Functional analysis of Cobra Venom Factor/human C3 chimeras transiently expressed in mammalian cells.
The complement activating venom component Cobra Venom Factor (CVF), a functional and structural homologue of the human complement component C3, forms a stable CVF-dependent C3 convertase complex, which, in contrast to C3-dependent convertase effects continuous activation of the complement and, thereby, decomplementation. In order to elucidate the mechanism underlying the enhanced activity of CVF compared to human C3, we generated two CVF/C3 chimeras and established different affinity-based assay systems for functional analysis of these constructs. To allow for convenient expression and subsequent functional characterisation, the CVF/C3 chimeras as well as CVF and C3 were transiently expressed in mammalian cells. Problems due to the low concentration of the recombinant proteins in the supernatants of transient expressions were circumvented by fusion to peptide tags enabling their efficient immobilisation onto suitable surfaces and subsequent characterisation. In an alternative approach monoclonal antibody fragments generated from a semisynthetic phage display scFv library were employed for concentrating the recombinant proteins by immunoprecipitation. Utilising both approaches all transiently expressed proteins could be characterised for their complement consumption activity. The data obtained with the CVF/C3 chimeras demonstrate that the increased stability of the CVFBb complex is independent of the domains in CVF corresponding to binding sites of factor B and H and the cleavage sites of factor I in the human C3 molecule.
Kölln
Johanna
J
Institut für Biochemie und Lebensmittelchemie, Abteilung für Biochemie und Molekularbiologie, Universität Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany.
Matzas
Mark
M
Jänner
Nathalie
N
Mix
Thorsten
T
Klensang
Katrin
K
Bredehorst
Reinhard
R
Spillner
Edzard
E
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D003176:Complement C3 / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*; D003169:Complement Inactivator Proteins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*; D006224:Cricetinae; D003412:Cricetulus; D004546:Elapid Venoms / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D016415:Sequence Alignment; D014162:Transfection
D003176:Complement C3; D003169:Complement Inactivator Proteins; D004546:Elapid Venoms; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C016310:cobra venom factor