N- and O-glycosylation Analysis of Human C1-inhibitor Reveals Extensive Mucin-type O-Glycosylation

Human C1-inhibitor (C1-Inh) is a serine protease inhibitor and the major regulator of the contact activation pathway as well as the classical and lectin complement pathways. It is known to be a highly glycosylated plasma glycoprotein. However, both the structural features and biological role of C1-Inh glycosylation are largely unknown. Here, we performed for the first time an in-depth site-specific N- and O-glycosylation analysis of C1-Inh combining various mass spectrometric approaches, including C18-porous graphitized carbon (PGC)-LC-ESI-QTOF-MS/MS applying stepping-energy collision-induced dissociation (CID) and electron-transfer dissociation (ETD). Various proteases were applied, partly in combination with PNGase F and exoglycosidase treatment, in order to analyze the (glyco)peptides. The analysis revealed an extensively O-glycosylated N-terminal region. Five novel and five known O-glycosylation sites were identified, carrying mainly core1-type O-glycans. In addition, we detected a heavily O-glycosylated portion spanning from Thr82-Ser121 with up to 16 O-glycans attached. Likewise, all known six N-glycosylation sites were covered and confirmed by this site-specific glycosylation analysis. The glycoforms were in accordance with results on released N-glycans by MALDI-TOF/TOF-MS/MS. The comprehensive characterization of C1-Inh glycosylation described in this study will form the basis for further functional studies on the role of these glycan modifications. Human C1-inhibitor (C1-Inh) is a serine protease inhibitor and the major regulator of the contact activation pathway as well as the classical and lectin complement pathways. It is known to be a highly glycosylated plasma glycoprotein. However, both the structural features and biological role of C1-Inh glycosylation are largely unknown. Here, we performed for the first time an in-depth site-specific N- and O-glycosylation analysis of C1-Inh combining various mass spectrometric approaches, including C18-porous graphitized carbon (PGC)-LC-ESI-QTOF-MS/MS applying stepping-energy collision-induced dissociation (CID) and electron-transfer dissociation (ETD). Various proteases were applied, partly in combination with PNGase F and exoglycosidase treatment, in order to analyze the (glyco)peptides. The analysis revealed an extensively O-glycosylated N-terminal region. Five novel and five known O-glycosylation sites were identified, carrying mainly core1-type O-glycans. In addition, we detected a heavily O-glycosylated portion spanning from Thr82-Ser121 with up to 16 O-glycans attached. Likewise, all known six N-glycosylation sites were covered and confirmed by this site-specific glycosylation analysis. The glycoforms were in accordance with results on released N-glycans by MALDI-TOF/TOF-MS/MS. The comprehensive characterization of C1-Inh glycosylation described in this study will form the basis for further functional studies on the role of these glycan modifications. Human C1-inhibitor (C1-Inh) 1The abbreviations used are: AmBiC, ammonium bicarbonate; C1-Inh, C1-Inhibitor; ENeuAc, α2,6-linked N-acetylneuraminic acid; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; ETD, electron-transfer dissociation; FA, formic acid; Fuc, fucose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; Hereditary angioedema, HAE; Hex, hexose; HexNAc, N-acetylhexosamine; HILIC, hydrophilic interaction liquid chromatography; HOBt, hydroxybenzotriazol hydrate; IAA, iodoacetamide; IPQ score, isotopic pattern quality score; IT, ion trap; LNeuAc, α2,3-linked N-acetylneuraminic acid; Na2HPO4x2H2O, sodium hydrogen phosphate dihydrate; NaOH, sodium hydroxide; NeuAc, N-acetylneuraminic acid; NP-40, Nonidet P-40; PGC, porous graphitized carbon; PNGase F, peptide-N-glycosidase F; RP, reversed-phase; sDHB, 2-hydroxy-5-methoxy-benzoic acid and 2,5-dihydroxybenzoic acid; Serpin, serine protease inhibitor; SPE, solid phase extraction. 1The abbreviations used are: AmBiC, ammonium bicarbonate; C1-Inh, C1-Inhibitor; ENeuAc, α2,6-linked N-acetylneuraminic acid; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; ETD, electron-transfer dissociation; FA, formic acid; Fuc, fucose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; Hereditary angioedema, HAE; Hex, hexose; HexNAc, N-acetylhexosamine; HILIC, hydrophilic interaction liquid chromatography; HOBt, hydroxybenzotriazol hydrate; IAA, iodoacetamide; IPQ score, isotopic pattern quality score; IT, ion trap; LNeuAc, α2,3-linked N-acetylneuraminic acid; Na2HPO4x2H2O, sodium hydrogen phosphate dihydrate; NaOH, sodium hydroxide; NeuAc, N-acetylneuraminic acid; NP-40, Nonidet P-40; PGC, porous graphitized carbon; PNGase F, peptide-N-glycosidase F; RP, reversed-phase; sDHB, 2-hydroxy-5-methoxy-benzoic acid and 2,5-dihydroxybenzoic acid; Serpin, serine protease inhibitor; SPE, solid phase extraction. is a serine protease inhibitor (serpin) and the major regulator of the contact activation pathway via inhibition of factor XIIa, kallikrein and factor XIa, as well as the classical and lectin complement pathways via C1s, C1r, and MASP (1.Caliezi C. Wuillemin W.A. Zeerleder S. Redondo M. Eisele B. Hack C.E. C1-esterase inhibitor : an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema.Pharmacol. Rev. 2000; 52: 91-112PubMed Google Scholar). A C1-Inh deficiency, either because of decreased or dysfunctional expression, is associated with hereditary angioedema (HAE), which results in vascular permeability causing tissue swelling (1.Caliezi C. Wuillemin W.A. Zeerleder S. Redondo M. Eisele B. Hack C.E. C1-esterase inhibitor : an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema.Pharmacol. Rev. 2000; 52: 91-112PubMed Google Scholar). For treatment and prophylaxis of HAE human plasma-derived C1-Inh replacement therapy is commonly applied (2.Bowen T. Cicardi M. Farkas H. Longhurst H.J. Zuraw B. Aygoeren-Pürsün E. Craig T. Binkley K. Hebert J. Ritchie B. Bouillet L. Betschel S. Cogar D. Dean J. Devaraj R. Hamed A. Kamra P. Keith P.K. Lacuesta G. Leith E. Lyons H. Mace S. Mako B. Neurath D. Poon M.C. Rivard G.E. Schellenberg R. Rowan D. Rowe A. Stark D. Sur S. Tsai E. Warrington R. Waserman S. Ameratunga R. Bernstein J. Björkander J. Brosz K. Brosz J. Bygum A. Caballero T. Frank M. Fust G. Harmat G. Kanani A. Kreuz W. Levi M. Li H. Martinez-Saguer I. Moldovan D. Nagy I. Nielsen E.W. Nordenfelt P. Reshef A. Rusicke E. Smith-Foltz S. Späth P. Varga L. Xiang Z.Y. 2010 International consensus algorithm for the diagnosis, therapy and management of hereditary angioedema.Allergy, Asthma, Clin. Immunol. 2010; 6: 24Crossref PubMed Google Scholar). Also other diseases, such as inflammatory diseases, sepsis and endotoxic shock, may be targeted by C1-Inh therapy (1.Caliezi C. Wuillemin W.A. Zeerleder S. Redondo M. Eisele B. Hack C.E. C1-esterase inhibitor : an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema.Pharmacol. Rev. 2000; 52: 91-112PubMed Google Scholar, 3.Davis A.E. Lu F. Mejia P. C1 inhibitor, a multi-functional serine protease inhibitor.Thromb. Haemost. 2010; 104: 886-893Crossref PubMed Scopus (164) Google Scholar). To meet this need, recombinant C1-Inh formats are currently being developed (4.Koles K. van Berkel P.H.C. Mannesse M.L.M. Zoetemelk R. Vliegenthart J.F. Kamerling J.P. Influence of lactation parameters on the N-glycosylation of recombinant human C1 inhibitor isolated from the milk of transgenic rabbits.Glycobiology. 2004; 14: 979-986Crossref PubMed Scopus (27) Google Scholar, 5.Koles K. van Berkel P.H.C. Pieper F.R. Nuijens J.H. Mannesse M.L. Vliegenthart J.F. Kamerling J.P. N- and O-glycans of recombinant human C1 inhibitor expressed in the milk of transgenic rabbits.Glycobiology. 2004; 14: 51-64Crossref PubMed Scopus (93) Google Scholar, 6.Bos I.G.A. De Bruin E.C. Karuntu Y.A. Modderman P.W. Eldering E. Hack C.E. Recombinant human C1-inhibitor produced in Pichia pastoris has the same inhibitory capacity as plasma C1-inhibitor.Biochim Biophys Acta. 2003; 1648: 75-83Crossref PubMed Scopus (27) Google Scholar, 7.Lamark T. Ingebrigtsen M. Bjørnstad C. Melkko T. Mollnes T.E. Nielsen E.W. Expression of active human C1 inhibitor serpin domain in Escherichia coli.Protein Expr. Purif. 2001; 22: 349-358Crossref PubMed Scopus (36) Google Scholar). C1-Inh is considered as one of the most heavily glycosylated proteins in human plasma (1.Caliezi C. Wuillemin W.A. Zeerleder S. Redondo M. Eisele B. Hack C.E. C1-esterase inhibitor : an anti-inflammatory agent and its potential use in the treatment of diseases other than hereditary angioedema.Pharmacol. Rev. 2000; 52: 91-112PubMed Google Scholar). The protein consists of 478 amino acids and the calculated molecular mass of C1-Inh is ∼53 kDa without glycans, whereas a much higher apparent molecular mass was observed on SDS-PAGE (>80kDa) because of its heavy glycosylation S. D. of the and of C1 inhibitor of human complement by PubMed Scopus Google Scholar, J.H. Hack C.E. of plasma C1 inhibitor in Clin. PubMed Scopus Google Scholar). It has C1-Inh six N- and up to O-glycosylation sites S. D. of the and of C1 inhibitor of human complement by PubMed Scopus Google Scholar, A. G. J. characterization of O-glycosylation sites and glycan of human PubMed Scopus Google Scholar, K. Nielsen E. C1 and PubMed Scopus Google Scholar, G. H. of of inhibitor (C1-Inh) by R. Google Scholar, H.J. A. A. the O-glycosylation of human and PubMed Scopus Google Scholar). the with A. G. J. characterization of O-glycosylation sites and glycan of human PubMed Scopus Google Scholar, K. Nielsen E. C1 and PubMed Scopus Google Scholar, H.J. A. A. the O-glycosylation of human and PubMed Scopus Google Scholar). The protein consists of the domain which of the six N-glycosylation the inhibition of C1-Inh and is to other and the N-terminal which consists of amino acid amino acids in the the N- and all O-glycosylation sites K. Nielsen E. C1 and PubMed Scopus Google Scholar, and molecular of Google Scholar). protein glycosylation has a on biological protein and protein the of Rev. PubMed Scopus Google Scholar, E. and of N-glycans in N- PubMed Google the structural features as well as biological role of C1-Inh glycosylation is largely unknown. 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G. J. characterization of O-glycosylation sites and glycan of human PubMed Scopus Google Scholar, H.J. A. A. the O-glycosylation of human and PubMed Scopus Google Scholar, A. J. W. S. P. B. H. Human analysis of N- and by and PubMed Scopus Google and were confirmed in Likewise, was confirmed from human plasma H.J. A. A. the O-glycosylation of human and PubMed Scopus Google Scholar). and are known from amino acid studies K. Nielsen E. C1 and PubMed Scopus Google and were for the first time confirmed by mass spectrometric The of the sites and has revealed by amino acid and in the O-glycosylation of and K. Nielsen E. C1 and PubMed Scopus Google Scholar). the and with and one are largely with mass spectrometric of up to 16 of the O-glycosylation sites in the the analysis of Thr82-Ser121 for the first time the O-glycosylation of this region. to the of the O-glycosylation sites as well as the O-glycosylation are for the first In a study on plasma O-glycosylation was as an O-glycosylation H.J. A. A. the O-glycosylation of human and PubMed Scopus Google which was confirmed by the glycoprotein. However, we the glycosylation of this In the we mainly and O-glycans and of O-glycans for the glycosylation sites It be the other O-glycosylation sites of which were be with and because these sites by A. G. J. characterization of O-glycosylation sites and glycan of human PubMed Scopus Google Scholar, H.J. A. A. the O-glycosylation of human and PubMed Scopus Google Scholar, A. J. W. S. P. B. H. Human analysis of N- and by and PubMed Scopus Google Scholar). 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Influence of lactation parameters on the N-glycosylation of recombinant human C1 inhibitor isolated from the milk of transgenic rabbits.Glycobiology. 2004; 14: 979-986Crossref PubMed Scopus (27) Google Scholar, S. D. of the and of C1 inhibitor of human complement by PubMed Scopus Google Scholar, G. H. of of inhibitor (C1-Inh) by R. Google Scholar, of of C1-inhibitor and of PubMed Scopus Google Scholar). In with we confirmed the glycosylation of all six N-glycosylation and C1-Inh N-glycans S. D. of the and of C1 inhibitor of human complement by PubMed Scopus Google Scholar, G. H. of of inhibitor (C1-Inh) by R. Google and of and glycan S. D. of the and of C1 inhibitor of human complement by PubMed Scopus Google Scholar). is in accordance with the major detected in of and glycan detected by on site-specific N-glycosylation of C1-Inh has A detected on J. A. C. E. G. G. of for glycan and 6: PubMed Scopus Google whereas we detected of than of glycan this The role of C1-Inh glycosylation has and with to its functional in a The of as of the protein in from in because of the in the The role of acid in the functional and the of Immunol. Google Scholar). However, of human C1-Inh with PNGase F, or both on its protease inhibition A. and of human C1 on functional PubMed Scopus Google Scholar, A. P. L. D. C. C1 as a The of on its and Immunol. PubMed Scopus Google Scholar). were for the of C1-Inh glycosylation in the inhibition of the and human C1-Inh its to the further protein this A. P. L. D. C. C1 as a The of on its and Immunol. PubMed Scopus Google Scholar). results the of N-terminal O-glycosylation in C1-Inh protease inhibition in the contact In studies the protease inhibition of recombinant C1-Inh and with of the first or amino the highly glycosylated N-terminal the same interaction with C1s, kallikrein or factor acid including the recombinant in P. pastoris and E. I.G.A. D. J.P. Hack C.E. Eldering E. The functional of the serpin domain of C1-inhibitor on the N-terminal as revealed by a 2003; PubMed Scopus Google Scholar, M. A.E. analysis of the serpin domain of C1 Immunol. Google Scholar). The has described to be a because of the of the S. D. of the and of C1 inhibitor of human complement by PubMed Scopus Google Scholar, K. Nielsen E. C1 and PubMed Scopus Google Scholar, E. H. and of human PubMed Scopus (27) Google Scholar). In the N-terminal either because of its has to with the interaction of C1-Inh with kallikrein and factor S. T.E. M. of plasma kallikrein by role of and the domain of Haemost. 2004; PubMed Scopus Google Scholar, I. of factor to its by the C1-esterase J. 2003; PubMed Scopus Google Scholar). It may be the N- and O-glycosylation the and and as well as of the N-terminal the of we the highly O-glycosylated with up to sites was to protease treatment, glycosylation C1-Inh from In we applied a for in-depth site-specific characterization of C1-Inh glycosylation O-glycosylation sites carrying mainly with five of being In addition, we a heavily O-glycosylated portion of C1-Inh spanning from Thr82-Ser121 with up to 16 O-glycans attached. Likewise, we covered all six N-glycosylation sites of C1-Inh by site-specific glycosylation analysis. and on C1-Inh glycosylation will to functional studies and is for targeted studies to the role of glycosylation of this plasma glycoprotein. we plasma-derived human C1-Inh which will be to further the glycosylation of expressed are the for the with