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

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

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

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

^*Corresponding author for this work

Research output: Contribution to Journal › Article › Academic › peer-review

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.

Original language	English
Pages (from-to)	1225-1238
Number of pages	14
Journal	Molecular and Cellular Proteomics
Volume	17
Issue number	6
Early online date	12 Dec 2017
DOIs	https://doi.org/10.1074/mcp.RA117.000240
Publication status	Published - 1 Jun 2018

Funding

* H. Mehmet Kayılı is grateful for financial support of scholarship program (2214) of TUBITAK and VU University Amsterdam. This project was supported by the European Union (Seventh Framework Programme HighGlycan project, grant number: 278535). R.E., D.W., and S.Z. are employed by Sanquin. □S This article contains supplemental material. ¶¶ To whom correspondence should be addressed: Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands. Tel.: +31 71 5268701; Fax: +31 71 5266907; E-mail: [email protected]. ‖‖ Authors equally contributed to this work.

Funders	Funder number
TUBITAK
Seventh Framework Programme	278535
European Commission
European Geosciences Union
Vrije Universiteit Amsterdam
Türkiye Bilimsel ve Teknolojik Araştirma Kurumu	2214
Seventh Framework Programme

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title = "N- and O-glycosylation Analysis of Human C1-inhibitor Reveals Extensive Mucin-type O-Glycosylation*□S",

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.",

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AU - Holst, Stephanie

AU - Koeleman, Carolien A.M.

AU - Engel, Ruchira

AU - Wouters, Diana

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AU - Salih, Bekir

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

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