Production and functional activity of a recombinant von Willebrand factor-A domain from human complement factor B.
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.
Williams
S C
SC
MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.
Hinshelwood
J
J
Perkins
S J
SJ
Sim
R B
RB
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003179:Complement C3b / Q000378:metabolism; D011415:Complement Factor B / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000302:isolation & purification; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis; D014841:von Willebrand Factor / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*
D010446:Peptide Fragments; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D003179:Complement C3b; D011415:Complement Factor B