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

Hui, Kwok-Min and Orriss, George L. and Schirmer, Tilman and Magnadóttir, Bergljót and Schifferli, Jürg A. and Inal, Jameel M.. (2005) Expression of functional recombinant von Willebrand factor-A domain from human complement C2 : a potential binding site for C4 and CRIT. The Biochemical Journal, Vol. 389, Pt. 3. pp. 863-868.

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Official URL: http://edoc.unibas.ch/dok/A5258242

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Abstract

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.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Schirmer)
UniBasel Contributors:Schirmer, Tilman
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:Portland Press
ISSN:0264-6021
Note:Publication type according to Uni Basel Research Database: Journal article
Last Modified:07 Aug 2015 12:05
Deposited On:22 Mar 2012 13:20

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