Post-translational Modification of Thrombospondin Type-1 Repeats in ADAMTS-like 1/Punctin-1 by C-Mannosylation of Tryptophan
2009; Elsevier BV; Volume: 284; Issue: 44 Linguagem: Inglês
10.1074/jbc.m109.038059
ISSN1083-351X
AutoresLauren W. Wang, Christina Leonhard-Melief, Robert S. Haltiwanger, Suneel Apte,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoProtein C-mannosylation is the attachment of α-mannopyranose to tryptophan via a C-C linkage. This post-translational modification typically occurs within the sequence motif WXXW, which is frequently present in thrombospondin type-1 repeats (TSRs). TSRs are especially numerous in and a defining feature of the ADAMTS superfamily. We investigated the presence and functional significance of C-mannosylation of ADAMTS-like 1/punctin-1, which contains four TSRs (two with predicted C-mannosylation sites), using mass spectrometry, metabolic labeling, site-directed mutagenesis, and expression in C-mannosylation-defective Chinese hamster ovary cell variants. Analysis of tryptic fragments of recombinant human punctin-1 by mass spectrometry identified a peptide derived from TSR1 containing the 36WDAWGPWSECSRTC49 sequence of interest modified with two mannose residues and a Glc-Fuc disaccharide (O-fucosylation). Tandem mass spectrometry (MS/MS) and MS/MS/MS analysis demonstrated the characteristic cross-ring cleavage of C-mannose and identified the modified residues as Trp39 and Trp42. C-Mannosylation of TSR1 of the related protease ADAMTS5 was also identified. Metabolic labeling of CHO-K1 cells or Lec35.1 cells demonstrated incorporation of d-[2,6-3H]mannose in secreted punctin-1 from CHO-K1 cells but not Lec35.1 cells. Quantitation of punctin-1 secretion in Lec35.1 cells versus CHO-K1 cells suggested decreased secretion in Lec35.1 cells. Replacement of mannosylated Trp residues in TSR1 with either Ala or Phe affected punctin secretion efficiency. These data demonstrate that TSR1 from punctin-1 carries C-mannosylation in close proximity to O-linked fucose. Together, these modifications appear to provide a quality control mechanism for punctin-1 secretion. Protein C-mannosylation is the attachment of α-mannopyranose to tryptophan via a C-C linkage. This post-translational modification typically occurs within the sequence motif WXXW, which is frequently present in thrombospondin type-1 repeats (TSRs). TSRs are especially numerous in and a defining feature of the ADAMTS superfamily. We investigated the presence and functional significance of C-mannosylation of ADAMTS-like 1/punctin-1, which contains four TSRs (two with predicted C-mannosylation sites), using mass spectrometry, metabolic labeling, site-directed mutagenesis, and expression in C-mannosylation-defective Chinese hamster ovary cell variants. Analysis of tryptic fragments of recombinant human punctin-1 by mass spectrometry identified a peptide derived from TSR1 containing the 36WDAWGPWSECSRTC49 sequence of interest modified with two mannose residues and a Glc-Fuc disaccharide (O-fucosylation). Tandem mass spectrometry (MS/MS) and MS/MS/MS analysis demonstrated the characteristic cross-ring cleavage of C-mannose and identified the modified residues as Trp39 and Trp42. C-Mannosylation of TSR1 of the related protease ADAMTS5 was also identified. Metabolic labeling of CHO-K1 cells or Lec35.1 cells demonstrated incorporation of d-[2,6-3H]mannose in secreted punctin-1 from CHO-K1 cells but not Lec35.1 cells. Quantitation of punctin-1 secretion in Lec35.1 cells versus CHO-K1 cells suggested decreased secretion in Lec35.1 cells. Replacement of mannosylated Trp residues in TSR1 with either Ala or Phe affected punctin secretion efficiency. These data demonstrate that TSR1 from punctin-1 carries C-mannosylation in close proximity to O-linked fucose. Together, these modifications appear to provide a quality control mechanism for punctin-1 secretion. The ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin type-1 repeats) superfamily (1Hurskainen T.L. Hirohata S. Seldin M.F. Apte S.S. J. Biol. Chem. 1999; 274: 25555-25563Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) consists of 19 secreted metalloproteases (ADAMTS proteases) and six ADAMTS-like proteins in humans. ADAMTS-like proteins closely resemble the ancillary domains of ADAMTS proteases and like them have a conserved modular organization containing one or more thrombospondin type-1 repeats (TSRs) 2The abbreviations used are: TSRthrombospondin repeatTSR1thrombospondin type-1 repeatHPLChigh pressure liquid chromatographyDol-P-Mandolichyl-phosphate mannoseCHOChinese hamster ovary. 2The abbreviations used are: TSRthrombospondin repeatTSR1thrombospondin type-1 repeatHPLChigh pressure liquid chromatographyDol-P-Mandolichyl-phosphate mannoseCHOChinese hamster ovary. (2Ahram D. Sato T.S. Kohilan A. Tayeh M. Chen S. Leal S. Al-Salem M. El-Shanti H. Am. J. Hum. Genet. 2009; 84: 274-278Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 3Hall N.G. Klenotic P. Anand-Apte B. Apte S.S. Matrix Biol. 2003; 22: 501-510Crossref PubMed Scopus (45) Google Scholar, 4Hirohata S. Wang L.W. Miyagi M. Yan L. Seldin M.F. Keene D.R. Crabb J.W. Apte S.S. J. Biol. Chem. 2002; 277: 12182-12189Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 5Koo B.H. Goff C.L. Jungers K.A. Vasanji A. O'Flaherty J. Weyman C.M. Apte S.S. Matrix Biol. 2007; 26: 431-441Crossref PubMed Scopus (42) Google Scholar). TSRs are modules of ∼50 amino acids having a characteristic six-cysteine signature. The prototypic ADAMTSL, ADAMTSL1, also referred to as punctin-1 because of its punctate distribution in the substratum of transfected cells, is a 525-residue glycoprotein containing four TSRs (4Hirohata S. Wang L.W. Miyagi M. Yan L. Seldin M.F. Keene D.R. Crabb J.W. Apte S.S. J. Biol. Chem. 2002; 277: 12182-12189Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). A longer punctin-1 variant arising from alternative splicing, containing 13 TSRs and homologous to ADAMTSL3, is predicted by the human genome sequencing project (NM_001040272) but has not yet been physically cloned and expressed. The function of ADAMTSL1/punctin-1 is unknown. Recently, ADAMTSL2 and ADAMTSL4 mutations were identified in the genetic disorders geleophysic dysplasia (6Le Goff C. Morice-Picard F. Dagoneau N. Wang L.W. Perrot C. Crow Y.J. Bauer F. Flori E. Prost-Squarcioni C. Krakow D. Ge G. Greenspan D.S. Bonnet D. Le Merrer M. Munnich A. Apte S.S. Cormier-Daire V. Nat. Genet. 2008; 40: 1119-1123Crossref PubMed Scopus (169) Google Scholar) and recessive isolated ectopia lentis, respectively (2Ahram D. Sato T.S. Kohilan A. Tayeh M. Chen S. Leal S. Al-Salem M. El-Shanti H. Am. J. Hum. Genet. 2009; 84: 274-278Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). In genome-wide analysis, the ADAMTSL3 locus has been associated with variation in human height (7Weedon M.N. Lango H. Lindgren C.M. Wallace C. Evans D.M. Mangino M. Freathy R.M. Perry J.R. Stevens S. Hall A.S. Samani N.J. Shields B. Prokopenko I. Farrall M. Dominiczak A. Johnson T. Bergmann S. Beckmann J.S. Vollenweider P. Waterworth D.M. Mooser V. Palmer C.N. Morris A.D. Ouwehand W.H. Zhao J.H. Li S. Loos R.J. Barroso I. Deloukas P. Sandhu M.S. Wheeler E. Soranzo N. Inouye M. Wareham N.J. Caulfield M. Munroe P.B. Hattersley A.T. McCarthy M.I. Frayling T.M. Nat. Genet. 2008; 40: 575-583Crossref PubMed Scopus (637) Google Scholar). Thus, in addition to known genetic disorders caused by ADAMTS mutations (8Apte S.S. Int. J. Biochem. Cell Biol. 2004; 36: 981-985Crossref PubMed Scopus (213) Google Scholar, 9Porter S. Clark I.M. Kevorkian L. Edwards D.R. Biochem. J. 2005; 386: 15-27Crossref PubMed Scopus (618) Google Scholar), ADAMTSL family members are now also implicated in human disease. Among the ADAMTS proteases, ADAMTS5 and ADAMTS4 are strongly associated with cartilage destruction in arthritis (10Arner E.C. Curr. Opin. Pharmacol. 2002; 2: 322-329Crossref PubMed Scopus (134) Google Scholar, 11Glasson S.S. Askew R. Sheppard B. Carito B. Blanchet T. Ma H.L. Flannery C.R. Peluso D. Kanki K. Yang Z. Majumdar M.K. Morris E.A. Nature. 2005; 434: 644-648Crossref PubMed Scopus (1003) Google Scholar, 12Stanton H. Rogerson F.M. East C.J. Golub S.B. Lawlor K.E. Meeker C.T. Little C.B. Last K. Farmer P.J. Campbell I.K. Fourie A.M. Fosang A.J. Nature. 2005; 434: 648-652Crossref PubMed Scopus (756) Google Scholar). thrombospondin repeat thrombospondin type-1 repeat high pressure liquid chromatography dolichyl-phosphate mannose Chinese hamster ovary. thrombospondin repeat thrombospondin type-1 repeat high pressure liquid chromatography dolichyl-phosphate mannose Chinese hamster ovary. Like most secreted proteins, the ADAMTS superfamily members undergo post-translational modification and are predicted to contain N-linked oligosaccharides. In addition, TSRs of ADAMTS superfamily members, by virtue of high sequence similarity to the corresponding motifs in thrombospondin 1 and properdin, are predicted to contain two uncommon types of glycosylation. Specifically, TSRs often contain the sequence motifs W0XXW+3 and C1X2–3(S/T)C2XXG, which are consensus sites for protein C-mannosylation of the W0 residue and O-fucosylation (of Ser/Thr) respectively, in close proximity to each other (13Gonzalez de Peredo A. Klein D. Macek B. Hess D. Peter-Katalinic J. Hofsteenge J. Mol. Cell. Proteomics. 2002; 1: 11-18Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 14Hofsteenge J. Huwiler K.G. Macek B. Hess D. Lawler J. Mosher D.F. Peter-Katalinic J. J. Biol. Chem. 2001; 276: 6485-6498Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). In recently published work, it was shown that ADAMTSL1 and ADAMTS13 are modified by O-fucosylation, the covalent attachment to Ser or Thr residues of fucose or a fucose-glucose disaccharide (15Ricketts L.M. Dlugosz M. Luther K.B. Haltiwanger R.S. Majerus E.M. J. Biol. Chem. 2007; 282: 17014-17023Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 16Wang L.W. Dlugosz M. Somerville R.P. Raed M. Haltiwanger R.S. Apte S.S. J. Biol. Chem. 2007; 282: 17024-17031Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Punctin-1 contains consensus sequences for O-fucosylation in all four of its TSRs, but the presence of the glycans was previously only confirmed on TSR2, -3, and -4 (16Wang L.W. Dlugosz M. Somerville R.P. Raed M. Haltiwanger R.S. Apte S.S. J. Biol. Chem. 2007; 282: 17024-17031Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Addition of O-fucose is mediated by protein O-fucosyltransferase 2 (POFUT2), which is a distinct transferase from that which catalyzes addition of O-linked fucose to epidermal growth factor-like repeats (POFUT1) (17Luo Y. Nita-Lazar A. Haltiwanger R.S. J. Biol. Chem. 2006; 281: 9385-9392Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 18Luo Y. Koles K. Vorndam W. Haltiwanger R.S. Panin V.M. J. Biol. Chem. 2006; 281: 9393-9399Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). A β3-glucosyltransferase subsequently adds glucose to the 3′-OH of the fucose (19Kozma K. Keusch J.J. Hegemann B. Luther K.B. Klein D. Hess D. Haltiwanger R.S. Hofsteenge J. J. Biol. Chem. 2006; 281: 36742-36751Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 20Sato T. Sato M. Kiyohara K. Sogabe M. Shikanai T. Kikuchi N. Togayachi A. Ishida H. Ito H. Kameyama A. Gotoh M. Narimatsu H. Glycobiology. 2006; 16: 1194-1206Crossref PubMed Scopus (60) Google Scholar). It was further demonstrated that O-fucosylation, which occurs after completion of TSR folding, was rate-limiting for secretion of punctin-1 and ADAMTS13 (15Ricketts L.M. Dlugosz M. Luther K.B. Haltiwanger R.S. Majerus E.M. J. Biol. Chem. 2007; 282: 17014-17023Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 16Wang L.W. Dlugosz M. Somerville R.P. Raed M. Haltiwanger R.S. Apte S.S. J. Biol. Chem. 2007; 282: 17024-17031Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). This role was inferred from the following two experimental observations. 1) Expression of wild-type punctin-1 and ADAMTS13 in Lec13 cells, which do not fucosylate proteins, led to their decreased secretion (15Ricketts L.M. Dlugosz M. Luther K.B. Haltiwanger R.S. Majerus E.M. J. Biol. Chem. 2007; 282: 17014-17023Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 16Wang L.W. Dlugosz M. Somerville R.P. Raed M. Haltiwanger R.S. Apte S.S. J. Biol. Chem. 2007; 282: 17024-17031Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). 2) Mutation of the modified Ser or Thr residues greatly reduced secretion of punctin-1 and ADAMTS13 (15Ricketts L.M. Dlugosz M. Luther K.B. Haltiwanger R.S. Majerus E.M. J. Biol. Chem. 2007; 282: 17014-17023Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 16Wang L.W. Dlugosz M. Somerville R.P. Raed M. Haltiwanger R.S. Apte S.S. J. Biol. Chem. 2007; 282: 17024-17031Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Protein C-mannosylation is the attachment of an α-mannopyranosyl residue to the indole C-2 of tryptophan via a C-C linkage (14Hofsteenge J. Huwiler K.G. Macek B. Hess D. Lawler J. Mosher D.F. Peter-Katalinic J. J. Biol. Chem. 2001; 276: 6485-6498Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 21Hofsteenge J. Blommers M. Hess D. Furmanek A. Miroshnichenko O. J. Biol. Chem. 1999; 274: 32786-32794Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Unlike O-fucosylation, it can utilize protein primary structure rather than tertiary structure as the determinant, i.e. mannose is added to unfolded polypeptides or unstructured synthetic peptides (22Krieg J. Hartmann S. Vicentini A. Gläsner W. Hess D. Hofsteenge J. Mol. Biol. Cell. 1998; 9: 301-309Crossref PubMed Scopus (121) Google Scholar). C-Mannosylation uses dolichyl-phosphate mannose (Dol-P-Man) as the precursor and appears to be enzyme-catalyzed within the endoplasmic reticulum (23Doucey M.A. Hess D. Cacan R. Hofsteenge J. Mol. Biol. Cell. 1998; 9: 291-300Crossref PubMed Scopus (141) Google Scholar), but the responsible mannosyltransferase has not yet been identified. A variety of mammalian cell lines can perform this modification (24Krieg J. Gläsner W. Vicentini A. Doucey M.A. Löffler A. Hess D. Hofsteenge J. J. Biol. Chem. 1997; 272: 26687-26692Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Proteins known to be C-mannosylated include human RNase 2, interleukin 12, the mucins MUC5AC and MUC5B, and several proteins containing TSRs, such as thrombospondin-1, F-spondin, and components of complement (C6 and C7) and properdin (13Gonzalez de Peredo A. Klein D. Macek B. Hess D. Peter-Katalinic J. Hofsteenge J. Mol. Cell. Proteomics. 2002; 1: 11-18Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 21Hofsteenge J. Blommers M. Hess D. Furmanek A. Miroshnichenko O. J. Biol. Chem. 1999; 274: 32786-32794Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 25Doucey M.A. Hess D. Blommers M.J. Hofsteenge J. Glycobiology. 1999; 9: 435-441Crossref PubMed Scopus (80) Google Scholar, 26Furmanek A. Hess D. Rogniaux H. Hofsteenge J. Biochemistry. 2003; 42: 8452-8458Crossref PubMed Scopus (44) Google Scholar, 27Perez-Vilar J. Randell S.H. Boucher R.C. Glycobiology. 2004; 14: 325-337Crossref PubMed Scopus (87) Google Scholar). Krieg et al. (22Krieg J. Hartmann S. Vicentini A. Gläsner W. Hess D. Hofsteenge J. Mol. Biol. Cell. 1998; 9: 301-309Crossref PubMed Scopus (121) Google Scholar) proposed a model in which the C-mannosyltransferase bound directly to the WXXW+3 motif, analogous to the Asn-X-(Thr/Ser) motif for N-glycosylation, and later analysis showed that both the Trp residues in the W0XXW+3XXX motif and the sole Trp residue in a (F/Y1)XXW+3 motif could be modified (13Gonzalez de Peredo A. Klein D. Macek B. Hess D. Peter-Katalinic J. Hofsteenge J. Mol. Cell. Proteomics. 2002; 1: 11-18Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Based on meta-analysis of the C-mannosylation literature, Julenius (28Julenius K. Glycobiology. 2007; 17: 868-876Crossref PubMed Scopus (137) Google Scholar) used a neural network approach to develop a prediction algorithm for protein C-mannosylation, termed NetCGlyc. This analysis suggested that Cys was an acceptable substitute for Trp at the +3 position (i.e. permitting C-mannosylation of W0 in a W0SSC motif). Julenius (28Julenius K. Glycobiology. 2007; 17: 868-876Crossref PubMed Scopus (137) Google Scholar) reported a clear preference for small and/or polar residues (Ser, Ala, Gly, and Thr) at the +1 position, whereas Phe and Leu were not allowed. The NetCGlyc algorithm provides a useful guide for prediction of C-mannosylation sites, especially in the ADAMTS superfamily, which has a large number of TSRs (Table 1). Nonetheless, this modification has not been experimentally identified nor functionally characterized in any ADAMTS superfamily member. In general, the functional significance of C-mannosylation is unclear, although a previous analysis of the MUC5AC and MUC5B Cys subdomains suggested it could have a role in regulation of protein secretion (27Perez-Vilar J. Randell S.H. Boucher R.C. Glycobiology. 2004; 14: 325-337Crossref PubMed Scopus (87) Google Scholar). Here we specifically inquired whether the short form of punctin-1, the prototypic ADAMTSL, is modified by C-mannosylation, analyzed the role of Trp residues in the punctin TSRs, and investigated its possible functional significance in punctin-1 biosynthesis. By demonstrating that TSR1 of ADAMTS5 is also C-mannosylated, we extended the analysis to identify this unusual modification in an ADAMTS protease.TABLE 1Predicted C-mannosylation sitesa in the ADAMTS superfamilya The full-length human reference ADAMTS sequences from GenBank™ were analyzed at the NetCGly 1.0 server for prediction of C-mannosylation sites. For prediction of O-fucosylation sites in the same peptide, the consensus sequence C1X2–3(S/T)C2 XXG was utilized.b The sequence context in which the predicted modified Trp residue occurs is provided, and the residue with predicted modification is numbered. Ser/Thr residues predicted to be O-fucosylated based on the consensus sequence CXX(S/T)C are underlined.c Sequences containing predicted C-mannosylation sites that are not within TSRs are shown in italics. a The full-length human reference ADAMTS sequences from GenBank™ were analyzed at the NetCGly 1.0 server for prediction of C-mannosylation sites. For prediction of O-fucosylation sites in the same peptide, the consensus sequence C1X2–3(S/T)C2 XXG was utilized. b The sequence context in which the predicted modified Trp residue occurs is provided, and the residue with predicted modification is numbered. Ser/Thr residues predicted to be O-fucosylated based on the consensus sequence CXX(S/T)C are underlined. c Sequences containing predicted C-mannosylation sites that are not within TSRs are shown in italics. Mammalian expression plasmids for wild-type human punctin-1 or the N-glycosylation-defective punctin-1 mutant N251Q (punctin-NQ), each having a C-terminal tandem Myc and His6 tag, were described previously (16Wang L.W. Dlugosz M. Somerville R.P. Raed M. Haltiwanger R.S. Apte S.S. J. Biol. Chem. 2007; 282: 17024-17031Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Site-directed mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene, La Jolla, CA) was used to substitute Trp residues of interest within likely motifs (Trp36, Trp39, Trp42, Trp385, and Trp445) with Ala or Phe. The primers used for mutagenesis are available on request. The CHO cell mutants Lec15.2 or Lec35.1, which lack Dol-P-Man synthase activity or the ability to utilize Dol-P-Man, respectively (23Doucey M.A. Hess D. Cacan R. Hofsteenge J. Mol. Biol. Cell. 1998; 9: 291-300Crossref PubMed Scopus (141) Google Scholar, 29Anand M. Rush J.S. Ray S. Doucey M.A. Weik J. Ware F.E. Hofsteenge J. Waechter C.J. Lehrman M.A. Mol. Biol. Cell. 2001; 12: 487-501Crossref PubMed Scopus (74) Google Scholar), were kindly provided by Dr. Mark Lehrman, University of Texas Southwestern Medical Center. These mutants, and CHO cells, were routinely grown on tissue culture plastic in Ham's F-12 medium supplemented with 15 mm HEPES, pH 7.2, 2% fetal bovine serum, 8% calf serum, and antibiotics. Transient transfections of HEK293 cells, CHO-K1 cells (both from ATCC, Manassas, VA), Lec15.2, and Lec35.1 cells were done using FuGENE 6 (Roche Diagnostics) as per the manufacturer's directions. CHO-K1 or Lec35.1 cells transiently transfected with punctin-NQ were cultured in serum-free, low glucose (0.5 mm) Dulbecco's modified Eagle's medium supplemented with 0.3 mm l-proline and 30 mCi/ml d-[2,6-3H]mannose (23Doucey M.A. Hess D. Cacan R. Hofsteenge J. Mol. Biol. Cell. 1998; 9: 291-300Crossref PubMed Scopus (141) Google Scholar). After biosynthetic labeling for 18 h, the conditioned medium was collected, and punctin-NQ was affinity-purified using Ni2+-agarose as described previously (4Hirohata S. Wang L.W. Miyagi M. Yan L. Seldin M.F. Keene D.R. Crabb J.W. Apte S.S. J. Biol. Chem. 2002; 277: 12182-12189Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). 80% of the sample was used for reducing SDS-PAGE followed by gel autoradiography as described previously (16Wang L.W. Dlugosz M. Somerville R.P. Raed M. Haltiwanger R.S. Apte S.S. J. Biol. Chem. 2007; 282: 17024-17031Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) The remaining 20% was used for reducing SDS-PAGE and Western blotting with anti-Myc antibody to ensure that punctin-1 was indeed affinity-isolated. For quantitative comparison of cellular protein content and secreted levels of punctin-NQ and Trp mutants, HEK293F or CHO cells were co-transfected with the appropriate punctin-1 plasmid and the plasmid HIgG-pRK5 for expression of the Fc portion of human IgG (16Wang L.W. Dlugosz M. Somerville R.P. Raed M. Haltiwanger R.S. Apte S.S. J. Biol. Chem. 2007; 282: 17024-17031Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Following Western blotting with anti-Myc polyclonal antibody (for punctin detection) and anti-IgG, the levels of punctin-1 and IgG were determined by densitometry. Punctin-1 levels normalized with respect to IgG were used for comparative analysis as described previously (16Wang L.W. Dlugosz M. Somerville R.P. Raed M. Haltiwanger R.S. Apte S.S. J. Biol. Chem. 2007; 282: 17024-17031Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Cellular levels of punctin-1 were similarly normalized to intracellular glyceraldehyde-3-phosphate dehydrogenase. Purification of His-tagged wild-type punctin-1 using Ni2+-agarose was described previously (4Hirohata S. Wang L.W. Miyagi M. Yan L. Seldin M.F. Keene D.R. Crabb J.W. Apte S.S. J. Biol. Chem. 2002; 277: 12182-12189Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Recombinant ADAMTS5 produced in CHO cells and encompassing residues Ser262–Glu753 was a kind gift from Dr. Elisabeth Morris at Wyeth Pharmaceuticals. Purified proteins were subjected to in solution digest, adapted from the method of Stone and Williams (30Stone K.L. Williams K.R. A Practical Guide to Protein and Peptide Purification for Microsequencing.in: Matsudaira P. Academic Press, San Diego, CA1993: 43-69Crossref Google Scholar). Briefly, ∼1 μg of protein was precipitated with 4 volumes of acetone overnight at −20 °C. Air-dried proteins were suspended in 10 μl of 8 m urea, 10 mm tris(2-carboxyethyl)phosphine, and 0.4 m di-ammonium phosphate, pH 8.0, vortexed, and heated to 50 °C for 5 min. Samples were alkylated by adding 100 mm iodoacetamide, 50 mm Tris-HCl, pH 8.0, vortexed, and incubated in the dark for 30 min. The urea was diluted to 2 m, and samples were incubated overnight at 37 °C with 150 ng of trypsin. Digested samples were acidified (with 7 μl of 5% formic acid), and particulates were removed using a 0.22-μm spin filter. One- to 5-μl aliquots were analyzed by liquid chromatography/tandem mass spectrometry using an Agilent 6340 ion-trap mass spectrometer coupled to a nano-flow HPLC-CHIP system. Samples were separated using an Agilent (Zorbax 300SB) nano-CHIP C18 column at a flow rate of 450 nl/min with a 25-min linear gradient from 5 to 95% acetonitrile in 0.1% formic acid. Effluent from the CHIP was sprayed directly into the mass spectrometer. The drying gas (nitrogen) flow rate was 5 liters/min with a drying gas temperature of 325 °C. The capillary voltage was maintained at 1800 V. Full MS scans (m/z) 300-2200 were performed, and the three most abundant ions in each spectrum were selected for collision-induced dissociation (MS/MS). Peptides with O-fucose glycans were identified by neutral loss searches for the loss of glucose (hexose, 162 Da), fucose (deoxyhexose, 146 Da), or the sequential loss of glucose-fucose (308 Da) as described previously (16Wang L.W. Dlugosz M. Somerville R.P. Raed M. Haltiwanger R.S. Apte S.S. J. Biol. Chem. 2007; 282: 17024-17031Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Peptides identified as glycosylated by neutral loss were manually selected in subsequent runs for MS/MS/MS, in which the most abundant ion from MS/MS (usually the unglycosylated parent ion) is fragmented again, yielding high intensity b and y ions to match the predicted fragmentation of unglycosylated parent peptides. The mass of unglycosylated peptides was matched to predicted tryptic peptides which contain the consensus sequence C1X2–3(S/T)C2XXG. Peptides that differed in mass by 162 Da (for each mannose) from predicted tryptic fragments that contained a WXXW (WXXWXXW) motif, with or without the consensus sequence for O-fucosylation, were subjected to MS/MS and/or MS/MS/MS. C-Mannosylation of Trp is highly stable, so modification was confirmed by both identification of b and y ions where Trp residues may have the additional 162 Da, but also by loss of 120 Da in MS/MS spectra, a characteristic cross-ring fragmentation product of aromatic C-glycosides, as well as loss of water molecules (31Hofsteenge J. Müller D.R. de Beer T. Löffler A. Richter W.J. Vliegenthart J.F. Biochemistry. 1994; 33: 13524-13530Crossref PubMed Scopus (242) Google Scholar). On examination of the sequences of the four TSRs present in the short form of punctin-1, we noticed that TSR1 contained the classic consensus sequence WXXW for C-mannosylation in tandem (Fig. 1A). TSR3 and TSR4 each contain a Trp residue with a Cys residue at the +3 position (Fig. 1A). TSR2 does not contain either the WXXW consensus sequence for C-mannosylation nor the WXXC variant motif present in TSR3 and TSR4. Analysis of human punctin-1 (GenBank™ accession number AF176313) at the NetCGLyc 1.0 server predicted that Trp39 and Trp42 in TSR1 and Trp445 in TSR4 were likely to be modified (see Table 1). Surprisingly, in contrast to the predicted modification of 445WSPC, modification of the 385WTAC motif in TSR3 was not predicted. TSR1 contains tandem consensus C-mannosylation sequences, i.e. Trp36-Asp-Trp39-Gly-Pro-Trp42-Ser-Glu-Cys, such that Trp36, Trp39, or Trp42 could each serve as the target residue for C-mannosylation, and Trp39 or Trp42 could be the +3 residue for modification of Trp36 and Trp39, respectively. N251Q punctin-1 (punctin-NQ) was used for biosynthetic analysis because this mutation eliminates the possibility of incorporation of mannose into the single N-linked oligosaccharide present in punctin-1 (4Hirohata S. Wang L.W. Miyagi M. Yan L. Seldin M.F. Keene D.R. Crabb J.W. Apte S.S. J. Biol. Chem. 2002; 277: 12182-12189Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) (Fig. 1A), and thus reports exclusively the incorporation of radiolabeled mannose at other sites. This N-glycan-deficient mutant is secreted at a lower level than wild-type punctin-1 but is nevertheless stable and is readily detected by Western blot in conditioned medium of transfected cells using anti-Myc monoclonal antibody (16Wang L.W. Dlugosz M. Somerville R.P. Raed M. Haltiwanger R.S. Apte S.S. J. Biol. Chem. 2007; 282: 17024-17031Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Affinity purification of punctin-NQ was done using the medium of metabolically labeled control CHO-K1 or mutant CHO cells that cannot utilize Dol-P-Man (Lec35.1) (29Anand M. Rush J.S. Ray S. Doucey M.A. Weik J. Ware F.E. Hofsteenge J. Waechter C.J. Lehrman M.A. Mol. Biol. Cell. 2001; 12: 487-501Crossref PubMed Scopus (74) Google Scholar). This demonstrated incorporation of d-[2,6-3H]mannose in punctin-NQ secreted from CHO-K1 cells but not from Lec35.1 cells, despite successful isolation of punctin-NQ from the medium of both cell types (Fig. 1B). These data strongly suggested that mannose is incorporated into punctin-1 at one or more sites independent of N-glycosylation. For analysis by mass spectrometry, punctin-1 was purified by nickel-Sepharose chromatography, digested with trypsin, and subjected to liquid chromatography/tandem mass spectrometry. Analysis of tryptic peptides from recombinant human punctin-1 by MS identified an ion with m/z = 892.5 (Fig. 2A, MS). This ion was consistent with the mass of the triply charged form of the peptide 29EEDRDGLWDAWGPWSECSR47 derived from TSR1 and modified by two mannose residues. MS2 fragmentation of this peptide showed characteristic cross-ring cleavage of C-mannose, resulting in sequential losses of 120 Da (40 Da for a triply charged peptide), supporting the presence of two C-mannose residues on this peptide (Fig. 2A, MS/MS). Multiple losses of water molecules (indicated by *) are also consistent with the presence of C-mannose (31Hofsteenge J. Müller D.R. de Beer T. Löffler A. Richter W.J. Vliegenthart J.F. Biochemistry. 1994; 33: 13524-13530Crossref PubMed Scopus (242) Google Scholar). In addition, a series of y ions clearly reveals the presence of C-mannose on Trp39 and Trp42. In contrast, the b10 ion demonstrates Trp36 is unmodified. These data support C-mannosylation of Trp39 and Trp42 but not Trp36.FIGURE 2TSR1 of punctin-1 is mo
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