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G Jung

Publications and source records attributed to G Jung.

At least 253 records · Page 14Linked to original sources
PubMed1995

Requirements for peptide binding to the human transporter associated with antigen processing revealed by peptide scans and complex peptide libraries.

Antigenic peptides are translocated into the lumen of the endoplasmic reticulum by the action of the transporter associated with antigen processing (TAP), where they are subsequently needed for the correct assembly of major histocompatibility complex molecules. The transport function was reconstituted in insect cells by expression of both TAP genes. On the basis of this over-expression system, substrate selection was analyzed in detail by a direct biomolecular peptide binding assay. Competition assays with peptide variants, including substitutions of residues with alanine or structurally related amino acids, underline the broad peptide specificity of the human TAP complex. Steric requirements of the substrate-binding pocket were mapped using elongated peptides and scans with bulky, hydrophobic amino acids. Complex nonapeptide libraries were used to determine the contribution of each residue to stabilize peptide-TAP complexes. For the first time, this approach lets us directly evaluate the importance of peptide selection for the overall process of antigen presentation on the level of the peptide transporter.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

High resolution crystal structures of recombinant human renin in complex with polyhydroxymonoamide inhibitors.

The crystal structures of recombinant glycosylated human renin in complex with several polyhydroxymonoamide inhibitors have been determined at up to 1.8 A resolution. The high resolution structures permit a detailed analysis of the conformation of renin, the interactions between the inhibitors and renin, and the network of ordered water molecules. The polyhydroxymonoamide inhibitors are bound with their backbones in an extended conformation, and with their side-chains occupying the S3 to S1 pockets. The inhibited renin molecules are shown to exist in both the closed and the open conformations. Inhibitors bound to the two distinct forms of renin can assume different conformations at the P3 position.

Amides↗
PubMed1995

Decrypting the structure of major histocompatibility complex class I-restricted cytotoxic T lymphocyte epitopes with complex peptide libraries.

Complex synthetic peptide libraries with defined amino acids in one or more positions of the H-2Kb-restricted cytotoxic T lymphocyte (CTL) epitopes SIINFEKL and RGYVYQGL and mixtures of 19 amino acids in the remaining positions were used to analyze the structural requirements of peptide binding to MHC class I molecules and antigen recognition by CTLs. This approach provides means to assess semiquantitatively the contribution of every amino acid to the binding of peptides to major histocompatibility complex (MHC) molecules without biases introduced by naturally processed peptides. Primary and secondary anchor residues were defined for their major contribution to the binding efficiency of the peptides. In contrast to primary anchors, secondary anchor amino acids vary greatly in their side chains and position in the sequences. All amino acids in the octapeptide sequences were found to exhibit positive or negative influences on binding to the MHC molecules and on recognition of the resulting complexes by CTLs. Strong interdependence of the effects of the individual residues in the epitope sequences was demonstrated. CTL responses to peptide libraries were suppressed when residues were introduced; however, they were augmented when the critical residues for T cell recognition were fixed, suggesting a potential use of the peptide libraries for defining epitope sequences in general.

Amino Acid Sequence↗
PubMed1995

Time-resolved ligand exchange reactions: kinetic models for competitive inhibitors with recombinant human renin.

The on and off rate constants (kon and koff) were determined for a series of peptidomimetic, competitive inhibitors of human renin using a novel binding assay. The method entails analyzing a pair of ligand exchange reactions in which a dansylated inhibitor serves as the fluorescent probe. The first in the pair of reactions involves preincubating renin with the probe and initiating the reaction by addition of a sample inhibitor; the second reaction involves preincubating renin with the sample inhibitor and initiating the reaction by addition of probe. Both reactions yield progress curves which contain complementary information concerning the kon and koff of each ligand. The two curves are fitted simultaneously using models derived from the differential rate equations describing the ligand exchange process. The kon and koff rate constants for the probe were 6.85 x 10(6) M-1 s-1 and 2.96 x 10(-4) s-1, respectively, giving a calculated Kd of 43.2 pM. The Kd values for the inhibitor series varied over 2 orders of magnitude (27-2320 pM), while the individual kon (10(6)-10(7) M-1 s-1) and koff (10(-4)-10(-3) s-1) constants varied only over 1 order of magnitude.

Binding, Competitive↗
PubMed1995

Oxidative decarboxylation of peptides catalyzed by flavoprotein EpiD. Determination of substrate specificity using peptide libraries and neutral loss mass spectrometry.

The flavoprotein EpiD catalyzes the COOH-terminal oxidative decarboxylation of the lantibiotic precursor peptide EpiA. Variations of the COOH-terminal heptapeptide S1FNSYCC7 of EpiA were used for determining the substrate specificity of EpiD. When Cys7 was replaced by serine, cysteine-amide, homocysteine, or a thioether amino acid residue, no reaction with EpiD was observed. Heptapeptide libraries with one variable amino acid residue at positions 1-7 of the peptide substrate S1FNSYCC7 were incubated with EpiD, and the reaction products were identified by neutral loss mass spectrometry. When the penultimate cysteine residue Cys6 of the substrate peptide was replaced with Ser, Thr, Ala, or Val, the reaction still occurred. Tyr5 could be replaced with other hydrophobic amino acid residues. Mass spectrometry was used to compare the kinetics of the reaction of EpiD with various peptides. Peptide sequencing of the reaction products was performed by tandem mass spectrometry, confirming that the last cysteine residue was modified. The removal of the acid COOH-terminal carboxyl group was confirmed by determination of the isoelectric points of the reaction products. To study the interaction between EpiA and EpiD, EpiA was coupled to N-hydroxysuccinimide-activated Sepharose HiTrap material; EpiD was only retarded under reducing conditions.

Amino Acid Sequence↗
PubMed1995

CTLs from lymphoid organs recognize an optimal HLA-A2-restricted and HLA-B52-restricted nonapeptide and several epitopes in the C-terminal region of HIV-1 Nef.

In a previous analysis of HIV-1-specific CTLs in lymphoid organs from HIV-seropositive patients, we reported high frequencies of in vivo differentiated CTLs directed against two immunodominant regions in the central and in the C-terminal part of the HIV-1 Nef protein. The present study analyzes the epitopes recognized by CTLs in the carboxyl terminus of Nef (amino acids 182-205). In addition to several epitopes that are recognized in association with different HLA molecules (A1, A2, A25(10), B35, B52), we defined an optimal nonapeptide (190-198). This nonapeptide was recognized by CTLs down to nanomolar concentrations in the context of at least two HLA molecules, HLA-B52 and HLA-A2, including three HLA-A2 subtypes: HLA-A2.1, -A2.2, and -A2.4. We also determined the relative frequencies of effector CTLs directed against peptide 190-198 to be as high as 10(-4), as opposed to lower frequencies ranging between 5 x 10(-5) and 5 x 10(-6) observed for the other peptides recognized in the same region, thus confirming the optimal presentation of this nonapeptide in vivo. Molecular modeling of the interactions between HLA-A2.1 and Nef peptide 190-198 suggests the formation of a stable complex and allowed us to study sequence motifs that are important for the binding of the HIV-1 peptide in the pockets of the HLA-A2.1 molecule.

Base Sequence↗
PubMed1995

The tetracyclic lantibiotic actagardine. 1H-NMR and 13C-NMR assignments and revised primary structure.

The primary structure of the peptide lantibiotic actagardine was determined in (2H3)acetonitrile/H2O by homonuclear two- and three-dimensional NMR spectroscopy as well as 2D 1H(13C) correlation spectra at natural abundance. Actagardine was found to be a tetracyclic 19-residue peptide containing one lanthionine and three overlapping beta-methyllanthionine bridges. Sequential resonance assignment and location of the four thioether rings was accomplished by 2D NOESY, 3D NOESY-TOCSY and gradient-enhanced 1H(13C)-HMBC spectra. The C-terminal thioether bridge was shown to be oxidized to a sulfoxide. The NMR data were additionally confirmed by mass spectrometry and Edman degradation after chemical modification, which allowed sequencing of lanthionine and beta-methyllanthionine residues. Our studies clearly show, that the structure of actagardine as previously published by Kettenring et al. (1990) J. Antibiot. 43, 1082-1088 is not correct.

Alanine↗
PubMed1995

Supermotifs enable natural invariant chain-derived peptides to interact with many major histocompatibility complex-class II molecules.

Class II-associated invariant chain peptides (CLIPs) compete with natural allele-specific ligands for binding to several purified HLA-DR molecules. Truncation and substitution analysis showed that a minimal sequence of 13 amino acids is sufficient for excellent binding to DR17 and DR1. Hydrophobic residues at relative positions 1 and 9 (P1 and P9) which are shared among these DR-ligands, and are found to be anchored in complementary pockets by x-ray crystallography allow specific binding. Two flanking residues at either end next to the specific contact sites Met107 and Met115 contribute to binding irrespective of their side chains, suggesting H-bonds to the major histocompatibility complex (MHC) molecule. Thus, CLIPs behave like conventional ligands, however, lack their allele-specific contact sites. Introduction of the DR17-specific contact site aspartate at P4 dramatically improves invariant chain-peptide binding to DR17, but reduces DR1 binding. By contrast, binding to DR1, but not DR17 is strongly improved after introduction of the DR1-specific contact site alanine at P6. In addition, analyzing the fine specificity of the hydrophobic contact sites at P1 and P9, CLIP variants reflected the allele-specific preferences of DR17- or DR1-ligands, respectively, for aliphatic or aromatic residues. Alignment studies suggest that CLIPs are designed for promiscuous binding in the groove of many MHC class II molecules by taking advantage of one or more supermotifs. One such supermotif, for example, does not include the DR17-specific contact site aspartate at P4, which in conventional natural ligands like Apolipoprotein (2877-94) is necessary to confer a stable conformation. Introduction of aspartate at P4 generates a CLIP variant that is stable in the presence of sodium dodecyl sulfate, such as allele-specific ligands. Studying the stability of class II-CLIP complexes at pH 5, we found that CLIPs, similar to anchor-amputated ligands, can be released from class II molecules, in contrast to conventional natural ligands, which were irreversibly bound. Taken together, our data provide compelling evidence that CLIP peptides bind into the class II groove.

Amino Acid Sequence↗
PubMed1995

Structure-activity relationships of neuropeptide Y analogues with respect to Y1 and Y2 receptors.

Secondary structure investigations, affinities, and activities of neuropeptide Y analogues with respect to the Y1 and the Y2 receptor are reviewed. The results are discussed with respect to the different prerequisites for affinities to both receptor subtypes. The results from a systematic scanning of the hormone using L-alanine and from a large variety of discontinuous and cyclic analogs suggest that two different conformations of neuropeptide Y are adopted at the Y1 and Y2 receptors. Whereas a C-terminal turn structure is suggested for Y1 receptor affinity, an alpha-helical conformation of the C-terminus is afforded for good binding to the Y2 receptor.

Amino Acid Sequence↗
PubMed1995

Identification of the catalytic histidine residue participating in the charge-relay system of carboxypeptidase Y.

The essential histidine residue of carboxypeptidase Y (CPY) was modified by a site-specific reagent, a chloromethylketone derivative of benzyloxycarbonyl-L-phenylalanine. The single modified histidine residue was converted to N tau-carboxy-methyl histidine (cmHis) upon performic acid oxidation. A peptide containing cmHis was isolated from the tryptic-thermolytic digest. Based on the amino acid composition and sequence analysis, the peptide is shown to be Val-Phe-Asp-Gly-Gly-cmHis-MetO2-Val-Pro, which was derived from CPY cleaved by trypsin at Arg 391 and thermolysin at Phe 401, and thus His 397 was modified. This histidine residue has been implicated previously by X-ray analysis to participate in the charge-relay system of CPY.

Amino Acid Chloromethyl Ketones↗
PubMed1995

Transcriptional control of the Saccharomyces cerevisiae ADH1 gene by autonomously replicating sequence binding factor 1.

Autonomously replicating sequence (ARS)-binding factor 1 (ABF1) is a multifunctional protein involved in transcriptional activation and repression, as well as DNA replication, in yeast. The ADH1 gene, encoding alcohol dehydrogenase 1, contains two ABF1 consensus binding sites in the promoter and the coding regions. To examine the effect of ABF1 on expression of the ADH1 gene, we constructed an ADH1-lacZ fusion plasmid. Both ABF1 binding sites appeared to be transcriptional activators because deletions and mutations of these sites decreased transcriptional activity. The ABF1 binding sites also acted in an orientation-independent manner when a synthetic ABF1 binding site was inserted into the yeast CYC1 gene lacking its transcriptional activation region. A gel mobility shift assay showed that ABF1 bound in vitro to both ABF1 binding sites in the promoter and coding regions. In a glycerol medium the degree of activation by ABF1 was higher than in a glucose medium. The expression of ADH1 was activated synergistically by both ABF1 binding sites. These observations suggest that ABF1 transactivates the ADH1 gene through its binding sequences in both the promoter and coding regions.

Alcohol Dehydrogenase↗
PubMed1995

Intramolecular immunodominance and intermolecular selection of H2d-restricted peptides define the same immunodominant region of the measles virus fusion protein.

The generation of a sustained antibody response requires the participation of MHC class II-restricted T helper cells. We have identified class II-restricted sequences by immunizing BALB/c (H-2d) mice with 108 overlapping synthetic pentadecapeptides covering the whole sequence of the measles virus fusion protein (MV-F). Several strong T cell epitopes were found including a major cluster of H-2d-restricted peptides between amino acids 256 and 305. Some of these peptides including peptide F(421-435) and F(256-270) induced MV-specific T lymphocytes in vivo while other H2d-restricted MV-F sequences did not. Immunization with mixtures of selected peptides indicated a hierarchy among H2d-restricted sequences due to competition between peptides. The dominant peptide F(421-435) impaired the response to other T cell epitopes including F(256-270). The response to F(91-105) was obliterated by F(421-435) and F(256-270) but not by peptides devoid of a T cell epitope. When BALB/c mice were immunized with the MV, the immunodominant sequence F(421-445) was identified which included the synthetic peptide F(421-435). Our data suggest that competition during processing and/or presentation between H2d-restricted peptides defines the immunodominant sequence of the viral protein. Even though only a single immunodominant region was defined after immunization with the MV, peptides from other regions were able to induce MV-specific T cell responses. This finding is of interest for the design of subunit vaccines in general and for studying MV-specific T helper cells in an animal model in particular.

Amino Acid Sequence↗
PubMed1995

Contribution of proteasome-mediated proteolysis to the hierarchy of epitopes presented by major histocompatibility complex class I molecules.

Major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL) recognize peptide epitopes of protein antigens in a hierarchical fashion. We investigated whether proteolytic cleavage, in particular by proteasomes, is important in determining epitope hierarchy. Using highly purified 20S proteasomes, we find preferred cleavage sites directly adjacent to the N- and C-terminal ends of the immunodominant epitope of chicken ovalbumin, Ova257-264, while most of the subdominant epitope, Ova55-62, is destroyed by a major cleavage site located within this epitope. Moreover, we show that variations in amino acid sequences flanking these epitopes influence proteasomal cleavage patterns in parallel with the efficacy of their presentation. The results suggest that proteasomal cleavage within and adjacent to class I-restricted epitopes contributes to their level of presentation.

Amino Acid Sequence↗