Abstract
Human complement factor H (CFH) plays a central role in regulating activated C3b to protect host cells. CFH contain 20 short complement regulator (SCR) domains and eight N-glycosylation sites. The N-terminal SCR domains mediate C3b degradation while the C-terminal CFH domains bind to host cell surfaces to protect these. Our earlier study of Pichia-generated CFH fragments indicated a self-association site at SCR-17/18 that comprises a dimerization site for human factor H. Two N-linked glycans are located on SCR-17 and SCR-18. Here, when we expressed SCR-17/18 without glycans in an Escherichia coli system, analytical ultracentrifugation showed that no dimers were now formed. To investigate this novel finding, full-length CFH and its C-terminal fragments were purified from human plasma and Pichia pastoris respectively, and their glycans were enzymatically removed using PNGase F. Using size-exclusion chromatography, mass spectrometry, and analytical ultracentrifugation, SCR-17/18 from Pichia showed notably less dimer formation without its glycans, confirming that the glycans are necessary for the formation of SCR-17/18 dimers. By surface plasmon resonance, affinity analyses interaction showed decreased binding of deglycosylated full-length CFH to immobilized C3b, showing that CFH glycosylation enhances the key CFH regulation of C3b. We conclude that our study revealed a significant new aspect of CFH regulation based on its glycosylation and its resulting dimerization.