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

Abstract

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 μM, 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.

Nontransgenic Hyperexpression of a Complement Regulator in Donor Kidney Modulates Transplant Ischemia/Reperfusion Damage, Acute Rejection, and Chronic Nephropathy

“…The gene coding for the N-terminal SCR1-3 of LHR-A of CR1 with a codon for an additional C-terminal cysteine was constructed as described by Dodd and colleagues, 17 and a plasmid, pDB1081-1 constructed as described in Mossakowska and colleagues 11 for transformation into the expression host Escherichia coli BL21 (DE3). The protein product of the expression vector was purified and shown to inhibit antibody-sensitized red blood cell lysis by human serum, to inhibit C3a release, and to function as a co-factor for Factor I.…”

“…17 The analysis of activity revealed that the 21-kd SCR1-3 fragment was active against both the classical and alternative pathways and appeared to be correctly folded. 11,13,17 …”

“…11 The corresponding reduced (free cysteine) form lacking SCR1-3 and used as a control agent in this study was designated "m-p" (myristoylated peptide).…”

“…using a commercially available antibody-sensitized sheep red blood cell preparation (Diamedix, Miami, FL) and a dilution of human serum of 1:400. 11 …”

“…9 The active site in CR1 was initially reported to reside in the first four short consensus repeats (SCRs) in each long homologous repeat 10 but is probably bounded by the first three domains-at least in long homologous repeat (LHR)-A. 11 Each of the functions of CR1 have been identified in other complement control proteins, but the presence of multiple functions in CR1 makes it an ideal candidate for therapeutic tissue engineering. 12 To confer ability to localize the soluble SCR1-3 fragment on target cell surfaces, a membrane-binding moiety was designed for posttranslational chemical attachment to SCR1-3 ( Figure 1b).…”