Biomedical subjects
Publications and source records attributed to R A Lerner.
The specific hydrolysis of unactivated esters bearing an R or S enantiomeric alcohol has been achieved by two separate classes of catalytic antibodies induced to bind either the R or S substrates. The antibodies exhibit rate accelerations (10(3) to 10(5] above background hydrolysis that, coupled with their antipodal specificity, provide a novel set of reagents for use in synthesis.
Monoclonal antibodies have been induced that are capable of catalyzing specific hydrolysis of the Gly-Phe bond of peptide substrates at neutral pH with a metal complex cofactor. The antibodies were produced by immunizing with a Co(III) triethylenetetramine (trien)-peptide hapten. These antibodies as a group are capable of binding trien complexes of not only Co(III) but also of numerous other metals. Six peptides were examined as possible substrates with the antibodies and various metal complexes. Two of these peptides were cleaved by several of the antibodies. One antibody was studied in detail, and cleavage was observed for the substrates with the trien complexes of Zn(II), Ga(III), Fe(III), In(III), Cu(II), Ni(II), Lu(III), Mg(II), or Mn(II) as cofactors. A turnover number of 6 x 10(-4) per second was observed for these substrates. These results demonstrate the feasibility of the use of cofactor-assisted catalysis in an antibody binding site to accomplish difficult chemical transformations.
The T cell-mediated immune response of infectious mononucleosis (IM) patients to five Epstein-Barr virus (EBV)-determined nuclear antigens, EBNAs, and to the membrane antigen associated with growth-transformed cells (latent membrane protein, LMP) was measured by the leukocyte migration inhibition (LMI) assay. Two different antigen sources were used: extracts from cells that only expressed EBNA-1, EBNA-2, or LMP after transfection with the corresponding EBV-DNA fragment, and synthetic peptides deduced from the corresponding genes. Patients in the acute phase of the disease failed to respond to EBNA-1, -5, -6, and LMP, but became responsive during convalescence. The majority of the patients responded to EBNA-2 and/or EBNA-3 in the acute phase (9/15 and 12/15, respectively). The response to EBNA-2 and/or EBNA-3 in the acute phase (9/15 and 12/15, respectively). The response to EBNA-3 disappeared more often in convalescence than the response to EBNA-2: 6 of 15 patients were negative to EBNA-2 and 12 of 15 to EBNA-3 during recovery. In addition to its value in the assessment of host sensitization to virus EBV antigens, these studies and the derived hypotheses also provide certain predictions about the predominant antigen expression in the EBV-infected host under normal and pathological conditions that can be subjected to direct experimental tests.
Efficient generation of catalytic antibodies is uniquely dependent on the exact nature of the binding interactions in the antigen-antibody complex. Current methods for generation of monoclonal antibodies do not efficiently survey the immunological repertoire and, therefore, they limit the number of catalysts that can be obtained. We are exploring methods to clone and express the immunological repertoire in Escherichia coli. As the essential first step, we present here a method for the establishment of a highly diverse heavy chain variable region library. Consequently, it should now be possible to express and recombine the heavy and light chain variable region fragments to generate a large array of functional combining portions of the antibody molecule. This technology may provide an alternative to the hybridoma methodology for accessing the monoclonal antibody specificity of the immune system.
The advent of site-directed serology has provided a specific and simplified means of distinguishing antibody responses to the two types of human immunodeficiency virus (HIV) in an outbred population. Remarkably, human sera containing HIV antibodies and simian sera containing simian immunodeficiency virus (SIV) antibodies had a very narrow amino acid dependence in the 23 residue long peptide, 582Ala-604Cys, used as antigen. A single dominating antigenic site was demonstrated in the C-terminal part of the peptide--596Trp-602Gln for HIV-2 and SIV antibodies and 597Gly-602Leu for HIV-1. A minor fraction of HIV-1 antibodies also reacted with a second site, 588Lys and 589Asp. These data define the precise amino acid dependence of a uniquely immunogenic site in the folded transmembrane protein of HIV and facilitate the optimizing of peptide antigens for site-directed serology.
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Applications of sensitive new technologies, in particular, two-dimensional NMR spectroscopy, have allowed detection of folded structures in short peptide fragments of proteins in aqueous solution under conditions where native proteins fold. These structures are in rapid dynamic exchange with unfolded states. These observations provide evidence in support of models for protein folding which postulate localized regions of folded structure as initiation sites for the folding process. Since these initiation processes are expected to be rapid, such models are consistent with kinetic evidence that the rate-determining steps of protein folding occur late in the process and probably involve rearrangement of incorrectly folded intermediates.
Catalysis of amide bond hydrolysis is of singular importance in enzymology. An antibody was induced to an analog of a high-energy intermediate anticipated along the reaction coordinate of amide hydrolysis. This antibody is an amidase with high specificity and a large rate enhancement (250,000) relative to the uncatalyzed reaction. This reaction represents the kinetically most difficult hydrolysis reaction yet catalyzed by an antibody.
A systematic examination by 1H nuclear magnetic resonance of the population of beta-turn-containing conformers in several series of short linear peptides in water solution has demonstrated a dependence on amino acid sequence which has important implications for initiation of protein folding. The peptides consist of a number of variants of the sequence Tyr-Pro-Tyr-Asp, the trans isomer of which was previously shown to contain a reverse turn in water. Two-dimensional rotating-frame nuclear Overhauser effect spectroscopy provides unequivocal evidence that substantial populations of reverse turn conformations occur in water solutions of certain of these peptides. In the unfolded state, the peptides adopt predominantly extended chain (beta) conformations in water. It appears probable from the nuclear Overhauser effect connectivities observed that the reverse turns in the trans isomers are predominantly type II. The low temperature coefficient of the amide proton resonance of the residue at position 4 of the turn suggests the presence of an intramolecular hydrogen bond. The presence of the beta-turn conformation has been confirmed for certain peptides by circular dichroism measurements. Substitutions at positions 3 and 4 in the sequence Tyr-Pro-Tyr-Asp-Val can enhance or abolish the beta-turn population in the trans peptide isomers. The residue at position 3 of the turn is the primary determinant of its stability. A small amount of additional stabilization appears to result from an electrostatic interaction between the side-chain of residue 4 and the unblocked amino terminus. For peptides of the series Tyr-Pro-X-Asp-Val, where X represents all L-amino acid except Trp and Pro, the temperature coefficient of the Asp4 amide proton resonance provides a measure of the beta-turn population. The beta-turn populations in water solution measured in this way correlate with the beta-turn probabilities determined from protein crystal structures. This indicates that it is frequently the local amino acid sequence, rather than medium- to long-range interactions in the folded protein, that determines the beta-turn conformation in the folded state. Such sequences are excellent candidates for protein folding initiation sites. A high population of structured forms appears to be present in the cis isomer of certain of the peptides, as shown by a considerable increase in the proportion of the cis isomer and by measurement of nuclear Overhauser effects and 3JN alpha coupling constants.(ABSTRACT TRUNCATED AT 400 WORDS)
1H nuclear magnetic resonance experiments indicate formation of secondary structures in water solutions of a synthetic immunogenic peptide of sequence EVVPHKKMHKDFLEKIGGL corresponding to the C-helix (residues 69 to 87) of myohemerythrin. The conformational ensemble consists of a set of turn-like structures, distributed over the C-terminal half of the peptide and rapidly interconverting by way of unfolded states. These structures, termed nascent helix, are stabilized into helical structure with long-range order in water/trifluorethanol mixtures. Circular dichroism measurements confirm the presence of 50% helix in water/trifluoroethanol but show no evidence of helicity in water solutions of the peptide. It is apparent that no one member of the transient set of helical conformations which constitutes the nascent helix is sufficiently long to be detectable by circular dichroism experiments. No preferred conformations could be detected by nuclear magnetic resonance in the N-terminal half of the peptide, either in water or water/trifluoroethanol mixtures. This region of the peptide is stabilized in helix by long-range interactions in the folded protein. The possible role of nascent secondary structure in induction of antipeptide antibodies and in initiation of protein folding is discussed.
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Human B lymphocytes transformed by infection with the Epstein-Barr virus (EBV) express a new membrane protein of 63 kDa (latent membrane protein, LMP) encoded by the virus. The function of this protein in the virus-cell interaction is not known. In this work we have identified in EBV- human and mouse cell molecules which cross-react with LMP. Two types of reagents were employed: (a) antibodies against LMP-derived synthetic peptides, affinity purified from antisera against a fusion protein containing the carboxy half of the LMP molecule and (b) antisera prepared by immunizing rabbits directly with the peptide conjugates. Cross-reactions were determined by radioimmunoblotting experiments. At least six molecules (Mr = 110, 85, 63, 53, 45 and 23 kDa), present in a variety of human cells (peripheral blood lymphocytes, B cell lines and epithelial cell lines) were found to cross-react with the LMP-derived peptides. Cross-reacting proteins were also identified in normal mouse tissues. The specificity of the cross-reacting antibodies was confirmed by inhibition experiments with the corresponding peptide. Furthermore, antibodies eluted from individual bands were shown to bind to the same band when reacted with new blots of the same extracts. Our data suggest that normal cells contain a family of highly conserved proteins cross-reacting with the LMP molecule. If, indeed, these proteins share common functions, their study may lead the way to unraveling the function of LMP.
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A new strategy for designing synthetic vaccines is presented. In this approach synthetic peptides are conformationally restricted by replacing putative hydrogen bonds with covalent mimics. The chemistry for substituting a hydrazone-ethane link (N-N = CH-CH2-CH2) for an (i + 4)----i hydrogen bond in a pentapeptide with alpha-helical potential is reported. Chemically shaping peptides to mimic the three-dimensional surfaces of proteins may enhance their immunogenicity. To test this strategy, a potential synthetic vaccine for malaria, Cys-(Asn-Pro-Asn-Ala)3-NH2, was conformationally restricted by replacing putative hydrogen bonds between asparagine side chains with a covalent replacement, an ethylene bridge, to give first generation chemically shaped immunogens. Antibodies to one of the shaped malarial peptides show a strong reaction with living Plasmodium falciparum sporozoites, a form of malaria which infects hundreds of millions of people yearly.
We report a nonhydrolytic bimolecular aminolysis reaction catalyzed by an antibody. The stereospecific formation of an amide from racemic lactone plus an aromatic amine is described by a random equilibrium bireactant kinetic sequence. The observed turnover rate may be approximated from the measured difference between the binding of reactants and the transition state analog.
The transmembrane glycoprotein (gp32) of the simian immunodeficiency virus (SIV) contains a highly antigenic region that includes amino acid residues 606-628. A synthetic peptide representing this region was highly immunoreactive with sera from SIV-infected primates in a site-directed enzyme-linked immunosorbent assay (ELISA). This reactivity extended across four primate species from three genera and identified infection with at least two distinct isolates of SIV. This site-directed ELISA represents a simple, accessible method with broad specificity for screening large numbers of primates for antibodies against SIV.
We developed a method for immunoaffinity purification of Saccharomyces cerevisiae adenylyl cyclase based on creating a fusion with a small peptide epitope. Using oligonucleotide technology to encode the peptide epitope we constructed a plasmid that expressed the fusion protein from the S. cerevisiae alcohol dehydrogenase promoter ADH1. A monoclonal antibody previously raised against the peptide was used to purify adenylyl cyclase by affinity chromatography. The purified enzyme appeared to be a multisubunit complex consisting of the 200-kilodalton adenylyl cyclase fusion protein and an unidentified 70-kilodalton protein. The purified protein could be activated by RAS proteins. Activation had an absolute requirement for a guanine nucleoside triphosphate.