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Glycobiology Advance Access published online on June 25, 2007
Glycobiology, doi:10.1093/glycob/cwm060
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Site-Specific N-Glycan Characterization of Human Complement Factor H

François Fenaille, Maxime Le Mignon, Catherine Groseil, Christine Ramon, Sandrine Riandé, Laurent Siret and Nicolas Bihoreau*

Laboratoire français du Fractionnement et des Biotechnologies, Développement Préclinique, 3 avenue des Tropiques, BP305 Les Ulis, 91958 Courtaboeuf cedex, France

E-mail address of the corresponding author: bihoreau{at}lfb.fr, fax number: 00 33 1 69 82 71 70; phone number: 00 33 1 69 82 73 38

Received on April 12, 2007; accepted on June 2, 2007

Human complement factor H (CFH) is a plasma glycoprotein involved in the regulation of the alternative pathway of the complement system. A deficiency in CFH is a cause of severe pathologies like atypical hemolytic uremic syndrome. CFH is a 155 kDa glycoprotein containing nine potential N-glycosylation sites. In the current study, we present a quantitative glycosylation analysis of CFH using capillary electrophoresis and a complete site-specific N-glycan characterization using MALDI-TOF/TOF and LC-ESIMS/MS. A 17.9 kDa mass decrease, observed after glycosidase treatment, indicated that N-glycosylation is the major post-translational modification of CFH. This mass difference is consistent with CFH glycosylation by diantennary disialylated glycans of 2204 Da on eight sites. CFH was not sensitive to endoglycosidase H deglycosylation, indicating the absence of hybrid and oligomannose structures. Quantitative analysis showed that CFH is mainly glycosylated by complex, diantennary disialylated, non-fucosylated glycans. Disialylated fucosylated and monosialylated non-fucosylated oligosaccharides were also identified. Mass spectrometry analysis allowed complete characterization of the protein backbone, verification of the glycosylation sites and site-specific N-glycan identification. The absence of glycosylation at Asn199 of the NGSP sequence of CFH is shown. Asn511, Asn700, Asn784, Asn804, Asn864, Asn893, Asn1011 and Asn1077 are glycosylated essentially by diantennary disialylated structures with a relative distribution varying between 45% for Asn804 to 75% for Asn864. Diantennary monosialylated glycans and triantennary trisialylated fucosylated and non-fucosylated structures have been also identified. Interestingly, the sialylation level along with the amount of triantennary structures decrease from the N- to the C-terminal side of the protein.

Key words: Complement Factor H / Glycosylation / Mass Spectrometry


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[Abstract] [Full Text] [PDF]


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