A Soluble Recombinant Multimeric Anti-Rh(D) Single-Chain Fv/CR1 Molecule Restores the Immune Complex Binding Ability of CR1-Deficient Erythrocytes

Abstract

CR1 (CD35, the C3b/C4b receptor) is a widely distributed membrane glycoprotein with a unique cluster conformation on the surface of erythrocytes (E). CR1 on E is responsible for the transport of immune complexes (IC) to liver and spleen. As a cofactor of the C3b cleavage by factor I, CR1 is also a potent inhibitor of C activation and inflammation. In some diseases (systemic lupus erythematosus, hemolytic anemia, AIDS, etc.) an acquired low level of CR1 on E has been observed, leading to an impaired clearance of IC. The aim of this study was to design a heterofunctional molecule that will bind to E and restore a normal or a supranormal CR1 density on E that could mimic the unique distribution pattern of CR1 on normal E. For that purpose a new multimerizing system based on the properties of the C-terminal part of the α-chain of the C4 binding protein (C4bp) was used. We first produced a multimeric soluble CR1 that proved to be a better inhibitor of in vitro C activation than the monomeric form of CR1, then a heteromultimeric molecule made of CR1 and single-chain Fv anti-Rh(D) valences able to attach E and providing E with as much as a 10-fold increase in CR1 density with the same CR1 distribution pattern as native E. CR1/single-chain Fv anti-Rh(D)-treated E were able in vitro to attach as many opsonized IC as native E. These data open the way for future use of multimeric and heteromultimeric forms of soluble recombinant CR1 as therapy of IC diseases.

The Oligomerization Domain of C4-Binding Protein (C4bp) Acts as an Adjuvant, and the Fusion Protein Comprised of the 19-Kilodalton Merozoite Surface Protein 1 Fused with the Murine C4bp Domain Protects Mice against Malaria

“…The IF tag functions like a histidine tag and facilitates purification; however, the murine C4bp domain was not well expressed using this tag, and it was replaced by the DsbA protein. 19 alone). Antibody titers were determined by IFA or by ELISA using pooled serum samples collected from each group 3 weeks after the third immunization.…”

“…Fusion protein glutathione S-transferase (GST)-P. yoelii MSP1 19 was purified by affinity chromatography (GE Healthcare); the GST tag was separated from the antigen by cleavage with thrombin, and the MSP1 19 (IMX269) was purified using Superdex S75 equilibrated in PBS. This form of MSP1 19 proved to be much simpler to prepare than IMX183.…”

“…The last exon of C4bp alpha chain genes encodes the only domain in the protein which does not belong to the complement control protein family. This non-complement control protein domain contains 57 amino acid residues in humans and 54 amino acid residues in mice and is both necessary (12) and sufficient (5,15,19,21) for the oligomerization of C4bp or other polypeptides fused to it. Related oligomerization sequences have also been found in other proteins (18).…”

Deciphering Complement Receptor Type 1 Interactions with Recognition Proteins of the Lectin Complement Pathway

“…The template containing the DNA coding sequence for the soluble part of CR1 was a generous gift from Prof. J. Cohen (Université de Reims Champagne-Ardennes, Reims, France) (29). First, BglII and KpnI restriction sites were introduced, respectively, at the 59 and 39 ends of the CCP 22-30 coding sequence.…”

CR1 genotype and haplotype involvement in coronary artery disease: The pivotal role of hypertension and dyslipidemia

“…It is involved in different activities of the complement system (13). In particular, erythrocyte CR1 mediates the transport of ICs throughout the bloodstream to phagocytes in the liver and spleen, and it has been proposed that the rate of IC clearance from the circulation can be directly correlated to the number of CR1 molecules expressed on erythrocytes (CR1/E ratio) (14). Proteolytic cleavage may be responsible for loss of CR1 on the cell surface (15), but quantitative expression of CR1 on erythrocytes is also genetically determined (16).…”