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J P Atkinson

Publications and source records attributed to J P Atkinson.

At least 19 recordsLinked to original sources
PubMed1992

Membrane cofactor protein (CD46) protects cells from complement-mediated attack by an intrinsic mechanism.

The cleavage of C3 is a critical step for complement (C) activation in the classical and alternative pathways. This reaction is controlled by the regulators of C activation protein family. Membrane cofactor protein (MCP) is a cofactor for the factor I-mediated inactivation of C3b and C4b. As a widely distributed membrane protein, MCP may protect host cells from inadvertent C activation. Human MCP has recently been shown to protect transfected rodent cells from human C-mediated lysis. In this report the relationship of MCP expression to C3b deposition and cytoprotection was examined using NIH/3T3 cells transfected with human MCP and exposed to human serum as a source of C and naturally occurring anti-mouse antibody. MCP inhibited C3b deposition in a dose-dependent fashion and inhibited lysis of the mouse cells expressing it. MCP did not inhibit lysis on bystander cells. These results demonstrate the protective role of MCP, at the cellular level, by an intrinsic mechanism.

3T3 Cells↗
PubMed1992

Identification and characterization of membrane cofactor protein of human spermatozoa.

Membrane cofactor protein (MCP) regulates C activation by serving as a cofactor for the cleavage of C3b and C4b by the serine protease factor I. An MCP-like molecule on the inner acrosomal membrane of human spermatozoa has been characterized. Three mAb and a rabbit polyclonal antibody against MCP recognized the sperm protein. On SDS-PAGE, it migrated as a single band with a molecular mass of 38,000 and 44,000 Da under nonreducing or reducing conditions, respectively. The molecular mass was 10,000 to 20,000 Da less than the two forms of MCP expressed on others cells. The electrophoretic pattern, by one- and two-dimensional gel analysis, and the isoelectric point profile (4.5 to 5.0) of the sperm protein were similar among multiple individuals. In contrast to MCP of other cells, digestion with endoglycosidases did not alter either the m.w. or the pI of the protein, suggesting that it is a poorly or nonglycosylated form of MCP. The solubilized sperm protein bound C3 with broken thioester bond to Sepharose and possessed cofactor activity for factor I-mediated cleavage of C3 with the broken bond. A mAb that blocks the regulatory function of MCP inhibited the cofactor activity of the sperm lysate. Thus, the sperm protein is an antigenic and functional homologue of MCP but has the distinct structural features of a lower m.w. and an apparent lack of glycosylation. MCP may play an essential role in the survival of the acrosome-reacted spermatozoa by modulating C activation in the female genital tract.

Acrosome↗
PubMed1992

Membrane cofactor protein.

MCP serves to down-regulate the activation of complement on host tissue. It performs this function by serving as a cofactor for the factor I-mediated cleavage of C3b and C4b. MCP is most likely an intrinsic regulator, i.e., it primarily protects its home cell. The wide tissue distribution of MCP mirrors this critical function of host cell protection. With the exception of erythrocytes, every cell and tissue examined expresses this protein. MCP is represented as two broad heterogeneous bands on SDS-PAGE with M(r)s of 51,000-58,000 and 59,000-68,000. The quantity of each form expressed is inherited in an autosomal codominant fashion. In most cells and cell lines, four isoforms of MCP predominate and arise by alternative splicing of a single MCP gene. All forms possess four repeating modules of--60 aminoacids, an area enriched in serines, threonines, and prolines [(STP), probable site of O-linked glycosylation], a short area of unknown function, a transmembrane domain, and a cytoplasmic tail. The isoforms differ, however, in the length and composition of the STP region and in the cytoplasmic tail. Alternative splicing of a single exon within the STP region determines the protein phenotype. Alternative splicing at the COOH_terminus gives rise to two distinct cytoplasmic tails. The biological significance of these structural variations in the STP and cytoplasmic tail regions is being investigated.

Animals↗
PubMed1992

Analysis of the human regulators of complement activation (RCA) gene cluster with yeast artificial chromosomes (YACs).

The human regulators of complement activation gene cluster (RCA cluster) have been partially characterized with yeast artificial chromosomes (YACs). While the data confirm many points previously elucidated, the finer resolution of YAC mapping has allowed the discovery and/or localization of partial gene duplications, the determination of gene orientations, and the measurement of gaps between known genes. Here nine overlapping YACs that encompass a genomic region of 800 kb, encoding four RCA genes and three gene-like elements, are described. The encoded genes and two of the gene-like elements share the same orientation and are ordered (5' to 3') DAF, CR2, CR1, MCP-like, CR1-like, and MCP. A C4bp-like region lies upstream from DAF and is likely to correspond to one recently observed by F. Pardo-Manuel, J. Rey-Campos, A. Hillarp, B. Dahlback, and S. Rodriguez de Cordoba (1990, Proc. Natl. Acad. Sci. USA 87: 4529-4533). MCP-like, a new genetic element, was discovered and found to be homologous to the 5' portion of the MCP gene. Two large gaps of 85 kb (between CR2 and DAF) and 110 kb (between DAF and the C4bp-like element) could carry additional RCA genes. The arrangement of CR1, MCP-like, CR1-like, and MCP, in that order, strongly suggests that this region was generated by a single duplication of neighboring CR1/CR1-like and MCP/MCP-like forerunners. The RCA YACs will now serve as convenient DNA sources for the subcloning and further characterization of this region.

Amino Acid Sequence↗
PubMed1992

Complement receptors.

Recently, a number of exciting developments have increased our understanding of complement receptors. These advances include determination of the spatial organization of the short consensus repeat unit, analysis of active sites within short consensus repeats, downregulation in vivo of the complement system by a recombinant receptor, elucidation of the structure of mouse receptors and their relationship to human molecules, and the cloning of the human C5a receptor.

Amino Acid Sequence↗
PubMed1992

Antiglobulin testing for CR1-related (Knops/McCoy/Swain-Langley/York) blood group antigens: negative and weak reactions are caused by variable expression of CR1.

The Knops, McCoy, Swain-Langley and York antigens have recently been identified as being on complement receptor type 1 (CR1, CD35, C3b/C4b receptor). We examined the relationship between CR1 expression and the reactivity of the CR1-related blood group antigens with their specific antibodies. RBC from donors of selected phenotypes were tested by hemagglutination using two monoclonal antibodies to CR1, as well as anti-Kna, -McCa, -S1a, -'Kn/McC' and -Yka. Monoclonal antibodies 3D9 and E11 required approximately 250 and approximately 400 CR1/RBC to obtain a positive reaction. Agglutination of antigen-positive cells by human polyclonal antisera was related to the CR1/RBC: thus, cells expressing 20-100 CR1/RBC were negative and included the previously designated null phenotypes for this collection, 100-150 were weak or negative, and greater than 200 were usually positive. One RBC sample carried Yka on the 190,000 dalton (A or F allele), but not the 220,000 dalton (B or S allele) variant of CR1, and gave inconsistent reactions with Yka antisera. These data provide an explanation for certain of the serologic characteristics of the CR1-related blood group antigen system.

Alleles↗
PubMed1992

Genetics of the complement system and rheumatic diseases.

The complement system, especially the early components of the classic pathway, are critically involved in immune complex processing. The deposition of clusters of complement component C3b on a target marks it for elimination, primarily through an interaction with complement receptors. Not surprisingly, total and partial deficiencies of certain complement components and C3b receptors are associated with rheumatic diseases, particularly systemic lupus erythematosus. This predisposition is explicable, based on the critical role complement plays in immune complex handling.

Complement Factor B↗
PubMed1991

Contribution of the repeating domains of membrane cofactor protein (CD46) of the complement system to ligand binding and cofactor activity.

Membrane cofactor protein (MCP) (CD46) of the C system binds to C3b and C4b, functions as a cofactor for their cleavage, and protects autologous cells from C-mediated injury. The predominant structural motif of MCP is the short consensus repeat (SCR), a repeating domain involved in ligand binding of other related C regulatory proteins. SCR deletion mutants were constructed to determine which of the four SCR of MCP contribute to ligand binding and cofactor activity. ELISA were developed to evaluate binding efficiency of mutants to ligand. Analysis of the deletion mutants indicated that the third and fourth SCR were important for both ligand binding and cofactor activity of C3b (iC3) and C4b. In addition, the same SCR were required for efficient binding of an mAb known to inhibit MCP function. The mutant deleted of SCR-2 bound but lacked cofactor activity for iC3. It did not bind or possess cofactor activity for C4b. Deletion of the first (amino-terminal) SCR had a minimal effect on iC3 binding and cofactor activity but reduced the efficiency of C4b binding. The results identify the SCR of MCP that contribute to ligand binding and cofactor activity. The data also suggest the presence of distinguishable iC3 and C4b binding sites and provide evidence that iC3 binding is not always sufficient for cofactor activity.

Antibodies, Monoclonal↗
PubMed1991

Membrane cofactor protein of the complement system: alternative splicing of serine/threonine/proline-rich exons and cytoplasmic tails produces multiple isoforms that correlate with protein phenotype.

Membrane cofactor protein (MCP) is a complement regulatory protein that is expressed on human cells and cell lines as two relatively broad species with Mr of 58,000-68,000 and 48,000-56,000. The structure of a previously reported cDNA clone indicated that MCP was a type 1 membrane glycoprotein and a member of the regulators of complement activation gene/protein cluster. However, it did not provide an explanation for the unusual phenotypic pattern of MCP. Therefore, in parallel with an analysis of the gene, additional cDNAs were cloned and characterized. Six different MCP cDNA classes were identified. All encode the same 5' untranslated signal peptide, four SCRs, transmembrane domain, and basic amino acid anchor. However, they differ in the length and composition of an extracellular serine/threonine/proline (STP)-rich area, a site of heavy O-glycosylation, and cytoplasmic tail. Analysis of the MCP gene demonstrated that the variation in cDNA structure was a result of alternative splicing. Peripheral blood cells and cell lines predominantly expressed four of the six isoforms. These varied by the presence or absence of an STP-rich segment of 15 amino acids (STPB) and by the use of one of two cytoplasmic domains. Analysis by polymerase chain reaction, Northern blots, and transfection indicated that the predominance of MCP cDNA isoforms with STPB correlated with the high molecular weight protein phenotype, while the predominance of isoforms without STPB correlated with the lower molecular weight phenotype. The expression in a single cell of four distinct protein species with variable STP-rich regions and cytoplasmic tails represents an interesting example of the use of alternative splicing to provide variability in a mammalian protein.

Amino Acid Sequence↗
PubMed1991

Sites within the complement C3b/C4b receptor important for the specificity of ligand binding.

Cysteine-rich repeated units of 40-70 amino acids are building blocks of many mammalian proteins, including 12 proteins of the complement system. Human complement arranged motifs, designated short consensus repeats (SCRs), which constitute the entire extracellular portion of this protein. Klickstein et al. [Klickstein, L. B., Bartow, T. J., Miletic, V., Rabson, L. D., Smith, J. A. & Fearon, D. T. (1988) J. Exp. Med. 168, 1699-1717 (abstr.)] localized a C4b binding domain to SCR-1 and/or SCR-2 and a C3b binding domain to SCR-8 and/or SCR-9. These SCRs bind different ligands, although SCR-1 and SCR-8 are 55% homologous and SCR-2 and SCR-9 are 70% homologous. To examine if one or two SCRs are required for ligand binding and to define sites within the SCRs that determine specificity of binding, mutagenesis analysis of a truncated, secreted form of CR1, called CR1-4 by Hourcade et al. [Hourcade, D., Meisner, D. R., Atkinson, J. P. & Holers, V. M. (1988) J. Exp. Med. 168, 1255-1270], was undertaken. The latter, composed of the first eight and one-half amino-terminal SCRs of CR1, efficiently bound C4b but not iC3. SCR-1 and SCR-2 were necessary for this interaction. Analysis of the mutant CR1-4 proteins, in which amino acids in SCR-1 and SCR-2 were substituted a few at a time with the homologous amino acids of SCR-8 and SCR-9, led to the identification of one amino acid in SCR-1 and three amino acids in SCR-2 important for C4b binding. Furthermore, five amino acids at the end of SCR-9, if placed in the homologous positions of SCR-2, conferred iC3 binding and are likely essential for ligand binding activity of SCR-8 and SCR-9. This iC3 binding occurred only if SCR-1 was present, indicating that two contiguous SCRs are necessary for this interaction. These results provide identification of amino acids within SCRs that are important for ligand binding.

Amino Acid Sequence↗
PubMed1991

The C3b/C4b receptor is recognized by the Knops, McCoy, Swain-langley, and York blood group antisera.

Erythrocytes (E) lacking high incidence blood group antigens were screened by an antiglobulin test with a monoclonal antibody to human complement receptor type 1 (CR1; C3b/C4b receptor; CD35). Some examples of E lacking Knops, McCoy, Swain-Langley, and York antigens, a serologically related group, were not agglutinated. Moreover, E of the null phenotype for these same antigens were nonreactive. To further explore this relationship, E expressing these antigens were surface labeled, solubilized, and incubated with the corresponding blood group-specific antisera. CR1 was immunoprecipitated, indicating that the epitopes recognized by each of these antisera are expressed on CR1. E of two individuals, putative null phenotypes for the Knops, McCoy, and Swain-Langley blood group antigens, expressed a very low number of CR1 (less than 30/E; approximately 10% of the normal mean). This observation accounts for their lack of reactivity in the antiglobulin test and their prior designation as null phenotypes. Also, the previously reported low as well as variable expression of CR1 on E explains prior difficulties in the serologic analyses of these blood group antigens.

Alleles↗
PubMed1991

Membrane cofactor protein of the complement system. A HindIII restriction fragment length polymorphism that correlates with the expression polymorphism.

An RFLP was found in the DNA of 25 unrelated persons, two families, and five cell lines that correlated with their membrane cofactor protein phenotype. If restricted with HindIII, DNA derived from upper band predominant protein (U) phenotypes had a band at 2 kb, whereas DNA of lower band predominant (L) phenotypes had a 4-kb band. The equal band protein phenotype, in which equal quantities of the two species are expressed, had bands at both 4 and 2 kb. The polymorphic HindIII site was localized to an intron within the membrane cofactor protein gene between exon 1 (codes for 5'UT/signal peptide) and exon 2 (codes for the first short consensus repeat). Using the polymerase chain reaction (PCR), sequences around this site were amplified and a single band of 260 bp was produced. In the U phenotype, the PCR product was restricted with HindIII into 200- and 60-bp fragments. In the L phenotype, there was no change in the size of 260 bp upon restriction with HindIII. For the equal band protein phenotype, the PCR product was partially cleaved. The 260-bp PCR product was subcloned and sequenced. DNA from the U phenotype demonstrated an intact HindIII site (AAGCTT), whereas in the DNA of the L phenotype, this site was altered because a "G" was substituted for a "C" (AAGGTT).

Alleles↗
PubMed1991

Preferential inactivation of the C5 convertase of the alternative complement pathway by factor I and membrane cofactor protein (MCP).

Human C3b bound to the ghost of sheep erythrocytes (E*) via activation of the alternative complement pathway (E*AC3b) consists of four major constituents on SDS-PAGE of 350, 260, 210 and 180 kDa. 350 kDa C3b is a dimeric form of C3b in which the alpha' chain of one C3b binds covalently to that of the other C3b. This complex is presumed to serve as a core for the alternative pathway C5 convertase. The other C3b populations are monomers complexed with membrane proteins or sugars. Using E*AC3b (C3b labeled) as a substrate, we have investigated functional properties of membrane cofactor protein (MCP), which is an integral membrane protein with C3b-binding and factor I-dependent cofactor activities. In conjunction with factor I, MCP was found to degrade the protein-bound C3b preferentially including the 350 kDa dimer. There was a similar but lesser tendency of this selective cleavage of C3b-dimer by CR1 but not by factor H or C4bp. In contrast to CR1 and factor H, detergent solubilization of EAC3b was required for MCP to fully express its cofactor activity for this selective degradation of C3b. We next separated the C3b dimer from the monomers and assessed their ability to assemble the alternative C5 convertase. The C3b dimer but not the monomers expressed C5 convertase activity following the addition of factors B and D, C5 and Ni2+. Kinetic analysis of the degradation of the C3b dimer by MCP and factor I suggested that only one C3b was efficiently converted to C3bi and this occurred concomitant with a decrease in C5 convertase activity. These results suggest that MCP has the ability to more efficiently interact with protein-bound C3b and that this may relate as well to its preferential ability to irreversibly inactivate the C5 convertase.

Antigens, CD↗
PubMed1991

Differential expression of complement regulatory proteins on subpopulations of human trophoblast cells.

Trophoblast cells forming the reactive interface between the mother and her semiallogeneic fetus risk attack by cellular and humoral elements of the maternal immune system. Biochemical, molecular, and immunohistologic studies have identified membrane cofactor protein (MCP) and decay accelerating factor (DAF) on trophoblast cells, which could assist in preventing lysis of the cells by complement-activating maternal antibodies. In this immunocytochemical study, differential expression of these two members of the family of complement regulatory proteins on subpopulations of human trophoblast cells and other types of cells in first and third trimester placentas was demonstrated. Staining with anti-MCP was particularly strong on villous cytotrophoblast cells and giant cells in first trimester tissues in comparison with other types of cells. In contrast, staining with anti-DAF was strong on proliferating cytotrophoblast in first trimester tissues, and on basal plate cytotrophoblast and decidual cells in term tissues. Placental villous mesenchymal cells but not trophoblast cells expressed a third regulatory protein, complement receptor 1. These observations support the postulate that complement regulatory proteins are critical to protection of the fetal allograft, and suggest specific requirements for trophoblast cells according to stage of differentiation and anatomic location.

Antigens, CD↗
PubMed1991

Evolution of the complement system.

The ancestral form of the alternative pathway of complement activation probably originated as a primitive independent immune system. Subsequent evolution of an adaptive immune response drove the specialization of the classical pathway to connect antibody-mediated nonself recognition to the complement-dependent effector mechanisms. In this article Timothy Farries and John Atkinson consider how the contemporary complexity arose by a succession of credible alterations at the genetic level, and the selective advantages provided at each step.

Biological Evolution↗
PubMed1991

Characterization of three monoclonal antibodies to membrane co-factor protein (MCP) of the complement system and quantification of MCP by radioassay.

MCP is a widely distributed regulatory glycoprotein of the complement system which binds C3b and C4b and has factor I-dependent co-factor activity. Monoclonal antibodies raised to lymphocytes (E4.3), chorionic microvilli (GB24) and an embryonal carcinoma cell line (TRA-2-10) recognize MCP (CD46). GB24 inhibited both the binding of MCP to its ligand iC3 and co-factor activity; E4.3 and TRA-2-10 did not. The binding of GB24 to cells bearing MCP was not cross-inhibited by E4.3 or TRA-2.10, but TRA-2-10 blocked binding and displaced pre-bound E4.3. Using these antibodies, we developed a radioassay for quantifying the number of MCP molecules/cells. Human peripheral blood mononuclear (PBMC) and polymorphonuclear cells (PMN) had about 10,000 MCP cell; platelets had about 600/cell, and no MCP was found on erythrocytes. Neoplastic hematopoietic cell lines, of myelocytic and T lymphocytic origin, had several-fold more (20-60,000) molecules cell than peripheral blood cells or B cell lines (about 12,000). Malignant epithelial cell lines. HeLa (about 100,000/cell) and HEp-2 (about 250,000 cell) had the highest MCP expression of any cells examined. These monoclonal antibodies--especially GB24, which blocks MCP function--and the direct binding assay will facilitate the further analysis of the biology of this complement regulatory protein.

Antibodies, Monoclonal↗