Recombinant generation of two fragments of the rat complement inhibitory factor H [FH(SCR1-7) and FH(SCR1-4)] and their structural and functional characterization in comparison to FH isolated from rat serum.
Factor H (FH) is the predominant soluble inhibitor of the complement system. With a concentration of 200-800 microg/ml in human and rat plasma it acts as a cofactor for the soluble factor I (FI)-mediated cleavage of the component C3b to iC3b. Furthermore it competes with factor B for binding to C3b and C3(H2O) and promotes the dissociation of the C3bBb complex. FH is a monomer of about 155 kDa which comprises 20 short consensus repeats (SCR), each of which is composed of approximately 60 amino acid (aa) residues. Two functional fragments of FH comprising the SCR1-4 or SCR1-7 were generated using either the Baculovirus system or stably transfected human embryonal kidney cells, respectively. These fragments, as well as FH purified from rat serum, were first analyzed for their relative molecular weights (Mr) using non-reducing or reducing SDS-PAGE. The Mr of the FH variants differed by about 20% depending on the experimental conditions employed. Only the Mr of proteins separated under reducing conditions were in accordance with the MW calculated from the aa sequence. Analyses of the glycosylation patterns using PAS-staining showed a lack of staining of the recombinant variants (SCR1-4 and SCR1-7) in contrast to FH(SCR1-20) from serum. Using a complement hemolysis assay (CH50-assay) all three variants exhibited a molar complement inhibitory activity of FH(1-20)/FH(1-7)/FH(1-4) of about 3/1/1. These data support the postulated model of FH bearing three binding sites for its ligand C3b, from which one is located in the SCR1-4, whereas the other two are located in the SCR8-20.
Demberg
T
T
Department of Immunology, Georg-August University of Göttingen, Germany.
Heine
I
I
Götze
O
O
Altermann
W W
WW
Seliger
B
B
Schlaf
G
G
D000595:Amino Acid Sequence; D000818:Animals; D003167:Complement Activation; D003179:Complement C3b / Q000378:metabolism; D017242:Complement Factor H / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology*; D051056:Complement Inactivating Agents; D004274:DNA, Recombinant; D006031:Glycosylation; D006461:Hemolysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology*; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification
D051056:Complement Inactivating Agents; D004274:DNA, Recombinant; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003179:Complement C3b; D017242:Complement Factor H