Genetic C3 variants reveal functional independence between extracellular and intracellular forms of complement C3

Complement component C3 is the central effector of extracellular complement activation, but recent studies have identified intracellular forms of C3 with distinct non-canonical functions. How pathogenic C3 variants affect intracellular C3 biology remains unknown. Here, we investigated two previously described C3 deficiency-associated variants, c.168_169del and c.1682G > A (p.G561D), linked to severe plasma C3 depletion. Unexpectedly, both variants remained detectable intracellularly despite markedly reduced extracellular C3 levels observed in the patients. The c.168_169del frameshift variant, predicted to abolish protein expression, generated a smaller non-secreted pro-C3 species lacking glycosylation and localized exclusively to the cytosol. Cellular fractionation, antigenic characterization, and split-GFP localization assays using cell models demonstrated that this protein resembles a non-canonical cytosolic C3 isoform generated through alternative translation initiation downstream of the mutation site. These findings provide clinical support for the existence of cytosol-restricted C3 generated independently of the canonical secretory pathway. In contrast, the p.G561D variant displayed normal intracellular expression, antigenicity, glycosylation, and secretion but showed impaired alternative pathway convertase activity, increased susceptibility to factor H/factor I-mediated degradation, and enhanced spontaneous turnover in serum, explaining the severe plasma C3 depletion observed in the patient. Collectively, our results demonstrate that pathogenic C3 variants can exert fundamentally different effects on extracellular and intracellular C3 pools. These findings support the concept that intracellular and extracellular C3 represent functionally independent compartments and highlight the importance of integrating intracellular complement biology into the interpretation of complement-associated diseases.