Search PubMed⌕ Search

Biomedical subjects

A Holm

Publications and source records attributed to A Holm.

At least 55 records · Page 3Linked to original sources
PubMed1998

Photoaffinity labeling of the human receptor for urokinase-type plasminogen activator using a decapeptide antagonist. Evidence for a composite ligand-binding site and a short interdomain separation.

Binding of urokinase-type plasminogen activator (uPA) to its cellular receptor (uPAR) renders the cell surface a favored site for plasminogen activation. Recently, a 15-mer peptide antagonist of the uPA-uPAR interaction, with an IC50 value of 10 nM, was identified using phage display technology [Goodson, R. J., Doyle, M. V., Kaufman, S. E., and Rosenberg, S. (1994) Proc. Natl. Acad. Sci. 91, 7129-7133]. In the present study, the molecular aspects of the interaction between this peptide and uPAR have been investigated. We have characterized the real-time receptor binding kinetics for the antagonist using surface plasmon resonance and identified critical residues by alanine replacements. The minimal peptide antagonist thus derived (SLNFSQYLWS) was rendered photoactivatable by replacing residues important for uPAR binding with photochemically active derivatives of phenylalanine containing either (trifluoromethyl)diazirine or benzophenone. These peptides incorporated covalently into purified soluble uPAR upon photoactivation, and this was inhibited by preincubation with receptor binding derivatives of uPA. The intact three-domain structure of uPAR was essential for efficient photoaffinity labeling. Proteolytic domain mapping using chymotrypsin revealed a specific labeling of both uPAR domain I and domains II + III dependent on the position of the photoprobe in the antagonist. On the basis of these studies, we propose the existence of a composite ligand binding site in uPAR combined of residues located in distinct structural domains. According to this model, a close spatial proximity between uPAR domain I and either domains II or III in intact uPAR is required for the assembly of this composite binding site. Since the receptor binding properties of the peptide antagonist closely mimic those of uPA itself, these two ligands presumably share coincident binding site in uPAR.

Amino Acid Sequence↗
PubMed1998

The P9 pocket of HLA-DQ2 (non-Aspbeta57) has no particular preference for negatively charged anchor residues found in other type 1 diabetes-predisposing non-Aspbeta57 MHC class II molecules.

Susceptibility and resistance to type 1 diabetes are associated with MHC class II alleles that carry non-Asp and Asp at residue 57 of their beta chain respectively. The effect of Asp or non-Aspbeta57 may relate to a differential ability of distinct class II molecules to bind specific immuno-pathogenic peptides. Recent studies in man and mouse have revealed that some type 1 diabetes-predisposing non-Aspbeta57 class II molecules (i.e. DQ8, DR4Dw15 and I-Ag7) preferentially bind peptides with a negatively charged anchor residue at P9. It has been suggested that this is a common feature of type 1 diabetes-predisposing class II molecules. The molecular explanation for such a phenomenon could be that class II beta chains with Aspbeta57 form a salt bridge between Aspbeta57 and a conserved Arg of the a chain, whereas in non-Aspbeta57 molecules the Arg is unopposed and free to interact with negatively charged P9 peptide anchor residues. We have investigated the specificity of the P9 pocket of the type 1 diabetes-associated DQ2 molecule and in particular examined for charge effects at this anchor position. Different approaches were undertaken. We analyzed binding of a high-affinity binding ligand and P9-substituted variants of this peptide, and we analyzed the binding of a set of synthetic random peptide libraries. The binding analyses were performed with wild-type DQ2 and a mutated DQ2 with Ala at beta57 substituted with Asp. Our results indicate that the wild-type DQ2 (non-Aspbeta57) prefers large hydrophobic residues at P9 and that there is no particular preference for binding peptides with negatively charged residues at this position. The specificity of the P9 pocket in the mutated DQ molecule is altered, indicating that the beta57 residue contributes to determining the specificity of the P9 pocket. Our data do not lend support to the hypothesis that all non-Asp beta57 class II molecules predispose to development of disease by binding peptides with negatively charged P9 anchor residues.

Alanine↗
PubMed1998

Sequence-assisted peptide synthesis (SAPS).

In solid-phase peptide synthesis (SPPS) the growing peptide chain may undergo chain aggregation which can cause serious synthetic problems. A number of investigations concerning this problem have been reported in the chemical literature. During a study of such "difficult sequences" using the Fmoc-protection strategy, we have observed that peptide-chain aggregation may be significantly reduced when certain amino acid sequences are incorporated C-terminally (1). Thus, synthesis of the difficult poly-alanine, (Ala)n, sequence (n < or = 20) has been investigated with (Lys(Boc))m (m < or = 6) and (Glu(tBu))m (m > or = 6) as pre-sequences. With m > or = 3, peptides are obtained as single, homogeneous products while a complex mixture of deletion peptides and corresponding Fmoc-protected peptides is formed (n > or = 6) without the pre-sequence. A mixed pre-sequence, (Lys(Boc)Glu(tBu))3, has a similar favorable effect on the synthetic results, but the positive effect seems confined to a rather narrow framework of amino acids and side-chain protecting groups in the pre-sequence as discussed in the article. Among other reputedly difficult sequences the synthesis of H-(Thr-Val)5-OH, H-Val-Asn-Val-Asn-Val-Gln-Val-Gln-Val-Asp-OH, the Acyl Carrier Protein (65-74) and the human insulin B-chain has been investigated. In all cases introduction of a pre-sequence gives rise to satisfactory synthetic results. In the latter case, the lysine pre-sequence may be cleaved enzymatically to give the desB30 insulin B-chain. NIR-FT Raman studies of the synthesis of the poly-alanine, (Ala)n, sequences have shown that the pre-sequence (Lys(Boc))6 shifts the conformation of the growing peptide chain from a beta-structure (n > or = 6) to a random coil conformation (1c). This result is in agreement with the general observation that SPPS proceeds optimally under random coil conditions.

Acyl Carrier Protein↗
PubMed1998

B-cell epitopes and quantification of the ESAT-6 protein of Mycobacterium tuberculosis.

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.

Amino Acid Sequence↗
PubMed1998

The dominant epitope of Borrelia garinii outer surface protein C recognized by sera from patients with neuroborreliosis has a surface-exposed conserved structural motif.

Epitope mapping of outer surface protein C (OspC) by using sera from patients with neuroborreliosis led to the identification of one single major immunodominant epitope within the C-terminal 10 amino acid residues. Peptide binding studies and alanine replacement scanning of the C-terminal decapeptide, PVVAESPKKP, revealed a critical role for the PKKP sequence and its terminal carboxyl group for the binding of immunoglobulin M (IgM) antibodies from patients with Lyme borreliosis. Electron microscopy of antibody-labeled spirochetes indicated that the C-terminal region is exposed on the surface of the spirochete. Based on homology to proteins of known function, this region most probably adopts a polyproline II-like helix, which is found in surface-exposed structures involved in protein-protein interactions. This structural motif is highly conserved in Borrelia species causing Lyme borreliosis and subjected to purifying selection. We suggest that the abundance of the C-terminal region of OspC on the surface of B. burgdorferi allows a multimeric high-avidity interaction between the spirochete and surface Igs on B cells. The resulting cross-linking of surface Igs on B cells may induce a T-cell-independent B-cell activation without IgM-to-IgG switching, thus explaining the lack of IgG antibodies to OspC in neuroborreliosis.

Adolescent↗
PubMed1998

Peptide-based OspC enzyme-linked immunosorbent assay for serodiagnosis of Lyme borreliosis.

Sera from 210 patients with Lyme borreliosis (LB) were studied by an enzyme-linked immunosorbent assay (ELISA) based on a synthetic peptide (pepC10) comprising the C-terminal 10-amino-acid residues of OspC of Borrelia burgdorferi. We found that 36.3 and 45.0% of the serum samples from patients with erythema migrans (EM) and neuroborreliosis (NB), respectively, displayed immunoglobulin M (IgM) anti-pepC10 reactivities, while these samples rarely (</=8%) displayed IgG antibody reactivities. Sera from patients with acrodermatitis chronica atrophicans did not contain anti-pepC10 antibodies. The diagnostic performance of this newly developed peptide ELISA was compared with those of an ELISA based on the full-length recombinant OspC protein (rOspC) and a commercially available ELISA based on the B. burgdorferi flagellum (Fla). The sensitivity of the IgM pepC10 ELISA was slightly lower (P < 0.04) than that of the rOspC ELISA for EM patients (36.3 versus 43.8%), while there was no difference for NB patients (45.0 versus 48.0%). However, the optical density values obtained by the pepC10 ELISA were generally higher than those obtained by the rOspC ELISA, leading to a significantly better quantitative discrimination between seropositive patients with NB and controls (P < 0.008). The specificity of the pepC10 ELISA was similar to those of the rOspC ELISA and the Fla ELISA for relevant controls including patients with syphilis and mononucleosis. Although the overall diagnostic sensitivity of the Fla ELISA was superior, 8.8 and 12.0% of the EM and NB patients, respectively, were antibody positive only by the pepC10 ELISA. Thus, use of a diagnostic test for LB based on the detection of IgM antibodies to pepC10 and Fla has increased sensitivity for the diagnosis of early LB.

Adolescent↗
PubMed1997

Peptomers: a versatile approach for the preparation of diverse combinatorial peptidomimetic bead libraries.

This report describes a versatile approach in the generation of peptidomimetic bead libraries. The method is based on the preparation of peptide-peptoid hybrids using the portioning-mixing procedure, which gives diverse peptidomimetic bead libraries composed of peptides, peptoids and peptide-peptoid hybrids. We term these peptomers, from peptide-peptoid hybrid polymers. The synthesis of the peptomers is easily accomplished by adapting the peptoid synthesis strategy, in which a primary amine reacts with bromoacetic acid, and we combine this methodology with conventional peptide synthesis. The sequence of the active compound is deduced by conventional microsequencing using Edman degradation chemistry, thus avoiding the synthesis of a coding structure or the addition of molecular tags. We demonstrate the utility of the peptomer approach by the synthesis of a bead library together with the identification of novel peptidomimetic ligands binding to the macromolecular targets streptavidin and the insulin receptor.

Binding Sites↗
PubMed1997

Characterization of the delayed type hypersensitivity-inducing epitope of MPT64 from Mycobacterium tuberculosis.

Mycobacterium tuberculosis secretes several proteins into the extracellular environment, some of which are restricted to the M. tuberculosis complex. One of these antigens is MPT64. Recently, the authors showed that native as well as recombinant MPT64 is able to distinguish between an M. tuberculosis infection and a BCG Danish 1331 vaccination. Improved distinction between tuberculin purified protein derivative (PPD) sensitivity conferred by an M. tuberculosis infection and that induced by a BCG vaccination or infection with environmental mycobacteria would be useful in the control of tuberculosis. In this study, the authors report the mapping and characterization of a Dth-inducing epitope by the use of synthetic peptides in guinea-pigs vaccinated with BCG Danish 1331 or Tokyo. Studies with overlapping synthetic peptides have pinpointed the biological activity to a single Dth-inducing epitope at the carboxyterminal region of MPT64 consisting of 15 residues between amino acids Gly-173 and Ala-187, the core epitope (CE15). A fine mapping using truncated versions of CE15 indicates the epitope is restricted to 13 residues between amino acids Val-174 to Glu-186. However, the optimal Dth reactivity is obtained by CE15. Different modifications of CE15 revealed that a lysine tree construction improves the skin reactivity to a maximum level approaching that of the reactivity to tuberculin PPD.

Amino Acid Sequence↗
PubMed1996

Key epitopes on the ESAT-6 antigen recognized in mice during the recall of protective immunity to Mycobacterium tuberculosis.

The recall of long-lived immunity in a mouse model of tuberculosis (TB) is defined as an accelerated accumulation of reactive T cells in the target organs. We have recently identified Ag 85B and a 6-kilodalton early secretory antigenic target, designated ESAT-6, as key antigenic targets recognized by these cells. In the present study, preferential recognition of the ESAT-6 Ag during the recall of immunity was found to be shared by five of six genetically different strains of mice. Overlapping peptides spanning the sequence of ESAT-6 were used to map two T cell epitopes on this molecule. One epitope recognized in the context of H-2b,d was located in the N-terminal part of the molecule, whereas an epitope recognized in the context of H-2a,k covered amino acids 51 to 60. Shorter versions of the N-terminal epitope allowed the precise definition of a 13-amino acid core sequence recognized in the context of H-2b. The peptide covering the N-terminal epitope was immunogenic, and a T cell response with the same fine specificity as that induced during TB infection was generated by immunization with the peptide in IFA. In the C57BL/6j strain, this single epitope was recognized by an exceedingly high frequency of splenic T cells (approximately 1:1000), representing 25 to 35% of the total culture filtrate-reactive T cells recruited to the site of infection during the first phase of the recall response. These findings emphasize the relevance of this Ag in the immune response to TB and suggest that immunologic recognition in the first phase of infection is a highly restricted event dominated by a limited number of T cell clones.

Amino Acid Sequence↗
PubMed1996

Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex.

Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide-MHC complexes have been less well characterized. We have used a complete set of singly substituted analogs of a mouse MHC class I, Kk-restricted peptide, influenza hemagglutinin (Ha)255-262, to address the binding specificity of this MHC molecule. Using the same peptide-MHC complexes we determined the fine specificity of two Ha255-262-specific, Kk-restricted T cells, and of a unique antibody, pSAN, specific for the same peptide-MHC complex. Independently, a model of the Ha255-262-Kk complex was generated through homology modeling and molecular mechanics refinement. The functional data and the model corroborated each other showing that peptide residues 1, 3, 4, 6, and 7 were exposed on the MHC surface and recognized by the T cells. Thus, the majority, and perhaps all, of the side chains of the non-primary anchor residues may be available for T-cell recognition, and contribute to the stringent specificity of T cells. A striking similarity between the specificity of the T cells and that of the pSAN antibody was found and most of the peptide residues, which could be recognized by the T cells, could also be recognized by the antibody.

Amino Acid Sequence↗
PubMed1996

Synthesis of stereoisomers and isoforms of a tryptic heptapeptide fragment of human growth hormone and analysis by reverse-phase HPLC and capillary electrophoresis.

The amino acid sequence Asn-Gly has at pH 7 a tendency to induce deamidation of asparagine to aspartic acid via the formation of a cyclic imide. This imide opens up to yield Asp-Gly or the isoaspartic acid (isoAsp) form, isoAsp-Gly. Both isomers may be found in their L-form or D-form. Like Asn-Gly, the sequence Asp-Gly has a tendency for isomerization and racemization via the formation of a cyclic imide intermediate. When human growth hormone is digested with trypsin, one of the fragments is a heptapeptide (amino acid residues 128-134) containing the amino acid sequence Asp-Gly (amino acid residues 130 and 131). This heptapeptide, as well as stereoisomers and isoforms where L-Asp was replaced by D-Asp, L-isoAsp, D-isoAsp or the L-cyclic imide, respectively, has been synthesized and used as a standard to achieve separation of the five forms by capillary electrophoresis and by reverse-phase HPLC. Capillary electrophoresis analysis was performed in uncoated capillaries by the use of aspartic acid/cyclodextrin buffers at low pH. The elution order of the aspartic-acid-containing heptapeptides was D-Asp, L-Asp, L-isoAsp, D-isoAsp and L-cyclic imide. Reverse-phase HPLC analysis was performed on a C18 column by the use of a shallow acetonitrile gradient in trifluoroacetic acid/water. The elution order was D-isoasp, L-isoASp, L-Asp, D-Asp and L-cyclic imide. Human growth hormone samples were degraded by incubation at high temperature and analyzed for their potential content of isomerization and racemization products. Only L-forms of aspartic acid and isoaspartic acid of the heptapeptide fragment were found.

Amino Acid Sequence↗
PubMed1996

Peptide binding specificity of major histocompatibility complex class I resolved into an array of apparently independent subspecificities: quantitation by peptide libraries and improved prediction of binding.

Considerable interest has focused on understanding how major histocompatibility complex (MHC) specificity is generated and characterizing the specificity of MHC molecules with the ultimate goal being to predict peptide binding. We have used a strategy where all possible peptides of a particular size are distributed into positional scanning combinatorial peptide libraries (PSCPL) to develop a highly efficient, universal and unbiased approach to address MHC specificity. The PSCPL approach appeared qualitatively and quantitatively superior to other currently used strategies. The average effect of any amino acid in each position was quantitated, allowing a detailed description of extended peptide binding motifs including primary and secondary anchor residues. It also identified disfavored residues which were found to be surprisingly important in shaping MHC class I specificity. Assuming that MHC class I specificity is the result of largely independently acting subsites, the binding of unknown peptides could be predicted. Conversely, this argues that MHC class I specificities consist of an array of subspecificities acting in a combinatorial mode.

Amino Acid Sequence↗
PubMed1996

The effect of first written language on the acquisition of English literacy.

The relationship between first and second language literacy was examined by identifying the skills and processes developed in the first language that were transferred to the second language. The performance of 40 university students from The People's Republic of China, Hong Kong, Vietnam and Australia were compared on a series of tasks that assessed phonological awareness and reading and spelling skills in English. The results indicated that the Hong Kong students (with non-alphabetic first language literacy) had limited phonological awareness compared to those students with alphabetic first language literacy. The reading and spelling tasks showed no differences between the groups on real word processing. However, the students from Hong Kong had difficulty processing nonwords because of their poor phonological awareness. The results supported the hypothesis that people learning English as a second language (ESL) transfer their literacy processing skills from their first language to English. When the phonological awareness required in English had not been developed in the first language, ESL students were limited to a whole-word, visual strategy. The findings indicate that students from non-alphabetic written language backgrounds might have difficulties with new, or unfamiliar words when attending universities where English is the medium of instruction.

Adult↗
PubMed1996

Photochemical conjugation of peptides to carrier proteins using 1,2,3-thiadiazole-4-carboxylic acid. Immunoreactivity of free C-terminal epitope with specific antibodies.

A new heterobifunctional cross-linking reagent, 1,2,3-thiadiazole-4-carboxylic acid, for the photochemical conjugation of peptides to proteins is described. The title compound can be coupled directly to a protected peptide resin during solid-phase peptide synthesis (SPPS) using standard coupling procedures. The probe is stable to TFA deprotection/cleavage mixtures containing ethanedithiol commonly used in Fmoc-SPPS. Furthermore, tritium may easily be introduced into the thiadiazole ring by base-catalyzed hydrogen-exchange. Upon irradiation at 245-300 nm, parent 1,2,3-thiadiazole rapidly eliminates N2, generating very reactive thioketene which reacts with amines to give a thioamide in high yield, even when the photolysis is carried out in hydroxylic solvents. In order to investigate the potential of the title compound as a heterobifunctional cross-linking reagent a model study with angiotensin II (AII) was conducted. The photoreactive peptide N2-4-carbonyl-1,2,3-thiadiazole-AII (TDA-AII) was synthesized by Fmoc-SPPS and conjugated to bovine serum albumin (BSA) by photolysis at 245 and 300 nm. By use of a capture competition ELISA, the C-terminal Pro-Phe epitope of photoconjugated AII with the sequence DRVYIHPF was shown to bind specifically to antiAII antibodies (anti-AII abs), although antibodies against both the C- and N-terminal epitopes were present in the assay. A dipeptide His-Leu carboxy-extension form of AII, angiotensin I (AI), only bound to anti-AII abs at 100-200 times higher concentrations, showing that the C-terminal epitope was blocked by the dipeptide.

Angiotensin II↗
PubMed1996

Amino monothio acids in solid-phase synthesis of peptide thioamides.

Peptides containing backbone thioamides (endothiopeptides) have been synthesized utilizing thioacylation under solid-phase conditions. The thioacylations were performed by activating N-protected amino monothio acids with the phosphorus-containing coupling reagent 6-nitrobenzotriazol-l-yloxytris(pyrrolidino)phosphoniu m hexafluorophosphate (PyNOP). This method avoids the use of P4S10-based O/S-exchange reagents, and it is thus amendable to amino acids with side-chain amides. Synthesis of endothio analogs of biologically active peptide such a pGlu-psi [CSNH]-His-Pro-NH2 (TRH) and Leu-Gln-psi [CSNH]-Leu-Lys demonstrated this feature. Proton and carbon NMR spectra of the TRH analog verified the sequential position of its thioamide function. Compatibility of endothiopeptides with allylprotecting groups was studied, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was evaluated as a substitute for piperidine.

Acylation↗