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R L Coppel

Publications and source records attributed to R L Coppel.

At least 127 records · Page 7Linked to original sources
PubMed1991

Primary structure of a Plasmodium falciparum rhoptry antigen.

The high-molecular-weight rhoptry complex of Plasmodium falciparum consists of 3 non-covalently associated polypeptides of 150, 135 and 105 kDa. We present the complete nucleotide sequence of the 105-kDa (RhopH3) component of this complex derived from analysis of genomic and cDNA clones. The genomic structure is unusually complex for P. falciparum, consisting of 7 exons including 2 mini-exons of 19 and 21 amino acids. The sequence lacks tandem repeats and is conserved among several parasite isolates. B cell epitopes that induce antibody responses during natural infection were mapped to five different regions of the polypeptide.

Amino Acid Sequence↗
PubMed1991

The identification and cloning of the murine genes encoding the liver specific F alloantigens.

The liver specific F alloantigen is a highly conserved abundant protein found in hepatic cytoplasm; smaller amounts are detected in renal tubule cells and the perikaryon cells of the central nervous system. Although the biological function of the F alloantigen is unknown, the immune response to F has been extensively studied as a murine model of tolerance and autoimmunity. Murine F exists in two allelic forms, designated F type 1 and type 2, each of approximately 43 kDa. The immune response to the allotypic form is restricted to mouse strains of I-Ak. Responding strains immunized with allotypic F break tolerance and produce precipitating antibody that reacts with both allelic forms, i.e., immunogen and self. Thus an autoantibody is produced. Using the previously isolated rat F cDNA as a probe, we report the cloning and sequencing of the two murine F allotypes. These two alleles are nearly homologous except at the extremes of the coding sequence. There are a number of regions within the F sequence that are similar to peptides that interact specifically with I-Ak. In particular, there is a sequence near the carboxy terminus, where the two allotypes differ, that has homology to the I-Ak restricted malarial antigen peptide of the ring-infected erythrocyte surface antigen (RESA).

Amino Acid Sequence↗
PubMed1991

Specific reactivity of recombinant human PDC-E1 alpha in primary biliary cirrhosis.

The mitochondrial autoantigens recognized by autoantibodies in patients with primary biliary cirrhosis have been identified as components of related multi-enzyme complexes, including acyltransferases of the pyruvate dehydrogenase complex (PDC), the branched-chain alpha-keto acid dehydrogenase complex (BCODH), the alpha-ketoglutarate dehydrogenase complex (OGDC), protein X and pyruvate dehydrogenase (PDC) E1 alpha and E1 beta. The major autoantigens, PDC-E2, BCODH-E2 and OGDC-E2, share some sequence homology; the epitopes on these antigens appear to be close to, or identical with, the lipoic acid binding site. Furthermore, all three antigens share some structural homology. In contrast, antibodies to PDC-E1 alpha are present in lower titers, and have been more difficult to detect. PDC-E1 alpha also differs from the three major autoantigens in that it lacks any covalently bound lipoic acid. PDC-E1 alpha cannot be purified in large quantities and becomes unstable in the absence of PDC-E1 beta. To address these problems, we have subcloned recombinant human PDC-E1 alpha to pGEX, pGEX is a vector which produces a recombinant polypeptide fused to glutathione S-transferase. The resultant E1 alpha fusion protein is stable and has a low background in immunoassays. Using the recombinant protein, we have developed an ELISA that allows rapid and reproducible quantification of antibodies to human PDC-E1 alpha. Finally, we demonstrate that a major epitope on PDC-E1 alpha is within a 300 amino acid region that contains the enzyme functional sites, namely the phosphorylation site and the TPP binding site.

Autoantibodies↗
PubMed1990

Mapping of multiple B cell epitopes on the 70-kilodalton autoantigen of the U1 ribonucleoprotein complex.

High titer IgG autoantibodies to the 70-kDa polypeptide component (p70) of the U1 ribonucleoprotein (RNP) complex occur in the sera of patients with mixed connective tissue disease, SLE, and related rheumatic diseases. To gain insight into the pathogenesis and diversity of this antibody response we have used recombinant DNA technology to map the linear B cell epitopes on p70. A full length 1.7-kb cDNA clone encoding p70 was isolated from a human placental library and restriction fragments or polymerase chain reaction-generated fragments of the gene subcloned into the bacterial expression vector pGEX. Purified fusion proteins representing specific regions of p70 were immunoblotted with a panel of 70 anti-(U1)RNP+ sera containing anti-p70 antibodies. Six epitopes, four major (A, B, C, and F) and two minor (D and E) were mapped and were located throughout the molecule. The anti-(U1)RNP sera displayed heterogeneity in their pattern of reactivity to the six epitopes although reactivity to epitope C was more frequently associated with SLE rather than mixed connective tissue disease. The identification of multiple B cell epitopes on p70 is consistent with the concept that this self Ag drives the autoantibody response.

Autoantigens↗
PubMed1990

The mature-parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum associates with the erythrocyte membrane skeletal protein, band 4.1.

Several proteins synthesized by mature asexual stages of Plasmodium falciparum interact with the erythrocyte membrane skeleton. One of these is the mature-parasite-infected erythrocyte surface antigen (MESA; also called PfEMP2), a phosphoprotein of 250-300 kDa, which is found on the internal face of the erythrocyte membrane. When MESA is precipitated with anti-MESA antibodies, another phosphoprotein of 80 kDa is co-precipitated. This 80-kDa phosphoprotein was identified by peptide mapping as the erythrocyte membrane component band 4.1. Thus, MESA is apparently anchored at the erythrocyte membrane through an association with band 4.1. Band 4.1 is more intensely phosphorylated in infected erythrocytes and is increased in relative molecular mass in erythrocytes infected by isolates of P. falciparum that cytoadhere.

Animals↗
PubMed1990

The relative affinity of recombinant dihydrolipoamide transacetylase for autoantibodies in primary biliary cirrhosis.

In normal individuals there is an adaptive immune response to a foreign antigen in which antibodies of increasing affinity are produced with time. This is not always true of an autoimmune response. However, because only a limited number of autoantigens have been cloned or purified, this issue has not been studied well. In primary biliary cirrhosis the predominant manifestation of autoimmunity is antimitochondrial antibodies that react with dihydrolipoamide transacetylase. The availability of recombinant dihydrolipoamide transacetylase and the development of a rapid and reproducible enzyme-linked immunosorbent assay for autoantibodies has allowed us to address the affinity of autoantibodies using thiocyanate inhibition. Thiocyanate is a chaotropic compound known to inhibit antigen-antibody binding in a concentration-dependent manner. We used this property to inhibit the binding by enzyme-linked immunosorbent assay of human recombinant dihydrolipoamide transacetylase with serum autoantibodies from 55 patients with primary biliary cirrhosis. The relative affinity and serum autoantibody titers were then compared with the histological stage of the liver biopsy sample. Interestingly, we found a considerable heterogeneity of relative affinities. These relative affinities did not correlate with the histological stage or the serum titer of antimitochondrial antibodies. However, the ability of serum autoantibodies to inhibit intact primary biliary cirrhosis enzyme activity was found to correlate highly (R2 = 0.751) with the relative affinity. Thus there are profound differences between patients with respect to qualitative expression of autoantibodies. The significance of this data will be unclear until more is determined regarding the nature of the epitope that drives T cells and leads to B-cell responses.

Acetyltransferases↗
PubMed1990

Inhibition of alpha-ketoglutarate dehydrogenase activity by a distinct population of autoantibodies recognizing dihydrolipoamide succinyltransferase in primary biliary cirrhosis.

Sera from patients with primary biliary cirrhosis contain autoantibodies that recognize mitochondrial proteins. Five of the target autoantigens have now been identified as enzymes of three related multienzyme complexes: the pyruvate dehydrogenase complex, the branched chain alpha-ketoacid dehydrogenase complex and the alpha-ketoglutarate dehydrogenase complex. Each complex consists of component enzymes designated E1, E2 and E3. In this report, we confirm that primary biliary cirrhosis sera react with dihydrolipoamide succinyltransferase, the E2 component of alpha-ketoglutarate dehydrogenase complex. Seventy-three of 188 (39%) primary biliary cirrhosis sera reacted with alpha-ketoglutarate dehydrogenase complex-E2 when immunoblotted against purified alpha-ketoglutarate dehydrogenase complex; one of these sera also reacted with the E1 component. In addition, primary biliary cirrhosis sera possessing alpha-ketoglutarate dehydrogenase complex-E2 reactivity specifically inhibited enzyme function of alpha-ketoglutarate dehydrogenase complex. Enzyme activity was not affected by primary biliary cirrhosis sera that contained autoantibodies to pyruvate dehydrogenase complex-E2 and/or branched chain alpha-ketoacid dehydrogenase complex-E2, which lacked alpha-ketoglutarate dehydrogenase complex-E2 reactivity. Furthermore, affinity-purified primary biliary cirrhosis sera against alpha-ketoglutarate dehydrogenase complex-E2 inhibited only alpha-ketoglutarate dehydrogenase complex activity but did not alter enzyme activity of either pyruvate dehydrogenase complex or branched chain alpha-ketoacid dehydrogenase complex. Finally, alpha-ketoglutarate dehydrogenase complex-E2 specific affinity-purified antisera did not react on immunoblot with any component enzymes of pyruvate dehydrogenase complex or branched chain alpha-ketoacid dehydrogenase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1990

Analysis of hepatic T lymphocyte and immunoglobulin deposits in patients with primary biliary cirrhosis.

The histological findings in patients with primary biliary cirrhosis have been well-defined and are often used in the clinical staging of disease. However, it has only been with the development of reagents that phenotypically characterize the lymphoid infiltrate that attempts have been made to correlate pathophysiology with immune effector populations. Indeed, the inflammatory hepatic lesions in primary biliary cirrhosis have been described as containing CD4-positive and CD8-positive T cells. Less clear, however, have been the T cell receptors in these lesions. Further, the data on immunoglobulin deposits in hepatic lesions have been less well-defined; this deficit may be a result of the quality of polyspecific sera and difficulties in background. To address these issues, we have used a battery of well-defined monospecific and polyspecific reagents to phenotypically define the occurrence of lymphoid cells in the livers of patients undergoing transplantation. Furthermore, we have defined these same markers on T cell lines derived from liver, regional lymph node and peripheral blood. The predominant cell type in the mononuclear infiltrate is the CD3+, CD4+ T lymphocyte bearing the T cell receptor alpha beta. T cell lines from the same patients demonstrate similar findings. Of special importance, however, was the detection of CD20+ B cells and Ig+ cells in the lymphoid infiltrate. Indeed, we also readily demonstrated the presence of immunoglobulin on the surface of biliary epithelium. These data suggest that mechanisms involved in the pathophysiology of primary biliary cirrhosis may include both T cell and antibody mechanisms. The results also underscore the need to develop a functional, and not just a phenotypical, assay of the inflammatory infiltrate.

Antibodies, Monoclonal↗
PubMed1990

Site-directed mutagenesis of lysine within the immunodominant autoepitope of PDC-E2.

The major autoantigens of PBC have been identified as the four closely related mitochondrial enzymes PDC-E2, BCKD-E2 OGDC-E2 and protein X. A major structural similarity of these enzymes is the presence of one or more lipoyl domains. The immunodominant epitope of each autoantigen has either been postulated or been demonstrated to be located within the lipoate binding region. However, it is not clear whether the binding of lipoic acid to the epitope is necessary for autoantibody recognition. To address this issue we have constructed by oligonucleotide site-directed mutatagenesis three mutants in the lipoyl domain of human PDC-E2. Because lipoic acid is covalently bound to the zeta-amino group of the lysine residue of PDC-E2, the mutants were designed to replace the lysine residue in the lipoyl domain with glutamine, a negatively charged amino acid; histidine, a positively charged amino acid; and tyrosine, an aromatic amino acid. Binding reactivity of sera from patients with PBC were analyzed by enzyme-linked immunosorbent assay, immunoblotting and specific absorption against each of the three mutants and control clones. All data were compared with parallel studies with a control recombinant clone, the liver-specific F alloantigen. We believe the recognition of the lipoyl domain is a reflection of the surface-exposed, hydrophilic and relatively mobile nature of this region of the autoantigen. Further studies on direct assay for the presence of lipoic acid will be needed to clarify these issues.

Autoantigens↗
PubMed1990

Structural diversity in the 45-kilodalton merozoite surface antigen of Plasmodium falciparum.

An integral membrane protein associated with the merozoite surface of Plasmodium falciparum termed merozoite surface antigen 2 (the 45-kDa merozoite surface antigen), occurs in antigenically diverse forms. Here we report the sequences of the MSA 2 gene from two other isolates of P. falciparum. The 43 N-terminal residues and the 74 C-terminal residues of all three MSA 2 sequences are highly conserved, but between these conserved regions there are dramatic differences among the alleles. Instead of the two copies of a 32-amino-acid repeat present in the MSA 2 of isolate FC27, MSA 2 from clone 3D7 and isolate Indochina 1 contain 5 and 12 copies respectively of the four amino acid sequence Gly Gly Ser Ala. The sequences flanking the repeats also differ among the three antigens. The repeats in MSA 2 appear to be immunodominant during natural infection, and antibodies to the repeat regions of different alleles react with a restricted number of parasite isolates.

Amino Acid Sequence↗
PubMed1990

T-cell epitopes on the 70-kDa protein of the (U1)RNP complex in autoimmune rheumatologic disorders.

High-titre IgG antibodies against the immunodominant 70-kDa protein of the (U1)ribonucleoprotein (RNP) complex are present in virtually 100% of patients with mixed connective tissue disease (MCTD), and less commonly in a variety of other autoimmune rheumatic diseases. As T-cell 'help' is assumed to be required for this potentially pathogenic form of immune response, investigations to define T-cell epitopes on the 70-kDa protein were undertaken. In prior studies we expressed the 70-kDa protein and a number of its fragments, spanning most of the molecule, as recombinant fusion proteins using the pGEX expression-vector system. These fusion proteins were used as antigens in the epitope mapping studies reported here. PBMC were isolated from patients with (U1)RNP-positive rheumatic diseases and from both normal controls and rheumatologic patients with other autoantibody reactivities, including those to Ro, La and dsDNA. Reactivity to the purified 70-kDa protein was assayed by thymidine incorporation and was evident only in anti-(U1)RNP positive patients but was not restricted to MCTD patients, being present also in patients with SLE and rheumatoid arthritis. The stimulation indices (SIs) observed were in the two- to five-fold range. Using the 70-kDa protein fragments, a T-cell stimulatory epitope was localized to the C-terminal 63 amino acids of the autoantigen. A T-cell line, derived from PBMC of a (U1)RNP positive patient with MCTD, also reacted predominantly with this C-terminal fragment but with an SI of approximately 15-fold. Thus, we have demonstrated the presence and specificity of autoreactive T lymphocytes to a defined peptide epitope in systemic rheumatic disease.

Arthritis, Rheumatoid↗
PubMed1989

Antimitochondrial antibodies of primary biliary cirrhosis recognize dihydrolipoamide acyltransferase and inhibit enzyme function of the branched chain alpha-ketoacid dehydrogenase complex.

Antimitochondrial antibodies (AMA) recognizing the acetyltransferase (E2) of the pyruvate dehydrogenase (PDH) complex have been previously well-documented and the immunodominant epitope mapped. In this study, we demonstrate that sera from patients with primary biliary cirrhosis (PBC) react with another lipoic acid containing acyltransferase enzyme, namely the E2 of the branched chain alpha-ketoacid dehydrogenase (BCKD) complex. Indeed, 85/120 (71%) sera from patients with PBC reacted with BCKD-E2 by immunoblotting against purified BCKD complex. In contrast, sera from patients with chronic active hepatitis or progressive sclerosing cholangitis as well as sera from healthy volunteers did not react with any component enzymes of the BCKD complex. More importantly, BCKD enzyme activity was inhibited after incubation of the BCKD complex with either PBC sera against BCKD-E2 or with affinity purified antisera to BCKD-E2. Enzyme activity was unaltered by control sera or with PBC sera that reacted with PDH-E2 but not BCKD-E2. Furthermore, immunoblots of purified mitochondria probed with PBC sera absorbed with BCKD-E2 demonstrated that BCKD-E2 and PDH-E2 are each recognized by distinct AMA populations which do not cross-react. In addition, affinity purified PBC sera against BCKD-E2 did not react with PDH-E2 nor inhibit PDH enzyme activity, thus providing further evidence that BCKD-E2 and PDH-E2 are recognized by separate AMA. These data further suggest that the BCKD-E2 epitope recognized by AMA contains, or is close to, a functional domain of this enzyme. The availability of cDNA clones encoding BCKD-E2 and PDH-E2 will allow the study of how key metabolic enzymes may be involved in the immunology and pathology of PBC.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1989

Reactivity of primary biliary cirrhosis sera with a human fetal liver cDNA clone of branched-chain alpha-keto acid dehydrogenase dihydrolipoamide acyltransferase, the 52 kD mitochondrial autoantigen.

Antimitochondrial autoantibodies recognizing 68 to 74 and 50 to 52 kD inner membrane mitochondrial antigens are characteristically present in sera of patients with primary biliary cirrhosis. The biochemical identification of the antigens, however, has remained elusive. We report herein that the 52 kD antigen is the dihydrolipoamide acyltransferase of the branched-chain alpha-keto acid dehydrogenase complex. This was demonstrated by three experiments through the use of recombinant fusion protein expressed in Escherichia coli from a cDNA insert encoding the human autoantigen. First, 33 of 37 primary biliary cirrhosis patients exhibiting reactivity toward the 50 to 52 kD mitochondrial antigen by immunoblotting also showed reactivity toward the recombinant fusion protein. Second, absorption of primary biliary cirrhosis sera with recombinant fusion protein, but not with an irrelevant recombinant clone, the F-specific rat liver antigen, was effective in absorbing out reactivity against the 50 to 52 kD mitochondrial antigen but not the 68 to 74 kD antigen. Third, complete removal of reactivity toward all four different isoelectric point polypeptides at 50 to 52 kD was observed in two-dimensional gel analysis. Furthermore, primary biliary cirrhosis sera were analyzed with mitochondria from three sources, rat liver, human placenta and bovine heart, in order to compare reactivity patterns and to determine precisely the comparative molecular weights of the autoantigens in the three species. The availability of recombinant autoantigens will provide improved diagnostic tests and, more importantly, will allow definite issues in primary biliary cirrhosis to be studied, including identification of immunodominant epitopes, the significance of autoantigen recognition and the establishment of autoreactive T cell clones.

Acyltransferases↗
PubMed1989

Immunization of experimental animals with dihydrolipoamide acetyltransferase, as a purified recombinant polypeptide, generates mitochondrial antibodies but not primary biliary cirrhosis.

The availability of recombinant mitochondrial autoantigens may permit the experimental study of the pathophysiology of primary biliary cirrhosis. Previously, we demonstrated that high-titer antibodies to the 74 kD mitochondrial autoantigen dihydrolipoamide acetyltransferase could be generated when BALB/c mice were immunized with purified recombinant protein. Based on these data, we attempted an 8-month study to induce antibodies and liver dysfunction by immunizing AKR/J, C3H/J and CBA/HeJ mice as well as rats, guinea pigs, rabbits and rhesus monkeys with purified recombinant human dihydrolipoamide acetyltransferase. Antibodies to dihydrolipoamide acetyltransferase were readily induced and detected in all species of experimental animals with species and strain differences in the titer of the responses. Of particular interest, rabbits and guinea pigs produced antibodies which were specifically reactive with the functional site of dihydrolipoamide acetyltransferase, whereas the other strains and species produced antibodies to other epitopes on the molecule. Finally, similar to data on humans with primary biliary cirrhosis, the pyruvate dehydrogenase enzyme pathway was inhibited in the presence of immunized animal sera. These data imply that features other than simply an antibody response to mitochondrial enzymes are required for the development of primary biliary cirrhosis. Further studies will be necessary to determine the mechanisms by which mitochondrial proteins elicit an immune response.

Acetyltransferases↗
PubMed1989

Antimitochondrial autoantibodies in primary biliary cirrhosis recognize cross-reactive epitope(s) on protein X and dihydrolipoamide acetyltransferase of pyruvate dehydrogenase complex.

Antimitochondrial autoantibodies are characteristically present in sera of patients with primary biliary cirrhosis. The antimitochondrial autoantibodies recognize four major antigens from beef heart mitochondria at relative molecular weights of 74, 56, 52 and 48 kD. In the present study, we report that the 56 kD antigen is the protein X of pyruvate dehydrogenase complex and that it possesses cross-reactive antimitochondrial autoantibody epitope(s) with the 74 kD antigen, the acetyltransferase (E2) of the pyruvate dehydrogenase complex. This was demonstrated by comparing the specificities of primary biliary cirrhosis sera with a protein X-specific rabbit antiserum and by absorbing primary biliary cirrhosis sera with recombinant pyruvate dehydrogenase-E2 fusion protein. In the two-dimensional gel analysis, primary biliary cirrhosis sera and protein X-specific rabbit antiserum reacted to the same two isoelectric point polypeptides at 56 kD molecular weight. The absorption of primary biliary cirrhosis sera with the human recombinant pyruvate dehydrogenase-E2 removed reactivity toward both the 74 and 56 kD antigens. Furthermore, analysis of 82 antimitochondrial autoantibody-positive primary biliary cirrhosis sera by immunoblotting did not reveal any sera which reacted solely against either the 74 or 56 kD antigen. Finally, primary biliary cirrhosis sera recognized protein X from human, bovine and porcine sources but not protein X from rat or mouse origin. The identification of protein X as another major target of the autoimmune response in primary biliary cirrhosis suggests that the pyruvate dehydrogenase complex may have a central role in the induction of this enigmatic disease.

Acetyltransferases↗
PubMed1988

The autoepitope of the 74-kD mitochondrial autoantigen of primary biliary cirrhosis corresponds to the functional site of dihydrolipoamide acetyltransferase.

Autoantibodies to mitochondrial antigens are characteristic of the autoimmune liver disease primary biliary cirrhosis (PBC), but the precise antigenic determinants recognized by these antibodies have not been defined. Recently, our laboratory identified a 1,370-bp rat liver cDNA clone that coded for a polypeptide recognized specifically by sera from patients with PBC but not by sera from patients with other forms of liver disease. This recombinant protein was identified as the 74-kD M2 mitochondrial inner membrane autoantigen, now known to be dihydrolipoamide acetyltransferase. In the present study, we have identified a 603-bp fragment that codes for a polypeptide containing all of the autoreactivity of the original clone. In addition, based on hydrophobicity/hydrophilicity plots of the amino acid sequence of this polypeptide segment, several peptides were synthesized and tested for reactivity by an inhibition assay using sera from patients with PBC. One peptide, defined by the amino acids AEIETDKATIGFEVQEEGYL, absorbed serum reactivity to the protein product of the original clone. Of particular interest was the finding that this peptide contains the lipoic acid binding site KATIGF of the dihydrolipoamide acetyltransferase found in the inner mitochondrial membrane. Thus, it appears that for this autoantigen, the target of the autoantibodies corresponds to a functional site of the dihydrolipoamide acetyltransferase.

Acetyltransferases↗