Higher-order structure and proteoforms of co-occurring C4b-binding protein assemblies in human serum

The complement is an evolutionarily conserved proteolytic cascade that plays a central role in the innate immune system. To maintain a delicate equilibrium preventing excessive complement activation, complement inhibitors are essential alongside complement activators. One of the major fluid-phase complement inhibitors is C4b-binding protein (C4BP). Human C4BP is a macromolecular glycoprotein composed of three distinct subunits, namely C4BPα, C4BPβ, and vitamin K-dependent protein S (ProS), which form an ensemble of coexisting higher-order structures. Here, we characterize these co-occurring higher-order assemblies of human serum C4BP. We resolve, quantify and characterize isoforms of purified human serum C4BP using distinct single-particle detection techniques: charge detection mass spectrometry, and mass photometry accompanied by high-speed atomic force microscopy. Combining cross-linking mass spectrometry, glycoproteomics, and structural modeling, we report comprehensive glycoproteoform profiles and full-length structural models of the endogenous co-occurring C4BP assemblies, expanding knowledge of this key complement inhibitor’s structure and composition. Finally, we reveal that increased C4BPα to C4BPβ ratio coincides with elevated C-reactive protein levels in patient serum samples. This observation highlights C4BP isoform variation and affirms the distinct role of co-occurring distinct C4BP assemblies upon acute phase inflammation.