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

Kathrin Stavenhagen,H Mehmet Kayili,Stephanie Holst,Carolien A.M Koeleman,Ruchira Engel,Diana Wouters,Sacha Zeerleder,Bekir Salih,Manfred Wuhrer

doi:10.1074/mcp.ra117.000240

Abstract

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.

Highlights

To address this, we here present a detailed site-specific Nand O-glycosylation characterization of plasma derived C1Inh using a panel of mass spectrometric approaches
To further decrease sample heterogeneity and enhance O-glycosylation site identification a portion of these digests were de-N-glycosylated by PNGase F and partially processed with exoglycosidases such as sialidase and galactosidase. This approach aimed to trim down short mucin-type O-glycans, to obtain O-glycopeptides with a single N-acetylhexosamine (HexNAc) or HexNAchexose (Hex) moiety attached to the O-glycosylation site, which allowed a more reliable site-specific analysis
Samples were analyzed by nanoLC-MS/MS analysis applying different tandem MS modes to obtain more structural information of the glycopeptides

Summary

Introduction

We here present a detailed site-specific Nand O-glycosylation characterization of plasma derived C1Inh using a panel of mass spectrometric approaches. The C1-Inh glycosylation as studied here will inform further func-. From the ‡Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands; §Division of BioAnalytical Chemistry, VU University Amsterdam, Amsterdam, The Netherlands; ¶Department of Chemistry, Cankırı Karatekin University, Cankırı, Turkey; ʈDepartment of Nutrition and Dietetics, Karabuk University, Karabuk, Turkey; **Department of Chemistry, Hacettepe University, Ankara, Turkey; ‡‡Department of Immunopathology, Sanquin Research and Landsteiner Laboratory of the AMC, Amsterdam, The Netherlands; §§Department of Hematology, Academic Medical Center, University of Amsterdam, The Netherlands. Human C1-Inhibitor Glycosylation tional studies in order to understand glycan involvement in C1-Inh function. This plasma-derived human C1Inh glycosylation will serve as a benchmark for evaluating the glycosylation profiles of recombinant C1-Inh

Methods

Results

Conclusion