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PubMed1999

Orientation of LamB signal peptides in bilayers: influence of lipid probes on peptide binding and interpretation of fluorescence quenching data.

The orientation in lipid bilayers of the signal sequence of the bacterial protein LamB was studied using binding, circular dichroism, and fluorescence quenching experiments. Measurements were made of binding modifications caused by the incorporation of lipid probes (brominated or nitroxide-labeled phospholipids) used in the parallax fluorescence quenching method of determining peptide penetration depth [Abrams, F. S., and London, E. (1992) Biochemistry 31, 5312-5322]. The signal peptide bound to a similar extent to neutral bilayers composed of either egg phosphatidylcholine (EPC) or phosphatidylcholines brominated at various positions on their acyl chains. The fluorescence of a tryptophan in either the 18 or 24 position of the peptide was quenched more by bromines in the 6 and 7 than in the 9 and 10 positions on the lipid hydrocarbon chain. Parallax calculations showed that tryptophan-18 was located only 4 A from the hydrocarbon-water interface, consistent with the peptide adopting a "hammock" configuration in the bilayer, with both termini exposed to the aqueous phase and the central alpha-helix located near the hydrocarbon-water interface. In contrast, the incorporation of 10% nitroxide-labeled lipids into EPC bilayers modified peptide binding in a manner dependent on the position of the nitroxide on the hydrocarbon chain; 7-Doxyl PC reduced the percent peptide bound by about one-half, whereas 12-Doxyl PC had little effect on binding. These binding differences modified tryptophan quenching by these probes, making parallax analysis problematical. In the presence of the positively charged LamB peptide, the incorporation of negatively charged phospholipids into EPC bilayers increased the level of peptide binding and modified tryptophan quenching by nitroxide probes. These results suggest that the nitroxide probe could be partially excluded from negatively charged lipid domains where the peptide preferentially bound. Quite different binding and quenching results were obtained with a negatively charged peptide analogue, showing that the charge on both the peptide and bilayer affects peptide-nitroxide probe interactions.

Amino Acid Sequence↗

PubMed2004

HCV core protein-expressing DNA vaccine induces a strong class I-binding peptide DTH response in mice.

The hepatitis C virus (HCV) core protein-encoding sequence (HCcAg) is the most conserved gene in HCV genome and therefore may be useful to study broadly reacting T-cell epitopes. In this study BALB/c and C57BL/6 mice were immunized with a DNA based vaccine expressing the first 176 aa of HCcAg (pIDKCo). After i.m or i.p injection of pIDKCo in BALB/c mice, a detectable INF-gamma secreting response to the relevant class I-binding peptide DLMGYIPLVGA (P1) (aa 132-142) was detected suggesting the induction of HCcAg specific CD8(+) T-cell effectors. CD8(+) T-cell responses were also monitored in vivo by T-cell-mediated DTH reactions after subcutaneous injection of class I-binding viral peptide P1. pIDKCo induced a strong P1-specific DTH response in both i.m and i.p immunized mice. To evaluate the T-cell response induced by pIDKCo in C57BL/6 mice, an HCcAg epitope was predicted based upon it containing the H-2K(b) binding motif XXXXF/YXXL (DLMGYIPL (P2)). pIDKCo induced a strong P2-specific DTH response with similar kinetics of swelling response to that observed in BALB/c mice. Previously, it had been demonstrated that only activated and protective CD8(+) effector T cells could mediate a specific DTH in footpads of virally infected mice after local injection of viral class I-binding peptides. Hence, pIDKCo could prime a strong HCcAg-specific T-cell response in mice with the potential capacity to exert their specific effector functions in peripheral tissues.

Amino Acid Sequence↗

PubMed1999

Enhanced peptide-binding capacities of small intestinal brush border membranes in celiac disease.

In pathogenesis of celiac disease, the significance of prolamin peptide interactions with enterocytes is controversial. Changes in cellular metabolism induced by gliadin peptides, as well as uptake and presentation by enterocytes, are discussed. We analyzed peptide binding to enterocytic membranes as a potential key event. Binding capacities of brush border membranes isolated from small intestinal biopsies of untreated (n = 49) and treated celiac patients on a gluten-free diet (n = 30), as well as control subjects (n = 43), were measured with a dot blot chemiluminescence assay. Synthetic gliadin peptides comprising amino acid position 8-19 (G XIV) and 30-41 (G XI) of alpha-gliadins, a peptic-tryptic digest of gliadin (PT-GLI), and a synthetic zein peptide were used. Comparing treated celiac patients with controls, we observed significantly enhanced membrane-binding of PT-GLI [mean 122.4 densitometric units/microg (95% confidence interval 116.0-128.9) vs 108.9 (102.1-115.7)] and of zein peptide [50.2 (38.4-61.9) vs 28.8 (13.4-44.2)], but only slightly increased binding of the synthetic gliadin peptides G XIV [65.5 (60.6-70.5) vs 62.4 (56.3-68.5) and G XI [75.2 (69.8-80.6) vs 65.9 (55.2-76.5)]. Independent of patient group, membrane-binding capacities for celiac-active gliadin peptides exceeded those of the zein peptide. Thus, interaction of gliadin peptides with the apical enterocytic membrane was not found exclusively in celiac disease. Furthermore, increased binding capacities in treated celiac disease were not confined to celiac-active peptides. Quantitative differences in gliadin peptide binding as a primary characteristic in celiac disease might contribute to pathogenetic effects exerted on small intestinal epithelial cells.

Adolescent↗

PubMed1999

Overlapping peptide-binding specificities of HLA-B27 and B39: evidence for a role of peptide supermotif in the pathogenesis of spondylarthropathies.

OBJECTIVE: Previous studies indicated the increase of HLA-B39 among HLA-B27 negative patients with spondylarthropathies (SpA). This study was performed to examine whether the natural ligands of HLA-B27 are capable of binding to HLA-B39. METHODS: Peptides were synthesized according to the sequences of known natural ligands of HLA-B27 or B39 and were tested for their binding to HLA-B*3901 and B*2705 by quantitative peptide binding assay, using a TAP-deficient RMA-S cell line transfected with human beta2-microglobulin and HLA class I heavy chain genes. RESULTS: Four of the 10 HLA-B27 binding peptides significantly bound to HLA-B*3901. All 4 peptides had hydrophobic/aromatic amino acids (Leu or Phe) at the C-terminus. In contrast, peptides with basic residues (Lys, Arg) or Tyr at the C-terminus did not bind to B*3901. In parallel experiments, 1 of the 2 natural ligands of HLA-B*3901 was found to bind to B*2705. CONCLUSION: A subset of natural HLA-B27 ligands was capable of binding to B*3901. In addition to Arg at position 2 (Arg2), hydrophobic/aromatic C-terminal residues, such as Leu or Phe, seemed to be crucial for the cross-specificity. These results suggested that HLA-B27 and B39 recognize overlapping peptide repertoires, supporting the hypothesis that the peptides presented by both of these class I antigens play a role in the pathogenesis of SpA.

Amino Acid Sequence↗

PubMed2000

Stability and peptide binding specificity of Btk SH2 domain: molecular basis for X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton's tyrosine kinase (Btk). The absence of functional Btk leads to failure of B-cell development that incapacitates antibody production in XLA patients leading to recurrent bacterial infections. Btk SH2 domain is essential for phospholipase C-gamma phosphorylation, and mutations in this domain were shown to cause XLA. Recently, the B-cell linker protein (BLNK) was found to interact with the SH2 domain of Btk, and this association is required for the activation of phospholipase C-gamma. However, the molecular basis for the interaction between the Btk SH2 domain and BLNK and the cause of XLA remain unclear. To understand the role of Btk in B-cell development, we have determined the stability and peptide binding affinity of the Btk SH2 domain. Our results indicate that both the structure and stability of Btk SH2 domain closely resemble with other SH2 domains, and it binds with phosphopeptides in the order pYEEI > pYDEP > pYMEM > pYLDL > pYIIP. We expressed the R288Q, R288W, L295P, R307G, R307T, Y334S, Y361C, L369F, and 1370M mutants of the Btk SH2 domain identified from XLA patients and measured their binding affinity with the phosphopeptides. Our studies revealed that mutation of R288 and R307 located in the phosphotyrosine binding site resulted in a more than 200-fold decrease in the peptide binding compared to L295, Y334, Y361, L369, and 1370 mutations in the pY + 3 hydrophobic binding pocket (approximately 3- to 17-folds). Furthermore, mutation of the Tyr residue at the betaD5 position reverses the binding order of Btk SH2 domain to pYIIP > pYLDL > pYDEP > pYMEM > pYEEI. This altered binding behavior of mutant Btk SH2 domain likely leads to XLA.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1998

Lyme disease-causing Borrelia species encode multiple lipoproteins homologous to peptide-binding proteins of ABC-type transporters.

To identify cell envelope proteins of Borrelia burgdorferi, the causative agent of Lyme disease, we constructed a library of B. burgdorferi genes fused to the Escherichia coli phoA gene, which expresses enzymatically active alkaline phosphatase. One such gene, oppA-1, encodes a predicted polypeptide with significant similarities to various peptide-binding proteins of ABC-type transporters. Immediately downstream of oppA-1 are two genes, oppA-2 and oppA-3, whose predicted polypeptide products show strong similarities in their amino acid sequences to OppA-1, including a sequence that resembles the most highly conserved region in peptide-binding proteins. By labeling with [3H]palmitate, OppA-1, OppA-2, and OppA-3 were shown to be lipoproteins. DNA hybridization analysis showed that the oppA-1 oppA-2 oppA-3 region is located on the linear chromosome of B. burgdorferi, and the genes are conserved among different Borrelia species that cause Lyme disease (B. burgdorferi, B. garinii, and B. afzelli), suggesting that all three homologous genes are important to the maintenance of Lyme disease spirochetes in one or more of their hosts.

ATP-Binding Cassette Transporters↗

PubMed1996

Peptide binding function and the paradox of HLA disease associations.

Many studies have found associations between HLA loci and susceptibility or resistance to both infectious and autoimmune diseases, but often subsequent studies fail to find the same association. Such inconsistencies are not surprising if we consider what is known of the population biology and evolution of HLA genes, especially the consequences of natural selection favouring heterozygosity in the peptide binding regions (PBR) of HLA molecules. Because of past recombination event and/or convergent evolution, alleles that are not closely related in overall sequence may come to resemble each other in the PBR. Because peptide binding is likely to be important for the role of HLA molecules in both infectious and autoimmune disease, a strategy of searching for HLA disease associations that groups alleles in functional categories based on PBR motifs may prove more successful than conventional strategies. Likewise, in developing approaches for molecular typing, it may be advisable to develop methods that group alleles on the basis of shared PBR motifs rather than simply on the basis of shared primer sites in less functionally important regions.

Animals↗

PubMedDate not supplied

[Interaction of a synthetic zinc-binding peptide with DNA].

Synthesis, DNA- and zinc ion-binding activities of the synthetic 23-residue peptide, forming a part of the DNA-binding domain of yeast transcription activator GAL-4, are reported. In presence of zinc ions considerable changes in the shapes of the fluorescence and CD spectra of the peptide are observed. It is shown that the peptide forms complexes with zinc ions containing one metal ion per peptide molecule with association constants on the order of (1-2) x 10(6) M-1. Using gel filtration on a TSK-gel column we have shown that in aqueous solution at concentrations of 10(-4)-10(-6) M the peptide exists predominantly in the dimeric form. Dimerization constants were found to be 5 x 10(6) M-1 and 1.7 x 10(7) M-1 in the absence and in the presence of zinc ions, respectively. It is shown that the peptide binds to DNA. The binding approaches saturation when one peptide molecule is bound approximately to five base pairs of DNA. The shapes of the titration curves obtained from binding of the peptide to DNA show that the peptide can bind to DNA both in the monomeric and self-associated forms (dimer or tetramer). Increasing DNA concentration and decreasing the peptide/DNA molar ratio lead to a shift in the equilibria between self-associated peptide species and monomers toward the formation of monomer peptide complexes.

Amino Acid Sequence↗

PubMed1983

DNA-binding peptides from rat liver and Novikoff hepatoma cells: quantitative level and possible biochemical differences.

DNA isolated from rat liver by intensive deproteinization with chloroform/isoamyl alcohol and phenol contains low molecular weight peptides in a quantity of about 20 micrograms/mg DNA. These peptides show high specific activity in inhibiting transcription in a reconstituted cell-free system with prokaryotic and eukaryotic RNA polymerase. Their level is markedly decreased in DNA prepared from Novikoff hepatoma cells. Moreover the amino acid analysis and the pattern of analytical separation by high performance liquid chromatography (HPLC) show some biochemical differences between DNA-binding peptides extracted from rat liver and Novikoff hepatoma cells. The possibility that carcinogenesis may involve mechanisms which lead to selective removal of some components of the DNA-binding peptides, is discussed.

Amino Acids↗

PubMed2000

C1q-binding peptides share sequence similarity with C4 and induce complement activation.

Two peptide motifs that bind to C1q have been identified from phage displayed libraries. A first panning cycle recovered phage that displayed a [N/S]PFxL motif. A synthetic peptide with that motif blocked those phage from binding to C1q. A second panning cycle was conducted with the [N/S]PFxL motif peptide present, leading to recovery of phage displaying a different motif, SHY. The two motifs are specific for C1q and are competed by DNA and the cognate synthetic peptide but not by immunoglobulins. Phage displayed peptide sequences containing the [N/S]PFxL have significant sequence similarity to a region of complement component C4, suggesting a possible site of interaction between C4, or one of its processed forms, and C1q. The SHY motif peptide induces C4 consumption in a hemolytic assay, suggesting that it activates C1 independent of immune complexes. This peptide may activate C1 by a mechanism similar to the beta-amyloid peptides found in Alzheimer's disease.

Animals↗

PubMed1994

Phosphatidyl inositol-linked forms of a murine class I MHC molecule expressed on Chinese hamster ovary cells retain peptide binding capability and alloreactivity.

A gene encoding a phosphatidyl inositol-linked form of the murine class I MHC molecule H-2Kd was constructed and the protein expressed in Chinese hamster ovary cells together with murine or human beta 2-microglobulin (beta 2m). The resulting lipid-linked class I heterodimers can be efficiently converted into a soluble form by treatment of transfected cells with a phospholipase. Cells expressing Kd heterodimers were characterized with respect to heavy chain levels at the cell surface, peptide binding, and recognition by Kd-specific antibodies and alloreactive cytotoxic T cells. All transfectants bound a 3H-labeled Kd-restricted nonamer peptide, although more peptide bound to cells expressing the Kd/human beta 2m combination, perhaps because of a greater number of empty molecules at the cell surface. A dissociation constant of 5 x 10(-8) M derived by Scatchard analysis is within the range expected for interactions of peptides with class I MHC molecules. Alloreactive cytotoxic T cells which recognize wild-type Kd on murine cells lysed the hamster cells expressing lipid-linked Kd without regard to the species of the beta 2m light chain. These results indicated that the engineered lipid-linked Kd molecule is expressed at the cell surface, is recognized by antibodies and T cells, and functions to bind peptide.

Amino Acid Sequence↗

PubMed1993

Identification of H-2Kb-, Db- and Dd-binding peptides derived from amino acid sequences of polyoma virus T antigens.

Viral and tumor antigens are presented to cytotoxic T cells (CTL) in the form of short peptides. The peptide antigen is transported to the cell surface in conjunction with molecules encoded by the major histocompatibility complex (MHC). Different methods have recently been described for the analysis of the MHC-class-I binding ability of synthetic peptides. Here, we describe a protocol, based on intact RMA-S cells cultured at 26 degrees C in the presence of synthetic peptides, which gives an allele-specific peptide binding pattern with high resolution. This allowed an analysis of the H-2Kb-, Db- and Dd-binding capacity of a panel of synthetic peptides with amino acid (aa) sequences derived from polyoma (py) virus large-, middle- and small-T (LT, MT and ST) antigens, previously used in immunization experiments against py-virus-induced tumors. Eight single aa mutants and a deletion mutant of one peptide with an ability to bind both to H-2Kb and to Db were also analyzed. We foresee that the present protocol, or variants thereof, may serve as a simple and rapid assay for the systematic screening of the class-I binding ability of large sets of synthetic peptides in vitro. Such analysis may facilitate the search for viral or tumor peptide epitopes that are recognized by CTL.

Alleles↗

PubMed1993

Evidence that transporters associated with antigen processing translocate a major histocompatibility complex class I-binding peptide into the endoplasmic reticulum in an ATP-dependent manner.

We have investigated the role of the putative peptide transporters associated with antigen processing (TAP) by using a permeabilized-cell system. The main objective was to determine whether these molecules, which bear homology to the ATP-binding cassette family of transporters, translocate antigenic peptides across the endoplasmic reticulum membrane for assembly with major histocompatibility complex (MHC) class I molecules and beta 2-microglobulin light chain. The pore-forming toxin streptolysin O was used to generate permeabilized cells, and peptide translocation was determined by measuring the amount of added radiolabeled peptide bound to endogenous class I molecules. No radiolabeled peptide was associated with MHC class I glycoproteins from unpermeabilized cells. We found that efficient peptide binding to MHC class I molecules in permeabilized cells is both transporter dependent and ATP dependent. In antigen-processing mutant cells lacking a functional transporter, uptake occurs only through a less-efficient transporter and ATP-independent pathway. In addition, short peptides (8-10 amino acids) known to bind MHC class I molecules compete efficiently with a radiolabeled peptide for TAP-dependent translocation, whereas longer peptides and a peptide derived from an endoplasmic reticulum signal sequence do not compete efficiently. This result indicates that the optimal substrates for TAP possess the characteristics of MHC-binding peptides.

Adenosine Triphosphate↗

PubMed1997

Sequence convergence in the peptide-binding region of primate and rodent MHC class Ib molecules.

In addition to the universally expressed and highly polymorphic class Ia genes, the major histocompatibility complex (MHC) of placental mammals includes class Ib genes that are characterized by restricted expression and low levels of sequence polymorphism. The functional importance of class Ib genes as well as their actual function has long been controversial. Phylogenetic analyses have suggested that there are no orthologous relationships among class Ib loci of mammals belonging to different orders, suggesting that these loci have evolved independently since the placental mammals diverged. Here, we present evidence of convergent evolution at the molecular sequence level in the putative peptide-binding regions (PBRs) of human and mouse class Ib genes. So far, there are few if any convincing examples of convergent evolution at the amino acid sequence level, and such evolution is believed to be likely to occur only as a result of strong positive selection. Because the present case involves the functionally important PBR and because the primate and rodent molecules are known to bind similar peptides, this study represents both a convincing case of molecular-level convergence and evidence that MHC class Ib molecules, although not orthologous, may evolve similar functions convergently.

Amino Acid Sequence↗

PubMed1984

Formation of cadmium-binding peptide allomorphs in fission yeast.

It has been reported that two kinds of Cd-binding peptide (Cd-BP1 and Cd-BP2) are induced in fission yeast upon exposure to Cd, and that they consist of the same unit peptide (cadystin), but Cd-BP1 binds 1.5 times more Cd atoms per cadystin than Cd-BP2 (Murasugi, A., Wada, C., & Hayashi, Y. (1981) J. Biochem. 90, 1561-1564). The relative amount of each allomorphic Cd-BP in the cell varied with time after induction and with the concentration of Cd in the induction medium. Further, the production of acid-labile sulfide in the cell increased greatly upon exposure to Cd and varied with time after Cd addition and with Cd concentration in the medium, as in the case of Cd-BP1. Since Cd-BP1 contains labile sulfide, the increase of labile sulfide production together with the increase of cellular Cd concentration may be the driving force to form Cd-BP1, resulting in the increase of the relative amount of Cd-BP1.

Ascomycota↗

PubMed1995

Interference of distinct invariant chain regions with superantigen contact area and antigenic peptide binding groove of HLA-DR.

In the endoplasmic reticulum, MHC class II alpha beta dimers associate with the trimeric invariant chain (li), generating a nine-subunit (alpha beta li)3 complex. In the presence of li, the peptide binding groove is blocked, so that loading with self or antigenic peptides can only occur after proteolytic removal of li in specialized post-Golgi compartments. The class II-associated invariant chain peptide region of li (about residues 81-104) is known to mediate binding to class II molecules and blockade of the groove, but this does not exclude additional contact sites for li. Using a set of overlapping li peptides and recombinant soluble li, we demonstrate here that a large segment of li encompassing approximately residues 71 to 128 interacts with HLA-DR molecules. The N- and C-terminal regions of this li segment appear to bind outside the peptide groove to the contact area for the staphylococcal superantigen Staphylococcus aureus enterotoxin B on the alpha 1 domain. The core region of this segment (residues 95-108) prevents binding of antigenic peptides, probably by interaction with the peptide groove. Occupation of the groove with antigenic peptides abolishes binding not only of the core region, but also that of those li peptides that bind outside the groove. These findings suggest the existence of distinct conformational states of class II molecules, with li binding preferentially to one form.

Amino Acid Sequence↗

PubMed2002

Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library.

Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaM-dependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain. A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca2+-dependent manner. The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding. SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif. These motifs are classified based on the positions of conserved hydrophobic residues. To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments. The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants. Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.

Amino Acid Motifs↗

PubMed1987

Contribution of a p75 interleukin 2 binding peptide to a high-affinity interleukin 2 receptor complex.

There are at least two forms of cellular receptors for interleukin 2 (IL-2); one with a very high affinity and the other with a lower affinity. We identified a non-Tac IL-2 binding peptide with a relative molecular weight of 75,000 (p75). Cell lines bearing either the p55 Tac or the p75 peptide alone manifested low-affinity IL-2 binding, whereas a cell line bearing both peptides manifested both high- and low-affinity receptors. After the internalization of labeled IL-2 through high-affinity receptors, the p75 peptide could not be detected by cross-linking studies. Furthermore, fusion of cell membranes from low-affinity IL-2 binding cell lines bearing the Tac peptide alone with membranes from a cell line bearing the p75 peptide alone generated hybrid membranes bearing high-affinity receptors. These results suggest a multichain model for the high-affinity IL-2 receptor in which high-affinity receptors would be expressed when both Tac and p75 IL-2 binding peptides are present and associated in a receptor complex.

Cell Line↗