Title :
Site-specific N-glycan characterization of human
complement factor H . Human
complement factor H (
CFH ) is a plasma
glycoprotein involved in the regulation of the alternative pathway of the complement system
Abstract :
- A deficiency in CFH is a cause of severe pathologies like atypical haemolytic uraemic syndrome (aHUS)
- 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 matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography-electrospray ionization tandem mass spectrometry (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 (Endo 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
- MS 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 and 75% for Asn864
- Diantennary monosialylated glycans and triantennary trisialylated fucosylated and non-fucosylated structures have also been identified
- Interestingly, the sialylation level along with the amount of triantennary structures decreases from the N- to the C-terminal side of the protein