Expression of functional recombinant von Willebrand factor-A domain from human complement C2: a potential binding site for C4 and CRIT

• Published in: Biochemical journal Vol. 389; no. Pt 3; pp. 863 - 868
• Main Authors: Hui, Kwok-Min, Orriss, George L, Schirmer, Tilman, Magnadóttir, Bergljót, Schifferli, Jürg A, Inal, Jameel M
• Format: Journal Article
• Language: English
• Published: England Portland Press Ltd 01.08.2005
• Subjects: Amino Acid Motifs; Binding Sites; Carrier Proteins - metabolism; Complement C2 - chemistry; Complement C2 - metabolism; Complement C4 - metabolism; Complement Inactivator Proteins - metabolism; Humans; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins; von Willebrand Factor - chemistry; von Willebrand Factor - metabolism
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/BJ20050183

CRIT (complement C2 receptor inhibitor trispanning) is a newly described transmembrane molecule that is capable of binding C2 via its first extracellular domain (ed1). CRIT competes with C4b for the binding of C2. Previous experiments have suggested that a major binding site for C2 is located on short, almost identical peptide sequences of CRIT-ed1 and the beta-chain of C4. The C2 domains involved in binding, however, remain unknown. We cloned the vWFA (von Willebrand factor-A) domain of C2, as it is a region likely to be involved in interactions with other proteins, and were able to functionally express the 25 kDa human complement C2 vWFA domain (amino acids 224-437). The recombinant vWFA protein fixed on MagneHis Ni-Particles bound C4 in normal human serum. The C4 alpha, beta and gamma chains were separated by SDS/PAGE and purified separately by electro-elution. The purified C4 chains were then used in a sandwich ELISA, which showed the vWFA to bind C4 only via the C4beta chain. In a haemolytic assay, the recombinant vWFA protein inhibited complement activation by the classical pathway in a dose-dependent manner by competing with native C2 for binding to C4b. vWFA bound the ed1 peptide of CRIT as well, and specifically to the 11-amino-acid peptide fragment of ed1 that is known to interact with whole C2. These findings show that the vWFA domain is centrally involved in the C2-CRIT and C2-C4b bindings. The cloned vWFA domain will allow us to dissect out the fine interactions between C2 and CRIT or C4b.