Biomedical subjects

D G Klapper

Publications and source records attributed to D G Klapper.

At least 37 records · Page 2Linked to original sources

PubMed1991

Sequence polymorphism of Amb a I and Amb a II, the major allergens in Ambrosia artemisiifolia (short ragweed).

Two of the major allergens in the pollen of short ragweed are Amb a I and Amb a II (formerly antigen E and antigen K, respectively). The genes for Amb a I and Amb a II have recently been cloned, and it was shown that Amb a I is a family of proteins with at least three distinct polymorphic family members. This study addresses the number of individual Amb a I and Amb a II family members, the polymorphism in each family member and the expression of these genes in ragweed plants from different geographical locations. This work led to the cloning and characterization of a fourth Amb a I family member, designated Amb a I.4.

Allergens↗

PubMed1991

Receptor- and G-protein-regulated 150-kDa avian phospholipase C: inhibition of enzyme activity by isoenzyme-specific antisera and nonidentity with mammalian phospholipase C isoenzymes established by immunoreactivity and peptide sequence.

A 150-kDa phospholipase C previously was purified from turkey erythrocytes and shown to be a P2Y-purinergic receptor- and guanine nucleotide-binding protein-regulated enzyme [J. Biol. Chem. 265:13508-13514 (1990)]. The relationship of this enzyme to the 150-kDa mammalian phospholipase C isoenzymes, termed phospholipase C-beta and -gamma, has been examined. Four antisera to the turkey erythrocyte phospholipase C recognized the avian enzyme in immunoblots but failed to recognize phospholipase C-gamma; one of the these weakly recognized phospholipase C-beta. Antibodies to phospholipase C-beta and -gamma failed to recognize the turkey erythrocyte phospholipase C. However, two antibodies raised against peptide sequence in regions of conserved sequence common to mammalian phospholipase C isoenzymes recognized the 150-kDa turkey erythrocyte phospholipase C. Antisera against the native form of the turkey erythrocyte phospholipase C inhibited the activity of this enzyme against phosphatidylinositol 4,5-bisphosphate presented as a component of mixed phospholipid vesicles or of mixed phospholipid and sodium cholate micelles; inhibition occurred as a decrease in Vmax, with no apparent change in Km for substrate or in the Ca2+ dependence of phospholipase C activity. Catalytic activity of phospholipase C-beta or -gamma against exogenous substrate was unaffected by antisera to the turkey erythrocyte enzyme. Antisera against the native form of the turkey erythrocyte phospholipase C also partially inhibited (50-60% inhibition) the capacity of AIF4- or adenosine 5'-O-(beta-thio) diphosphate plus guanosine 5'-O-(gamma-thio) triphosphate to stimulate phosphoinositide hydrolysis in ghosts prepared from [3H]inositol-prelabeled turkey erythrocytes. Moreover, the capacity of the purified 150-kDa enzyme to reconstitute receptor and G-protein-regulated phospholipase C activity in purified turkey erythrocyte plasma membranes devoid of this activity was completely inhibited by antisera to the turkey erythrocyte enzyme. Five peptides that were purified by high performance liquid chromatography from a tryptic digest of the turkey erythrocyte 150-kDa phospholipase C had no recognizable sequence homology with any deduced sequence of the mammalian phospholipase C isoenzymes. One turkey erythrocyte phospholipase C-derived peptide had clear homology with sequence in the first (X-domain) conserved region common to at least three of the mammalian phospholipase C isoenzymes, and another 16-amino acid peptide had partial sequence homology with the second (Y-domain) conserved region common to the mammalian enzymes. An 8-amino acid peptide from the tryptic digest had 75% homology with a sequence near the carboxyl terminus of mammalian phospholipase C-beta.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1990

Maturation of Escherichia coli maltose-binding protein by signal peptidase I in vivo. Sequence requirements for efficient processing and demonstration of an alternate cleavage site.

Comparative analyses of a number of secretory proteins processed by eukaryotic and prokaryotic signal peptidases have identified a strongly conserved feature regarding the residues positioned -3 and -1 relative to the cleavage site. These 2 residues of the signal peptide are thought to constitute a recognition site for the processing enzyme and are usually amino acids with small, neutral side chains. It was shown previously that the substitution of aspartic acid for alanine at -3 of the Escherichia coli maltose-binding protein (MBP) signal peptide blocked maturation by signal peptidase I but had no noticeable effect or MBP translocation across the cytoplasmic membrane of its biological activity. This identified an excellent system in which to undertake a detailed investigation of the structural requirements and limitations for the cleavage site. In vitro mutagenesis was used to generate 14 different amino acid substitutions at -3 and 13 different amino acid substitutions at -1 of the MBP signal peptide. The maturation of the mutant precursor species expressed in vivo was examined. Overall, the results obtained agreed fairly well with statistically derived models of signal peptidase I specificity, except that cysteine was found to permit efficient processing when present at either -3 and -1, and threonine at -1 resulted in inefficient processing. Interestingly, it was found that substitutions at -1 which blocked processing at the normal cleavage site redirected processing, with varying efficiencies, to an alternate site in the signal peptide represented by the Ala-X-Ala sequence at positions -5 to -3. The substitution of aspartic acid for alanine at -5 blocked processing at this alternate site but not the normal site. The amino acids occupying the -5 and -3 positions in many other prokaryotic signal peptides also have the potential for constituting alternate processing sites. This appears to represent another example of redundant information contained within the signal peptide.

ATP-Binding Cassette Transporters↗

PubMed1989

Identification and localization of allergenic determinants on grass group I antigens using monoclonal antibodies.

Epitopes recognized by five mAb which block the binding of human IgE antibodies to grass group I (GpI) Ag were characterized and partially mapped. Site specificity studies defined four apparently non-overlapping blocking antibody binding sites on the meadow fescue GpI molecule, Fes e I. One of these sites (site A) was localized to a 14,000 m.w. fragment designated P3 generated by CNBr cleavage of purified Fes e I. The P3 peptide possessed human IgE binding sites as well as other epitopes (non-site A) defined by 19 other anti-GpI mAb. All of the P3 reactive antibodies recognized cross-reactive determinants found on GpI Ag isolated from five different grasses suggesting that P3 is a conserved portion of grass GpI molecules. The P3 fragment from Fes e I was used to immunize mice and induced antibodies which reacted with intact GpI Ag from all 5 different grasses currently being studied in this laboratory.

Animals↗

PubMed1989

Isolation and characterization of a major cross-reactive grass group I allergenic determinant.

A series of peptides has been isolated from the Group I (GpI) allergen from five species of grasses currently under study in this laboratory. These peptides were generated by performing a limited tryptic digestion on each extensively reduced and alkylated GpI allergen and fractionating the digest by molecular sieve, ion-exchange and HPLC reverse-phase chromatography. Material in the elution profiles was assayed with a monoclonal antibody known to cross-react with all GpI allergens in this study. In addition, this particular monoclonal antibody has been previously shown to inhibit the ability of human IgE to bind to these GpI allergens. The peptides identified and isolated in this way were found to be highly homologous to one another.

Allergens↗

PubMed1988

Purification of a 31,000-dalton insulin-like growth factor binding protein from human amniotic fluid. Isolation of two forms with different biologic actions.

Human amniotic fluid has been shown to contain a protein that binds insulin-like growth factor I and II (IGF-I and IGF-II). Partially purified preparations of this protein have been reported to inhibit the biologic actions of the IGFs. In these studies our laboratory has used a modified purification procedure to obtain a homogeneous preparation of this protein as determined by polyacrylamide gel electrophoresis and amino acid sequence analysis. During purification the ion exchange chromatography step resulted in two peaks of material with IGF binding activity termed peaks B and C. Each peak was purified separately to homogeneity. Both peaks were estimated to be 31,000 daltons by polyacrylamide gel electrophoresis and their amino acid compositions were nearly identical. Amino acid sequence analysis showed that both peaks had identical N-terminal sequences through the first 28 residues. Neither protein had detectable carbohydrate side chains and each had a similar affinity for radiolabeled IGF-I (1.7-2.2 x 10(10) liters/mol). In contrast, these two forms had marked differences in bioactivity. Concentrations of peak C material between 2 and 20 ng/ml inhibited IGF-I stimulation of [3H]thymidine incorporation into smooth muscle cell DNA. In contrast, when peak B (100 ng/ml) was incubated with IGF-I there was a 4.4-fold enhancement of stimulation of DNA synthesis. Additionally, pure peak B was shown to adhere to cell surfaces, whereas peak C was not adherent. The non-adherent peak C inhibited IGF-I binding to its receptor and to adherent peak B. We conclude that human amniotic fluid contains two forms of IGF binding protein that have very similar physiochemical characteristics but markedly different biologic actions. Since both have similar if not identical amino acid compositions, N-terminal sequences, and do not contain carbohydrate, we conclude that they differ in some other as yet undefined post-translational modification.

Amino Acid Sequence↗

PubMed1988

Phospholipid binding properties of bovine prothrombin peptide residues 1-45.

The present study investigates the unique contribution of the NH2-terminal 33 residues of prothrombin, the gamma-carboxyglutamic acid (Gla) domain, to the Ca(II) and phospholipid-binding properties of prothrombin. Two Gla domain peptides, 1-42 and 1-45, produced by chymotryptic cleavage of prothrombin fragment 1 (residues 1-156 of the amino terminus of bovine prothrombin) and isolated by anion-exchange chromatography were utilized to characterize the Gla domain of prothrombin. This investigation utilized several experimental approaches to examine the properties of the Gla domain peptides. These studies were somewhat hampered by the metal ion-induced insolubility of the peptides. However, the 1-45 peptide was specifically radioiodinated, which facilitated the study of this peptide at low concentrations. In contrast to prothrombin fragment 1, the intrinsic fluorescence of both 1-42 and 1-45 was not quenched upon the addition of 1 mM Ca(II) or any concentration of Mg(II). Equilibrium dialysis studies revealed that the 1-42 peptide bound three Ca(II) ions noncooperatively, whereas fragment 1 binds seven Ca(II) ions in a positive cooperative manner. Ca(II)-promoted conformational changes are observed by comparison of electrophoretic mobility changes in the presence of increasing Ca(II) concentrations. Prothrombin, fragment 1, and the Gla domain peptides 1-42 and 1-45 exhibited similar electrophoretic mobility behavior in the presence of Ca(II) ions. The radiolabeled 1-45 peptide was found to comigrate with phospholipid vesicles on gel permeation chromatography in the presence of Ca(II). Fragment 1 was shown to inhibit this Ca(II)-dependent phospholipid binding of 1-45, demonstrating that the 1-45 peptide does possess the necessary phospholipid-binding structure. Furthermore, a metal ion-dependent conformational monoclonal antibody, F9.29, was inhibited from binding fragment 1 by the 1-42 peptide.

Amino Acid Sequence↗

PubMed1988

Monoclonal antibodies to denatured ragweed pollen allergen Amb a I: characterization, specificity for the denatured allergen, and utilization for the isolation of immunogenic peptides of Amb a I.

The epitope structure of short ragweed allergen Amb a I (formerly antigen E or AgE) has been investigated by characterizing monoclonal antibodies (MAbs) from mice immunized with Amb a I. The MAbs to Amb a I that we previously produced and characterized (HaA1, HaB1 and HaC1) reacted with determinants which were conformationally dependent. Denaturation of Amb a I, which is a prerequisite for peptide isolation and hence epitope localization, destroyed these determinants. We report here the isolation of 15 MAbs produced against denatured Amb a I and present their reactivities with native Amb a I, denatured Amb a I, and peptide fragments of Amb a I. None of the 15 MAbs to denatured Amb a I bind to native Amb a I, as judged by their lack of binding to Amb a I in immune complex with HaA1 and HaC1. All 15 MAbs reacted with the extensively reduced and alkylated form of denatured Amb a I and 14 of the 15 were shown by Western blotting techniques and ELISA to have activity against the isolated alpha chain or beta chains of Amb a I, but not both. In addition, several MAbs react with tryptic fragments of Amb a I which allowed us to isolate and sequence two tryptic peptides of Amb a I. Antisera raised against KLH-coupled synthetic analogs of these peptides reacted well with intact, denatured Amb a I. Finally, during chromatographic purification of Amb a I from fresh pollen extracts, various MAbs began to react with Amb a I as a function of stage of isolation, suggesting that some proportion of Amb a I spontaneously denatures during purification, converting from the allergenic form possessing two major B cell epitopes to an allergenically inactive form.

Allergens↗

PubMed1988

Reversible phase variation of expression of Neisseria meningitidis class 5 outer membrane proteins and their relationship to gonococcal proteins II.

Neisseria meningitidis class 5 proteins are major outer membrane proteins that share many properties with the proteins II (P.II) of Neisseria gonorrhoeae. We generated two bactericidal monoclonal antibodies, each of which bound specifically to one of the two identified class 5 proteins produced by N. meningitidis FAM18. The monoclonal antibodies also bound to class 5 proteins of a limited number of other meningococcal strains. Using the bactericidal activity of the monoclonal antibodies, we demonstrated that expression of both class 5 proteins was subject to reversible phase variation in vitro. The N-terminal amino acid sequence of a purified class 5 protein revealed striking similarity to the N-terminal amino acid sequence of gonococcal P.II proteins. Using a cloned class 5 gene, we identified three potential class 5 gene loci in N. meningitidis FAM18. These class 5 sequences also had homology with gonococcal P.II gene sequences and contained the CTCTT repeat sequence believed to be important in the regulation of gonococcal P.II expression.

Amino Acid Sequence↗

PubMed1987

Fast atom bombardment/tandem mass spectrometry of physalaemin-like peptides.

Fragment ion spectra obtained from collision-induced decomposition of protonated molecular ions have been used to determine amino acid sequences of several physalaemin-like peptides which were recently purified from rabbit stomach. This technique was chosen because the peptides were available in microgram quantities and were anticipated to contain pyroglutamate as the blocked N-terminal residue. Such spectra of several synthetic analogs of the naturally occurring peptides were obtained and analyzed to confirm the veracity of this peptide sequencing strategy. In addition, methyl ester derivatives of these synthetic peptides provided a crucial test for the spectral interpretation via the mass shifts thus induced.

Amino Acid Sequence↗

PubMed1987

Genetic and biochemical analyses of protein II.

We compared the structure of P.II proteins of gonococcal strain FA1090 by N-terminal sequence analysis of purified proteins and by DNA sequencing of cloned P.II genes. Regulation of P.II gene expression does not involve major DNA rearrangements, but may involve generation of frame-shifts in unexpressed P.II genes. There are probably 8 or 9 P.II genes, each possessing a common leader sequence, in the gonococcal chromosome.

Bacterial Outer Membrane Proteins↗

PubMed1987

Cross-reactive and unique grass group I antigenic determinants defined by monoclonal antibodies.

As part of a study to probe the immunochemical basis for allergenic cross-reactivity among grass pollens, a series of useful reagents has been prepared. The major grass-pollen allergen, designated group I (GpI), was isolated from five grass pollens (meadow fescue, June grass, sweet vernal grass, redtop grass, and perennial ryegrass). The purified GpI antigens were used to immunize individual groups of BALB/c mice. A total of 123 hybridoma-derived anti-GpI monoclonal antibodies (mAbs) was produced. These mAbs were used to evaluate the antigenic relationship among the GpI antigens by means of two types of ELISA. The experiments revealed a high level of epitope diversity and demonstrated a wide range of antibody specificities. A cross-reactivity ELISA was used to identify and compare antigenic determinants on the GpI molecules, and it was possible to define mAbs with specificities unique for the immunizing allergen and other mAbs that cross-reacted with one or more other members of the test panel of allergens. These murine mAbs reflect the range of specificities present in sera from grass-allergic individuals.

Animals↗

PubMed1987

A new protocol to digest human IgM with papain that results in homogeneous Fab preparations that can be routinely crystallized.

A method has been developed for the routine production of Fab fragments from human IgM in high yield. After the IgM is purified at physiological pH, it is digested with papain in the presence of cysteine at room temperature for 16 hours. The Fab fragments are purified initially by gel filtration and then by ion exchange chromatography. The yield of Fab has been 60-80%. Some heterogeneity in the size of the Fabs from the different monoclonal IgMs has been observed. Fab fragments from four different IgM rheumatoid factors (RF) have been crystallized after such digestion and purification, in a variety of conditions including phosphate buffer alone or with the precipitating agents ammonium sulfate, polyethylene glycol or methylpentanediol. This modified papain digestion method has also been used for another non-RF monoclonal human IgM with equally good yield. Biological activity can be detected in the purified Fab fragment indicating that this procedure does not destroy the native conformation of the molecule.

Amino Acid Sequence↗

PubMed1987

Localization of a conserved epitope and an azurin-like domain in the H.8 protein of pathogenic Neisseria.

The pathogenic neisseriae, Neisseria gonorrhoeae and Neisseria meningitidis, possess an outer membrane protein, H.8, which contains a conserved monoclonal antibody (MAb)-binding epitope in all strains tested. We have cloned and sequenced a meningococcal H.8 gene, and determined the characteristics of the predicted protein. The predicted signal peptide has features characteristic of a prokaryotic lipoprotein. The region at the N-terminal end of the mature protein (39 amino acids) is primarily composed of alanine, glutamate and proline residues arranged in imperfect repeats with the consensus sequence AAEAP. The epitope for H.8 MAb-binding was localized to a 20-amino-acid sequence within this region. The remainder of the predicted amino acid sequence shows extensive homology to azurins, which are small blue copper-binding proteins found in a limited number of species of pathogenic bacteria.

Amino Acid Sequence↗

PubMed1987

Antigenic and structural differences among six proteins II expressed by a single strain of Neisseria gonorrhoeae.

Gonococci express a family of related outer membrane proteins designated protein II (P.II), which undergo both phase and antigenic variation. Six P.II proteins have been identified in strain FA1090. We developed monoclonal antibodies specific for each P.II protein. Using these antibodies as probes, we purified the six different P.II proteins of this strain. Despite the relatedness of the proteins, we could not purify all of them by a single purification scheme. Four P.II proteins were purified by chromatofocusing, and the remaining two proteins were purified by hydrophobic interaction chromatography on phenyl-Sepharose. The N-terminal amino acid sequence of the proteins showed a high degree of sequence conservation. However, there was variability at specific amino acid residues, giving each P.II protein a unique N-terminal amino acid sequence. Thus P.II proteins of one strain differ among themselves not only in antigenic determinants and primary structure, but also in other characteristics affecting their properties in different chromatographic systems.

Amino Acid Sequence↗

PubMed1986

Purification and properties of an oestrogen-stimulated mouse uterine glycoprotein (approx. 70 kDa).

An oestrogen-induced secretory protein from mouse uterine luminal fluid was purified by CM-Affi-Gel Blue chromatography and reverse-phase h.p.l.c. This protein has an apparent molecular mass of approx. 70 kDa both by SDS/polyacrylamide-gel electrophoresis (with or without 2-mercaptoethanol) and by gel-filtration column chromatography, indicating that it exists as a single-chain polypeptide. Further analysis of the protein revealed that it is highly basic (pI greater than or equal to 10) and is a glycoprotein. The N-terminus appears to be blocked to Edman degradation. The partial amino acid sequence of a fragment was obtained by cleavage with CNBr; no sequence homology was apparent between the analysed fragment and other known sequences. The incorporation of [35S]methionine into uterine proteins in vitro revealed that oestrogen treatment of immature mice stimulates both synthesis and secretion of the 70 kDa protein. An enzyme-linked immunosorbent assay with polyclonal antibody was used to determine the tissue distribution of the protein. Tissues such as lung, brain, spleen, muscle, intestine, liver, kidney and ovary of oestrogen-treated mice did not have detectable amounts of the 70 kDa protein. Immunoreactivity was present in uterine and vaginal tissues from oestrogen-treated animals. The 70 kDa protein was not induced by testosterone or progesterone. Although the function of this protein is unknown, it is useful as a marker for the study of oestrogen action in the mammalian uterus as well as regulation of gene expression at the molecular level.

Amino Acid Sequence↗

PubMed1986

Two major human allergenic sites on ragweed pollen allergen antigen E identified by using monoclonal antibodies.

Murine monoclonal antibodies specific for antigen E (AgE), the major allergen isolated from short ragweed pollen, have been produced and characterized. These monoclonal antibodies, when coupled to Sepharose and used as immunoadsorbents, specifically bound AgE when a crude pollen extract was passed through the column. Three antigenic sites (A, B, and C) on AgE were identified by using five of these monoclonal antibodies in both inhibition and double-bind solid-phase ELISA. These three antigenic sites appear to be nonoverlapping and nonrepeated, that is, present only once on each AgE molecule. Site C on AgE could readily be bound by the monoclonal antibody specific for that site, but only when AgE was in solution or "presented" by an anti-site A or anti-site B antibody. Site C appears to be only marginally available for binding when AgE is directly adsorbed to polyvinyl chloride microtiter wells. The majority of monoclonal antibodies isolated after immunization of BALB/c mice were specific for site A on AgE. In addition, the binding to AgE of pooled BALB/c polyclonal, hyperimmune antisera against AgE was blocked approximately 80% by a monoclonal antibody directed against site A, but was only blocked approximately 20% by an anti-site B monoclonal antibody. This suggests that site A on AgE is the predominant antigenic site in the BALB/c immune response and that site B represents a less dominant site. The binding of IgE in pooled human serum from ragweed-allergic individuals is blocked approximately 50% by a monoclonal antibody directed to site A on AgE and also approximately 50% by a monoclonal antibody directed against site B. A series of individual human short ragweed allergic antisera also showed significant, although varied, inhibition of IgE binding to AgE by both anti-site A and anti-site B monoclonal antibodies. Simultaneous addition of anti-site A and anti-site B was somewhat additive and inhibited up to 80% of the binding of human IgE specific for AgE. The conclusion from these data is that site A and site B defined by two murine monoclonal antibodies represent two very major allergenic sites in the human response to this molecule.

Allergens↗

PubMed1986

Physalaemin-like immunoreactive peptides from rabbit stomach.

Two physalaemin (PHY)-like immunoreactive peptides, designated PHLIPs, have been purified from extracts of rabbit stomach tissue. Fast atom bombardment/mass spectrometry (FAB/MS) indicated that the m/z values for the PHLIP protonated molecular ions were 867.419 and 796.4. FAB/tandem MS spectra, coupled with a knowledge of the amino acid composition and the aid of a computerized fragment-matching program, indicated the amino acid sequences to be: (formula; see text) The sequences of PHLIPs-7 and -8 were confirmed with synthetic peptides. The PHY-antiserum cross-reactivity of the PHLIPs reflects homology at amino acid residues 1, 3, 4 and 5 for the mammalian and amphibian residues.

Amino Acid Sequence↗