Glycoproteomic and genetic analysis of N-glycosylation of complement component C3 reveals immune pathway regulation.

BACKGROUND Post-translational modifications represent a critical layer of protein regulation, yet their genetic control and population variation remain poorly characterized. Here we present a comprehensive analysis integrating population-scale glycoproteomics with genome-wide association study (GWAS) to uncover genetic regulatory networks controlling N-glycosylation of complement component C3. RESULTS Through LC-MS glycopeptide analysis of 816 Croatian adults, we characterize site-specific N-glycosylation at two C3 sites (N63 and N917), confirming an unusual phenotype consisting of exclusive high-mannose structures rather than complex glycans typical of secreted proteins. GWAS identifies six genetic loci significantly associated with C3 N-glycosylation patterns, including genes encoding proteins involved in maintenance of the protein secretory pathway, proteins involved in the complement pathway or regulation of complement, and regulators of gene expression. Using colocalization analysis, we discover shared causal variants between C3 glycosylation and immune diseases, particularly rheumatoid arthritis and inflammatory bowel disease, suggesting glycosylation as a mechanistic link between genetic variation and disease susceptibility. C3 N-glycoprofiling reveals significant associations with sex, age, and metabolic parameters, indicating integration of genetic and environmental factors. Structural modeling provides mechanistic insights, revealing how protein architecture constrains glycan processing and enables functional glycan-mediated interactions. CONCLUSIONS Our multiomic approach establishes a framework for understanding how genetic variation shapes post-translational modifications at population scale, demonstrating C3 N-glycosylation as both a genetically and environmentally regulated checkpoint in complement activation with implications for precision medicine approaches in immune and metabolic diseases.