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• W2049668747 abstract "Core 2 β1,6-N-acetylglucosaminyltransferase I (C2GnT-I) plays a pivotal role in the biosynthesis of mucin-type O-glycans that serve as ligands in cell adhesion. To elucidate the three-dimensional structure of the enzyme for use in computer-aided design of therapeutically relevant enzyme inhibitors, we investigated the participation of cysteine residues in disulfide linkages in a purified murine recombinant enzyme. The pattern of free and disulfide-bonded Cys residues was determined by liquid chromatography/electrospray ionization tandem mass spectrometry in the absence and presence of dithiothreitol. Of nine highly conserved Cys residues, under both conditions, one (Cys217) is a free thiol, and eight are engaged in disulfide bonds, with pairs formed between Cys59–Cys413, Cys100–Cys172, Cys151–Cys199, and Cys372–Cys381. The only non-conserved residue within the β1,6-N-acetylglucosaminyltransferase family, Cys235, is also a free thiol in the presence of dithiothreitol; however, in the absence of reductant, Cys235 forms an intermolecular disulfide linkage. Biochemical studies performed with thiolreactive agents demonstrated that at least one free cysteine affects enzyme activity and is proximal to the UDP-GlcNAc binding site. A Cys217 → Ser mutant enzyme was insensitive to thiol reactants and displayed kinetic properties virtually identical to those of the wild-type enzyme, thereby showing that Cys217, although not required for activity per se, represents the only thiol that causes enzyme inactivation when modified. Based on the pattern of free and disulfide-linked Cys residues, and a method of fold recognition/threading and homology modeling, we have computed a three-dimensional model for this enzyme that was refined using the T4 bacteriophage β-glucosyltransferase fold. Core 2 β1,6-N-acetylglucosaminyltransferase I (C2GnT-I) plays a pivotal role in the biosynthesis of mucin-type O-glycans that serve as ligands in cell adhesion. To elucidate the three-dimensional structure of the enzyme for use in computer-aided design of therapeutically relevant enzyme inhibitors, we investigated the participation of cysteine residues in disulfide linkages in a purified murine recombinant enzyme. The pattern of free and disulfide-bonded Cys residues was determined by liquid chromatography/electrospray ionization tandem mass spectrometry in the absence and presence of dithiothreitol. Of nine highly conserved Cys residues, under both conditions, one (Cys217) is a free thiol, and eight are engaged in disulfide bonds, with pairs formed between Cys59–Cys413, Cys100–Cys172, Cys151–Cys199, and Cys372–Cys381. The only non-conserved residue within the β1,6-N-acetylglucosaminyltransferase family, Cys235, is also a free thiol in the presence of dithiothreitol; however, in the absence of reductant, Cys235 forms an intermolecular disulfide linkage. Biochemical studies performed with thiolreactive agents demonstrated that at least one free cysteine affects enzyme activity and is proximal to the UDP-GlcNAc binding site. A Cys217 → Ser mutant enzyme was insensitive to thiol reactants and displayed kinetic properties virtually identical to those of the wild-type enzyme, thereby showing that Cys217, although not required for activity per se, represents the only thiol that causes enzyme inactivation when modified. Based on the pattern of free and disulfide-linked Cys residues, and a method of fold recognition/threading and homology modeling, we have computed a three-dimensional model for this enzyme that was refined using the T4 bacteriophage β-glucosyltransferase fold. Mucin-type O-glycans expressed on the cell surface of leukocytes figure prominently in controlling cell adhesion events and, in this respect, have been shown to play a role during the initial course of the inflammatory cascade (1Fukuda M. Biochim. Biophys. Acta. 2002; 1573: 394-405Crossref PubMed Scopus (113) Google Scholar, 2Lowe J.B. Immunol. Rev. 2002; 186: 19-36Crossref PubMed Scopus (200) Google Scholar). These sugars serve as ligands for the selectins, a family of structurally related cell-surface glycoproteins that mediate tethering and rolling of leukocytes on activated endothelial cells (3Vestweber D. Blanks J.E. Physiol. Rev. 1999; 79: 181-213Crossref PubMed Scopus (823) Google Scholar, 4Patel K.D. Cuvelier S.L. Wiehler S. Semin. Immunol. 2002; 14: 73-81Crossref PubMed Scopus (169) Google Scholar). There are several types of O-glycans, which are classified based on their core structure (5Schachter H. Brockhausen I. Allen H.J. Kisailus E.C. Glycoconjugates: Composition, Structure, and Function. Marcel Dekker, Inc., New York1992: 263-332Google Scholar). Those with a β1,6-GlcNAc branch, namely the Core 2-type, may be extended with poly(N-acetyllactosamine) and capped with the Lewisx antigen, an α2–3-sialylated, α1–3-fucosylated tetrasaccharide that represents the minimal carbohydrate epitope recognized by P-, E-, and L-selectins (6Foxall C. Watson S.R. Dowbenko D. Fennie C. Lasky L.A Kiso M. Hasegawa A. Asa D. Brandley B.K. J. Cell Biol. 1992; 117: 895-902Crossref PubMed Scopus (650) Google Scholar).The biosynthesis of Core 2-branched oligosaccharides is associated with the activity of the Golgi enzyme UDP-Glc-NAc:Galβ1,3GalNAc-R β1,6-N-acetylglucosaminyltransferase (C2GnT), 1The abbreviations used are: C2GnT-I, core 2 β1,6-N-acetylglucosaminyltransferase I; IGnT, I-branching β1,6-N-acetylglucosaminyltransferase; SLex, sialyl-Lewis X; LC/ESI-MS/MS, liquid chromatography/electrospray ionization-tandem mass spectrometry; BSA, bovine serum albumin; MES, 2-(N-morpholino)ethanesulfonic acid; pNp, para-nitrophenyl; ProtA-C2GnT-I, NH2-Protein A-C2GnT-I-COOH chimera; M-biotin, polyethylene oxide-maleimide-activated biotin; PNGase F, peptide N-glycosidase F; SpsA, Bacillus subtilis nucleoside-diphospho-sugar glycosyltransferase, T4BGT, bacteriophage T4 β-glucosyltransferase; DTT, dithiothreitol; IA, iodoacetamide; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); NEM, N-ethylmaleimide; aa, amino acids.1The abbreviations used are: C2GnT-I, core 2 β1,6-N-acetylglucosaminyltransferase I; IGnT, I-branching β1,6-N-acetylglucosaminyltransferase; SLex, sialyl-Lewis X; LC/ESI-MS/MS, liquid chromatography/electrospray ionization-tandem mass spectrometry; BSA, bovine serum albumin; MES, 2-(N-morpholino)ethanesulfonic acid; pNp, para-nitrophenyl; ProtA-C2GnT-I, NH2-Protein A-C2GnT-I-COOH chimera; M-biotin, polyethylene oxide-maleimide-activated biotin; PNGase F, peptide N-glycosidase F; SpsA, Bacillus subtilis nucleoside-diphospho-sugar glycosyltransferase, T4BGT, bacteriophage T4 β-glucosyltransferase; DTT, dithiothreitol; IA, iodoacetamide; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); NEM, N-ethylmaleimide; aa, amino acids. EC 2.4.1.102, which converts core 1 (Galβ1,3Gal-NAcβ-O) into core 2 (Galβ1,3[GlcNAcβ1,6]GalNAcβ-O) structures (7Williams D. Schachter H. J. Biol. Chem. 1980; 255: 11247-11252Abstract Full Text PDF PubMed Google Scholar). The action of this enzyme defines an important regulatory step responsible for the structural diversity (8Piller F. Piller V. Fox R.I. Fukuda M. J. Biol. Chem. 1988; 263: 15146-15150Abstract Full Text PDF PubMed Google Scholar, 9Yousefi S. Higgins E. Daoling Z. Pollex-Kruger A. Hindsgaul O. Dennis J.W. J. Biol. Chem. 1991; 266: 1772-1782Abstract Full Text PDF PubMed Google Scholar, 10Whitehouse C. Burchell J. Gschmeissner S. Brockhausen I. Lloyd K.O. Taylor-Papadimitriou J. J. Cell Biol. 1997; 137: 1229-1241Crossref PubMed Scopus (93) Google Scholar) and functional significance of these sugars, as evidenced by a number of physiological and pathological events, such as immune responses (Refs. 8Piller F. Piller V. Fox R.I. Fukuda M. J. Biol. Chem. 1988; 263: 15146-15150Abstract Full Text PDF PubMed Google Scholar, 11Higgins E.A. Siminovitch K.A. Zhuang D. Brockhausen I. Dennis J.W. J. Biol. Chem. 1991; 266: 6280-6290Abstract Full Text PDF PubMed Google Scholar, 12Tsuboi S. Fukuda M. J. Biol. Chem. 1998; 273: 30680-30687Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar and reviewed in Ref. 13Tsuboi S. Fukuda M. BioEssays. 2001; 23: 46-53Crossref PubMed Google Scholar), diabetic cardiomyopathy (14Koya D. Dennis J.W. Warren C.E. Takahara N. Schoen F.J. Nishio Y. Nakajima T. Lipes M.A. King G.L. FASEB J. 1999; 13: 2329-2337Crossref PubMed Scopus (21) Google Scholar), and cancer (9Yousefi S. Higgins E. Daoling Z. Pollex-Kruger A. Hindsgaul O. Dennis J.W. J. Biol. Chem. 1991; 266: 1772-1782Abstract Full Text PDF PubMed Google Scholar, 15Shimodaira K. Nakayama J. Nakamura N. Hasebe O. Katsuyama T. Fukuda M. Cancer Res. 1997; 57: 5201-5206PubMed Google Scholar, 16Machida E. Nakayama J. Amano J. Fukuda M. Cancer Res. 2001; 61: 2226-2231PubMed Google Scholar). Interestingly, a common element of these conditions is the altered dynamics of cell-cell or cell-matrix interactions caused by the differential expression of C2GnT and cognate O-glycans.Three isoforms of C2GnT have been identified and cloned to date. Two of them, the widely expressed leukocyte-type (C2GnT-I) (17Bierhuizen M.F.A. Fukuda M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9326-9330Crossref PubMed Scopus (279) Google Scholar, 18Bierhuizen M.F. Maemura K. Kudo S. Fukuda M. Glycobiology. 1995; 5: 417-425Crossref PubMed Scopus (32) Google Scholar) and the thymus-associated enzyme (C2GnTIII) (19Schwientek 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 (94) Google Scholar), exhibit exclusive core 2 acceptor specificity, whereas C2GnT-II, which is distributed in mucin-secreting tissues along the gastrointestinal tract, can form core 2, core 4, and I-branching structures (20Yeh J.-C. Ong E. Fukuda M. J. Biol. Chem. 1999; 274: 3215-3221Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 21Schwientek T. Nomoto M. Levery S.B. Merkx G. van Kessel A.G. Bennett E.P. Hollingsworth M.A. Clausen H. J. Biol. Chem. 1999; 274: 4504-4512Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Cloning of C2GnTs from different sources revealed a high degree of sequence identity across species (Fig. 1). When aligned to rat and bovine C2GnT-I, the murine protein exhibits sequence identities of 92 and 79%, respectively. Similarly, there is significant homology with the human C2GnT isoforms, C2GnT-I (84%), C2GnT-II (54%), C2GnT-III (44%), and human I-GnT (37%). In addition, a bovine herpesvirus enzyme homologous to human C2GnT-II was identified based on 81.1% amino acid sequence similarity (22Vanderplasschen A. Markine-Goriaynoff 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 (45) Google Scholar), with a 57% homology to murine C2GnT-I. Interestingly, all members of the β1,6-Core2/I-GnT family share nine conserved Cys residues, whose structural/functional role has not yet been elucidated.A number of in vitro studies confirm a fundamental requirement for C2GnT-I activity in the biosynthesis of ligands with effective binding to P- (23Kumar R. Camphausen R.T. Sullivan F.X. Cumming D.A. Blood. 1996; 88: 3872-3879Crossref PubMed Google Scholar, 24Li F. Wilkins P.P. Crawley S. Weinstein J. Cummings R.D. McEver R.P. J. Biol. Chem. 1996; 271: 3255-3264Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 25Ellies L.G. Tsuboi S. Petryniak B. Lowe J.B. Fukuda M. Marth J.D. Immunity. 1998; 9: 881-890Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 26Snapp K.R. Heitzig C.E. Ellies L.G. Marth J.D. Kansas G.S. Blood. 2001; 97: 3806-3811Crossref PubMed Scopus (77) Google Scholar) and L-selectins (25Ellies L.G. Tsuboi S. Petryniak B. Lowe J.B. Fukuda M. Marth J.D. Immunity. 1998; 9: 881-890Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 27Hiraoka N. Petryniak B. Nakayama J. Tsuboi S. Suzuki M. Yeh J.-C. Izawa D. Tanaka T. Miyasaka M. Lowe J.B. Fukuda M. Immunity. 1999; 11: 79-89Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 28Bernimoulin M.P. Zeng X.L. Abbal C. Giraud S. Martinez M. Michielin O. Schapira M. Spertini O. J. Biol. Chem. 2003; 278: 37-47Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 29Mitoma J. Petryniak B. Hiraoka N. Yeh J.-C. Lowe J.B. Fukuda M. J. Biol. Chem. 2003; 278: 9953-9961Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar); conversely, it is not certain whether the enzyme plays a prominent role in the functional decoration of E-selectin ligands (26Snapp K.R. Heitzig C.E. Ellies L.G. Marth J.D. Kansas G.S. Blood. 2001; 97: 3806-3811Crossref PubMed Scopus (77) Google Scholar). In vivo studies using C2GnT-I null mice showed a severe, albeit incomplete, deficit in neutrophil recruitment following induction of peritonitis by thioglycollate (25Ellies L.G. Tsuboi S. Petryniak B. Lowe J.B. Fukuda M. Marth J.D. Immunity. 1998; 9: 881-890Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar), which is consistent with the sharp reduction in P- (30Sperandio M. Thatte A. Foy D. Ellies L.G. Marth J.D. Ley K. Blood. 2001; 97: 3812-3819Crossref PubMed Scopus (108) Google Scholar), E- (30Sperandio M. Thatte A. Foy D. Ellies L.G. Marth J.D. Ley K. Blood. 2001; 97: 3812-3819Crossref PubMed Scopus (108) Google Scholar), and L-selectin-dependent (31Sperandio M. Forlow S.B. Thatte J. Ellies L.G. Marth J.D. Ley K. J. Immunol. 2001; 167: 2268-2274Crossref PubMed Scopus (34) Google Scholar) leukocyte rolling that was observed in cremaster muscle venules via intravital microscopy. Based on these results, small molecule, orally available inhibitors of C2GnT-I activity can be expected to be of therapeutic use for the treatment of overzealous inflammatory responses that lead to pathological conditions such as reperfusion injury, rheumatoid arthritis, asthma, and inflammatory bowel disease. In this regard, the development of SLex mimics as anti-inflammatory drugs has already become an active area of research (reviewed in Ref. 32Kaila N. Thomas IV, B.E. Med. Res. Rev. 2002; 22: 566-601Crossref PubMed Scopus (93) Google Scholar), and soluble SLex tetrasaccharide was shown to block neutrophil invasion and acute inflammation in animal models of injury (33Mulligan M.S. Paulson J.C. De Frees S. Zheng Z.L. Lowe J.B. Ward P.A. Nature. 1993; 364: 149-151Crossref PubMed Scopus (309) Google Scholar, 34Mulligan M.S. Warner R.L. Rittershaus C.W. Thomas L.J. Ryan U.S. Foreman K.E. Crouch L.D. Till G.O. Ward P.A. J. Immunol. 1999; 162: 4952-4959PubMed Google Scholar). However, the short serum half-life and low affinity of the monomeric sugar ligand in solution has, thus far, limited success in discovering viable drug therapies (32Kaila N. Thomas IV, B.E. Med. Res. Rev. 2002; 22: 566-601Crossref PubMed Scopus (93) Google Scholar).In support of a program focused on the development of drug-like C2GnT-I inhibitors, we initially computed two possible three-dimensional protein models of the catalytic domain of the enzyme for use in both virtual screenings and optimization of lead inhibitor candidates identified via high-throughput enzyme assay efforts (35Donovan R.S. Datti A. Baek M.-G. Wu Q. Sas I.J. Korczak B. Berger E.G. Roy R. Dennis J.W. Glycoconj. J. 1999; 16: 607-615Crossref PubMed Scopus (20) Google Scholar). In order to select and refine the most likely protein fold, we conducted Cys analysis by LC/ESIMS/MS in a murine recombinant form of C2GnT-I.This study shows that of the nine highly conserved Cys residues shared by all members of the β1,6-Core2/I-GnT family, eight are engaged in disulfide bonds and one is a free thiol that, although not required for enzyme activity, is responsible for inactivation of the enzyme if modified. We also noted that the only unconserved Cys residue forms an intermolecular bridge under non-reducing conditions, thereby causing dimerization of the enzyme protein. Finally, our results show that the spatial constraints defined by the disulfide bonds, together with evidence for lack of divalent cations in the core protein, argue for a protein fold predicated upon that of T4 bacteria phage β-glucosyltransferase.EXPERIMENTAL PROCEDURESMaterials—Materials used for methods in molecular biology, enzymology, and cell biology were routinely purchased from reputable suppliers (specified in the text for most items) and were of the highest quality available. For the radiometric enzyme assay, UDP-GlcNAc and BSA (essentially free of fatty acids and globulins, used as a stabilizer) were from Sigma; MES (free acid) was from Calbiochem; UDP-[3H]Glc-NAc (12 Ci/mmol) was from Toronto Research Chemicals (Toronto, Canada), and the acceptor substrate Galβ1–3GalNAcα-pNp was from Rose Scientific (Edmonton, Canada). Sep-Pak C18 columns (500 mg) were purchased from Waters, and scintillation fluid (Scintisafe™ 30%) was from Fisher. Protein quantitation was performed with the bicinchoninic acid (BCA) kit from Pierce.Murine, Recombinant C2GnT-I—The procedures followed to obtain a murine C2GnT-I fusion protein have been described previously (35Donovan R.S. Datti A. Baek M.-G. Wu Q. Sas I.J. Korczak B. Berger E.G. Roy R. Dennis J.W. Glycoconj. J. 1999; 16: 607-615Crossref PubMed Scopus (20) Google Scholar). A truncated cDNA fragment encoding amino acids 38–428 was prepared by PCR and fused in-frame to pPROTA vector (36Sanchez-Lopez R. Nicholson R. Gesnel M.C. Matrisian L.M. Breathnach R. J. Biol. Chem. 1988; 263: 11892-11899Abstract Full Text PDF PubMed Google Scholar) for expression as a secreted NH2-ProteinA-C2GnT-I-COOH chimera (ProtA-C2GnT-I). The expression vector was co-transfected into Chinese hamster ovary cells, along with pSV2neo, in a 10:1 molar ratio, using the calcium phosphate method. Cells were cultured in the presence of 800 μg/ml geneticin (antibiotic G418, Invitrogen), and resistant cell clones were selected and tested for C2GnT activity in culture medium. The representative clone 614 C2 showed stable expression of C2GnT activity and was selected for enzyme production. The cells were routinely propagated in minimum essential medium (Invitrogen) containing 5% fetal bovine serum (Invitrogen) and G418 (0.2 mg/ml). To partially purify the enzyme, IgG-Sepharose Fast Flow™ beads (Amersham Biosciences) were added in a ratio of 5 μl of a 50% bead slurry, 2.5 μl of 2 m Tris·HCl, pH 8.0, and 5 μl of 10% Tween 20 per ml of culture medium. Following incubation on a rocking platform at 4 °C for 20 h, the beads were collected by centrifugation, washed with 10 volumes of TNT buffer (50 mm Tris·HCl, pH 8.0, 150 mm NaCl, 0.05% Tween 20) and 2 volumes of 5 mm NH4Ac, pH 5.0. The recombinant ProtA-C2GnT-I enzyme was then eluted with 1 volume 0.5 m acetic acid, pH 3.4, and immediately resuspended in 3 volumes of 0.5 m MES, pH 7.5. The enzyme preparation was routinely passed through a 0.22-micron filter prior to storage at 4 °C in 30 mm MES buffer, pH 6.7. Purity of the ProtA-C2GnT-I protein was ∼95%, based on Coomassie Blue staining following SDS-PAGE.Production of a Murine (Cys217 → Ser) C2GnT-I Mutant—A truncated cDNA fragment encoding amino acids 34–428 of murine C2GnT-I was amplified by PCR and ligated in-frame into pFLAG-CMV™-3 vector (Sigma) using BglII and XbaI sites, for expression as a secreted fusion protein NH2-FLAG(DYKDDDDKLAAANSSIDL)-C2GnTI-COOH.The cDNA was mutated at base pair 650 where a G nucleotide was exchanged for a C nucleotide, thereby resulting in a segment encoding a mutant protein in which Cys217 was replaced with Ser. Site-directed mutagenesis was performed using the QuikChange® kit (catalog number 200518, Stratagene). The mutated construct was transiently transfected into Chinese hamster ovary-S (CHO-S) cells (Invitrogen) using LipofectAMINE™ 2000 (Invitrogen) following the manufacturer's instructions. Briefly, CHO-S cells were grown in Dulbecco's modified Eagle's medium/F-12 (Invitrogen) supplemented with 10% fetal bovine serum and 0.1 mm non-essential amino acids (growth medium). cDNA (200 μg) was added to 3 ml of reduced serum medium (Opti-MEM® I, Invitrogen) and then mixed with a second 3-ml aliquot of Opti-MEM® containing 130 μl of LipofectAMINE™ 2000. After 20 min of incubation at room temperature, the cDNA/LipofectAMINE mixture was added to cells (90% confluence) that were seeded in flasks that had been treated previously for 24 h with 30 ml of growth medium. After 16 h of culture, medium was removed and tested to confirm enzyme activity. Cells were washed twice with Dulbecco's phosphate-buffered saline (Invitrogen) and then cultured in 50 ml of serum-free medium (CHO-S-SFM II, Invitrogen) for 24 h. Medium was harvested and stored at 4 °C if not promptly used in the subsequent enzyme purification step. Cells were washed again in Dulbecco's phosphate-buffered saline, resuspended in 70 ml of CHO-S-SFM II, and cultured for an additional 36–48 h, after which medium was again collected.Purification of the FLAG-C2GnT-I (Cys217 → Ser) chimera was performed via affinity chromatography, using the ANTI-FLAG® M1 monoclonal antibody-agarose affinity gel (Sigma) according to the manufacturer's instructions (5 ml of gel beads per 400 ml of medium). The column was washed 4 times with 50 ml of a solution made of 50 mm Tris·HCl, pH 7.4, 150 mm NaCl, and 5 mm CaCl2 prior to elution of the fusion protein in 30 ml of 50 mm Tris·HCl, pH 7.4, containing 2 mm EDTA and 0.05% Tween 20. Eluates were concentrated using Centricon plus-80 centrifugal filter units (30,000 molecular weight cut-off, Millipore), followed by dialysis in 30 mm MES buffer, pH 6.7. Each sample was routinely passed through a 0.22-micron filter prior to storage at 4 °C. Purity of the FLAG-(Cys217 → Ser) C2GnT-I fusion protein exceeded 98%, based on Coomassie Blue staining after SDS-PAGE.Determination of C2GnT-I Activity—A radiometric enzyme assay for C2GnT-I was performed essentially as described previously (9Yousefi S. Higgins E. Daoling Z. Pollex-Kruger A. Hindsgaul O. Dennis J.W. J. Biol. Chem. 1991; 266: 1772-1782Abstract Full Text PDF PubMed Google Scholar, 37Datti A. Orlacchio A. Siminovitch K.A. Dennis J.W. Anal. Biochem. 1992; 206: 262-266Crossref PubMed Scopus (9) Google Scholar), using a mixture of 30 mm MES buffer, pH 6.7, 1 mg/ml BSA, 1 mm UDP-GlcNAc, 1 μCi of UDP-[3H]GlcNAc (12 Ci/mmol), 0.5 mm Galβ1–3GalNAcα-pNp as the acceptor substrate, and 10 μl of recombinant C2GnT-I enzyme (15–20 × 10-6 units), 2One unit is the amount of enzyme required to transform 1 μmol of substrate per min. in a total reaction volume of 30 μl. Reactions were incubated for 2 h at 37 °C, followed by C18 Sep-Pak processing to separate the reaction product, which was then quantified based on radioactive emissions. In all instances, measurements were performed in duplicate; background was calculated under identical conditions except for the presence of enzyme and regularly subtracted from the signal.Enzyme Treatment with Thiol-reactive Agents—A fresh preparation of ProtA-C2GnT-I was divided into identical aliquots, each with ∼1 μm protein. Samples were treated with varying concentrations of iodoacetamide, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), or N-ethylmaleimide. A control sample was prepared identically, except for the addition of thiol-specific reagents (replaced by the same volume of water). Each sample, in a total volume of 20 μl, was preincubated at 37 °C for various time points, after which 3-μl aliquots were removed from each solution and diluted with 600 μl of 30 mm MES, pH 6.7. Ten μl of the diluted protein solution (∼3.2 ng) was added to 20 μl of assay mixture to determine enzyme activity (38Kitz R. Wilson I.B. J. Biol. Chem. 1962; 237: 3245-3249Abstract Full Text PDF PubMed Google Scholar).Substrate Protection Experiments—Aliquots of ProtA-C2GnT-I protein (1 μm) were preincubated at 37 °C for 5 min with various concentrations of either donor or acceptor substrate, prior to treatment with thiol-reactive agents. Controls were prepared in the same way, except for the addition of thiol reactants (replaced by an identical volume of water). The total volume in each sample was 20 μl. At various time points, 3-μl aliquots were removed from each tube and added to 600 μl of 30 mm MES, pH 6.7. Ten μl of the diluted solution, corresponding to ∼3.2 ng of enzyme protein, was used as the source of activity in the enzyme assay.Alkylation of Cys Residues for LC/ESI-MS/MS Analyses—A ProtAC2GnT-I preparation (0.14 μg/μl in 10 mm Tris·HCl, pH 7.5, containing 50 mm KCl) was split into 2 aliquots (200 μl each), one of which was treated with 1 mm DTT for 8 h at room temperature. Both samples were subjected to alkylation in the presence of 10 mm polyethylene oxide-maleimide-activated biotin (M-biotin, Pierce), which was incubated for 60 h in the dark at room temperature. M-biotin-labeled ProtA-C2GnT-I samples were denatured with 8 m urea for 1 h, after which they were transferred to a Microcon YM-30 (Millipore) unit and ultrafiltered (8,000 × g) three times with 200 μl of 20 mm Tris·HCl, pH 7.5, to remove unreacted M-biotin. Prior to PNGase F digestion, the enzyme protein preparations were reconstituted in 80 μl of 50 mm Tris·HCl, pH 7.2.Peptide-N-Glycosidase (PNGase) F Digestion—Lyophilized PNGase F (ProZyme) was dissolved in 10 mm Tris·HCl buffer, pH 7.2, containing 15 mm NaCl and 1 mm EDTA, at a concentration of 1250 units/ml. Two μl of this preparation was used to treat each enzyme sample overnight at 37 °C.Digestion with Trypsin, Chymotrypsin, or Endoproteinase Glu-C—Following PNGase F digestion, the ProtA-C2GnT-I sample was split into identical aliquots and digested with either trypsin (sequencing grade, Promega), chymotrypsin (sequencing grade, Roche Applied Science), or endoproteinase Glu-C (sequencing grade, Roche Applied Science). The reactions were set using a 1:20 to 1:40 ratio (w/w) of proteolytic enzyme versus ProtA-C2GnT-I. Incubations were carried out overnight at 37 °C.Identification of Free Cys Residues and Disulfide Bond Pairs—Liquid Chromatography/Electrospray Ionization-Tandem Mass Spectrometry (LC/ESI-MS/MS) analyses were performed using a LCQ™ Classical ion trap mass spectrometer (Thermo Finnigan) with a modified electrospray ionization source (39Yen T.-Y. Yan H. Macher B.A. J. Mass Spectrom. 2002; 37: 15-30Crossref PubMed Scopus (73) Google Scholar). A positive voltage of 1.8 kV was applied to the electrospray needle, and the temperature of the stainless steel heating capillary was maintained at 220 °C. The voltages at the exit end of the heating capillary and the tube lens were held at 17 and 3 V, respectively, to minimize source-induced dissociation and optimize the ESI signal of the analyte. Ion injection was controlled by automatic gain control to avoid space charge effects. The full scan mass spectrum was acquired from m/z 300 to m/z 2000. MS/MS experiments were carried out with a relative collision energy of 38%. The LC/MS analysis was conducted using a Micro-LC system (Micro-Tech Scientific) coupled to the ion trap mass spectrometer. The mobile phase was subjected to splitting prior to injection, after which a flow rate of 0.3 μl/min was established through the capillary C18 column (75 μm × 90 mm). The enzymatically digested peptides were eluted from the column using 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B) using a three-step linear gradient, where phase B was 10–25% during the first 35 min, 25–35% over the next 10 min, and 35–50% in the final 10 min. The LC/ESI-MS/MS analysis was conducted using an automated data acquisition procedure, based on a cyclic use of full, zoom, and MS/MS scan modes. The most intense peak (signal >1.5 × 105 counts) in a full scan was selected as the precursor ion, upon which a high resolution zoom and an MS/MS scan were performed to determine charge state and structural fragment ions, respectively. The resulting MS/MS spectra were then screened against a protein data base using the program Sequest to confirm the sequences of the tryptic peptides.Fold Recognition and Comparative Molecular Modeling—Two glycosyltransferases were used as templates for homology modeling of C2GnT-I: (i) bacteriophage T4 β-glucosyltransferase (T4BGT, Protein Data Bank code 1JIV), which transfers glucose from UDP-glucose to DNA, and (ii) rabbit N-acetylglucosaminyltransferase I (GnT I, Protein Data Bank code 1FOA), a medial Golgi enzyme that catalyzes the addition of N-acetylglucosamine in β1,2-linkage from UDP-GlcNAc to Man5GlcNAc2. Initially, the lack of homology with T4BGT and GnT I prompted us to align the amino acid sequence of murine C2GnT-I with that of both templates, using each fold for threading. Manual adjustments were performed based on conformational constraints dictated by pairing of Cys residues in disulfide bonds. Alignments were further adjusted by inspection of the Protein Data Bank structures, with insertions and deletions that were moved into proximal loop regions whenever they occurred inside secondary structure elements. The predicted secondary structure for C2GnT-I was obtained using the PHD server (University of Columbia) (40Rost B. Methods Enzymol. 1996; 266: 525-539Crossref PubMed Google Scholar). The MODELER module in Insight II (Accelrys) was used for the homology modeling; the coordinates of homologous regions were transferred from each template structure to C2GnT-I following final alignments. Optimization was performed locally to remove steric side chain clashes. Three models were generated for each alignment, and the one with the lowest objective function was chosen for research applications.Sample Preparation for Inductively Coupled Plasma Atomic Emission Spectrometry—One liter of stock solution of partially purified mouse ProtA-C2GnT-I in 30 mm MES, pH 6.7, was concentrated to a volume of 5" @default.
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• W2049668747 title "Highly Conserved Cysteines of Mouse Core 2 β1,6-N-Acetylglucosaminyltransferase I Form a Network of Disulfide Bonds and Include a Thiol That Affects Enzyme Activity" @default.
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