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PubMed1995

Delivery of nascent MHC class II-invariant chain complexes to lysosomal compartments and proteolysis of invariant chain by cysteine proteases precedes peptide binding in B-lymphoblastoid cells.

The intracellular trafficking, proteolysis, and dissociation of invariant chain (li) associated with nascent class II molecules was examined in B-lymphoblastoid cells. Metabolic labeling and Percoll gradient centrifugation was used to assess the kinetics of delivery and processing of class II-li complexes within the endocytic pathway. Catabolism of class II-li complexes rapidly followed their delivery from post-Golgi compartments to dense lysosome-like compartments distinct from early and late endosomes. Direct peptide binding assays revealed that class II molecules associated with even small N-terminal fragments of li failed to bind peptide. Cysteine protease inhibitors alone blocked li proteolysis/dissociation and accumulation of class II-li biosynthetic intermediates within lysosome-containing compartments. Active-site labeling of cysteine proteases in B cells was used to identify cysteine proteases capable of mediating li proteolysis within endosomal compartments. Our results indicate rapid, possibly direct, transport of nascent class II-li complexes from the Golgi/trans-Golgi network to dense lysosomal compartments wherein cysteine protease(s), likely including cathepsin B, mediate complete removal of li. Inhibition of cysteine protease activity results in the accumulation of incompletely processed class II-li complexes, which lack peptide binding ability, within lysosomal compartments.

Amino Acid Sequence↗

PubMed1999

Ovalbumin(323-339) peptide binds to the major histocompatibility complex class II I-A(d) protein using two functionally distinct registers.

Proteins of the class II major histocompatibility complex (MHC) bind antigenic peptides that are subsequently presented to T cells. Previous studies have shown that most of the residues required for binding of the chicken ovalbumin (Ova) 323-339 peptide to the I-A(d) MHC class II protein are contained within the shorter 325-336 peptide. This observation is somewhat inconsistent with the X-ray structure of the Ova peptide covalently attached to I-A(d) ( structure) in which residues 323 and 324 form binding interactions with the protein. A second register for the Ova(325-336) peptide is proposed where residues 326 and 327 occupy positions similar to residues 323 and 324 in the structure. Two Ova peptides that minimally encompass the and alternate registers, Ova(323-335) and Ova(325-336), respectively, were found to dissociate from I-A(d) with distinct kinetics. The dissociation rates for both peptides were enhanced when the His81 residue of the MHC beta-chain was replaced with an asparagine. In the structure the betaH81 residue forms a hydrogen bond to the backbone carbonyl of I323. If the Ova(325-336) peptide were also bound in the register, there would be no comparable hydrogen-bond acceptor for the betaH81 side chain that could explain this peptide's sensitivity to the betaH81 replacement. The Ova(323-335) peptide that binds in the register does not stimulate a T-cell hybridoma that is stimulated by Ova(325-336) bound in the alternate register. These results demonstrate that a single peptide can bind to an MHC peptide in alternate registers producing distinct T-cell responses.

Animals↗

PubMed1995

Detailed motifs for peptide binding to HLA-A*0201 derived from large random sets of peptides using a cellular binding assay.

Extensive sets of in total about 2000 synthetic peptides were investigated for their binding affinities to HLA-A*0201. Comparisons of the amino acid compositions of binding to nonbinding sets of peptides provided new information concerning the rules for 9-, 10-, and 11-mer peptide binding at the amino acid level. Preferred primary anchors were shown to depend on peptide length, longer peptides being more demanding in this respect. A clear preference exists for certain amino acids at several nonanchor positions. In addition, the presence of particular amino acids at those positions almost completely precludes peptide binding. We found no evidence for preferred anchor pairs. From these results new and detailed HLA-A*0201 peptide-binding motifs for 9-, 10-, and 11-mer peptide binding were deduced. The motifs are in accordance with earlier reports but include new findings, including C as a C-terminal anchor, the importance of D at positions 4 for binding, and the deleterious effect of R at position 5 (in 9-mers). The motifs are presented in such a way that they can be used to predict peptide binding to HLA-A*0201 by computer analysis (see accompanying paper [56]).

Alleles↗

PubMed1996

Definition of an HLA-A3-like supermotif demonstrates the overlapping peptide-binding repertoires of common HLA molecules.

An HLA-A3-like supertype (minimally comprised of products from the HLA class I alleles A3, A11, A31, A*3301, and A*6801) has been defined on the basis of (a) structural similarities in the antigen-binding groove, (b) shared main anchor peptide-binding motifs, (c) the identification of peptides cross-reacting with most or all of these molecules, and (d) the definition of an A3-like supermotif that efficiently predicts highly cross-reactive peptides. Detailed secondary anchor maps for A3, A11, A31, A*3301, and A*6801 are also described. The biologic relevance of the A3-like supertype is indicated by the fact that high frequencies of the A3-like supertype alleles are conserved in all major ethnic groups. Because A3-like supertype alleles are found in most major HLA evolutionary lineages, possibly a reflection of common ancestry, the A3-like supermotif might in fact represent a primeval human HLA class I peptide-binding specificity. It is also possible that these phenomena might be related to optimal exploitation of the peptide specificity by human TAP molecules. The grouping of HLA alleles into supertypes on the basis of their overlapping peptide-binding repertoires represents an alternative to serologic or phylogenetic classification.

Alleles↗

PubMed1998

A region of conformational variability outside the peptide-binding site of a class I MHC molecule.

Peptide binding is known to influence the conformation of the surface of class I molecules as detected with mAbs and TCR. A new conformationally sensitive epitope on the mouse class I molecule Kb is defined by mAb AF6-88.5. The recognized structure is affected by amino acid substitutions in any of the three external domains of the class I heavy chain and, in addition, is influenced by the substitution of human for mouse beta2-microglobulin. Interestingly, the epitope for this Ab is not affected by mutations within the peptide-binding cleft or by the nature of the peptide bound. These findings indicate that the effect of a change in one domain of class I can radiate to other parts of the molecule. Furthermore, the existence of conformationally sensitive structures outside of the peptide-binding site suggests the possibility that class I molecules may change their structure in response to binding by receptors and ligands such as the TCR and the coligand CD8. Such structural changes may represent signals that can influence cellular activation events.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Two complementary methods for predicting peptides binding major histocompatibility complex molecules.

Peptides that bind to major histocompatibility complex products (MHC) are known to exhibit certain sequence motifs which, though common, are neither necessary nor sufficient for binding: MHCs bind certain peptides that do not have the characteristic motifs and only about 30% of the peptides having the required motif, bind. In order to develop and test more accurate methods we measured the binding affinity of 463 nonamer peptides to HLA-A2.1. We describe two methods for predicting whether a given peptide will bind to an MHC and apply them to these peptides. One method is based on simulating a neural network and another, called the polynomial method, is based on statistical parameter estimation assuming independent binding of the side-chains of residues. We compare these methods with each other and with standard motif-based methods. The two methods are complementary, and both are superior to sequence motifs. The neural net is superior to simple motif searches in eliminating false positives. Its behavior can be coarsely tuned to the strength of binding desired and it is extendable in a straightforward fashion to other alleles. The polynomial method, on the other hand, has high sensitivity and is a superior method for eliminating false negatives. We discuss the validity of the independent binding assumption in such predictions.

Artificial Intelligence↗

PubMed2006

Selective photoaffinity labeling identifies the signal peptide binding domain on SecA.

SecA, an ATPase crucial to the Sec-dependent translocation machinery in Escherichia coli, recognizes and directly binds the N-terminal signal peptide of an exported preprotein. This interaction plays a central role in the targeting and transport of preproteins via the SecYEG channel. Here we identify the signal peptide binding groove (SPBG) on SecA addressing a key issue regarding the SecA-preprotein interaction. We employ a synthetic signal peptide containing the photoreactive benzoylphenylalanine to efficiently and specifically label SecA containing a unique Factor Xa site. Comparison of the photolabeled fragment from the subsequent proteolysis of several SecAs, which vary only in the location of the Factor Xa site, reveals one 53 residue segment in common with the entire series. The covalently modified SecA segment produced is the same in aqueous solution and in lipid vesicles. This spans amino acid residues 269 to 322 of the E. coli protein, which is distinct from a previously proposed signal peptide binding site, and contributes to a hydrophobic peptide binding groove evident in molecular models of SecA.

Adenosine Triphosphatases↗

PubMed2004

Determination of the peptide binding motif and high-affinity ligands for HLA-DQ4 using synthetic peptide libraries.

Juvenile idiopathic arthritis (JIA) is considered to be an autoimmune disease. Various human leukocyte antigen (HLA) associations for different subgroups of this heterogeneous disease have been found. For early-onset pauciarticular arthritis (now oligoarthritic JIA), a strong association with the HLA class II haplotype DQA1*0401/DQB1*0402 (DQ4) has been described. We determined the peptide-binding specificities of this HLA-DQ molecule by screening a synthetic acetylated nonapeptide amide library with one defined and eight random sequence positions. A characteristic binding motif could be deduced. By use of these data, we designed defined specific nonapeptides and identified high-affinity ligands binding to HLA-DQ4. The peptide binding motif of HLA-DQ4 is very similar to the motif of HLA-DQ7, also associated with oligoarthritic JIA. It is, however, different from binding motifs of neutral or protective HLA-DQ molecules. Our results further support the idea of differential peptide presentation in the pathogenesis of oligoarthritic JIA.

Amino Acid Motifs↗

PubMed2005

A novel pesticide-induced conformational state of the oestrogen receptor ligand-binding domain, detected by conformation-specific peptide binding.

The diverse effects of different natural and synthetic oestrogen receptor ligands depend on induction of different receptor conformations, allowing differential interactions with other transcription factors. Different conformations of the oestrogen receptor ligand binding domains can be monitored by conformation-specific binding to peptides selected from phage-displayed peptide libraries. We now report that a group of chlorinated pesticides, including 2,4-dichlorodiphenyl-dichloroethylene, induces a peptide recognition pattern different from those induced by any one of the classical oestrogen receptor ligands. The pesticide-complexed oestrogen receptors recognized peptides reacting with the receptors complexed both with the natural oestrogen 17beta-oestradiol and with the synthetic partial antagonist 4-hydroxy-tamoxifen, respectively, indicating that the pesticide-induced conformation shares features with both the 17beta-oestradiol- and the 4-hydroxy-tamoxifen-induced conformations. The substitution H524A in the ligand binding domain conferred the pesticide-specific peptide recognition pattern and transactivation activity to the oestradiol- and the 4-hydroxy-tamoxifen-complexed receptors, indicating that one important determinant of the pesticide-induced conformation is a lack of stabilisation of any one particular receptor conformation by ligand interaction with H524, which is known to interact with both oestradiol and 4-hydroxy-tamoxifen. Thus, peptide binding analyses of oestrogen receptor conformations induced by environmental endocrine disruptors can give novel information about molecular mechanisms of oestrogen action in general.

Amino Acid Substitution↗

PubMed1992

N-linked oligosaccharides of murine major histocompatibility complex class II molecule. Role in antigenic peptide binding, T cell recognition, and clonal nonresponsiveness.

To evaluate the functional role of the N-linked oligosaccharides of major histocompatibility complex (MHC) class II molecules, affinity-purified murine IAs class II molecules were deglycosylated in the presence of asparagine amidase enzyme. The deglycosylated IAs molecules were characterized by 12% SDS-polyacrylamide gel analysis under reduced and native conditions and the complete enzymatic removal of all three N-linked sugar components from the alpha/beta heterodimer was confirmed by lectin-link Western blot analysis. Like the native IAs molecules, the deglycosylated IAs molecules were fully capable of binding an antigenic peptide from myelin basic protein MBP(89-101). The kinetics of dissociation of preformed complexes of IAs.MBP(89-101) and deglycosylated IAs.MBP(89-101) were compared at 4 and at 37 degrees C. Both complexes were equally stable at 4 degrees C; however, at 37 degrees C the deglycosylated IAs.MBP(89-101) complexes showed an increased rate of dissociation as compared with the native IAs.MBP(89-101) complexes. When tested for their ability to recognize the T cell receptor on T cells, both complexes bound to cloned HS-1 T cells that recognize and respond to IAs.MBP(89-101). Finally, the complexes of deglycosylated IAs.MBP(89-101) were tested for the induction of in vitro nonresponsiveness and compared with native IAs.MBP(89-101) complexes. Both complexes were capable of inducing 95-100% nonresponsiveness in a proliferation assay. These results suggest that the N-linked oligosaccharide of MHC class II molecules may not be essential for either antigenic peptide binding or T cell recognition. In addition results obtained here provide evidence that the carbohydrate moities of MHC class II molecules may not be involved in induction of T cell clonal anergy.

Amidohydrolases↗

PubMed1991

In vitro peptide binding to the heavy chain of the class I molecule of the major histocompatibility complex molecule HLA-A2.

The heavy chain of class I molecules of the major histocompatibility complex forms the binding site for antigenic peptides. We describe the binding of a synthetic peptide to the purified heavy chain of the human major histocompatibility complex molecule HLA-A2. The peptide binding capacity is found to be markedly increased if the protein is first partly denatured by reduction of its disulfide bonds in detergent and subsequently renatured by reoxidation. In the presence of certain detergents, the heavy chain binds peptides even when the protein is partly unfolded.

Amino Acid Sequence↗

PubMed1981

Isolation of the membrane-binding peptide of NADPH-cytochrome P-450 reductase. Characterization of the peptide and its role in the interaction of reductase with cytochrome P-450.

A peptide identified as the membrane-associated segment of NADPH-cytochrome P-450 reductase has been generated by steapsin protease treatment of vesicle-incorporated reductase and isolated by preparative gel electrophoresis. This peptide remains associated with vesicles when steapsin protease digests of vesicle-incorporated reductase were fractionated by Sepharose 4B chromatography, confirming its identity as the membrane-binding peptide. The molecular weight of the membrane-binding peptide was 6400 as determined by gel filtration in 8 M guanidine hydrochloride, and its amino acid content was not especially hydrophobic. The activity of reconstituted hydroxylation systems consisting of reductase, cytochrome P-446, and dilauroyl phosphatidylcholine was not inhibited by large molar excesses of purified membrane-binding peptide. Moreover, when purified reductase and cytochrome P-446 were added to liposomes which contained the membrane-binding peptide, it was determined that mixed function oxidase activity was reconstituted as effectively as when vesicles without the membrane-binding peptides were used. Similar results were obtained with reductase, cytochrome P-450, and detergent-solubilized liposomes (with or without the membrane-binding peptide). Thus, the membrane-binding peptide does not appear to interact with either of these two forms of the hemoprotein in a site-specific manner to prevent reconstitution of hydroxylation activity.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1996

The peptide binding specificity of the MHC class II I-A molecule of the Lewis rat, RT1.BI.

The specificity of peptide binding to MHC molecules is defined by binding motifs composed of several relatively conserved anchor positions. The peptide binding motifs of murine MHC class II I-A molecules are functionally important but poorly characterized. Here we use peptide binding studies and isolation of naturally presented peptides to characterize the peptide binding motif of the MHC class II I-A molecule, RT1.BI, a molecule that is involved in experimental autoimmunity in the Lewis rat. We now report that, similar to other class II motifs, the RT1.BI motif consists of a nonamer sequence with four major anchor positions (P1, P4, P6 and P9). Residues at P4 and P9, rather than at P1, appeared to be particularly important for binding. Negatively charged residues were favored at P9, consistent with the presence of a serine at position 57 of the RT1.BI beta chain. This RT1.BI motif could be observed in the dominant autoantigenic T cell epitopes mapped previously in the Lewis rat. These results highlight a general similarity and some important differences in the organization of MHC class II peptide binding motifs. The reported RT1.BI motif should facilitate the prediction and design of T cell epitopes for the induction and control of experimental autoimmune diseases in Lewis rat models.

Amino Acid Sequence↗

PubMed2000

Identification of novel prostate-specific antigen-binding peptides modulating its enzyme activity.

Prostate-specific antigen (PSA) is a serine protease with highly prostate-specific expression. Measurement of PSA in serum is widely used for diagnosis and monitoring of prostate cancer. PSA dissolves the seminal gel forming after ejaculation. It has been suggested to mediate invasion and metastasis of prostate cancer but also to exert antiangiogenic activity. We have identified peptides specific for PSA by screening cyclic phage display peptide libraries. PSA-binding peptides were isolated from four different libraries and produced as a fusion protein with glutathione S-transferase (GST). The phage and fusion proteins were shown to bind to PSA specifically as indicated by lack of binding to other serine proteinases. A peptide with four cysteines showed the highest affinity for PSA. Zn2+, an inhibitor of PSA activity, increased the affinity of the peptides to PSA. The binding specificity was characterized by cross-inhibition using monoclonal anti-PSA antibodies of known epitope specificities. The peptides bound to the same region as mAbs specific for free PSA indicating that they bind close to the active site of the enzyme. The peptides enhanced the enzyme activity of PSA against a chromogenic substrate. These results show that peptides binding to PSA and modulating its enzyme activity can be developed by phage display technique. The peptides have the potential to be used for identification of PSA variants and for imaging and targeting of prostatic tumors.

Amino Acid Sequence↗

PubMed1994

Antigenic peptide binding to MHC class II molecules at increased peptide concentrations.

Affinity-purified major histocompatibility complex (MHC) class II molecules are known to bind antigenic peptides in vitro. The percentage of MHC class II molecules occupied with such peptides is usually very low and varies significantly depending upon the sequence and size of a given antigenic peptide. The present study describes a method by which complete saturation of affinity-purified MHC class II with antigenic peptide can be achieved by simply incubating purified MHC class II molecules at neutral pH in the presence of several 100-fold molar excess of antigenic peptide. Complexes of human HLA-DR2 and a peptide analog from human myelin basic protein MBP (83-102)Y83 were selected for this study. The on-rate kinetic results showed saturation of MHC class II occupancy at 300-500-fold molar excess peptide concentrations. The specificity of the MBP (83-102)Y83 peptide binding to HLA-DR2 at higher peptide concentration was demonstrated by incubating an equivalent amount of another epitope from myelin basic protein [MBP (1-14) peptide] as well as by competitive binding assays. The quantitation of bound peptide was carried out using biotinylated-MBP (83-102)Y83 peptide which showed 100-125% occupancy of HLA-DR2 with a recovery of 100%. The presence of a single peptide entity in purified complexes was confirmed by reverse-phase narrowbore HPLC analysis of the acid extracted supernatant and by mass spectrometry analysis. Two-dimensional gel electrophoresis (IEF/SDS) of purified HLA-DR2 and DR2.MBP (83-102)Y83 complexes showed the absence of various endogenous polypeptides in 100% loaded complexes. These results demonstrate that higher peptide concentrations can be useful in generating MHC class II-peptide complexes of defined composition. Such complexes of MHC class II occupied with a single peptide may have significant clinical relevance for antigen-specific therapy of various autoimmune diseases and may provide better understanding of MHC-peptide-TCR interactions.

Amino Acid Sequence↗

PubMed1988

A novel arginine vasopressin-binding peptide that blocks arginine vasopressin modulation of immune function.

We report on a novel peptide that blocks the neuroendocrine hormone arginine vasopressin (AVP) helper signal for IFN-gamma production by direct interaction with the hormone. The AVP-binding nonapeptide has the sequence Thr-Met-Lys-Val-Leu-Thr-Gly-Ser-Pro (binding peptide). AVP and its 6-amino acid N-terminus cyclic ring pressinoic acid (PA) are both capable of replacing the IL-2 requirement for IFN-gamma production by mouse splenic lymphocytes. We show that the AVP-binding peptide specifically and reversibly blocks AVP help in IFN-gamma production, but fails to block the helper signal of PA. Thus the intact AVP molecule and not just the N-terminal cyclic ring is important for interaction with the binding peptide. AVP interacts with the binding peptide with an apparent KD of approximately 50 nM. The AVP-binding peptide does not inhibit AVP interaction with its receptor on lymphocytes. Interestingly, whereas the AVP-binding peptide does not block the PA helper signal for IFN-gamma induction, the complex of AVP and binding peptide does reversibly block the PA signal. The AVP family of hormones requires conformational flexibility for signal transduction. Thus, we hypothesize that the AVP-binding peptide restricts this flexibility and converts AVP into an antagonist of its own action.

Adjuvants, Immunologic↗

PubMed2001

Soluble heparin-binding peptides regulate chemokinesis and cell adhesive forces.

The ability of a soluble heparin-binding peptide sequence derived from fibronectin to modulate the adhesion and chemokinetic migration behavior of arterial smooth muscle cells was assessed using a novel glass microsphere centrifugation assay and automated time-lapse fluorescence videomicroscopy, respectively. Treatment of cells grown on fibronectin-coated substrates with the soluble heparin-binding peptide resulted in the disassembly of focal adhesions, as assessed by immunohistochemical staining. These observations were consistent with an observed dose-dependent two- to fivefold reduction in cell-substrate adhesive strength (P < 0.001) and a biphasic effect on migration speed (P < 0.05). Moreover, heparin-binding peptides induced a twofold reduction (P < 0.01) in two-dimensional cell dispersion in the presence of a non-heparin-binding growth factor, platelet-derived growth factor-AB (PDGF-AB). Heparin-binding peptides were unable to mediate these effects when cells were grown on substrates lacking a heparin-binding domain. These data support the notion that competitive interactions between cell surface heparan sulfates with heparin-binding peptides may modulate chemokinetic cell migration behavior and other adhesion-related processes.

Animals↗

PubMed2004

Characterization of natural peptide ligands from HLA-DP2: new insights into HLA-DP peptide-binding motifs.

Although natural peptide ligands of HLA-DR and HLA-DQ molecules have been extensively studied, information about peptides naturally bound to HLA-DP is limited. Here we describe HLA-DP2 peptide ligands corresponding to 24 different source proteins that were identified by peptide pool elution and mass spectrometry sequencing from HLA-DP2 molecules expressed on EBV-LCLs. Sequencing analysis led to the identification of both promiscuous and allele-specific peptides. Moreover, the alignment of the natural ligands for HLA-DP2 described here, combined with previous results from our group and others concerning HLA-DP2 antigen presentation and HLA-DP molecular modelling, provide a better understanding of HLA-DP2 peptide-binding motifs.

Amino Acid Motifs↗