Mapping of the C3b-binding site of CR1 and construction of a (CR1)2-F(ab')2 chimeric complement inhibitor

J Exp Med. 1991 Dec 1;174(6):1451-60. doi: 10.1084/jem.174.6.1451.

Abstract

CR1/CR2 chimeric receptors in which various short consensus repeats (SCRs) of CR1 were attached to CR2 were transiently expressed on COS cells, and assessed for the binding of polymerized C3b (pC3b) and anti-CR2 by immunofluorescence. Of COS cells expressing chimeras containing SCR 1-4, 1-3, 2-4, 1-2, and 2-3 of the long homologous repeats (LHRs) -B or -C, 96%, 66%, 23%, 0%, and 0%, respectively, bound pC3b. K562 cells were stably transfected with wild-type CR1, deletion mutants of CR1, and the CR1/CR2 chimeras, respectively, and assayed for binding of 125I-pC3b. The dissociation constants (Kd) for pC3b of wild-type CR1 and the LHR-BD and -CD constructs were in the range of 1.0-2.7 nM, and of the CR1/CR2 chimeras containing SCRs 1-4, 1-3, and 2-4 of LHR-B or -C were 1.8-2.4, 6-9, and 22-36 nM, respectively. The factor I-cofactor function of the CR1/CR2 chimeras paralleled the C3b-binding function of the constructs. A CR1/immunoglobulin (Ig) chimeric protein was prepared by fusing SCRs 1-4 of LHR-B to the heavy chains of a murine F(ab')2 anti-nitrophenacetyl (NP) monoclonal antibody. The (CR1)2-F(ab')2 chimera, which retained its specificity for NP, was as effective as soluble, full-length CR1 in binding pC3b, serving as a cofactor for factor I-mediated cleavage of C3b, and inhibiting activation of the alternative pathway, indicating that the bivalent expression of these SCRs reconstitutes the alternative pathway inhibitory function of CR1. The feasibility of creating CR1/Ig chimeras makes possible a new strategy of targeting complement inhibition by the use of Ig fusion partners having particular antigenic specificities.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antigens, Differentiation, B-Lymphocyte / metabolism
  • Base Sequence
  • Binding Sites
  • Complement C3b / metabolism*
  • Complement Inactivator Proteins / pharmacology*
  • Complement Pathway, Alternative
  • Humans
  • Immunoglobulin Fab Fragments / physiology*
  • Mice
  • Molecular Sequence Data
  • Receptors, Complement / metabolism*
  • Receptors, Complement 3b
  • Receptors, Complement 3d
  • Recombinant Fusion Proteins / pharmacology*
  • Repetitive Sequences, Nucleic Acid

Substances

  • Antigens, Differentiation, B-Lymphocyte
  • Complement Inactivator Proteins
  • Immunoglobulin Fab Fragments
  • Receptors, Complement
  • Receptors, Complement 3b
  • Receptors, Complement 3d
  • Recombinant Fusion Proteins
  • Complement C3b