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

• Published in: Molecular & cellular proteomics Vol. 17; no. 6; pp. 1225 - 1238
• Main Authors: Stavenhagen, Kathrin, Kayili, H. Mehmet, Holst, Stephanie, Koeleman, Carolien A.M., Engel, Ruchira, Wouters, Diana, Zeerleder, Sacha, Salih, Bekir, Wuhrer, Manfred
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2018; American Society for Biochemistry and Molecular Biology; The American Society for Biochemistry and Molecular Biology
• Subjects: Complement activation; Complement C1 Inhibitor Protein - metabolism; Complement system; Energy of dissociation; Glycan; Glycomics; Glycoproteins; Glycoproteomics; Glycosylation; Graphitization; Humans; Mass Spectrometry; Mucin; N-glycans; O-glycosylation; Peptides; Plasma or serum analysis; Polysaccharides; Polysaccharides - metabolism; Post-translational modifications; Protease inhibitors; Proteinase inhibitors; Serine; Serine proteinase; Spectrometry; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tandem Mass Spectrometry; Glycoproteomics; Post-translational modifications; Mass Spectrometry; Glycomics; O-glycosylation; Glycoproteins; Complement system; Plasma or serum analysis
• ISSN: 1535-9476; 1535-9484; 1535-9484
• DOI: 10.1074/mcp.RA117.000240

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.