Expression of functional recombinant von Willebrand factor-A domain from human complement C2: a potential binding site for C4 and CRIT.
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.
Hui
Kwok-Min
KM
University Hospital Basel, Immunonephrology, Department of Research, Hebelstrasse 20, CH-4031 Basel, Switzerland. kwok-min.hui@unibas.ch
Orriss
George L
GL
Schirmer
Tilman
T
Magnadóttir
Bergljót
B
Schifferli
Jürg A
JA
Inal
Jameel M
JM
D020816:Amino Acid Motifs; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D003175:Complement C2 / Q000737:chemistry* / Q000378:metabolism; D003181:Complement C4 / Q000378:metabolism*; D003169:Complement Inactivator Proteins / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D014841:von Willebrand Factor / Q000737:chemistry* / Q000378:metabolism*
C505022:CRIT protein, human; D002352:Carrier Proteins; D003175:Complement C2; D003181:Complement C4; D003169:Complement Inactivator Proteins; D011994:Recombinant Proteins; D014841:von Willebrand Factor