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R L Coppel

Publications and source records attributed to R L Coppel.

At least 145 records · Page 8Linked to original sources
PubMed1988

Characteristics and epitope mapping of a cloned human autoantigen La.

The La (SS-B) polypeptide is a ribonucleoprotein against which high titer antinuclear antibodies (ANA) react in the human autoimmune disease primary Sjögren's syndrome. To identify the autoepitopes with which the ANA anti-La (anti-SS-B) reacts, we isolated a 1.4-kb cDNA clone for La from a lambda gt10 library made from a human Burkitt's cell line. This clone contained an open reading frame of 1065 bp, encoding a 40.1-kDa polypeptide that corresponded to the carboxyl-terminal end of the La protein. The predicted polypeptide sequence of the recombinant protein was highly charged and unrelated to any previously published sequence. We also compared this clone to a previously published cDNA sequence for La and demonstrated significant differences, particularly that the open reading frame in our cDNA continued for 926 additional bases 3' to a putative termination codon in the previously reported sequence. The recombinant La protein was expressed in Escherichia coli and tested for reactivity with 200 sera containing ANA of various specificities. Only the sera containing anti-La antibodies reacted with the cloned La. By expressing subclones of the La cDNA as fusion proteins with beta-galactosidase, we have localized at least one epitope for the binding of anti-La antibodies to the carboxyl-terminal 103 amino acids of the La protein. No anti-La binding could be demonstrated to the region of the La protein that had previously been predicted to contain an autoepitope for the binding of anti-La (SS-B) antibodies. Studies of cloned autoepitopes could provide important clues to the role ANA play in disease and lead to targeted intervention in the treatment of primary Sjögren's syndrome.

Amino Acid Sequence↗
PubMed1988

Variation in the precursor to the major merozoite surface antigens of Plasmodium falciparum.

The major antigens on the surface of Plasmodium falciparum merozoites are derived from a single high molecular weight polypeptide. The precursor to the major merozoite surface antigen (PMMSA) has conserved and variable antigenic determinants and varies in size in different isolates. Since the protein is a candidate for a malaria vaccine, it is important to understand the molecular basis of this variation. We present the complete sequence of the PMMSA of the Papua New Guinea isolate FC27 and the partial sequence of the West African isolate NF7. The gene consists of blocks of sequence which are either conserved or variable between different isolates. Variable sequences fall into two distinct types, indicating that the PMMSA is encoded by dimorphic alleles that undergo recombination within conserved blocks at the 5' end of the gene. Genetic exchange is not apparent within the other two-thirds of the gene in 12 isolates, suggesting strong selection against such recombinants. The most variable block located near the 5' end contains repeats that are different in independent cloned isolates. This variation presumably accounts for much of the size and antigenic variability.

Alleles↗
PubMed1988

Autoantibodies to mitochondria in systemic sclerosis. Frequency and characterization using recombinant cloned autoantigen.

Mitochondrial autoantibodies, a hallmark of primary biliary cirrhosis (PBC), have been widely described for many years in patients with systemic sclerosis, and there have been several reports of the concurrence of systemic sclerosis and PBC. However, there is very little information with respect to the significance of these autoantibodies or any definitive evidence that the antigens involved represent the mitochondrial autoantigens (M2 complex) described in PBC. We have cloned and sequenced a rat complementary DNA which encodes for all the epitopes recognized by autoantibodies to the major, or 70-kd, mitochondrial autoantigen in patients with PBC. Using this recombinant fused autoantigen, as well as by immunoblotting with human placental mitochondria, we tested for antimitochondrial antibody specificity in sera from 250 patients with systemic sclerosis. Nineteen sera (7.6%), including those from patients with CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias) and diffuse scleroderma, had reactivity with human placental mitochondria proteins by immunoblot testing. All 19 sera reacted with the M2 complex. All sera that reacted with the 70-kd protein likewise reacted with the recombinant cloned autoantigen. The predominant autoantibody isotype to the 70-kd protein was IgG3. Interestingly, the 70-kd protein is 11% proline, an amino acid which is frequently preceded by hydrophobic amino acids.

Antibody Specificity↗
PubMed1988

A component of an antigenic rhoptry complex of Plasmodium falciparum is modified after merozoite invasion.

Human antibodies affinity purified on an adsorbent prepared from a cDNA clone (Ag44) expressing a portion of a rhoptry antigen were used to characterize the synthesis and fate of the antigen in the asexual blood stages of Plasmodium falciparum. The rhoptry antigen is synthesized in the mature trophozoite-stage parasites as a 103 kDa polypeptide, is present in the schizonts and merozoites as a 105 kDa polypeptide, is discharged from the rhoptries and found in the newly invaded red cells as a 110 kDa polypeptide. Anti-Ag44 antibodies immunoprecipitate the antigen and two additional polypeptides of 135 and 150 kDa from lysates of infected cells and from culture supernatants. The three polypeptides are associated in a non-covalent complex that persists in the newly invaded red cells. All the components of the high molecular weight rhoptry complex are antigenic and can be precipitated with immune human serum. The 135 kDa polypeptide is identical to a 140 kDa rhoptry antigen previously identified by a monoclonal antibody.

Animals↗
PubMed1988

MESA is a Plasmodium falciparum phosphoprotein associated with the erythrocyte membrane skeleton.

The mature-parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum is an antigenically variable, high molecular weight protein of trophozoites and schizonts that is located at the erythrocyte surface membrane. It is first synthesized at the late ring stage and continues to be synthesized until late schizogony. MESA cannot be detected on the external surface of erythrocytes infected by trophozoites and early schizonts but is located at the internal surface in association with the erythrocyte membrane skeleton. The degree of association with the membrane skeleton varies among parasite lines, being greater in knobby parasite lines. MESA is phosphorylated and is present in a similar location to another phosphoprotein, the ring-infected erythrocyte surface antigen (RESA). However, it differs from RESA in being detected at a later stage of asexual development of the parasite.

Animals↗
PubMed1988

Identification of two integral membrane proteins of Plasmodium falciparum.

We describe the isolation and cloning of two integral membrane protein antigens of Plasmodium falciparum. The antigens were isolated by Triton X-114 temperature-dependent phase separation, electrophoretically transferred to nitrocellulose, and used to affinity-purify monospecific human antibodies. These antibodies were used to isolate the corresponding cDNA clones from a phage lambda gt11-Amp3 cDNA expression library. Clone Ag512 corresponds to a Mr 55,000 merozoite rhoptry antigen, and clone Ag513 corresponds to a Mr 45,000 merozoite surface antigen. Both proteins can be biosynthetically labeled with [3H]glucosamine and [3H]myristic acid, suggesting that they may be anchored in membranes via a glycosylphosphatidylinositol moiety. Similarities in the C-terminal sequences of the Mr 45,000 merozoite surface antigen and the Trypanosoma brucei variant surface glycoproteins provides further evidence that this antigen has a glycosylphosphatidylinositol anchor.

Amino Acid Sequence↗
PubMed1988

Primary structure of the human M2 mitochondrial autoantigen of primary biliary cirrhosis: dihydrolipoamide acetyltransferase.

Primary biliary cirrhosis is a chronic, destructive autoimmune liver disease of humans. Patient sera are characterized by a high frequency (greater than 95%) of autoantibodies to a Mr 70,000 mitochondrial antigen, a component of the M2 antigen complex. We have identified a human cDNA clone encoding the complete amino acid sequence of this autoantigen. The predicted structure has significant similarity with the dihydrolipoamide acetyltransferase (EC 2.3.1.12) of the Escherichia coli pyruvate dehydrogenase multienzyme complex. The human sequence preserves the Glu-Thr-Asp-Lys-Ala motif of the lipoyl-binding site and has two potential binding sites. Expressed fragments of the cDNA react strongly with sera from patients with primary biliary cirrhosis but not with sera from patients with autoimmune chronic active hepatitis or sera from healthy subjects.

Acetyltransferases↗
PubMed1988

A second antigenic heat shock protein of Plasmodium falciparum.

We describe here an antigen of Plasmodium falciparum, defined by a cDNA clone designated Ag361. The antigen is a soluble cytoplasmic 70-kD polypeptide present in all isolates analyzed and in all stages of asexual development in the blood. The antigen is a natural immunogen, although it lacks repeating epitopes of many P. falciparum antigens. Ag361 shares extensive sequence homology with the hsp70 proteins of Xenopus laevis, Drosophila melanogaster, Escherichia coli, and man, as well as a previously isolated P. falciparum hsp70 protein. The genome of P. falciparum contains at least five hsp70-like genes, located on at lest four different chromosomes.

Amino Acid Sequence↗
PubMed1988

Third form of the precursor to the major merozoite surface antigens of Plasmodium falciparum.

The precursor to the major merozoite surface antigens of Plasmodium falciparum appears to be encoded by two distinctly different (dimorphic) alleles able to undergo limited recombination. We analyzed 18 previously uncharacterized P. falciparum isolates to test the dimorphic model. All but one, a Thailand isolate, conformed to the dimorphic model, and this isolate conformed to the dimorphic model in all but variable block 2. Sequence analysis revealed that block 2 of isolate CSL2 was a third form. Hence, the dimorphic model is not strictly correct. Recombination between alleles was found only within two conserved blocks near the 5' end of the gene.

Alleles↗
PubMed1988

Patterns of antigen expression in asexual blood stages and gametocytes of Plasmodium falciparum.

The stage specificity and localization of 12 Plasmodium falciparum antigens were determined by immunofluorescence using acetone-fixed parasites reacted with monospecific antibodies against cloned antigens. Antibodies were prepared by immunization of rabbits with recombinant proteins or by affinity purification of human plasma against cloned antigen adsorbents. Most of the antigens occurred predominantly in mature asexual parasites, two were abundant in ring stages and three were absent in rings. Four of the 12 antigens were detected in asexual stages but not in gametocytes. Grouping of antigens by localization within blood stages was difficult because of the complexity of fluorescence patterns observed. With some antibodies, fluorescence was apparently distributed evenly over the parasites, but in other cases label was concentrated within discrete compartments or organelles. Extraparasitic intraerythrocytic fluorescence was also observed.

Animals↗
PubMed1988

Immunization trials with the ring-infected erythrocyte surface antigen of Plasmodium falciparum in owl monkeys (Aotus vociferans).

A protocol was developed for the testing of blood stage vaccines against Plasmodium falciparum using Peruvian Aotus vociferans and the Indochina I/CDC strain of the parasite. Three different fused polypeptide vaccines containing elements of the ring-infected erythrocyte surface antigen molecule combined with Freund's complete and Freund's incomplete adjuvants were tested to determine their ability to protect against overwhelming infection following challenge with this highly virulent strain of P. falciparum, and to invoke antibody responses as measured by a standard indirect immunofluorescence technique. Nine of 14 immunized animals exhibited some protection. Presented are the test procedures developed for the conduct of such trials with New World monkeys and the analysis of results that led to the identification of variables selected for study in future trials.

Animals↗
PubMed1988

Variation in p126, a parasitophorous vacuole antigen of Plasmodium falciparum.

We describe a cDNA clone expressing part of p126, a parasitophorous vacuole antigen of Plasmodium falciparum that is processed to smaller fragments about the time of schizont rupture. The amino acid sequence includes determinants that are antigenic during the course of infection. The sequence contains a string of serine residues but no other repetitive elements. Comparison of the sequence to a previously published sequence demonstrates conserved and variable sequence elements. The genomic context of the single copy gene is conserved in six different isolates. p126 is shown to be identical to Pf140, an antigen previously demonstrated to partially protect Saimiri monkeys against P. falciparum infection.

Amino Acid Sequence↗
PubMed1987

Molecular cloning of the liver-specific rat F antigen.

F antigen is a 43-kDa widely conserved liver protein that has been intensively used in studies of immunogenicity and tolerance; two murine allotypes have been identified. Immunization of specific responder inbred strains with liver homogenates from the opposite allotype leads to precipitating antibody and cell-mediated immunity against F. The antibodies produced are autoantibodies as they react equally well with self. We have identified a cDNA clone from rat liver that reacts with alloantisera to F. The fused polypeptide produced by the clone was shown to correspond to F by several experiments. First, alloantisera to F antigen reacted with the cloned fused polypeptide, but not control recombinant clones. Second, mice immunized with the fused polypeptide generate an antibody response that reacts specifically with the 43-kDa protein of mouse liver homogenates and with highly purified F antigen. Finally, both anti-F allosera and sera from mice immunized with the fused polypeptide react with the same 43-kDa liver protein on two-dimensional immunoblots. The nucleotide and deduced amino acid sequence of the clone are presented and the sequence was found to have a significant homology with L28, an Escherichia coli ribosomal protein. The availability of recombinant F antigen will allow definitive questions to be addressed with respect to epitopes and specifically the identification of the T cell epitope which allows for autoimmune responses.

Amino Acid Sequence↗
PubMed1987

Serological diagnosis of primary Sjögren's syndrome by means of human recombinant La (SS-B) as nuclear antigen.

Human recombinant La nucleoprotein was purified from cultures of Escherichia coli containing a vector with a 1.4 kilobase cDNA encoding La; the nucleoprotein was used to test for antinuclear antibodies (ANA) to La. Serum samples from 260 patients with autoimmune diseases associated with ANA and 100 healthy subjects were tested by an enzyme-linked immunosorbent assay (ELISA). Samples from 47 (94%) of 50 patients with primary Sjögren's syndrome and 1 (7%) of 14 patients with secondary Sjögren's syndrome reacted with the recombinant La. No reactivity was demonstrated in 196 patients with other ANA-associated autoimmune diseases or in 100 healthy subjects. The study confirms the high correlation between ANA, anti-La, and primary Sjögren's syndrome and shows how gene cloning can provide large quantities of human autoantigens for use in highly specific and sensitive diagnostic assays.

Antibodies, Antinuclear↗
PubMed1987

Identification and specificity of a cDNA encoding the 70 kd mitochondrial antigen recognized in primary biliary cirrhosis.

Mitochondrial autoantibodies are characteristic of the disease primary biliary cirrhosis (PBC), but the immunoreactive mitochondrial antigens have not been defined. We used a rat liver cDNA library in lambda gt 11-Amp3 to clone a 1370-base pair insert that coded for a polypeptide reactive with PBC sera. This insert was subcloned for expression into pBTA224, a plasmid vector in the same reading frame as lambda-Amp3. A positive clone, designated pRMIT, that expressed a fused polypeptide of 160 kd, was recognized by 25 of 25 sera from patients with PBC and none of 96 sera from normal persons or patients with systemic lupus erythematosus, rheumatoid arthritis, or chronic active hepatitis. This fused polypeptide was shown to correspond with the 70 kd mitochondrial autoantigen by several experiments. First, lysates of pRMIT in J101 absorbed out the 70 kd reactivity of PBC sera when probed against fractionated placental mitochondria. Second, affinity-purified antisera reactive with the fused polypeptide also reacted with the 70 kd mitochondrial antigen. Third, such affinity-purified antisera produced the characteristic anti-mitochondrial pattern of immunofluorescence on tissue sections. Finally, immunization of BALB/c mice with the fused polypeptide elicited antibodies to mitochondria. These murine antibodies reacted with the 70 kd mitochondrial protein and also produced typical mitochondrial immunofluorescence on tissue sections. The nucleotide and amino acid sequence of the recombinant protein, which encodes for approximately a 48 kd protein, showed no significant homologies with known proteins, and there were no homologies with mitochondrial genomic DNA. The availability of a recombinant form of the 70 kd mitochondrial autoantigen will allow several definitive questions to be addressed in PBC, including identification of B cell epitopes, T cell recognition, and a model of PBC in mice.

Amino Acid Sequence↗
PubMed1987

Plasmodium falciparum: identification and localization of a knob protein antigen expressed by a cDNA clone.

Differential screening of cDNA libraries constructed from knobby and predominantly knobless Plasmodium falciparum isolates, identified the sequence SD17. Chromosome blotting experiments have shown that this sequence, which is located on chromosome 2 of most isolates, was deleted in the cloned parasite line E12 of the FCQ27/PNG isolate. Here we show that erythrocytes infected with the SD17-containing cloned line D10 have typical knob structures on their surfaces, whereas those infected with the line E12 lack knobs. An expression clone was constructed from SD17 and used to affinity purify antibodies from the sera of individuals living in areas of Papua New Guinea where malaria is endemic. The antibodies reacted in immunoblotting experiments with a single polypeptide that varied in Mr from 85,000 to 105,000 among different isolates. The antigen was not expressed in the knobless clone E12. Postembedding immunoelectron microscopy showed localization of the antigen over the knobs of FC27 and two other isolates, largely on the cytoplasmic side. We conclude that the parasite antigen corresponding to clone SD17 is a knob protein.

Animals↗