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

The complement is a conserved cascade that plays a central role in the innate immune system. To maintain a delicate equilibrium preventing excessive complement activation, complement inhibitors are essential. One of the major fluid-phase complement inhibitors is C4b-binding protein (C4BP). Human C4BP is a macromolecular glycoprotein composed of two distinct subunits, C4BPα and C4BPβ. These associate with vitamin K-dependent protein S (ProS) forming an ensemble of co-occurring higher-order structures. Here, we characterize these C4BP assemblies. We resolve and quantify 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 C4BP assemblies, expanding knowledge of this key complement inhibitor's structure and composition. Finally, we reveal that an increased C4BPα to C4BPβ ratio coincides with elevated C-reactive protein levels in patient plasma samples. This observation highlights C4BP isoform variation and affirms a distinct role of co-occurring C4BP assemblies upon acute phase inflammation. Synopsis: C4b-binding protein complement inhibitor forms co-occurring assemblies in human serum. Here, by using an integrative structural biology approach, structural and compositional details of C4BP are exposed, resulting in structural models of full-length glycosylated C4BP assemblies. Integrating high-speed atomic force microscopy and cross-linking mass spectrometry we present full-length structural models of C4BP, a spider-like assembly of C4BPα and C4BPβ, non-covalently interacting with vitamin K-dependent protein S. Dominant C4BP variants in healthy human serum are C4BP(β+) captured and analyzed by single molecule mass photometry and charge detection mass spectrometry. Quantitative serum proteomics reveals that C4BP variant composition changes during acute phase inflammation, with an increase in α7 variant abundance. Structual analysis of complement inhibitor C4BP uncovers full-length spider-like assemblies of C4BPα and C4BPβ subunits and composition changes during acute phase inflammation. [ABSTRACT FROM AUTHOR]