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J J Marchalonis

Publications and source records attributed to J J Marchalonis.

At least 73 records · Page 4Linked to original sources
PubMed1993

Natural human antibodies to synthetic peptide autoantigens: correlations with age and autoimmune disease.

Clinically healthy humans as well as patients suffering from various autoimmune diseases produce natural antibodies against a variety of self-components. Such antibodies have been proposed to carry out a physiologic role in maintaining the integrity of self, as well as potentially destructive roles in the generation of autoimmune diseases. Because human autoantigens, particularly membrane proteins, are usually present in extremely small amounts, it is generally impossible to obtain enough to carry out a detailed characterization of the antibodies or the antigenic determinants recognized. To circumvent this difficulty, we developed synthetic autoantigens predicted from the gene sequence of two functionally critical membrane proteins; the band 3 anion transport protein which is found on all cells, and the T-cell receptor (beta chain) which is the antigen-specific receptor on thymus-derived lymphocytes. We have investigated the natural human IgM and IgG antibody responses to peptides selected on the basis of predicted molecular surface exposure and previously known antigenicity, and correlate levels of binding with changes in age and by comparison with autoimmune diseases. We report that the IgM response to synthetic autoantigens tends to be higher than that of IgG molecules, but significant IgG binding occurs to some peptides. This situation is particularly noticeable in comparison of rheumatoid arthritis patients with normal individuals. Distinct peptide portions of individual molecules are recognized differently by the autochthonous immune system as manifested by age dependence of the response and differential levels of IgM and IgG activity. The synthetic autoantigens that tend to generate the highest amounts of natural antibody are those that are either exposed on the surface of the cell (band 3 peptides) or are exposed in the predicted 3-dimensional folding of the molecule (T-cell receptor beta peptides). Rheumatoid arthritis patients tend to give higher IgM reactivities to both band 3 and Tcr beta peptides than do normals, with this effect being less pronounced in the distinct autoimmune disease systemic lupus erythematosus. Studies of normal humans ranging in age from 20 to 90 years suggest two major patterns for the IgM natural antibody response to synthetic peptides giving high response. The first is that the level of IgM reactivity is high early in life and remains high throughout. The second pattern is one in which the reaction is high in younger individuals, but diminishes substantially in the latter decades of life.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed1993

Partial amino acid sequence of monoclonal extracellular antigen-specific T cell proteins.

Antigen-specific molecules secreted by murine T cell hybrids (TABM) specific for 4-hydroxy 3-nitrophenyl (NP) or azobenzenearsonate (ABA) were purified from ascitic fluid by ion exchange chromatography and/or affinity for antigen. Partial amino acid sequence of reduced Mr 72,000 NP-specific polypeptides and Mr 20,000 peptides prepared by treatment of the ABA-specific immunoprotein with cyanogen bromide was obtained and a septapeptide of the NP-specific TABM shared 3/7 residues with the ABA-specific TABM. Both TABM shared residues present in 95% T cell receptor for antigen (TCR) V alpha subgroup I and 83-96% murine immunoglobulin V kappa Fr3. These results provide evidence that extracellular antigen-specific T cell proteins are soluble analogues of TCR alpha chains and belong to the immunoglobulin supergene family.

Amino Acid Sequence↗
PubMed1993

Human autoantibodies to a synthetic putative T cell receptor beta-chain regulatory idiotype: expression in autoimmunity and aging.

We used synthetic peptides to analyze the human natural antibody response to V beta determinants. A major determinant recognized by IgM and IgG autoantibodies of clinically healthy individuals as well as those suffering from rheumatoid arthritis (RA) was a peptide corresponding to the first complementarity-determining region (CDR1). The natural IgM response of RA patients to this synthetic autoepitope was significantly elevated relative to that shown by healthy individuals. The levels of IgM reactivity to determinants corresponding to this region decreased with increasing age. By contrast, IgG autoantibodies to certain V beta CDR1 peptides increased markedly with age. In order to determine whether the CDR1 V beta determinant might be involved in immunization, we immunized rabbits with a human peptide that is greater than 80% identical to the homologous sequence derived from a rabbit V beta gene. As a control, the rabbits were immunized with a peptide of equal length derived from the N-terminus of the human V beta chain. Like humans, rabbits tended to have high levels of autoantibodies to the CDR1 peptide but not to the N-terminal segment. Following immunization, the rabbits produced strong IgG responses to the N-terminal peptide. By contrast, immunization with the CDR1 peptide resulted in levels of IgG antibody less than or equal to the natural activity in unimmunized rabbits. These studies indicate that the CDR1 region of Tcr V beta is a widely recognized autoantigenic portion of the Tcr that most probably functions as a regulatory epitope in man and other species.

Adult↗
PubMed1992

Human autoantibodies reactive with synthetic autoantigens from T-cell receptor beta chain.

We used mapping with synthetic overlapping peptides in combination with molecular modeling to analyze the IgG antibodies that humans naturally produce against human T-cell receptor beta chains and to localize the recognized peptide autoantigens in the three-dimensional structure of the molecule. Healthy individuals produce low levels of antibodies against T-cell receptor peptides, and these can be increased in autoimmune diseases. We characterized the reactivities in detail because IgG molecules reactive with self peptides occur in preparations of intravenous immunoglobulin and can be isolated by immunoaffinity chromatography. Natural IgG antibodies were directed against three major peptides. One corresponds to the first complementarity-determining region of the variable region. A second corresponds to the third framework of the variable region. The third is located in the constant region and is predicted to be a loop that extends out of the beta-barrel structure. This peptide is one that would give a characteristic structural distinction between the beta-chain constant region and the constant regions of immunoglobulin light chains to which beta chains are homologous. The capacity to bind these peptides is found in small fractions of normal polyclonal IgG, which contains both kappa chains and lambda chains. The activity is antibody-like in being confined to the Fab fragment and in its capacity to discriminate among homologous synthetic peptides corresponding to distinct beta-chain variable-region genes. We propose that a recognition and regulatory process naturally occurs that parallels the immune network for the regulation of the production of antibodies.

Adult↗
PubMed1992

Complete sequence of a cDNA clone specifying sandbar shark immunoglobulin light chain: gene organization and implications for the evolution of light chains.

A full-length cDNA clone specifying sandbar shark (Carcharhinus plumbeus) immunoglobulin light chain has been isolated and sequenced. By alignment with human lambda chains, the leader, framework, complementarity-determining, joining, and constant regions are clearly identified in the shark light chain. Approximately 40-50% identity is shared between the human and shark sequences in the variable and constant regions. We have performed sequence comparisons of the individual segments and constructed phylogenetic trees for the variable region. These studies identify the shark protein as a lambda chain. In addition, the sandbar shark light chain is only distantly related to that of horned shark (Heterodontus francisci) [Shamblott, M. J. & Litman, G. W. (1989) Proc. Natl. Acad. Sci. USA 86, 4684-4688], demonstrating that the long evolutionary time of divergence among shark species has led to the generation of substantial differences in sequence. The positions of the variable, joining, and constant gene segments in 14 genomic clones have been mapped. The segments are linked in individual clusters (variable, joining, constant) occupying 3-7 kilobases. Cluster arrangement can be grouped into two patterns based upon spacing between the genes in the individual clones. This arrangement is fundamentally different from that observed in higher vertebrates.

Amino Acid Sequence↗
PubMed1992

Antigenic mapping of a human lambda light chain: correlation with three dimensional structure.

Although the amino acid sequence and three-dimensional structure of human immunoglobulin light chains have been known for more than 15 years, the location of antigenic markers characteristic of lambda chains has not been determined. Here, we use a set of synthetic overlapping peptides to completely model the sequence of the lambda chain Mcg and test these for the binding of rabbit and goat antisera specific for lambda chain determinants. We assess peptide contributions to lambda-antigenic reactivity and also to identify a portion of C-region where conformational factors contribute to the antigenicity. Specific determinants occur both in the constant and variable (first and third framework) domains of the molecule. The fourth framework of the variable region, a segment specified by the joining gene, is also recognized and cross-reacts antigenically with the homologous region of T cell receptor beta chains. Major lambda specific determinants are localized in the N- and C-terminal segments, which are linear and devoid of major conformational folding. Other segments that are strongly antigenic, such as the third framework of the V region (residue 78-93) and a segment of the constant region (residues 177-192), show strong conformational dependence in antigenicity.

Amino Acid Sequence↗
PubMed1992

Antigenic cross-reactions among immunoglobulin of diverse vertebrates (elasmobranchs to man) detected using xenoantisera.

1. Antisera raised in rabbits and goats against intact immunoglobulins or their constituent light and heavy chains from man, mouse and the galapagos shark (Carcharhinus galapagenesis) were tested for their reactivity with immunoglobulins of elasmobranchs, other lower vertebrates and eutherian and prototherian mammals. 2. Xenoantisera directed against human heavy chain isotypes allowed the serological identification of IgM and IgG immunoglobulins in the echidna (Tachyglossus aculeatus), a monotreme which is one of the most primitive species of extant mammals. 3. The antisera to heavy chains reacted to varying degrees with purified immunoglobulins of non-mammalian species, including the chicken, teleost fish and elasmobranchs in a fashion that was specific for immunoglobulins, but was not related to defined human isotypic markers. 4. The reactions of some antisera seem to skip species known to possess homologous immunoglobulins. 5. Antisera directed against isotypic markers of human kappa and lambda light chains reacted with shark light chains in a manner that was specific for light chain determinants but was not isotype-related. 6. Antisera directed against heavy chains of either sharks or mammals reacted with heavy chains, but not with light chains of diverse species. 7. A rabbit antiserum specific for shark light chain reacted with human and murine monoclonal lambda chains and with two synthetic peptides corresponding to human V lambda Fr3 and Fr4 sequences. 8. These results establish that a variety of antigenic markers including conformational and linear determinants can be shared among immunoglobulins of vertebrates species that had an ancestral divergence more than 400 million years ago.

Amino Acid Sequence↗
PubMed1992

Purification of a novel heterodimer from shark (Carcharhinus plumbeus) serum by gel-immobilized metal chromatography.

1. Resolution of the fraction of sandbar shark (Carcharhinus plumbeus) serum that was soluble in 50% saturated ammonium sulfate by gel-immobilized metal-affinity chromatography allowed the isolation of a novel disulfide-bonded heterodimer of intact mass 70 kDa. 2. Following reduction, the molecule could be resolved into two chains of apparent mass 36 and 24 kDa. 3. The molecules were glycoproteins as determined by an observed reduction in molecular weight following enzymatic glycosylation. 4. The two separate chains were related to one another on the basis of amino-acid composition analysis and by comparison of the N-terminal amino acids (seven out of 10 identities). 5. The exact relationship of this molecule to characterized heterodimers of higher vertebrates is unknown. 6. Cross-linked agarose-acetate was synthesized and proved to be an efficient concentrating agent and also a hydrophobic interaction adsorbant.

Amino Acid Sequence↗
PubMed1992

Reactivity of anti-human C-reactive protein (CRP) and serum amyloid P component (SAP) monoclonal antibodies with limulin and pentraxins of other species.

Limulus polyphemus C-reactive protein (CRP) (limulin) has approximately 30% amino acid sequence homology and shares at least one idiotypic determinant associated with ligand-binding activity with human CRP (hCRP); limulin also shares amino acid sequence homology and lectin activity with human serum amyloid P component (hSAP). In the present study panels of 14 anti-hCRP monoclonal antibodies (mAb) directed to distinct hCRP epitopes and 11 anti-hSAP mAb directed to distinct epitopes of hSAP were tested for reactivity with limulin and pentraxins of other species including rabbit CRP (raCRP), rat CRP and hamster female protein (FP) by ELISA and Western blot analyses. None of the anti-human pentraxin mAb showed strong cross-reactivity with limulin; only five mAb reacted with limulin at all, and cross-reactivities of these mAb with the other pentraxins, when present, also were weak. Cross-reactivity of limulin with hCRP and hSAP was similar, and in light of comparable amino acid sequence homology, suggests this molecule can be considered the limulus SAP as well as the limulus CRP. Several anti-hCRP mAb cross-reacted strongly with rabbit CRP and rat CRP; a few anti-hSAP cross-reacted strongly with FP; and weak cross-reactions were observed between hCRP and hSAP, but cross-reactivities between the pentraxins generally were limited and weak. A rabbit polyclonal antibody raised to highly conserved limulin peptide 141-156 and strongly reactive with limulin reacted weakly with hCRP and raCRP but failed to react with rat CRP, hSAP or FP. These studies emphasize a limited but distinct antigenic similarity between limulin, hCRP and other pentraxins, and identify mAb reactive with potential regions of shared structure and/or function between pentraxins of different species.

Alpha-Globulins↗
PubMed1992

T-cell receptors of man and mouse studied with antibodies against synthetic peptides.

We used polyclonal rabbit antibodies directed against synthetic peptides predicted from the gene sequence of the human T-cell receptor (TCR) beta-chain YT35 to study the antigen receptor on human helper T-cell leukemia lines and on normal mouse thymocytes. Antibodies were raised to peptides corresponding to joining segment (J beta) and to a conserved stretch of sequence around the first cysteine in the constant region (C beta). These peptides were selected on the basis of homology with corresponding segments of immunoglobulin light chains. The specificity of the antibodies was established using synthetic overlapping peptides that modelled the complete TCR beta-chain. Western blot analysis was performed against detergent lysates of T cells. Both of the antibodies reacted strongly with 2-3 polypeptides in the mass range 40-45 kDa in mouse and human cells. Clearance experiments using monoclonal antibodies against murine TCR alpha- and beta-chains and against human TCR beta-chain and immunoprecipitations with monoclonal antibody to the murine T3 complex established that these components represented the alpha/beta heterodimer. An additional component around 31 kDa was detected by anti-J beta antibodies in murine thymus extracts. The use of the affinity-purified antipeptide antibody in two-dimensional Western blot analyses allows the clear discrimination between the characteristic individual receptors of monoclonal neoplastic T cells and the polydisperse patterns representative of heterogeneous normal populations. Antigenic cross-reactions between T-cell receptor beta-chains of man and mouse observed with monoclonal antibodies and rabbit antisera to peptides are consistent with the homology in gene sequence between the two species.

Amino Acid Sequence↗
PubMed1991

Hypothesis: synthetic aging antigen can be used to manipulate cellular lifespan.

Physiologic removal of old and damaged erythrocytes, platelets, and other terminally differentiated cells is initiated by the appearance of an aging antigen that marks them for death by initiating the binding of IgG autoantibody and subsequent removal by phagocytes. We have developed a synthetic aging antigen peptide that blocks binding of IgG to senescent cells in vitro. We hypothesize that the synthetic antigen can be used to prevent cell destruction in diseases such as autoimmune hemolytic anemias and idiopathic thrombocytopenia purpura, and that the antigen itself can be used to manipulate cellular lifespan in vivo.

Amino Acid Sequence↗
PubMed1990

Sequence analysis of homogeneous peptides of shark immunoglobulin light chains by tandem mass spectrometry: correlation with gene sequence and homologies among variable and constant region peptides of sharks and mammals.

Morphologically, sharks are living fossils that are remarkably similar to their Devonian ancestors of ca. 400 million years ago. If a parallel conservation in biochemical properties characterizes shark evolution, knowledge of the properties of shark immunoglobulins should provide information on the structure of primordial immunoglobulins and their genes. The problem of polyclonality of shark immunoglobulins has precluded detailed analysis of shark immunoglobulin light polypeptide chains. Here, we approach the problem of obtaining direct sequence information on polyclonal light chains of shark immunoglobulins by isolating homogeneous peptides from tryptic digests of shark light chains and sequencing these by tandem mass spectrometry. To confirm the location of the peptides, we isolated a complementary DNA (cDNA) clone from a sandbar shark cDNA library in the expression vector lambda gt11, identifying the clone by its ability to produce a peptide serologically detectable using rabbit antibody to purified shark light chain. The correspondence between peptide sequence and that derived from gene sequence provided direct proof that the gene studied was that of a major expressed serum light chain. Using this combined approach, we isolated homogeneous peptides from both constant and variable regions. The variable region peptides showed homology to corresponding sequences of mammalian V lambda and V kappa sequences. The constant region gene sequence we obtained was homologous to mammalian C lambda sequence. The four constant region tryptic peptides we sequenced corresponded exactly to stretches of the C lambda sequence derived from the DNA sequence. The combined approach described here shows that shark light chains exhibit heterogeneity at both the protein and gene level, but that the constant regions of these chains can be identified as homologs of mammalian lambda chains and that evolutionary conservation has occurred in V region sequences ranging from elasmobranchs to man.

Amino Acid Sequence↗
PubMed1990

Definition of a physiologic aging autoantigen by using synthetic peptides of membrane protein band 3: localization of the active antigenic sites.

Senescent cell antigen (SCA), an aging antigen, is a protein that appears on old cells and marks them for removal by the immune system in mammals. It is derived from band 3, a ubiquitous membrane transport protein found in diverse cell types and tissues. We have used synthetic peptides to identify aging antigenic sites on band 3, using a competitive inhibition assay and immunoblotting with IgG directed against the aging antigen on old cells. Results indicate that: (i) the active antigenic sites of the aging antigen reside on membrane protein band 3 residues that are extracellular regions implicated in anion transport (residues 538-554 and 788-827); (ii) a putative ankyrin-binding-region peptide is not involved in SCA activity; and (iii) carbohydrate moieties are not required for the antigenicity or recognition of SCA because synthetic peptides alone abolish binding of senescent cell IgG to erythrocytes. One of the putative transport sites that contributes to the aging antigen is located toward the carboxyl terminus. A model of band 3 is presented. Localization of the active antigenic site on the band 3 molecule facilitates definition of the molecular changes occurring during aging that initiate molecular as well as cellular degeneration.

Amino Acid Sequence↗
PubMed1989

Antibodies to synthetic peptides corresponding to variable-region first-framework segments of T cell receptor. Detection of T cell products and cross-reactions with classical immunoglobulins.

Recent studies at the gene level have shown that T cells express rearranged genes for four types of T cell receptors that are strongly homologous to classical immunoglobulins in the joining region and in the framework 1 (Fr1) and 3 segments of the variable region. Based upon the homologies in gene sequence, it follows that the gene products would show similarities in amino acid sequence and in the folding of the proteins so that cross-reactivities in antigenic determinants would be expected between variable regions of the T cell receptors and classical immunoglobulins. We have synthesized peptides corresponding to predicted protein sequences of the Fr1 residues of T cell receptor alpha, beta- and gamma-chains and have produced antibodies in rabbits against these synthetic peptides. Use of antisera and affinity-purified antipeptide antibodies indicated that high-titer antibodies could be raised that were specific for individual Fr1 peptides. Cross-reactions among Fr1 peptides of T cell receptors and immunoglobulin light chains were observed. In addition, some rabbit antisera raised against classical polyclonal immunoglobulins or affinity-purified immunoglobulin-like T cell receptors were found to exhibit binding activity against Fr1 peptides of T cell receptor beta- and gamma-chains. The sequence homology, although real among the Fr1 of T cell receptors and immunoglobulin light chains, is moderate and the antigenic cross-reaction must reflect the configuration and types of amino acids present. The development of antipeptide antibodies holds promise for the characterization of T cell receptors of various T cell sources and also offers a new means for the identification of molecules related to rearranging immunoglobulins.

Animals↗