available on the World Wide Web at www.pdb.org) with three-dimensional folds that match structural properties of the sequence of bC2GnT-M. Matchmaker examines propensities of amino acid residues from the protein sequence to be in a certain environment (solvent-exposed or buried), finds the optimal alignment (frozen or thawed mode) of the sequence to the “structural fingerprint” describing the three-dimensional environment at each residue position, and estimates pseudoenergy scores for different protein folds. Three sets of gap penalties corresponding to Standard, Restrictive or Permissive parameters were used to scan the structural data base. Matchmaker and SYBYL graphical interfaces were used to analyze results. Finally, the Biopolymer module of SYBYL was used to build and analyze structural model of the bC2GnT-M molecule. Purification and Characterization of the Recombinant bC2GnT-M Secreted into the Medium—We found that the recombinant enzyme secreted into the medium was fully active. However, the relative activity of the recombinant bC2GnT-M toward the three acceptors, core 1, core 3, and blood group i oligosaccharides, was changed from 0.7/1.0/0.4 in the wild-type bC2GnT-M (17Choi K.H. Osorio F.A. Cheng P.-W. Am. J. Respir. Cell Mol. Biol. 2004; 30: 710-719Crossref PubMed Scopus (10) Google Scholar) to 6.0/1.0/1.0 in the recombinant bC2GnT-M. Treatment with dithiothreitol (2.5 mm) and β-mercaptoethanol (10 mm) did not affect the enzyme activity. The yield of the recombinant C2GnT-M isolated from the serum-free conditioned medium by nickel-nitrilotriacetic acid affinity column was about 1.5 μg/ml. Coomassie Blue staining of the SDS-PAGE gel of the purified recombinant showed a single band of about 58 kDa (Fig. 1), which was larger than the calculated molecular mass (52,479 Da) of the recombinant protein. Western blot analysis using an anti-Myc antibody also showed one band. Treatment of the purified enzyme with N-glycanase with or without sialidase A plus O-glycanase decreased the size of the recombinant protein by about 4-5 kDa, suggesting that the recombinant protein was N-glycosylated at one or both of the two potential N-glycosylation sites, N-72 and N-108 (17Choi K.H. Osorio F.A. Cheng P.-W. Am. J. Respir. Cell Mol. Biol. 2004; 30: 710-719Crossref PubMed Scopus (10) Google Scholar). The lack of apparent change in size after treatment with sialidase A plus O-glycanase suggests either the absence or presence of a small amount of O-glycan T antigen with or without sialic acid in the recombinant bC2GnT-M. RP-HPLC Tryptic Map of Recombinant bC2GnT-M Labeled with [35S]Cysteine or [35]Methionine—The recombinant bC2GnT-M contains 10 cysteines, of which nine are conserved among all members of
Referência(s)