C-Mannosylation and O-Fucosylation of Thrombospondin Type 1 Repeats
2002; Elsevier BV; Volume: 1; Issue: 1 Linguagem: Inglês
10.1074/mcp.m100011-mcp200
ISSN1535-9484
AutoresAnne Gonzalez de Peredo, Dominique Klein, Boris Maček, Daniel Heß, Jasna Peter‐Katalinić, Jan Hofsteenge,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoThe final chemical structure of a newly synthesized protein is often only attained after further covalent modification. Ideally, a comprehensive proteome analysis includes this aspect, a task that is complicated by our incomplete knowledge of the range of possible modifications and often by the lack of suitable analysis methods. Here we present two recently discovered, unusual forms of protein glycosylation, i.e. C-mannosylation and O-fucosylation. Their analysis by a combined mass spectrometric approach is illustrated with peptides from the thrombospondin type 1 repeats (TSRs) of the recombinant axonal guidance protein F-spondin. Nano-electrospray ionization tandem-mass spectrometry of isolated peptides showed that eight of ten Trp residues in the TSRs of F-spondin are C-mannosylated. O-Fucosylation sites were determined by a recently established nano-electrospray ionization quadrupole time-of-flight tandem-mass spectrometry approach. Four of five TSRs carry the disaccharide Hex-dHex-O-Ser/Thr in close proximity to the C-mannosylation sites. In analogy to thrombospondin-1, we assume this to be Glc-Fuc-O-Ser/Thr. Our current knowledge of these glycosylations will be discussed. The final chemical structure of a newly synthesized protein is often only attained after further covalent modification. Ideally, a comprehensive proteome analysis includes this aspect, a task that is complicated by our incomplete knowledge of the range of possible modifications and often by the lack of suitable analysis methods. Here we present two recently discovered, unusual forms of protein glycosylation, i.e. C-mannosylation and O-fucosylation. Their analysis by a combined mass spectrometric approach is illustrated with peptides from the thrombospondin type 1 repeats (TSRs) of the recombinant axonal guidance protein F-spondin. Nano-electrospray ionization tandem-mass spectrometry of isolated peptides showed that eight of ten Trp residues in the TSRs of F-spondin are C-mannosylated. O-Fucosylation sites were determined by a recently established nano-electrospray ionization quadrupole time-of-flight tandem-mass spectrometry approach. Four of five TSRs carry the disaccharide Hex-dHex-O-Ser/Thr in close proximity to the C-mannosylation sites. In analogy to thrombospondin-1, we assume this to be Glc-Fuc-O-Ser/Thr. Our current knowledge of these glycosylations will be discussed. Ideally, the complete description of a proteome should include the definition of co- and post-translational modifications. The covalent attachment of carbohydrates is a wide spread feature of secreted, cytoplasmic, and nuclear proteins (1Varki A. Cummings C. Esko J.D. Freeze H. Hart G.W. Marth J.D. Essentials of Glycobiology. 1st Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York1999Google Scholar). Although N-glycosidic attachment of GlcNAc to Asn residues and GalNAc O-linked to Ser/Thr have been known for a relatively long time (2Gottschalk A. Gottschalk A. Historical Introduction in Glycoroteins. Elsevier Science Publishers B.V., Amsterdam1966: 1-19Google Scholar, 3Gottschalk A. Carbohydrate-Peptide Linkages in Glycoproteins and Methods for Their Elucidation in Glycoproteins. Elsevier Science Publishers B.V., Amsterdam1966: 273-295Google Scholar), only more recently the existence of a large variety of protein-carbohydrate linkages has been revealed (1Varki A. Cummings C. Esko J.D. Freeze H. Hart G.W. Marth J.D. Essentials of Glycobiology. 1st Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York1999Google Scholar). Here we present two of these, including their analysis by a combined mass spectrometric approach. The covalent attachment of an α-mannopyranosyl residue to the C-2 atom of the side chain of Trp was initially described in human RNase 2. The structure of this glycoconjugate (Fig. 1), which does not contain a typical glycosidic linkage but rather a C-C bond, has been based on MS 1The abbreviations used are: MS, mass spectrometry; CID, collision-induced dissociation; MSMS, tandem-mass spectrometry; Q-TOF, quadrupole time-of-flight; rF-spondin, recombinant F-spondin; TSP-1, thrombospondin-1; TSR, thrombospondin type 1 repeat, ESI, electrospray ionization; EGF, epidermal growth factor; LC, liquid chromatography; PMP-C, pars intercerebralis major peptide C. and NMR studies (4Hofsteenge J. Müller D.R. de Beer T. Löffler A. Richter W.J. Vliegenthart J.F.G. New type of linkage between a carbohydrate and a protein: C-glycosylation of a specific tryptophan residue in human RNase Us.Biochemistry. 1994; 33: 13524-13530Scopus (236) Google Scholar, 5de Beer T. Vliegenthart J.F.G. Löffler A. Hofsteenge J. The hexopyranosyl residue that is C-glycosidically linked to the side chain of tryptophan-7 in human RNase Us is α-mannopyranose.Biochemistry. 1995; 34: 11785-11789Google Scholar). The reaction is catalyzed by a microsomal transferase, which uses dolichyl-phosphate-mannose as the sugar donor and recognizes, in nearly all cases, the sequence Trp-X-X-Trp (6Krieg J. Hartmann S. Vicentini A. Gläsner W. Hess D. Hofsteenge J. The recognition signal for C-mannosylation of Trp-7 in RNase 2 consists of the sequence Trp-x-x-Trp.Mol. Biol. Cell. 1998; 9: 301-309Google Scholar, 7Doucey M.-A. Hess D. Cacan R. Hofsteenge J. Protein C-mannosylation is enzyme-catalysed and uses dolichyl-phosphate-mannose as a precursor.Mol. Biol. Cell. 1998; 9: 291-300Google Scholar). The transferase activity can be detected in organisms ranging from Caenorhabditis elegans to man (8Doucey, M.-A. (1998) La C-mannosylation du tryptophane: un nouveau type de glycosylation des protéines, Ph.D. thesis, Université Louis Pasteur de StrassbourgGoogle Scholar). Presently, a total of 49 C-mannosylated tryptophan residues have been identified, derived from 11 different proteins, performing a wide variety of functions (for a review see Ref. 9Furmanek A. Hofsteenge J. Protein C-mannosylation: facts and questions.Acta Biochim. Pol. 2000; 47: 781-789Google Scholar). Evidence for C-mannosylation and for its site of attachment can conveniently be obtained in CID MSMS experiments of isolated peptides. Here we show examples of this kind of analysis and summarize our current knowledge of this modification. Fucose was long considered to be a terminal sugar of oligosaccharides. This changed, however, when fucose O-linked to Ser or Thr was discovered in the EGF-like modules from proteins that play an important role in fibrinolysis and coagulation (for an overview, see Refs. 10Harris R.J. Spellman M.W. O-Linked fucose and other post-translational modification unique to EGF modules.Glycobiology. 1993; 3: 219-224Google Scholar and 11Vliegenthart J.F.G. Casset F. Novel forms of protein glycosylation.Curr. Opin. Struct. Biol. 1998; 8: 565-571Google Scholar). There exist at least two pathways for the extension of O-linked fucose (12Moloney D.J. Lin A.I. Haltiwanger R.S. The O-linked fucose glycosylation pathway.J. Biol. Chem. 1997; 272: 19046-19050Google Scholar). The first one leads to the formation of the tetrasaccharide NeuAc-α2–6-Gal-β1,4-GlcNac-β1,3-Fuc-α1-O-Ser/Thr, as found in factor IX and Notch (13Harris R.J. van Halbeek H. Glushka J. Basa L.J. Ling V.T. Smith K.J. Spellman M.W. Identification and structural analysis of the tetrasaccharide NeuAc α(2→6)Galβ(1→4)GlcNAcβ(1→3)Fucα1→O-linked to serine 61 of human factor IX.Biochemistry. 1993; 32: 6539-6547Google Scholar, 14Nishimura H. Takao T. Hase S. Shimonishi Y. Iwanaga S. Human factor IX has a tetrasaccharide O-glycosidically linked to Serine 61 through the fucose residue.J. Biol. Chem. 1992; 267: 17520-17525Google Scholar, 15Moloney D.J. Shair L.H. Lu F.M. Xia J. Locke R. Matta K.L. Haltiwanger R.S. Mammalian Notch 1 is modified with two unusual forms of O-linked glycosylation found on epidermal growth factor-like modules.J. Biol. Chem. 2000; 275: 9604-9611Google Scholar). For Notch, it has been shown that receptor-ligand interactions are modulated by extension of the of O-linked fucosyl residues (16Moloney D.J. Panin V.M. Johnston S.H. Chen J. Shao L. Wislon R. Wang Y. Stanley P. Irvine K.D. Haltiwanger R.S. Vogt T.F. Fringe is a glycosyltransferase that modifies notch.Nature. 2000; 406: 369-375Google Scholar, 17Brückner K. Perez L. Clausen H. Cohen S. Glycosyltransferase activity of Fringe modulates Notch-Delta interactions.Nature. 2000; 406: 411-415Google Scholar). The second pathway yields the disaccharide Glc-β1,3-Fuc-α1-O-Ser/Thr, first found as the amino acid glycoside in human urine (18Hallgren P. Lundblad A. Svensson S. A new type of carbohydrate-protein linkage in a glycopeptide from normal human urine.J. Biol. Chem. 1975; 250: 5312-5314Google Scholar). Recently, we discovered that all three TSRs in TSP-1 carry this disaccharide in close proximity to C-mannosylation sites. Nano-ESI Q-TOF mass spectrometry has proven to be very useful in the determination of O-fucosylation sites (19Macek B. Hofsteenge J. Peter-Katalinic J. Direct determination of glycosylation sites in O-fucosylated glycopeptides using nano-electrospray quadrupole time-of-flight mass spectrometry.Rapid Commun. Mass Spectrom. 2001; 15: 771-777Google Scholar, 20Hofsteenge J. Huwiler K.G. Macek B. Hess D. Lawler J. Mosher D.F. Peter-Katalinic J. C-Mannosylation and O-fucosylation of the thrombospondin type 1 module.J. Biol. Chem. 2001; 276: 6485-6498Google Scholar). Here we use the same approach in the analysis of rF-spondin. This protein also contains TSRs and is secreted by the floor plate during development of the nervous system (21Klar A. Baldassare M. Jessell T.M. F-sponding: a gene expressed at high levels in the floor pate encodes a secreted protein that promotes neural cell adhesion and neurite extension.Cell. 1992; 69: 95-110Google Scholar). It functions as a guidance protein for commissural neurons by attracting their axons to the midline during rostral growth. Recombinant F-spondin was expressed in COS-7 cells. 2Manuscript in preparation. Plasmid pSecF-spo-His, encoding residues 27–752 of rat F-spondin followed by a Myc tag and a His6 sequence, was a generous gift of Dr. A. Klar, Hebrew University-Hadassah Medical School, Jerusalem, Israel (21Klar A. Baldassare M. Jessell T.M. F-sponding: a gene expressed at high levels in the floor pate encodes a secreted protein that promotes neural cell adhesion and neurite extension.Cell. 1992; 69: 95-110Google Scholar). Peptides for the analysis of the C-mannosylation and O-glycosylation sites were obtained from aminoethylated and carbamidomethylated rF-spondin, respectively. The former was cleaved with endoproteinase Lys-C and trypsin, whereas the latter was digested with trypsin. Digests were fractionated by C8 reversed phase LC-ESI MS using a Sciex API 300 triple quadrupole mass spectrometer, as described previously (22Krieg J. Gläsner W. Vincentini A. Doucey M.-A. Löffler A. Hess D. Hofsteenge J. Protein C-mannosylation is an intracellular process, performed by a variety of cultured cells.J. Biol. Chem. 1997; 272: 26687-26692Google Scholar, 23Hofsteenge J. Blommers M.M. Hess D. Furmanek A. Miroshnichenko O. The four terminal components of the complement system are C-mannosylated on multiple tryptophan residues.J. Biol. Chem. 1999; 274: 32786-32794Google Scholar). Fractions were collected and analyzed using the same instrument. Peptides have been numbered according to their occurrence in mature F-spondin (21Klar A. Baldassare M. Jessell T.M. F-sponding: a gene expressed at high levels in the floor pate encodes a secreted protein that promotes neural cell adhesion and neurite extension.Cell. 1992; 69: 95-110Google Scholar). The analysis of C-mannosylated peptides by nano-ESI MSMS using a Sciex API 300 triple quadrupole mass spectrometer and Edman degradation has been described previously (23Hofsteenge J. Blommers M.M. Hess D. Furmanek A. Miroshnichenko O. The four terminal components of the complement system are C-mannosylated on multiple tryptophan residues.J. Biol. Chem. 1999; 274: 32786-32794Google Scholar). Determination of O-fucosylation sites was performed by low energy CID tandem-MS in the nanospray mode, using a Q-TOF instrument (Micromass, Manchester, United Kingdom), equipped with a Z-spray atmospheric pressure ionization source operated at 30 °C. The 2pressure of the collision gas (Ar) in the hexapole collision cell was 2.7 × 10−5 millibar, and the collision energy was typically 20 eV. Details of the method have been described (19Macek B. Hofsteenge J. Peter-Katalinic J. Direct determination of glycosylation sites in O-fucosylated glycopeptides using nano-electrospray quadrupole time-of-flight mass spectrometry.Rapid Commun. Mass Spectrom. 2001; 15: 771-777Google Scholar). C-Mannosylation occurs only on secreted proteins, 3T. Smilda, A. Ulvestad, J. Krieg, A. Vicentini, and J. Hofsteenge, manuscript in preparation. which, in addition to this modification, also often contain N- and/or O-linked oligosaccharides. This, and the fact that only a single mannosyl residue (mass, 162 Da) is added in the process of C-mannosylation, makes it difficult to detect (C2-Man-)Trp in the intact protein. Therefore, the isolation and analysis of relevant peptides is required. Toward that aim, a proteolytic digest of the protein is fractionated by reversed phase LC-MS. Subsequently, the MS data are extracted for the theoretical masses of the peptides containing the recognition sequence Trp-X-X-Trp, assuming the presence of one or more C-linked mannosyl residues. As an example, the detection of pep6tide KT81 (LVTEWGEWDDC545), one of the C-mannosylated peptides from rF-spondin, is shown in Fig. 2A. The peptide, containing a single C-mannosylated Trp residue (theoretical mass, 1396 Da, average mass), is identified eluting at 38.3 min. Strong indication for the presence of a (C2-Man-)Trp residue can already be obtained at this stage. Because of the stability of the C-C bond in aromatic C-glycosides, these compounds display a characteristic loss of 120 Da in low energy CID experiments (4Hofsteenge J. Müller D.R. de Beer T. Löffler A. Richter W.J. Vliegenthart J.F.G. New type of linkage between a carbohydrate and a protein: C-glycosylation of a specific tryptophan residue in human RNase Us.Biochemistry. 1994; 33: 13524-13530Scopus (236) Google Scholar, 24Li Q.M. van den Heuvel H. Dillen L. Claeys M. Differentiation of 6-C and 8-C-glycosidic flavonoids by positive ion fast atom bombardment and tandem-mass spectrometry.Biol. Mass Spectrom. 1992; 21: 213-221Google Scholar, 25Becchi M. Fraisse D. Fast atom bombardment and fast atom bombardment collision-activated dissociation/mass-analysed ion kinetic energy analysis of C-glycosidic flavonoids.Biomed. Environ. Mass Spectrom. 1989; 18: 122-130Google Scholar). Probably as a result from in-source CID, this phenomenon can also be observed in LC-MS experiments, as illustrated for peptide KT81 in Fig. 2B. This behavior clearly distinguishes C-mannosylated peptides from ones that contain O-linked mannose, which show a loss of 162 Da under similar LC-MS conditions (Fig. 3). Information on the position of the modified Trp residue can be obtained form an MSMS experiment. The results (Fig. 4) confirm the identity of the peptide and provide strong evidence for C-hexosylation of Trp539. Because all hexoses add a mass of 162 Da to the Trp residue, MS does not reveal the identity of the sugar involved. Initially the proof for a C-α-mannosyl residue has been obtained by NMR experiments on peptides from human RNase 2 (4Hofsteenge J. Müller D.R. de Beer T. Löffler A. Richter W.J. Vliegenthart J.F.G. New type of linkage between a carbohydrate and a protein: C-glycosylation of a specific tryptophan residue in human RNase Us.Biochemistry. 1994; 33: 13524-13530Scopus (236) Google Scholar), as well as on the intact protein (26Löffler A. Doucey M.-A. Jansson A.M. Müller D.R. de Beer T. Hess D. Meldal M. Richter W.J. Vliegenthart J.F.G. Hofsteenge J. Spectroscopic and protein chemical analyses demonstrate the presence of C-mannosylated tryptophan in intact human RNase 2 and its isoforms.Biochemistry. 1996; 35: 12005-12014Google Scholar). Furthermore, NMR analysis of peptides from interleukin 12β and complement factor C9 also yielded mannose as the sugar present (23Hofsteenge J. Blommers M.M. Hess D. Furmanek A. Miroshnichenko O. The four terminal components of the complement system are C-mannosylated on multiple tryptophan residues.J. Biol. Chem. 1999; 274: 32786-32794Google Scholar, 27Doucey M.-A. Hess D. Blommers M.J. Hofsteenge J. Recombinant human interleukin-12 is the second example of a C-mannosylated protein.Glycobiology. 1999; 9: 435-441Google Scholar). In addition, solid phase Edman degradation can be used for the analysis of smaller amounts of the peptides. Using an optimized elution protocol, phenylthiohydantoin-(C2-Man-)Trp elutes shortly after phenylthiohydantoin-Tyr (28Hofsteenge J. Löffler A. Müller D.R. Richter W.J. de Beer T. Vliegenthart J.F.G. Marshak D.R. Protein C-glycosylation in techniques in protein chemistry VII. Academic Press, New York1996: 163-171Google Scholar). The elution position of phenylthiohydantoin-(Hex)Trp, with hexoses other than Man, remains to be established.Fig. 3Comparison of the behavior of C- and O-linked mannose in LC-ESI MS experiments. The experiment was performed on an API 300 triple quadrupole mass spectrometer. A, spectrum of the peptide F-T-(C2-Man-)W-A isolated from human RNase 2. B, spectrum of the synthetic peptide F-(O-Man-)T-W-A.View Large Image Figure ViewerDownload (PPT)Fig. 4Low energy CID tandem-ESI MS of the C-mannosylated peptide KT61 from rF-spondin. The experiment was performed on an API 300 triple quadrupole mass spectrometer. The 120-Da loss, typical for aromatic C-glycosides, has been indicated with 120 and 60 for singly and doubly charged ions, respectively. Losses of H2O have been indicated with asterisks. W#, C-mannosylated tryptophan. Cysteine residues have been aminoethylated.View Large Image Figure ViewerDownload (PPT) Following this approach we have detected 48 (C2-Man-Trp) residues in 10 different proteins (Table I). In addition, it seems probable also that a neuropeptide from Carausius morosus is C-glycosylated (29Gäde G. Keller R. Rinehart K.L. Proefke M.L. A tryptophan-substituted member of the AKH/RPCH family isolated from a stick insect corpus cardiacum.Biochem. Biophys. Res. Comm. 1992; 189: 1303-1309Google Scholar).TABLE ISummary of C-mannosylated sequencesProteinFunctionNumber of (C2-Man-)TrpSequenceaC-Mannosylated Trp residues have been indicated in bold (fully modified) or bold, italic (partially modified). For estimates of the degree of modification, see the original references.ReferenceRNase 2Host defence (?)1TWAQWF4Hofsteenge J. Müller D.R. de Beer T. Löffler A. Richter W.J. Vliegenthart J.F.G. New type of linkage between a carbohydrate and a protein: C-glycosylation of a specific tryptophan residue in human RNase Us.Biochemistry. 1994; 33: 13524-13530Scopus (236) Google ScholarInterleukin 12βCytokine receptor homologue1SWSEWA27Doucey M.-A. Hess D. Blommers M.J. Hofsteenge J. Recombinant human interleukin-12 is the second example of a C-mannosylated protein.Glycobiology. 1999; 9: 435-441Google ScholarHypertrehalosemic hormoneNeuropeptide1 (?)bGäde et al. (29) hypothesize that the unknown hexose is attached to N1 of the indole.FTPNWGTG29Gäde G. Keller R. Rinehart K.L. Proefke M.L. A tryptophan-substituted member of the AKH/RPCH family isolated from a stick insect corpus cardiacum.Biochem. Biophys. Res. Comm. 1992; 189: 1303-1309Google ScholarProteins with TSRsC6Complement; forms, together with C5, C7, C8α,6DHYAWTQWT23Hofsteenge J. Blommers M.M. Hess D. Furmanek A. Miroshnichenko O. The four terminal components of the complement system are C-mannosylated on multiple tryptophan residues.J. Biol. Chem. 1999; 274: 32786-32794Google ScholarC8β, and C9, the membrane attack complexLLGDFGPWSQWGCWSSWSC7Complement4QWDFYAPWS23Hofsteenge J. Blommers M.M. Hess D. Furmanek A. Miroshnichenko O. The four terminal components of the complement system are C-mannosylated on multiple tryptophan residues.J. Biol. Chem. 1999; 274: 32786-32794Google ScholarGWSCWSSWSC8αComplement4QLSNWSEWT23Hofsteenge J. Blommers M.M. Hess D. Furmanek A. Miroshnichenko O. The four terminal components of the complement system are C-mannosylated on multiple tryptophan residues.J. Biol. Chem. 1999; 274: 32786-32794Google ScholarSWSCWSSWSC8βComplement4ELSSWSSWT23Hofsteenge J. Blommers M.M. Hess D. Furmanek A. Miroshnichenko O. The four terminal components of the complement system are C-mannosylated on multiple tryptophan residues.J. Biol. Chem. 1999; 274: 32786-32794Google ScholarKWNCWSNWSC9Complement2RMSPWSEWS23Hofsteenge J. Blommers M.M. Hess D. Furmanek A. Miroshnichenko O. The four terminal components of the complement system are C-mannosylated on multiple tryptophan residues.J. Biol. Chem. 1999; 274: 32786-32794Google ScholarProperdinPositive regulator of complement15RWSLWSTWA37Hartmann S. Hofsteenge J. Properdin, the positive regulator of the complement, is highly C-mannosylated.J. Biol. Chem. 2000; 275: 28569-28574Google ScholarGWSGWGPWEAWATWGPWTGWGPWGPVSEWDSWGEWSSWSEWSTWGThrombospondin-1TGF-β activation4GWSPWSEWT20Hofsteenge J. Huwiler K.G. Macek B. Hess D. Lawler J. Mosher D.F. Peter-Katalinic J. C-Mannosylation and O-fucosylation of the thrombospondin type 1 module.J. Biol. Chem. 2001; 276: 6485-6498Google ScholarAngiogenesis inhibitorGWSHWSPWSGWGPWSPWDF-spondinAxonal guidance8IYSNWSPWSFootnote 3T. Smilda, A. Ulvestad, J. Krieg, A. Vicentini, and J. Hofsteenge, manuscript in preparation.TMSEWITWSLVTEWGEWDLLSPWSEWSELSEWSQWSa C-Mannosylated Trp residues have been indicated in bold (fully modified) or bold, italic (partially modified). For estimates of the degree of modification, see the original references.b Gäde et al. (29Gäde G. Keller R. Rinehart K.L. Proefke M.L. A tryptophan-substituted member of the AKH/RPCH family isolated from a stick insect corpus cardiacum.Biochem. Biophys. Res. Comm. 1992; 189: 1303-1309Google Scholar) hypothesize that the unknown hexose is attached to N1 of the indole. Open table in a new tab The recent discovery of the O-linked disaccharide Glc-Fuc-O-Ser/Thr in TSP-1 raises the question whether the same modification also occurs in other proteins that contain TSRs. We examined recombinant rF-spondin and properdin and show results obtained with the former to illustrate the approach. Relevant peptides were produced by tryptic digestion of the carbamidomethylated protein. To simplify the analysis, a secondary digestion with an appropriate protease was performed when needed. Cleavage of peptide Thr61 with endoproteinase Asp-N yields peptide T61D (Asp-Asp-Cys-Ser-Ala-Thr548-Cys-Gly-Met-Gly-Met-Lys) with a monoisotopic mass of 1639.59 Da, which is 308 Da heavier than predicted from the cDNA sequence. Together with the partial loss of the substituent in low energy LC-MS experiments (data not shown), this strongly suggested the presence of O-linked sugars. Because nano-Q-TOF MSMS allows the direct determination of glycosylation sites in O-fucosylated glycopeptides (19Macek B. Hofsteenge J. Peter-Katalinic J. Direct determination of glycosylation sites in O-fucosylated glycopeptides using nano-electrospray quadrupole time-of-flight mass spectrometry.Rapid Commun. Mass Spectrom. 2001; 15: 771-777Google Scholar), we analyzed all potential O-fucosylation sites in rF-spondin by this method. As an example, the results obtained with the doubly charged molecular ion of peptide T61D are shown in Fig. 5. The observation of B fragment ions (nomenclature of Domon and Costello (30Domon B. Costello C.E. A systematic nomencalture for carbohydrate fragmentation in FAB-MS/MS spectra of glycoconjugates.Glycoconjugate J. 1988; 5: 397-409Google Scholar)) of the Hex-dHex disaccharide (m/z = 309.13), the Hex (m/z = 163.07), and dHex (m/z = 147.09), 4The resolution was sufficient to distinguish the sugar oxonium ion from the y1 ion. as well as ions corresponding to their neutral loss of water, provides evidence for the presence of a disaccharide, rather than two monosaccharides. Furthermore, the consecutive loss of the Hex− and dHex− residues strongly indicates that the disaccharide is attached through the dHex. In addition to the deglycosylated fragment ions that confirmed the identity of the peptide, ions y7, y9, and b11 were observed with Hex-dHex and dHex still attached (Fig. 5). Despite the sometimes low abundance of these ions, their signals displayed good signal-to-noise ratios and high resolution (Fig. 5, inset). The results strongly indicated that Thr548 carries the Hex-dHex disaccharide. Following this approach, the same O-glycosylations were also found in TSR-1, -2, and -4. Table II summarizes all O-fucosylation sites that have been found in TSRs to date. It should be noted that whereas for TSP-1 and properdin, the identity of the monosaccharides have been shown to be Glc and Fuc, this has not yet been determined for the peptides from rF-spondin.TABLE IIO-Fucosylation sites in the TSRs of TSP-1, properdin, and rF-spondina TSR1 of rF-spondin is O-glycosylated, but the exact site of attachment could not yet be determined. This has been indicated in lower case. Open table in a new tab a TSR1 of rF-spondin is O-glycosylated, but the exact site of attachment could not yet be determined. This has been indicated in lower case. The results presented here emphasize the abundance of C-mannosylation in TSRs. In fact, all 24 TSRs examined to date are modified in this way (Table I). However, other proteins, and possibly other protein families, also contain this glycosylation. For example, interleukin 12β is a member of the type 1 cytokine receptor superfamily and C-mannosylated on Trp319 (27Doucey M.-A. Hess D. Blommers M.J. Hofsteenge J. Recombinant human interleukin-12 is the second example of a C-mannosylated protein.Glycobiology. 1999; 9: 435-441Google Scholar). Interestingly, nearly all members of this superfamily contain the so-called "WSXWS" box (31Bazan J.F. Stuctural design and molecular evolution of a cytokine receptor superfamily.Proc. Natl. Acad. Sci. 1990; 87: 6934-6938Google Scholar). The analysis of these proteins will be of interest, because mutations in this box in the erythropoietin receptor cause its transport to the cell surface to stop at the endoplasmic reticulum-Golgi interface (32Hilton D.J. Watowich S.S. Katz L. Lodish H.F. Saturation mutagenesis of the WSXWS motif of the erythropoietin receptor.J. Biol. Chem. 1996; 271: 4699-4708Google Scholar). The stoichiometry of C-mannosylation shown in Table I differs for different Trp residues. In 35 cases full modification was observed, whereas in the remainder, it has been estimated to vary from <10 to 95% (see references in Table I for details). Estimating the degree of modification can be difficult. In case the modified and unmodified peptides are obtained in pure form from the initial LC-MS experiment, their ratio can be obtained by integrating their peaks in the UV trace. If contamination with other peptides occurs, this procedure cannot be used. An approximate ratio may be obtained from integration of the peaks in the total ion current trace. However, this will only give very approximate estimates, because of e.g. differences in ionization efficiency of modified and unmodified peptides (for a discussion see Ref. 23Hofsteenge J. Blommers M.M. Hess D. Furmanek A. Miroshnichenko O. The four terminal components of the complement system are C-mannosylated on multiple tryptophan residues.J. Biol. Chem. 1999; 274: 32786-32794Google Scholar). Clearly, more work is needed on this aspect of the analysis of C-mannosylation and covalent modifications in general. The results obtained with TSRs raise a question with respect to the specificity of C-mannosylation. Both studies in vivo with RNase 2 and in vitro studies using synthetic peptides have revealed that the recognition motif for the transferase is Trp-X-X-Trp, where only the first Trp becomes modified (6Krieg J. Hartmann S. Vicentini A. Gläsner W. Hess D. Hofsteenge J. The recognition signal for C-mannosylation of Trp-7 in RNase 2 consists of the sequence Trp-x-x-Trp.Mol. Biol. Cell. 1998; 9: 301-309Google Scholar, 7Doucey M.-A. Hess D. Cacan R. Hofsteenge J. Protein C-mannosylation is enzyme-catalysed and uses dolichyl-phosphate-mannose as a precursor.Mol. Biol. Cell. 1998; 9: 291-300Google Scholar, 22Krieg J. Gläsner W. Vincentini A. Doucey M.-A. Löffler A. Hess D. Hofsteenge J. Protein C-mannosylation is an intracellular process, performed by a variety of cultured cells.J. Biol. Chem. 1997; 272: 26687-26692Google Scholar, 27Doucey M.-A. Hess D. Blommers M.J. Hofsteenge J. Recombinant human interleukin-12 is the second example of a C-mannosylated protein.Glycobiology. 1999; 9: 435-441Google Scholar). This specificity does, however, not hold for the TSRs. Of the 24 C-mannosylated TSRs, 22 contain the sequence Trp-X-X-Trp-X-X-X, without a Trp or other aromatic residue at position +3 relative to the second Trp. In 7 of these, only the first Trp is modified, in agreement with the RNase 2 results. However, in the other 15, both Trp residues are modified. Similarly, 2 TSRs contain the C-mannosylated sequence φ-X-X-Trp (φ = Tyr, Phe) (Table I). A possible explanation for these observations is that features outside of the motif determine whether one or both Trps become modified. Alternatively, more than one C-mannosyltransferase might exist. Cloning of the enzyme(s) catalyzing this reaction and the study of their enzymological properties should resolve this issue. Based on antibody inhibition studies, it has been proposed that the activity of F-spondin in axonal guidance is located in the TSRs (33Burstyn-Cohen T. Tzarfaty V. Frumkin A. Feinstein Y. Stoeckli E. Klar A. F-Spondin is required for accurate pathfinding of commissural axons at the floor plate.Neuron. 1999; 23: 233-246Google Scholar). Whether the Trp-X-X-Trp motifs are involved is not known. By far the most studies on this motif have been performed with TSP-1. Synthetic peptides containing C-mannosylation motifs were shown to bind to and/or inhibit the interactions of TSP-1 with fibronectin, transforming growth factor-β, and heparin (34Adams J.C. Tucker R.P. The thrombospondin type 1 repeat (TSR) superfamily: diverse proteins with related roles in neuronal development.Dev. Dyn. 2000; 218: 280-299Google Scholar, 35Lawler J. The functions of thrombospondin-1 and -2.Curr. Opin. Cell Biol. 2000; 12: 634-640Google Scholar). It should be noted, however, that in all cases non-C-mannosylated peptides were used (for a review see Ref. 34Adams J.C. Tucker R.P. The thrombospondin type 1 repeat (TSR) superfamily: diverse proteins with related roles in neuronal development.Dev. Dyn. 2000; 218: 280-299Google Scholar). The two Trp residues were shown to be important for the interaction with heparin. Interestingly, the neurite-extending activity of F-spondin can be inhibited by this glycosaminoglycan. The gas phase instability of O-linked fucose poses a challenging problem for the determination of the exact attachment site in peptides and proteins by MS methods. Because of the high sensitivity and excellent signal-to-noise ratio of Q-TOF instruments, it is often possible to successfully characterize this modification in peptides. The sugar-containing b- and y-ions, needed for the identification, are always detected at 10–100-fold lower levels than the corresponding unglycosylated ones. Using low picomolar amounts of peptide, low collision energy, and long acquisition times (see "Experimental Procedures"), glycosylated ions can often unambiguously be identified with nano-ESI Q-TOF MSMS. Using this approach, we have identified three O-fucosylation sites in TSP-1, four in properdin, and four in rF-spondin (note that in this case the identity of the sugars remains to be determined). Whereas TSR2–4 in rF-spondin are fully modified at the sites indicated in Table II, TSR1 appears to be modified for less than 10%. In properdin, O-fucosylation was complete in TSR2–4, but it was only ∼50% in TSR1. Examination of the TSR superfamily reveals that 63 of 88 TSRs contain a Ser or Thr at the position corresponding to the O-fucosylation sites reported here (20Hofsteenge J. Huwiler K.G. Macek B. Hess D. Lawler J. Mosher D.F. Peter-Katalinic J. C-Mannosylation and O-fucosylation of the thrombospondin type 1 module.J. Biol. Chem. 2001; 276: 6485-6498Google Scholar). This suggests that the modification occurs in many more proteins. In all cases the O-fucosylation and C-mannosylation sites are in close proximity, yet it seems unlikely that one requires the other. Protein chemical studies on recombinant TSR3 from TSP-1 revealed the presence of (C2-Man-)Trp, Glc-Fuc-O-Thr, and Fuc-O-Thr, which occurred in all six possible combinations, strongly suggesting that the two glycosylations occur independently (20Hofsteenge J. Huwiler K.G. Macek B. Hess D. Lawler J. Mosher D.F. Peter-Katalinic J. C-Mannosylation and O-fucosylation of the thrombospondin type 1 module.J. Biol. Chem. 2001; 276: 6485-6498Google Scholar). Three forms of O-fucosylation are presently known, attachment of (i) a single fucosyl residue to the insect proteinase inhibitor PMP-C and EGF modules (10Harris R.J. Spellman M.W. O-Linked fucose and other post-translational modification unique to EGF modules.Glycobiology. 1993; 3: 219-224Google Scholar, 36Nakakura N. Hietter H. Van Dorselaer A. Luu B. Isolation and structural determination of three peptides from the instect Locusta migratoria. Identification of a deoxyhexose-linked peptide.Eur. J. Biochem. 1992; 204: 147-153Google Scholar), (ii) NeuAc-Gal-GlcNAc-Fuc- to EGF modules (15Moloney D.J. Shair L.H. Lu F.M. Xia J. Locke R. Matta K.L. Haltiwanger R.S. Mammalian Notch 1 is modified with two unusual forms of O-linked glycosylation found on epidermal growth factor-like modules.J. Biol. Chem. 2000; 275: 9604-9611Google Scholar), and (iii) Glc-Fuc- to TSR modules (20Hofsteenge J. Huwiler K.G. Macek B. Hess D. Lawler J. Mosher D.F. Peter-Katalinic J. C-Mannosylation and O-fucosylation of the thrombospondin type 1 module.J. Biol. Chem. 2001; 276: 6485-6498Google Scholar) (this work). In addition to the functional studies on Notch mentioned in the Introduction, those on EGF-like domains in other proteins suggest that O-fucosylation has a function. Urokinase-type plasminogen activator is mitogenic for a number of cells in culture, and this activity depends on its EGF-like domain that carries a fucosyl residue on Thr18. The defucosylated domain binds as tightly to cells as the fucosylated one, but it is not able to produce the mitogenic signal (38Rabbani S.A. Mazar A.P. Bernier S.M. Haq M. Bolivar I. Henkin J. Goltzman D. Structural requirements for the growth factor activity of the amino-terminal domain of urokinase.J. Biol. Chem. 1992; 267: 14151-14156Google Scholar, 39Schnaper H.W. Barnathan E.S. Mazar A. Maheshwari S. Ellis S. Cortez S.L. Baricos W.H. Kleinman H.K. Plasminogen activators augment endothelial cell organization in vitro by two distinct pathways.J. Cell. Physiol. 1995; 165: 107-118Google Scholar, 40Koopman J.L. Slomp J. de Bart A.C. Quax P.H. Verheijen J.H. Mitogenic effects of urokinase on melanoma cells are independent of high affinity binding to the urokinase receptor.J. Biol. Chem. 1998; 273: 33267-33272Google Scholar). The effect of O-linked fucose on protein structure has been examined in PMP-C (41Mer G. Hietter H. Lefevre J.-F. Stabilization of proteins by glycosylation examined by NMR analysis of a fucosylated inhibitor.Nat. Struct. Biol. 1996; 3: 45-52Google Scholar) and the EGF-like domain derived from blood coagulation factor VII (42Kao Y.H. Lee G.F. Wang Y. Starovasnik M.A. Kelley R.F. Spellman M.W. Lerner L. The effect of O-fucosylation on the first EGF-like domain from human blood coagulation factor VII.Biochemistry. 1999; 38: 7097-7110Google Scholar). In both cases little difference in structure was found between the protein with and without fucose. However, the dynamic fluctuations of PMP-C were decreased, and its stability was increased upon O-fucosylation. The overall structures of PMP-C and the EGF-like module from factor VII are different. However, both are all-β proteins with a central β-sheet and three disulfide bridges. TSR modules are likely to have a β-sheet structure, as well, based on their homology with the all-β proteins HB-GAM (heparin-binding growth-associated molecule) and midkine (43Kilpeläinen I. Kaksonen M. Kinnunen T. Avikainen H. Fath M. Linhardt R. Raulo E. Rauvala H. Heparin-binding growth-associated molecule contains two heparin-binding β-sheet domains that are homologous to the thrombospondin type 1 repeat.J. Biol. Chem. 2000; 275: 13564-13570Google Scholar). If this proposition is correct, the O-fucosylation site in TSRs is located in a loop between the first two β-sheets. What is the function of O-fucosylation in TSRs is not known yet. However, in a number of TSR proteins the sequence in which O-fucosylation takes place has been implicated in cell binding. For instance, the cell surface receptor CD36 that mediates the inhibition of neovascularization by TSP-1 in vivo has been proposed to bind to sequences CSVTCG (fucosylation site in bold; for a review see Ref. 34Adams J.C. Tucker R.P. The thrombospondin type 1 repeat (TSR) superfamily: diverse proteins with related roles in neuronal development.Dev. Dyn. 2000; 218: 280-299Google Scholar). It is important to note that unmodified synthetic peptides were used in these studies. The Golgi is the only secretory compartment containing a transporter for GDP-Fuc (44Abeijon C. Mandon E.C. Hirschberg C.B. Transporters of nucleotide sugars, nucleotide sulfate and ATP in the golgi apparatus.Trends Biochem. Sci. 1997; 22: 203-207Google Scholar). It seems likely that O-fucosylation of TSR modules, like that of EGF modules (10Harris R.J. Spellman M.W. O-Linked fucose and other post-translational modification unique to EGF modules.Glycobiology. 1993; 3: 219-224Google Scholar), takes place there. Leukocyte adhesion deficiency II has recently been attributed to an aberrant form of the GDP-Fuc transporter, causing a general lack of fucosylation of glycoconjugates (45Luhn K. Wild M.K. Eckhardt M. Gerardy-Schahn R. Vestweber D. The gene defective in leukocyte adhesion deficiency II encodes a putative GDP-fucose transporter.Nat. Genet. 2001; 28: 69-72Google Scholar, 46Lubke T. Marquardt T. Etzioni A. Hartmann E. von Figura K. Korner C. Complementation cloning identifies CDG-IIc, a new type of congenital disorders of glycosylation, as a GDP-fucose transporter deficiency.Nat. Genet. 2001; 28: 73-76Google Scholar). The leukocyte-associated defects in this disease can be understood by the lack of α1,3-linked fucose in Lewis X antigen-based selectin ligands (47Becker D.J. Lowe J.B. Leukocyte adhesion deficiency type II.Biochim Biophys. Acta. 1999; 1455: 193-204Google Scholar). However, the non-fucosylated glycoconjugates that cause the defects in mental development are unknown. It is, therefore, interesting to point out that the TSR proteins TSP-1, F-spondin, Unc-5, and the semaphorins 5A and B all play a role in neuronal development (34Adams J.C. Tucker R.P. The thrombospondin type 1 repeat (TSR) superfamily: diverse proteins with related roles in neuronal development.Dev. Dyn. 2000; 218: 280-299Google Scholar). Because O-fucosylation has now been found in the first two of these proteins, and several putative sites are also present in the others, analysis of their O-fucosylation status and functional properties in the context of the disease seems warranted. We thank Dr. Avihu Klar, Hebrew University, Jerusalem for providing the rF-spondin expression vector and Renate Matthies for help with protein sequencing.
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