Aberrant N-glycation of Complement Factor H Contributes to Alternative Pathway Activation and Disease Progression in Diabetic Kidney Disease

Background: An increasing body of evidence points to an essential role for the activation of complement alternative pathway (AP) in the pathogenesis of diabetic kidney disease (DKD). However, the precise mechanisms of AP activation in DKD are still poorly defined. Complement factor H (CFH), the key regulator of the AP, exerts its decay acceleration activity and cofactor activity to tightly control AP activation. Of note, CFH is a glycoprotein containing 9 N-glycosites, where N-glycation probably affects its 3D structure and function. Methods: Site-specific N-glycation profile of CFH, including glycosite occupancies, glycan compositions, structural types, and branching patterns, were analyzed using high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). MS data were processed using Byonic software for glycopeptide identification. Subsequently, the AP activity and decay acceleration activity of CFH were measured by ELISA, and the cofactor activity of CFH was evaluated by SDS-PAGE. Results: Among the 9 N-glycosites, 3 sites (Asn529, Asn718, and Asn822) were unoccupied in controls but occupied in DKD patients. The remaining 6 sites showed consistent N-glycation, with 4 of them exhibiting a higher occupancy rate in DKD. A total of 147 N-glycan compositions were identified in CFH of DKD patients, far exceeding the 25 N-glycans found in controls. CFH from DKD patients revealed increased relative normalized abundances of N-glycans compared to controls. Sialylation was a common branching pattern of CFH, with higher levels observed in DKD. Both the decay acceleration activity and cofactor activity of CFH were reduced, while levels of AP activity were elevated in DKD patients. The amounts of N-glycans in CFH was positively correlated to proteinuria and negatively correlated to eGFR. Conclusion: Advanced N-glycation of CFH reduced its regulatory capacities, leading to complement AP activation. Aberrantly N-glycated CFH was demonstrated in DKD patients and associated with the activity and severity of renal injury. Funding: Government Support – Non-U.S.N-glycation profile of CFH from DKD patients and healthy controls