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PubMed1990

Analysis of physical interactions between peptides and HLA molecules and application to the detection of human immunodeficiency virus 1 antigenic peptides.

The physical association of 40 antigenic peptides and purified HLA class I and class II molecules was monitored using a direct peptide binding assay (PBA) in solid phase and an inhibition peptide binding assay (IPBA) in which the competing peptide was present in a soluble phase. We also examined the ability of different peptides to inhibit the lytic activity of human antiviral cytolytic T cells towards cells incubated with the corresponding target peptide. Our results showed that: (a) Binding of a given human T cell-recognized peptide to several HLA class I and class II molecules occurred frequently. Nevertheless, preferential binding of peptides to their respective restriction molecules was also observed. (b) Binding of HLA molecules to peptides recognized by murine T cells occurred less frequently. (c) 11 of 24 (46%) randomly selected HIV-1 peptides contained agretopic residues allowing their binding to HLA molecules. (d) The kinetics of HLA/peptide association depended on the peptide tested and were faster than or similar to those reported for Ia molecules. Dissociation of these complexes was very low. (e) Peptide/HLA molecule binding was dependent on length, number of positive charges, and presence of hydrophobic residue in the peptide. (f) A correlation was demonstrated between a peptide inhibitory effect in the IPBA and its blocking effect in the cytolytic test. Our data indicated that the restriction phenomenon observed in T cell responses was not strictly related to either an elective HLA/peptide association, or a high binding capacity of a peptide to HLA molecules. These data also showed that the PBA and IPBA are appropriate for the detection of agretopic residues within HIV-1 proteins.

Amino Acid Sequence↗
PubMed1994

Peptide size selection by the major histocompatibility complex-encoded peptide transporter.

The major histocompatibility complex (MHC)-encoded heterodimeric TAP1/TAP2 transporter (TAP) translocates cytosolic peptides into the lumen of the endoplasmic reticulum (ER), where peptides of 8 to 11 amino acids long associate with MHC class I molecules. We have studied the selectivity of peptide translocation by TAP in streptolysin O-permeabilized cells using glycosylatable, radioiodinated model peptides to detect import into the ER lumen. TAP-dependent translocation of a radiolabeled nonamer peptide was most efficiently inhibited by unlabeled 9- to 11-mer peptides. Peptides between 7 and 40 amino acids long all could inhibit transport, the longer peptides being least effective. Also, peptides shorter than eight amino acids were inefficiently translocated. The use of directly labeled length variants in translocation assays and TLC analysis of the transported material revealed two pathways for translocation: short peptides (7 to 13 amino acids long) were translocated without prior modification. In contrast, transport of longer peptides was not effective. Instead such peptides were clipped by cytosolic peptidases before efficient transport. Our data suggest that TAP preferentially translocates peptides of appropriate length for class I binding. Furthermore, TAP-translocated peptides were rapidly released from the ER unless they were trapped there by being glycosylated or by binding to MHC class I molecules.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Presentation of a T cell receptor antagonist peptide by immunoglobulins ablates activation of T cells by a synthetic peptide or proteins requiring endocytic processing.

T cell receptor (TCR) antagonism is being considered for inactivation of aggressive T cells and reversal of T cell-mediated autoimmune diseases. TCR antagonist peptides silence aggressive T cells and reverse experimental allergic encephalomyelitis induced with free peptides. However, it is not clear whether free antagonist peptides could reverse natural disease where the antigen is presumably available for endocytic processing and peptides gain access to newly synthesized class II MHC molecules. Using an efficient endocytic presentation system, we demonstrate that a proteolipid protein (PLP) TCR antagonist peptide (PLP-LR) presented on an Ig molecule (Ig-PLP-LR) abrogates the activation of T cells stimulated with free encephalitogenic PLP peptide (PLP1), native PLP, or an Ig containing PLP1 peptide (Ig-PLP1). Free PLP-LR abolishes T cell activation when the stimulator is free PLP1 peptide, but has no measurable effect when the stimulator is the native PLP or Ig-PLP1. In vivo, Ig-PLP1 induces a T cell response to PLP1 peptide. However, when coadministered with Ig-PLP-LR, the response to PLP1 peptide is markedly reduced whereas the response to PLP-LR is normal. Free PLP-LR coadministered with Ig-PLP1 has no effect on the T cell response to PLP1. These findings indicate that endocytic presentation of an antagonist peptide by Ig outcompete both external and endocytic agonist peptides whereas free antagonist hinders external but not endocytic agonist peptide. Direct contact with antagonist ligand and/or trans-regulation by PLP-LR-specific T cells may be the operative mechanism for Ig-PLP-LR-mediated downregulation of PLP1-specific T cells in vivo. Efficient endocytic presentation of antagonist peptides, which is the fundamental event for either mechanism, may be critical for reversal of spontaneous T cell-mediated autoimmune diseases where incessant endocytic antigen processing could be responsible for T cell aggressivity.

Animals↗
PubMedDate not supplied

Peptide vaccination for patients with melanoma and other types of cancer based on pre-existing peptide-specific ctotoxic T-lymphocyte precursors in the periphery.

Identification of antigenic peptides expressed on cancer cells enables us to treat cancer patients with peptide-based immunotherapy. Although optimal protocols for peptide-based vaccines have not yet been elucidated, boosting the immune system could be a better approach than priming the immune system to elicit prompt and potent peptide-specific T-cell responses in cancer patients. With this possibility in mind, the authors undertook a clinical trial in which cancer patients were vaccinated with peptides (maximum 4) after confirmation of pre-existing peptide-specific cytotoxic T-lymphocyte (CTL) precursors in the periphery. Fourteen patients (seven with melanoma and seven with other types of cancer) positive for either HLA-A24 or HLA-A2 were enrolled in this study. Fourteen and 16 peptides were used to screen for HLA-A24+ and HLA-A2+ patients, respectively. The vaccination was well tolerated, and the only adverse effects were local pain and fever. Kinetic analysis revealed that peptide-reactive CTLs increased after peptide vaccination in 7 of 14 patients. Immunoglobulin G (IgG) reactive to the administered peptides was detected in 2 patients before vaccination, although it became detectable in 8 of the other 12 patients after the peptide vaccination. Stable disease for more than 6 months was observed in five patients (one with melanoma and four with other types of cancer); all of these patients showed increased levels of peptide-specific IgG. These results indicate that peptide vaccination of patients showing evidence of pre-existing peptide-specific CTL precursors can be applied in further clinical trials aimed at the treatment of melanoma and other types of cancer.

Adult↗
PubMed2002

Comparative study of peptides isolated from class I antigen groove of urological specimens. a further step toward the future of peptide therapy in bladder cancer patients.

BACKGROUND: The major histocompatibility (MHC) class I antigens act as associative molecules for interaction amongst immuno-competent cells. The grooves of class I antigens are normally loaded with self peptides of between 8 and 11 amino acids. However, when the cells transform to malignant state they may carry peptide(s) of non-self origin within these grooves. Using immuno-bead purification followed by high-performance liquid chromatography (HPLC), this study attempted to isolate peptides from class I antigens of various biological specimens RESULTS: The combination of immuno-bead purification (BP) and HPLC was reliable for peptide isolation. Class I antigens and associated peptides could be isolated from normal peripheral blood leukocyte (PBL). Under the same conditions, the PBL yielded almost twice as much peptide as that of tumour cell lines. The HPLC profile of peptides (range of 8-10 amino acid residues) isolated from a bladder and a cervical tumour cell line showed unique features. In the case of the bladder line there were at least 22 peptides. In addition, the class I-associated peptides could also be isolated from kidney tumour fragments of three individuals. In each the isolated peptides showed a unique HPLC peak profile with some similarities as well as differences. CONCLUSIONS: These data indicated a variation in the nature of peptides isolated from different specimens. The approach showed the feasibility of preparing peptide(s) from a relatively small number of cells. The data also showed that peptide isolation could also be carried out from tumour tissue biopsies paving the way for the future of peptide vaccination in cancer patients following the identification of putative tumour-specific replicate(s).

Chromatography, High Pressure Liquid↗
PubMed2006

PepDist: a new framework for protein-peptide binding prediction based on learning peptide distance functions.

BACKGROUND: Many different aspects of cellular signalling, trafficking and targeting mechanisms are mediated by interactions between proteins and peptides. Representative examples are MHC-peptide complexes in the immune system. Developing computational methods for protein-peptide binding prediction is therefore an important task with applications to vaccine and drug design. METHODS: Previous learning approaches address the binding prediction problem using traditional margin based binary classifiers. In this paper we propose PepDist: a novel approach for predicting binding affinity. Our approach is based on learning peptide-peptide distance functions. Moreover, we suggest to learn a single peptide-peptide distance function over an entire family of proteins (e.g. MHC class I). This distance function can be used to compute the affinity of a novel peptide to any of the proteins in the given family. In order to learn these peptide-peptide distance functions, we formalize the problem as a semi-supervised learning problem with partial information in the form of equivalence constraints. Specifically, we propose to use DistBoost, which is a semi-supervised distance learning algorithm. RESULTS: We compare our method to various state-of-the-art binding prediction algorithms on MHC class I and MHC class II datasets. In almost all cases, our method outperforms all of its competitors. One of the major advantages of our novel approach is that it can also learn an affinity function over proteins for which only small amounts of labeled peptides exist. In these cases, our method's performance gain, when compared to other computational methods, is even more pronounced. We have recently uploaded the PepDist webserver which provides binding prediction of peptides to 35 different MHC class I alleles. The webserver which can be found at http://www.pepdist.cs.huji.ac.il is powered by a prediction engine which was trained using the framework presented in this paper. CONCLUSION: The results obtained suggest that learning a single distance function over an entire family of proteins achieves higher prediction accuracy than learning a set of binary classifiers for each of the proteins separately. We also show the importance of obtaining information on experimentally determined non-binders. Learning with real non-binders generalizes better than learning with randomly generated peptides that are assumed to be non-binders. This suggests that information about non-binding peptides should also be published and made publicly available.

Algorithms↗
PubMed1995

Kaliuretic peptide and long acting natriuretic peptide as well as atrial natriuretic factor inhibit aldosterone secretion.

The present investigation was designed to determine whether atrial natriuretic peptides consisting of amino acids 1-30 (long acting natriuretic peptide), 31-67 (vessel dilator) and 79-98 (kaliuretic peptide) as well as 99-126 (atrial natriuretic factor (ANF)) of the 126 amino acid ANF prohormone inhibit aldosterone secretion. Thirty healthy human subjects were studied following infusion of 100 ng/kg body weight/min for 60 min of each of the respective peptides. Kaliuretic peptide decreased plasma aldosterone concentration by the greatest amount (6-fold) and plasma aldosterone was still significantly decreased (P < 0.001) three hours after stopping the infusion. In contrast, within 30 min of cessation of the ANF infusion, plasma aldosterone levels had returned to pre-infusion values. Long acting natriuretic peptide also significantly (P < 0.01) decreased plasma aldosterone levels which remained significantly (P < 0.001) decreased 3 h after cessation of infusion. Vessel dilator did not decrease plasma aldosterone levels. Kaliuretic peptide, ANF and long acting natriuretic peptide also decreased (P < 0.01) urinary aldosterone concentrations. None of these peptides changed the plasma potassium concentration. We conclude that two new peptide hormones (long acting natriuretic peptide and kaliuretic peptide) inhibit aldosterone secretion. The length of time that aldosterone secretion is inhibited following kaliuretic peptide and long acting natriuretic peptide infusion is significantly longer (P < 0.001) than following ANF infusion.

Adrenal Glands↗
PubMed1989

Comparison of peptide-coating conditions in solid phase plate assays for detection of anti-peptide antibodies.

Mice were immunized with 14 free (i.e. not conjugated to any carrier) synthetic peptides representing the entire human hemoglobin alpha-chain. Antibodies against each peptide were determined using solid phase radioimmunoassay, both with free peptides and peptides coupled to a protein carrier as the coating antigen. It has been demonstrated that large improvements in the ability to detect anti-peptide antibodies were achieved in some cases by precoating the assay wells with free peptides and in other cases by precoating with peptide-protein conjugates. Sodium carbonate buffer, pH 9.6, had a favorable effect on the coating of two of the free peptides when compared with phosphate-buffered saline, pH 7.2. The assay with the plates coated with optimum peptide form (free peptide or peptide-protein conjugate) was superior in the detection of antibody binding to 9 of the peptides when compared with the assay using chemically activated plates. The results suggest that the appropriate form (conjugated or free) and conditions for immobilizing small peptides to plastic supports are not universal but will have to be determined for each test peptide.

Amino Acid Sequence↗
PubMed1994

Immunity to TCR peptides in multiple sclerosis. II. T cell recognition of V beta 5.2 and V beta 6.1 CDR2 peptides.

The biased expression of V beta 5.2 and V beta 6.1 by T cells specific for myelin basic protein (BP) has led to our use of TCR peptides from these V gene sequences to induce anti-TCR immunity in patients with multiple sclerosis (MS). Injection of V beta 5.2-39-59 or V beta 6.1-39-59 peptides significantly increased the peptide specific T cell frequency in 7 of 11 MS patients, often with an accompanying delayed hypersensitivity reaction at the injection site. Here, we validate these cellular immune responses by characterizing TCR peptide specific T cells from an MS patient with biased V beta 5.2 expression in BP reactive T cells before treatment with TCR peptides, and from two MS patients in whom the frequencies of anti-TCR peptide specific T cells were significantly boosted after injection with low doses of TCR peptides. In both cases, T cell lines were established with relative ease, especially after boosting with the peptides. A V beta 5.2-39-59 reactive line responded selectively to the boosting peptide and was restricted by both MHC class I (HLA-B7) and MHC class II (HLA-DR2) molecules. Characterization of 22 clonal isolates revealed that the responding T cells were predominantly activated CD4+CD8lo, circulating memory cells restricted by either HLA-B7 or HLA-DR2, that utilized mainly V beta 4, V beta 6, V beta 12, and V beta 14, but not V beta 5.2 in their TCR. T cell isolates specific for V beta 6.1-39-59 possessed similar characteristics but contained specificities cross-reactive with an N-terminal sequence on V beta 5.2-39-59. Upon stimulation with peptide or Con A, the TCR peptide specific T cell lines had increased message production for IFN-gamma, GM-CSF, IL-4, IL-5, and to a lesser degree, IL-2. This lymphokine mRNA profile differed from a BP-specific T cell line that produced message for IFN-gamma and GM-CSF but low or absent levels of IL-4 and IL-5. The extensive parallels between human T cells specific for V beta 5.2 and V beta 6.1 CDR2 peptides and rat T cells specific for V beta 8.2 CDR2 peptide that are highly protective against experimental encephalomyelitis strengthen the rationale for the therapeutic use of TCR peptides in human autoimmunity.

Adult↗
PubMed1996

Probing HLA-B7 conformational shifts induced by peptide-binding groove mutations and bound peptide with anti-HLA monoclonal antibodies.

To determine the influence of peptide-binding groove residues and MHC-bound peptide on HLA-B7 conformation, we investigated the binding sites of nine locus- or allele-specific mAbs using a panel of 82 HLA-B7 variants. The functional mAb epitopes encircle the HLA-B7 peptide-binding groove. Three mAbs are affected by mutations at solvent-accessible peptide-binding groove mutations. Mutations in peptide-binding groove residues 45, 63, and 150 affect multiple nonoverlapping mAb epitopes, probably by interaction with other MHC residues or bound peptide. However, 18 of 24 peptide-binding groove mutations do not affect mAb binding, indicating that the conformation of solvent-accessible HLA-B7 structures is largely dissociated from changes in the peptide-binding groove. To test whether bound peptides alter HLA-B7 conformation, we loaded HLA-B7 heavy chains on acid-stripped cells with beta2-microglobulin and 20 individual synthetic peptides. Two of eight mAbs are sensitive to HLA-B7-bound peptides. A likely interpretation of these data is that the conformational flexibility of HLA-B7 is due to peptide-induced conformational shifts in MHC side chains, rather than major shifts in the MHC main chain. These results suggest that HLA-B7 conformation is largely maintained in the context of different bound peptides and different peptide-binding grooves.

Antibodies, Monoclonal↗
PubMed1996

Peptides modeled on the transmembrane region of the slow voltage-gated IsK potassium channel: structural characterization of peptide assemblies in the beta-strand conformation.

A 27-residue peptide, having a sequence corresponding to the transmembrane domain of the IsK protein with slow voltage-gated potassium channel activity, has been incorporated into synthetic saturated-chain phospholipid membranes. The peptide-lipid complexes have been characterized by attenuated-total-reflection Fourier-transform-infrared spectroscopy (ATR-FTIR), spin-label electron spin resonance (ESR) spectroscopy, 31P and 2H nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry, and low-angle X-ray diffraction. From FTIR spectroscopy, it is found that, when reconstituted into membranes by dialysis from 2-chloroethanol, the peptide has a predominantly beta-strand secondary structure in which the peptide backbone is oriented at an angle of approximately 56 degrees relative to the membrane normal in dry films of phosphatidylcholines. Hydration of the dry film in the gel phase does not appear to affect the orientation of the peptide backbone, and a relatively small change in orientation occurs when the bilayer undergoes the transition to the fluid phase. The ESR and NMR spectra from spin-labeled and 2H-labeled phospholipids, respectively, indicate that the incorporated peptide restricts the rotational motion of the lipids, without appreciably affecting the chain order, in a way similar to that found for integral membrane proteins. The characteristic two-component ESR spectra from spin-labeled lipids further indicate a selectivity in the interaction of anionic phospholipids with the peptide. The motional restriction of the chains of the spin-labeled phosphatidylcholine and the reduction in the enthalpy of the lipid chain-melting transition indicate that, on average, approximately two to three phospholipid molecules interact directly with each peptide monomer, which is consistent with a limited degree of aggregation of the beta-sheet structures. Both 31P NMR spectroscopy and X-ray diffraction indicate that the lipid-peptide complexes have a lamellar structure up to the highest peptide concentration studied (Rp = 0.2). The surface area occupied by lipid molecules (ca. 30 A2 per chain) in the peptide complexes, deduced from the lamellar repeat spacings at defined water content, is very similar to that in pure fluid lipid bilayers, consistent with the 2H NMR results. The additional membrane surface area contributed by the peptide is approximately 112 A2 per monomer. This large value for the peptide area in the fluid bilayer is consistent with the ATR studies of dry peptide/lipid films which suggest that the long axis of the beta-strand is strongly tilted with respect to the bilayer normal (56 degrees in the dry film).

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1993

Hydrolysis of human and pig brain natriuretic peptides, urodilatin, C-type natriuretic peptide and some C-receptor ligands by endopeptidase-24.11.

Endopeptidase-24.11 (E-24.11, EC 3.4.24.11) is widely believed to play a physiological role in metabolizing atrial natriuretic peptide (ANP). Since the discovery of ANP, new natriuretic peptides have been isolated and other peptides synthesized as receptor ligands. The hydrolysis in vitro of six related peptides by the endopeptidase has been studied, mainly by h.p.l.c. The initial attack on the 32-residue form of pig brain natriuretic peptide (pBNP-32) was shown to be at the Ser20-Leu21 bond, as had been previously shown for the 26-residue form. In contrast, human brain natriuretic peptide-32 (hBNP-32), which differs in ten residues from pBNP-32, was attacked first at the Met4-Val5 bond, releasing the N-terminal tetrapeptide, and only later at bonds within the ring: at Arg17-Ile18 and subsequently at four other sites. Urodilatin, which has a four-residue extension at the N-terminus compared with alpha-human atrial natriuretic peptide-28 (alpha-hANP), was degraded at about half the rate of the latter, though the C-terminal Phe-Arg-Tyr was released at the same rate. The 22-residue C-type natriuretic peptide was hydrolysed more rapidly than alpha-hANP, as were two C-receptor ligands (peptides with deletions within the ring): C-ANP4-23 (rANP4-23 des-Gln18,Ser19,Gly20,Leu21,Gly22) and SC 46542 (hANP5-28 des-Phe8,Gly9,Ala17,Gln18). Angiotensin-converting enzyme failed to hydrolyse pBNP-32, hBNP-32 or 125I-rat (r) ANP, even after prolonged incubation. Km and kcat values were determined for the hydrolysis of alpha-hANP, porcine BNP-26, porcine BNP-32 and 125I-rANP by E-24.11. Ki values were determined for six peptides, alpha-hANP, urodilatin, hBNP-32, C-type natriuretic peptide (CNP), SC 46542 and C-type natriuretic peptide (C-ANP4-23), in radiometric assays of E-24.11 with either [125I] insulin B chain or [125I] rANP as substrate. The Ki values (2.5-13 microM) for CNP were the lowest of any of the group, whereas those for hBNP-32 (151-172 microM) were the highest. The physiological significance of these results is discussed, especially in regard to the relative resistance of hBNP-32 to attack and the ability of the C-receptor ligands to compete with natriuretic peptides for hydrolysis by E-24.11.

Amino Acid Sequence↗
PubMed1994

Mutations inside but not outside the peptide binding cleft of the H-2 Ld molecule affect CTL recognition and binding of the nucleoprotein peptide from the lymphocytic choriomeningitis virus.

In order to investigate the role of residues inside and outside the peptide binding cleft of the Ld molecule in peptide presentation to cytotoxic T lymphocytes (CTL), we constructed a series of point mutations in the Ld gene. We determined the effects of the mutations in the Ld molecule on the binding and recognition of an Ld-restricted CTL epitope derived from the nucleoprotein (NP) of the lymphocytic choriomeningitis virus (LCMV). Each of the mutations within the Ld peptide binding cleft resulted in a complete loss of CTL recognition. Addition of the LCMV NP peptide to cells expressing these mutants did not increase surface Ld expression, suggesting that the mutations altered peptide binding. Mutations involving pockets D and E within the cleft affected LCMV peptide binding and recognition as drastically as those in pocket B, which was predicted to interact with a main anchor residue of the peptide. In striking contrast, the mutations located outside the cleft did not change either recognition or binding. These results demonstrate that the Ld residues in the peptide binding cleft are the main determinants dictating LCMV NP peptide binding, and that the residues in each of the pockets within the cleft play a role in this interaction. Surprisingly, one mutation outside the peptide binding cleft, T92S, abrogated CTL lysis of target cells treated with the LCMV NP peptide, but not virus-infected cells. These data show that this mutation selectively altered the presentation of the LCMV NP peptide introduced to the cell exogenously, but not endogenously. This implies that the pathway by which peptides associate with class I molecules within the cell differs from that of exogenous peptide binding.

Amino Acid Sequence↗
PubMed2005

HIV-1 MN Env 15-mer peptides better detect HIV-1 specific CD8 T cell responses compared with consensus subtypes B and M group 15-mer peptides.

OBJECTIVE: To compare the ability of three Env (15-mer) peptide sets derived from the HIV-1 MN, the subtype B consensus, and the group M consensus to detect HIV-1 specific interferon (IFN)-gamma responses in HIV-1 subtype B infected subjects. METHODS: Peripheral blood mononuclear cells were obtained from 17 HIV-1 subtype B seropositive and 5 HIV-1 seronegative subjects. Peptide matrices comprising each peptide set were used in IFN-gamma Elispot assays to screen for T cell epitopes. Following matrix deconvolution, individual peptides were analyzed by IFN-gamma intracellular cytokine-staining to confirm and characterize the responding cells. RESULTS: HIV specific IFN-gamma responses were detected in 17 of 17 HIV-1 seropositive and none of 5 HIV-1 seronegative subjects by Elispot. Within the 17 HIV-1 seropositives, 16, 14, and 11 subjects responded to MN, B consensus, and group M env peptides, respectively. Responses were confirmed by intracellular cytokine analysis in 14 subjects and were in the CD3CD8 compartment. Cross-recognition of 'equivalent' peptides (i.e., peptides mapping to the same sequence region from the three peptide sets) was observed in 9 of 17 subjects. Peptide set specific responses to individual peptides were also observed; 11, 1, and 1 subjects demonstrated peptide set specific responses to MN, B consensus, and consensus group M, respectively. CONCLUSION: MN derived Env peptides were better able to detect HIV-1 specific CD8 T cell responses, many of which were not detectable by the equivalent clade or group consensus peptides. No single peptide set detected all the IFN-gamma responses within an individual. These results demonstrate the importance of reagent selection for monitoring of HIV responses in HIV-1 infected individuals and subsequently vaccine recipients.

Adult↗
PubMed2003

Immunocytochemical and molecular data guide peptide identification by mass spectrometry: orcokinin and orcomyotropin-related peptides in the stomatogastric nervous system of several crustacean species.

In order to identify new orcokinin and orcomyotropin-related peptides in crustaceans, molecular and immunocytochemical data were combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In the crayfish Procambarus clarkii, four orcokinins and an orcomyotropin-related peptide are present on the precursor. Because these peptides are highly conserved, we assumed that other species have an identical number of peptides. To identify the peptides, immunocytochemistry was used to localize the regions of the stomatogastric nervous system in which orcokinins are predominantly present. One of the regions predominantly containing orcokinins was a previously undescribed olive-shaped neuropil region within the commissural ganglia of the lobsters Homarus americanus and Homarus gammarus. MALDI-TOF MS on these regions identified peptide masses that always occur together with the known orcokinins. Seven peptide ions occurred together in the peptide massspectra of the lobsters. Mass spectrometric fragmentation by MALDI-MS post-source decay (PSD) and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI Q-TOF MS) collision-induced dissociation (CID) were used in the identification of six of these masses, either as orcokinins or as orcomyotropin-related peptides and revealed three hitherto unknown peptide variants, two of which are [His13]-orcokinin ([M+H]+ = 1540.8 Da) and an orcomyotropin-related peptide FDAFTTGFGHN ([M+H]+ = 1213.5 Da). The mass of the third previously unknown orcokinin variant corresponded to that of an identified orcokinin, but PSD fragmentation did not support the suggested amino acid sequence. CID analysis allowed partial de novo sequencing of this peptide. In the crab Cancer pagurus, five orcokinins and an orcomyotropin-related peptide were unambigously identified, including the previously unknown peptide variant [Ser9-Val13]-orcokinin ([M+H]+ = 1532.8 Da).

Amino Acid Sequence↗
PubMed1994

Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains.

A method to predict the relative binding strengths of all possible nonapeptides to the MHC class I molecule HLA-A2 has been developed based on experimental peptide binding data. These data indicate that, for most peptides, each side-chain of the peptide contributes a certain amount to the stability of the HLA-A2 complex that is independent of the sequence of the peptide. To quantify these contributions, the binding data from a set of 154 peptides were combined together to generate a table containing 180 coefficients (20 amino acids x 9 positions), each of which represents the contribution of one particular amino acid residue at a specified position within the peptide to binding to HLA-A2. Eighty peptides formed stable HLA-A2 complexes, as assessed by measuring the rate of dissociation of beta 2m. The remaining 74 peptides formed complexes that had a half-life of beta 2m dissociation of less than 5 min at 37 degrees C, or did not bind to HLA-A2, and were included because they could be used to constrain the values of some of the coefficients. The "theoretical" binding stability (calculated by multiplying together the corresponding coefficients) matched the experimental binding stability to within a factor of 5. The coefficients were then used to calculate the theoretical binding stability for all the previously identified self or antigenic nonamer peptides known to bind to HLA-A2. The binding stability for all other nonamer peptides that could be generated from the proteins from which these peptides were derived was also predicted. In every case, the previously described HLA-A2 binding peptides were ranked in the top 2% of all possible nonamers for each source protein. Therefore, most biologically relevant nonamer peptides should be identifiable using the table of coefficients. We conclude that the side-chains of most nonamer peptides to the first approximation bind independently of one another to the HLA-A2 molecule.

Amino Acid Sequence↗
PubMed1997

Modified phage peptide libraries as a tool to study specificity of phosphorylation and recognition of tyrosine containing peptides.

Tyrosine phosphorylation and protein recognition, mediated by phosphotyrosine containing peptides, play an important role in determining the specific response of a cell, when stimulated by external signals. We have used peptide repertoires displayed by filamentous phage as a tool to study the substrate specificity of the protein tyrosine kinase (PTK) p55(fyn) (Fyn). Peptide libraries were incubated for a short time in the presence of Fyn and phages displaying efficiently phosphorylated peptides were selected by panning over anti-phosphotyrosine antibodies. The characterization of the peptides enriched after three phosphorylation/selection rounds allowed us to define a canonical substrate sequence for the kinase Fyn, E-(phi/T)YGx phi, where phi represents any hydrophobic residue. A peptide conforming to this sequence is a better substrate than a second peptide designed to be in accord with the consensus sequence recognised by the Fyn SH2 domain. When the library phosphorylation reaction is carried out in saturation conditions, practically all the tyrosine containing peptides are phosphorylated, irrespective of their context. These "fully modified" peptide libraries are a valuable tool to study the specificity of phosphotyrosine mediated protein recognition. We have used this new tool to identify a family of peptides that bind the PTB domain of the adapter protein Shc. Comparison of the peptide sequences permits us to confirm the essential role of N at position -3, while P often found at position -2 in natural targets is not absolutely required. Furthermore, our approach permits us to reveal an "extended" consensus indicating that residues that do not seem to influence binding in natural peptides can make productive contacts, at least in linear peptides.

Consensus Sequence↗
PubMed1998

Acidic peptide-mediated expression of the antimicrobial peptide buforin II as tandem repeats in Escherichia coli.

Antimicrobial peptides have received increasing attention as a new pharmaceutical substance, because of their broad spectrum of antimicrobial activities and the rapid development of multidrug-resistant pathogenic microorganisms. The main obstacle to the wide application of antimicrobial peptides has been the lack of a cost-effective, mass-production method. A novel mass-production method for an antimicrobial peptide of 21 amino acids, buforin II, which was isolated from the stomach of the amphibian Bufo bufo gargarizans, has been developed. This method is based on the neutralization of the positive charges of buforin II by fusing to an acidic peptide to avoid the lethal effect of the expressed antimicrobial peptide on the host cells. The fusion peptide was expressed in Escherichia coli as tandem repeats to increase the product yield. Multimers of the acidic peptide-buforin II fusion peptide were expressed at high levels without causing damage to the cells. The presence of cysteine residues in the acidic peptide was critical for the high level expression of the fusion peptide multimers. Multimers of this fusion peptide were expressed as inclusion bodies, and about 107 mg of pure buforin II was obtained from 1 L of E. coli culture by cleaving the multimers with CNBr. Recombinant buforin II had an antimicrobial activity identical to that of natural buforin II. These results may lead to a general, cost-effective solution to the mass production of antimicrobial peptides and other basic peptides which are lethal to the host strain.

Amino Acid Sequence↗