Production and functional activity of a recombinant von Willebrand factor-A domain from human complement factor B

Abstract

Factor B is a five-domain 90 kDa serine protease proenzyme which is part of the human serum complement system. It binds to other complement proteins C3b and properdin, and is activated by the protease factor D. The fourth domain of factor B is homologous to the type A domain of von Willebrand Factor (vWF-A). A full-length human factor B cDNA clone was used to amplify the region encoding the vWF-A domain (amino acids 229-444 of factor B). A fusion protein expression system was then used to generate it in high yield in Escherichia coli, where thrombin cleavage was used to separate the vWF-A domain from its fusion protein partner. A second vWF-A domain with improved stability and solubility was created using a Cys(267)-->Ser mutation and a four-residue C-terminal extension of the first vWF-A domain. The recombinant domains were investigated by analytical gel filtration, sucrose density centrifugation and analytical ultracentrifugation, in order to show that both domains were monomeric and possessed compact structures that were consistent with known vWF-A crystal structures. This expression system and its characterization permitted the first investigation of the function of the isolated vWF-A domain. It was able to inhibit substantially the binding of (125)I-labelled factor B to immobilized C3b. This demonstrated both the presence of a C3b binding site in this portion of factor B and a ligand-binding property of the vWF-A domain. The site at which factor D cleaves factor B is close to the N-terminus of both recombinant vWF-A domains. Factor D was shown to cleave the vWF-A domain in the presence or absence of C3b, whereas the cleavage of intact factor B under the same conditions occurs only in the presence of C3b.

The BON/CPN gene family represses cell death and promotes cell growth in Arabidopsis

“…C2 domains are calcium‐dependent phospholipid‐binding domains that confer calcium or lipid regulation activities on the proteins in which they reside (Rizo and Sudhof, 1998). The A domain of copines shows homology to the von Willebrand A domain found in integrin, and this domain is involved in protein–protein interaction (Williams et al. , 1999).…”

A Humidity-Sensitive Arabidopsis Copine Mutant Exhibits Precocious Cell Death and Increased Disease Resistance

“…The C2 domain appears to confer Ca 2 ϩ -dependent phospholipid binding activity to proteins, and mammalian copine I has Ca 2 ϩ -dependent phospholipid binding activity (Creutz et al, 1998). The A domain may be involved in extracellular protein-protein interactions, as in the case of the von Willebrand factor (Williams et al, 1999). However, available biochemical evidence indicates that copines are intracellular proteins (Creutz et al, 1998;Tomsig and Creutz, 2000).…”

Induction of Ocular Complement Activation by Inflammatory Stimuli and Intraocular Inhibition of Complement Factor D in Animal Models

“…C3b or C3H 2 O in C3FB complex was considered to be essential for FD to recognize FB and to cleave FB in in vitro studies. 54,55 Using FB breakdown product Ba to monitor FB activation in C3-deficient mice, our data provided in vivo evidence that C3 is required for FB activation. Cfb À/À mice exhibited a significant (P < 0.01) but not complete reduction in C3 breakdown products C3d and iC3b in eyes and plasma comparing between LPS-challenged Cfb À/À and WT animals (Figs.…”