Winnie W. Wong

Genetic regulation of a structural polymorphism of human C3b receptor.

Two forms of the human C3b receptor (C3bR), which have relative molecular weights (Mr) of 250,000 and 260,000 and are designated F and S, respectively, have been identified in specific immunoprecipitates from erythrocytes and leukocytes by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both forms of the receptor were visualized on gels by autoradiography of 125I-labeled antigen and by silver nitrate staining. Individual donors expressed one of three possible patterns of C3bR, either the F or S form alone or both, and these patterns represented stable phenotypic characteristics of their erythrocytes and polymorphonuclear and mononuclear leukocytes. Removal of N-linked oligosaccharides by endoglycosidase-F treatment decreased the Mr of both forms but did not abolish the difference in their electrophoretic mobilities. That both forms of the receptor were functional was indicated by the capacity of all antigenic C3bR sites on erythrocytes from individuals having any of the three phenotypes to bind dimeric C3b with affinities ranging from 3 to 5 X 10(7) M-1. Analyses of the occurrence of the F and S forms of C3bR in 76 individuals from 15 families revealed that this polymorphism was regulated by two alleles transmitted in an autosomal codominant manner. Of 111 normal unrelated individuals, 64.9% were homozygous for the F form (FF), 1.8% were homozygous for the S form (SS), and 33.3% were heterozygotes (FS). This distribution did not differ from that calculated by the Hardy-Weinberg equilibrium based on two codominant alleles that regulate the expression of the F and S forms and that have frequencies of 81.5 and 18.5%, respectively. The locus regulating structural polymorphism of C3bR is designated C3BRM (M for mobility or Mr), and is distinct from the recently described locus regulating the quantitative expression of C3bR on erythrocytes.

Rapid purification of the human C3b/C4b receptor (CR1) by monoclonal antibody affinity chromatography

The human C3b/C4b receptor (CR1) is a polymorphic glycoprotein that is expressed on erythrocytes, leukocytes and glomerular podocytes. Further structural analysis and molecular genetic studies would be facilitated by the availability of relatively larger amounts of purified CR1. Milligram quantities of CR1 were purified from erythrocyte membranes 10,000-fold with an average yield of 30-40% by a rapid procedure which utilized sequential chromatography on Matrex Red A and a monoclonal anti-CR1 antibody affinity column. The purified receptor was homogeneous by SDS-PAGE and consisted of the 2 most common alleles of CR1. Purified CR1 also retained its function of serving as a cofactor for the cleavage of C3b to iC3b, C3dg and C3c. The amino acid composition was typical of that of a globular protein and sequence analysis of the N-terminus of the purified CR1 revealed that it was blocked.

Human C3b/C4b Receptor (Cr1): Isolation, Protein Sequence Analysis, and Cloning of a Partial cDNA from Human Tonsil

The human receptor for the C3b/C4b fragments of complement, designated CR1, is a large glycoprotein comprised of a single polypeptide chain that exhibits genetically regulated structural and quantitative polymorphisms. The structural polymorphisms are manifested in an autosomal codominant expression of different allotypic proteins; all allotypic forms are capable of binding C3b1,2,3,4. The most common proteins have MW of 250,000 (F allotype) and 260,000 (S allotype) by NaDodSO/PAGE, although rarer forms differ by as much as 90,000 daltons4. Removal of the N-linked oligosaccharides does not eliminate this polymorphism, suggesting that the primary structures of the various allotypic forms are different1. CR1 is an integral membrane glycoprotein residing on erythrocytes, polymorphonuclear leukocytes, monocytes/macrophages,fiall B and some T lymphocytes, and renal glomerular epithelial cells5,6. Quantitative polymorphism refers to the number of receptor sites on erythrocytes, which varies by 10-fold among different individuals. Population and family studies have shown that there are two autosomal codominant alleles that determine the quantitative polymorphism.

Human receptor for C3b/C4b: complement receptor type I.

Publisher Summary Receptor function and protein are shown to reside on cells of the myelomonocytic series, mature B lymphocytes, glomerular podocytes, a small population of T lymphocytes, follicular dendritic cells, and even in plasma. The purification scheme to be described in the chapter has made available amounts of purified CR1 sufficient for amino acid sequencing of tryptic peptides. Synthetic oligonucleotides based on these sequences are used to screen a cDNA library and a partial cDNA clone specific for human CR1 has been identified. Analyses of this clone demonstrated the presence of long homologous repetitive nucleotide sequences in the CRI gene and revealed structural similarities of CR1 to factor H and C4-binding protein, two functionally analogous proteins to which CR1 in linked. This linkage group is localized to band q32 of chromosome 1 by in situhybridization to cDNA probes. The first purification scheme of CRI from erythrocyte membranes involved the use of ion-exchange chromatography, gel filtration, C3- and lentil lectin-Sepharose chromatography. The most recent scheme for the purification of CR1 to be discussed in the chapter is a combination of the dye matrix and YZ-1 monoclonal antibody (MAb) anti-CRl affinity chromatography.