Structure-activity relationships within the N-terminal short consensus repeats (SCR) of human CR1 (C3b/C4b receptor, CD35): SCR 3 plays a critical role in inhibition of the classical and alternative pathways of complement activation.
Genes coding for between one and four short consensus repeats (SCR) of the N-terminal region of human complement receptor 1 (CR1) were synthesized from oligonucleotides and those encoding SCR(1-2), SCR(1-3), SCR(1-4), SCR3 and SCR(3-4) were expressed as inclusion bodies in Escherichia coli. Following solubilization in urea, the proteins were partially purified and refolded and the activity of each protein was assessed in both classical and alternative pathway complement assays. All fragments showed a varying degree of activity with the general order being SCR(1-3) = SCR(1-4) > SCR(1-2). Addition of SCR3 to SCR(1-2) significantly improved potency, whereas the addition of SCR4 conferred no additional benefit. This observation, coupled with the ability of the single-domain SCR3 to inhibit classical pathway mediated lysis with an IH50% (inhibition of hemolysis by 50%) of 4.8 microM, demonstrates that SCR3 provides key binding interactions with activated complement components. SCR(1-3) was able to inhibit both classical and alternative pathways of complement activation, showing that the N-terminal SCR of CR1 retain the ability to interact with C3b. Assays for CR1-like cofactor activity for factor I using C4b-like C4 or C3b-like C3 as substrates showed that SCR(1-3) possessed such cofactor activity and that C4b-like C4 was a better substrate. When compared to full-length (30 SCR) soluble CR1 (sCR1), SCR(1-3) was significantly less potent in accord with a model involving multi-valent binding of C3b/C4b to CR1.
Mossakowska
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D
SmithKline Beecham Pharmaceuticals, Harlow, GB.
Dodd
I
I
Pindar
W
W
Smith
R A
RA
D000818:Animals; D001483:Base Sequence; D003170:Complement Pathway, Alternative; D003171:Complement Pathway, Classical; D016384:Consensus Sequence; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006461:Hemolysis; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011817:Rabbits; D017463:Receptors, Complement 3b / Q000737:chemistry* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D020449:Repetitive Sequences, Amino Acid; D013329:Structure-Activity Relationship
D017463:Receptors, Complement 3b; D011994:Recombinant Proteins; D004247:DNA