Cell surface expression of the C3b/C4b receptor (CR1) protects Chinese hamster ovary cells from lysis by human
S C Makrides1, S M Scesney, P J Ford
1T Cell Sciences, Inc., Cambridge, Massachusetts 02139.
Insights
Complement receptor type 1 (CR1) regulates complement activation. Specific domains of CR1 inhibit complement-mediated cell lysis and bind C3b and C4b, acting as an intrinsic complement regulator.
Area of Science:
- Immunology
- Complement System
- Molecular Biology
Background:
- Complement receptor type 1 (CR1, CD35) is a glycoprotein with repeating short consensus repeats (SCRs).
- CR1 is known to regulate extrinsic complement activation by binding and degrading C3b.
- The precise structural domains responsible for CR1's regulatory functions are not fully elucidated.
Purpose of the Study:
- To investigate the functional domains of CR1 responsible for inhibiting complement-mediated cell lysis.
- To determine the structural requirements for C3b and C4b binding to CR1.
- To assess CR1's role as an intrinsic regulator of complement activation.
Main Methods:
- Generation of recombinant CR1 proteins with deletions in SCR domains.
- Assay of recombinant CR1 proteins for inhibition of complement-mediated lysis of Chinese hamster ovary (CHO) cells.
- Measurement of binding affinities (Kd) for monomeric and dimeric C3b.
- Assessment of cofactor activity for C3b proteolysis by factor I.
Main Results:
- Full-length recombinant CR1 inhibited complement-mediated lysis of CHO cells, with efficiency proportional to receptor number.
- SCR domains 15-18, but not 15-16, inhibited lysis, bound C3b, and acted as a cofactor for C3b degradation.
- SCR domains 1-4, but not 1-2, inhibited lysis, indicating four SCRs are sufficient for C4b binding, analogous to C3b binding.
Conclusions:
- CR1 functions as an intrinsic regulator of complement activation by inhibiting complement-mediated lysis of the cell expressing it.
- Specific SCR domains (15-18 and 1-4) are critical for CR1's inhibitory and binding functions.
- The structural basis for C3b and C4b binding involves four SCR domains of CR1.
Abstract:
The C3b/C4b receptor, also known as complement receptor type 1 (CR1, CD35), is a single chain glycoprotein consisting of 30 repeating homologous protein domains known as short consensus repeats (SCR) followed by transmembrane and cytoplasmic domains. A series of recombinant proteins derived from CR1 has been prepared and assessed for the capacity to inhibit complement lysis of the host Chinese hamster ovary (CHO) cells. The full-length recombinant CR1 inhibited human complement-mediated CHO cell lysis, and the efficiency of inhibition was directly proportional to the number of receptors/cell. The SCR 15-18 of CR1, but not SCR 15-16, inhibited complement lysis of the host CHO cell, bound monomeric C3b (Kd,app = 6.5 x 10(-7) M), and dimeric C3b (Kd = 1.8 x 10(-8) M), and served as a cofactor in the proteolysis of C3b by factor I, confirming and extending the observations of Fearon and colleagues (Kalli, K. R., Hsu, P., Bartow, T. J., Ahearn, J. M., Matsumoto, A. K., Klickstein, L. B., and Fearon, D. T. (1991) J. Exp. Med. 174, 1451-1460). The SCR 1-4 of CR1, but not SCR 1-2, also inhibited complement lysis of the host CHO cell, indicating that more than two SCR are necessary and that four SCR are sufficient for optimal C4b binding to CR1. Thus, the structural requirements for C4b binding are analogous to those for C3b binding, namely, four SCR of CR1 form the binding sites for each of these proteins. CR1 has long been recognized to regulate extrinsic complement activation, that is, to bind to and promote the degradation of fluid phase C3b and of C3b attached to immune complex. These results demonstrate that CR1 is also an intrinsic regulator of complement activation in that, under appropriate conditions, CR1 inhibits complement-mediated lysis of the cell on which it is expressed.
