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PubMed1997

MHC affinity, peptide liberation, T cell repertoire, and immunodominance all contribute to the paucity of MHC class I-restricted peptides recognized by antiviral CTL.

MHC class I-restricted T cell responses to viral proteins focus on a limited set of peptides. To better understand this phenomenon, we examined all of the 26 nonameric peptides encoded by the influenza virus A/Puerto Rico/8/34 (PR8) conforming to the canonical Kd binding motif. Ten peptides bound strongly to Kd as assessed by a cell surface stabilization assay. Five of these 10 induced in vitro secondary CD8+ T cell responses from splenocytes derived from PR8-immunized mice. The strongest responses were induced by the two previously defined antigenic peptides, which ranked only second and fifth in relative binding affinity. To examine the limiting factors in the immunogenicity of Kd-binding peptides, we produced recombinant vaccinia viruses (rVVs) expressing cytosolic or endoplasmic reticulum (ER)-targeted peptides. rVVs expressing ER-targeted versions of the 7 peptides with the highest relative affinities for Kd rescued Kd cell surface expression in T2 cells, while those expressing the 3 lowest affinity peptides did not. The immunogenicity of several, but not all, of the highest affinity peptides was greatly enhanced when expressed as VV-encoded cytosolic or ER-targeted peptides as compared with full length proteins. We conclude that limitations in the immunogenicity of class I binding peptides reflects, in order of decreasing importance, peptide liberation by cellular proteases, T cell repertoire, and TAP-mediated peptide transport. We also observed an additional important contributing factor: suppression of T cell responses to nondominant peptides by an immunodominant peptide located in the same protein.

Animals↗
PubMed1997

Amylin, calcitonin gene-related peptide, calcitonin, and adrenomedullin: a peptide superfamily.

The calcitonin gene peptide superfamily consists of calcitonin (CT), calcitonin gene-related peptide (CGRP), and amylin. CT and CGRP derive from the CT/CGRP gene, which is encoded on chromosome 11. Alternative splicing of the primary RNA transcript leads to the translation of CGRP and CT peptides in a tissue-specific manner. CGRP (a 37-amino-acid neuropeptide) and its receptors are widely distributed in the body, and it is the most potent endogenous vasodilatory peptide discovered so far. CT (a 32-amino-acid peptide) is, however, a hormone primarily involved in protecting the skeleton during periods of "calcium stress" such as growth, pregnancy, and lactation. CT derives from the C cells of the thyroid gland and is the most potent peptide inhibitor of osteoclast-mediated bone resorption. Therefore, treatment with CT is highly effective for conditions associated with increased bone turnover such as Paget's disease, osteoporosis, Sudeck's atrophy, and hypercalcemia. Amylin (a 37-amino-acid peptide) is generated from a gene located on chromosome 12 (thought to be an evolutionary duplication of chromosome 11) and shares 46% amino acid sequence homology with CGRP and 20% with human CT. Amylin is predominantly located in the beta cells of the islets of the pancreas and may be involved in the pathogenesis of type II diabetes by deposition as amyloid within the pancreas, leading to beta cell destruction. Adrenomedullin, a recently discovered 52-amino-acid vasoactive peptide from adrenal tissue, shares 24% homology with CGRP and is also a member of this superfamily of peptides. A portion of the B-chain of insulin is strongly homologous to these four peptides. Not only does adrenomedullin (13-52) show 24% amino acid homology with CGRP, it also has a biological activity profile similar to that of CGRP.CGRP, CT, and amylin are related to the insulin gene superfamily of peptides, which may all have diverged from a common ancestral gene during evolution. When the crystallographic- and nuclear magnetic resonance-based molecular modeling of the three-dimensional structure of CGRP, CT, amylin, and adrenomedullin peptides and their receptors is available, it will lead to a greater understanding of the involvement of this family of peptides in pathophysiology. Together, CGRP, CT, amylin and adrenomedullin have overlapping biological effects owing to their structures and cross-reactivity between receptors. I propose that CT, CGRP, adrenomedullin, and amylin belong to a family of G-protein-coupled receptors (an "insulin superfamily" of peptides) and therefore share some of the characteristics of insulin, such as growth factor-like effects, and possible interaction at insulin receptor sites as an antagonist.

Adrenomedullin↗
PubMed2006

Biological assay using T cell response for Cry-consensus peptide designed for the peptide-based immunotherapy of Japanese cedar pollinosis.

INTRODUCTION: Cry-consensus peptide is a linearly linked peptide of T-cell epitopes for the management of Japanese cedar (JC) pollinosis and is expected to become a new drug for immunotherapy. However, the mechanism of T-cell epitopes in allergic diseases is not well understood, and thus, a simple in vitro procedure for evaluation of its biological activity is desired. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 27 JC pollinosis patients and 10 healthy subjects, and cultured in vitro for 4 days in the presence of Cry-consensus peptide and (3)H-thymidine. The relationship between growth stimulation (stimulation index; SI) and antigen-specific IgE levels in serum was also investigated in JC pollinosis patients. Moreover, to confirm the importance of the primary sequence in Cry-consensus peptide, heat-treated Cry-consensus peptide and a mixture of the amino acids of which Cry-consensus peptide is composed, and their (3)H-thymidine uptake was compared with Cry-consensus peptide. Finally, whether Cry-consensus peptide stimulates PBMCs from healthy subjects was investigated. RESULTS: The mean SI of JC patients showed a good correlation with Cry-consensus peptide concentration in the culture medium; however, the SI was independent of the anti-Cry j 1 IgE level. Heat-denatured Cry-consensus peptide retained a PBMC proliferation stimulatory effect comparable to the original Cry-consensus peptide, while the mixture of amino acids constituting Cry-consensus peptide did not stimulate PBMC proliferation. PBMCs from healthy subjects did not respond to Cry-consensus peptide at all. DISCUSSION: These data indicate that the PBMC response of patients suffering from JC pollinosis to Cry-consensus peptide is specific for the sequence of T cell epitopes thereof and may be useful for the evaluation of the efficacy of Cry-consensus peptide in vivo.

Adult↗
PubMed1989

Effect of insulin on ATP-citrate lyase phosphorylation: regulation of peptide A and peptide B phosphorylations.

Insulin decreases multifunctional protein kinase (MFPK) activity in rat adipose tissue [Ramakrishna, S., & Benjamin, W. B. (1988) J. Biol. Chem. 263, 12677-12681]. Insulin also decreases the phosphorylation of peptide B but increases the phosphorylation of peptide A of ATP-citrate lyase (ATP-CL). The mechanism for this increase in peptide A phosphorylation was studied with purified ATP-CL from control and insulin- and isoproterenol-treated fat pads by using MFPK and the catalytic subunit of cAMP-dependent protein kinase (A-kinase). ATP-CL purified from insulin-treated fat pads is a better substrate for phosphorylation by MFPK compared to controls. This result is consistent with the hypothesis that insulin action decreases peptide B phosphorylation. To determine if the degree of phosphorylation at peptide B affects the phosphorylation rate of peptide A by A-kinase, ATP-CL was prepared with determined phosphate contents of peptides A and B. ATP-CL with a low phosphate content at peptide B is a better substrate for phosphorylation at peptide A by A-kinase than is ATP-CL with a high phosphate content at peptide B. These results suggest that the insulin-induced increase in ATP-CL phosphorylation at peptide A is due to a decrease in peptide B phosphorylation. ATP-CL prepared from isoproterenol-treated fat pads is also a better substrate for phosphorylation at peptide B by MFPK than controls. This increase in phosphorylation at peptide B by MFPK is due to positive second-site regulation by the isoproterenol-induced increase in peptide A phosphorylation.

ATP Citrate (pro-S)-Lyase↗
PubMed2007

Observation of an unusually facile fragmentation pathway of gas-phase peptide ions: a study on the gas-phase fragmentation mechanism and energetics of tryptic peptides modified with 4-sulfophenyl isothiocyanate (SPITC) and 4-chlorosulfophenyl isocyanate (SPC) and their 18-crown-6 complexes.

Various peptide modifications have been explored recently to facilitate the acquisition of sequence information. N-terminal sulfonation is an interesting modification because it allows unambiguous de novo sequencing of peptides, especially in conjunction with MALDI-PSD-TOF analysis; such modified peptide ions undergo fragmentation at energies lower than those required conventionally for unmodified peptide ions. In this study, we systematically investigated the fragmentation mechanisms of N-terminal sulfonated peptide ions prepared using two different N-terminal sulfonation reagents: 4-sulfophenyl isothiocyanate (SPITC) and 4-chlorosulfophenyl isocyanate (SPC). Collision-induced dissociation (CID) of the SPC-modified peptide ions produced a set of y-series ions that were more evenly distributed relative to those observed for the SPITC-modified peptides; y(n-1) ion peaks were consistently and significantly larger than the signals of the other y-ions. We experimentally investigated the differences between the dissociation energies of the SPITC- and SPC-modified peptide ions by comparing the MS/MS spectra of the complexes formed between the crown ether 18-crown-6 (CE) and the modified peptides. Upon CID, the complexes formed between 18-crown-6 ether and the protonated amino groups of C-terminal lysine residues underwent either peptide backbone fragmentation or complex dissociation. Although the crown ether complexes of the unmodified ([M + CE + 2H]2+) and SPC-modified ([M* + CE + 2H]2+) peptides underwent predominantly noncovalent complex dissociation upon CID, the low-energy dissociations of the crown ether complexes of the SPITC-modified peptides ([M' + CE + 2H]2+) unexpectedly resulted in peptide backbone fragmentations, along with a degree of complex dissociation. We performed quantum mechanical calculations to address the energetics of fragmentations observed for the modified peptides.

Benzenesulfonates↗
PubMed2001

A study of peptide--peptide interaction by matrix-assisted laser desorption/ionization.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to study peptide-peptide interaction. The interaction was seen when 6-aza-2-thiothymine was used as a matrix (pH 5.4), but was disrupted with a more acidic matrix, alpha-cyano-4-hydroxycinnamic acid (pH 2.0). In the present study, we show that dynorphin, an opioid peptide, and five of its fragments that contain two adjacent basic residues (Arg6-Arg7), all interact noncovalently with peptides that contain two to five adjacent acidic residues (Asp or Glu). Two other nonrelated peptides containing two (Arg6-Arg7) or three (Arg1-Lys2-Arg3) adjacent basic amino acid residues were studied and exhibited the same behavior. However, peptides containing adjacent Lys or His did not form noncovalent complexes with acidic peptides. The noncovalent bonding was sufficiently stable that digestion with trypsin only cleaved Arg and Lys residues that were not involved in hydrogen bonding with the acidic residues. In an equimolar mixture of dynorphin, dynorphin fragments (containing the motif RR), and an acidic peptide (minigastrin), the acidic peptide preferentially complexed with dynorphin. If the concentration of minigastrin was increased 10 fold, noncovalent interaction was seen with dynorphin and all its fragments containing the motif RR. In the absence of dynorphin, minigastrin formed noncovalent complexes with all dynorphin fragments. These findings suggest that conformation, equilibrium, and concentration do play a role in the occurrence of peptide-peptide interaction. Observations from this study include: (1) ionic bonds were not disrupted by enzymatic digests, (2) conformation and concentration influenced complex formation, and (3) the complex did not form with fragments of dynorphin or unrelated peptides that did not contain the motifs RR or RKR, nor with a fragment of dynorphin where Arg7 was mutated to a phenylalanine residue. These findings strongly suggest that peptide-peptide interaction does occur, and can be studied by MALDI if near physiologic pH is maintained.

Amino Acid Sequence↗
PubMed1996

The serpin-enzyme complex receptor recognizes soluble, nontoxic amyloid-beta peptide but not aggregated, cytotoxic amyloid-beta peptide.

There is now extensive evidence that amyloid-beta peptide is toxic to neurons and that its cytotoxic effects can be attributed to a domain corresponding to amyloid-beta 25-35, GSNKGAIIGLM. We have shown recently that the serine proteinase inhibitor (serpin)-enzyme complex receptor (SEC-R), a receptor initially identified for binding of alpha1-antitrypsin (alpha1-AT) and other serine protease inhibitors, also recognizes the amyloid-beta 25-35 domain. In fact, by recognizing the amyloid-beta 25-35 domain, SEC-R mediates cell surface binding, internalization, and degradation of soluble amyloid-beta peptide. In this study, we examined the possibility that SEC-R mediates the neurotoxic effect of amyloid-beta peptide. A series of peptides based on the sequences of amyloid-beta peptide and alpha1-AT was prepared soluble in dimethyl sulfoxide or insoluble in water and examined in assays for SEC-R binding, for cytotoxicity in neuronal PC12 cells and murine cortical neurons in primary culture, and for aggregation in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The results show that amyloid-beta peptide 25-35 and amyloid-beta peptide 1-40 prepared soluble in dimethyl sulfoxide compete for binding to SEC-R, are nontoxic, and migrate as monomers in SDS-PAGE analysis. In contrast, the same peptides aged in water did not compete for binding to SEC-R but were toxic and migrated as aggregates in SDS-PAGE. An all-D-amyloid-beta 25-35 peptide was not recognized at all by SEC-R but retained full toxic/aggregating properties. Using a series of deleted, substituted, and chimeric ambeta/alpha1-AT peptides, toxicity correlated well with aggregation but poorly with SEC-R recognition. In a subclone of PC12 cells which developed resistance to the toxic effect of aggregated amyloid-beta 25-35 there was a 2.5-3-fold increase in the number of SEC-R molecules/cell compared with the parent PC12 cell line. These data show that SEC-R does not mediate the cytotoxic effect of aggregated amyloid-beta peptide. Rather, SEC-R could play a protective role by mediating clearance and catabolism of soluble, monomeric amyloid-beta peptide, if soluble amyloid-beta peptide proves to be an in vivo precursor of the insoluble, toxic peptide.

Amino Acid Sequence↗
PubMed1998

Determinants of recombinant production of antimicrobial cationic peptides and creation of peptide variants in bacteria.

Cationic peptides possessing antibacterial activity are virtually ubiquitous in nature, and offer exciting prospects as new therapeutic agents. We had previously demonstrated that such peptides could be produced by fusion protein technology in bacteria and several carrier proteins had been tested as fusion partners including glutathione-S-transferase, S. aureus protein A, IgG binding protein and P. aeruginosa outer membrane protein OprF. However these fusion partners, while successfully employed in peptide expression, were not optimized for high level production of cationic peptides (Piers, K., Brow, M. L., and Hancock, R. E. W. 1993, Gene 137, 7-13). In this paper we took advantage of a small replication protein RepA from E. coli and used its truncated version to construct fusion partners. The minimal elements required for high level expression of cationic peptide were defined as a DNA sequence encoding a fusion protein comprising, from the N-terminus, a 68 amino acid carrier region, an anionic prepro domain, a single methionine and the peptide of interest. The 68 amino acid carrier region was a block of three polypeptides consisting of a truncated RepA, a synthetic cellulose binding domain and a hexa histidine domain. The improved system showed high level expression and simplified downstream purification. The active peptide could be yielded by CNBr cleavage of the fusion protein. This novel vector was used to express three classes of cationic peptides including the alpha-helical peptide CEMA, the looped peptide bactenecin and the extended peptide indolicidin. In addition, mutagenesis of the peptide gene to produce peptide variants of CEMA and indolicidin using the improved vector system was shown to be successful.

Amino Acid Sequence↗
PubMed1992

Application and limitations of the multiple antigen peptide (MAP) system in the production and evaluation of anti-peptide and anti-protein antibodies.

The multiple antigen peptide (MAP) system has been proposed as a novel and valuable approach for eliciting antibodies to peptides and developing synthetic vaccines. The MAP system consists of a small immunogenically inert core matrix of lysine residues with alpha- and epsilon-amino groups for anchoring multiple copies of the same or different synthetic peptides. Several MAP systems, each containing eight copies of 6-15 residue-long peptides derived from the terminal and central regions of various proteins were analyzed in this study. The immunogenicity of MAPs was compared to that of the same peptides linked to carrier protein by means of conventional conjugation procedures. The various peptide antisera were tested in ELISA with homologous peptides conjugated to a carrier protein via their C terminal (as in the MAP system) or their N terminal end, or with their parent proteins. The antigenic properties of MAPs were studied with anti-peptide sera obtained by classical methods and with anti-protein sera. The results showed that the MAP system was an efficient antigen in ELISA except when the peptide corresponded to a C terminal epitope. However, the value of MAPs for raising anti-peptide antibodies cross-reactive with the cognate protein appeared much more limited. In the case of one N terminal peptide, the MAP construction was not immunogenic while the conventionally conjugated peptide induced antibodies that reacted strongly with the corresponding protein. In the case of the two C terminal peptides tested, the antibodies raised against MAP constructs reacted well with homologous MAPs but did not cross-react with the whole protein. Only in the case of a peptide from an internal domain of histone H2A did immunization with a MAP generate antibodies that cross-reacted with the protein.

Amino Acid Sequence↗
PubMed1998

Selection of phage displayed peptides from a random 10-mer library recognising a peptide target.

BACKGROUND: Peptide display libraries are powerful tools in the search for detailed information about protein-protein interactions. Usual targets for isolation of phage displayed peptide ligands include antibodies, various receptors, other full size proteins or larger fragments thereof. Smaller protein fragments such as synthetic peptides have not been reported as targets for screening of peptide display libraries. OBJECTIVES: To investigate whether a protein target used for screening of a peptide display library could be scaled down to peptide size. As the peptide target we wanted to use a sequence derived from the cytosolic tail of MHC class II associated invariant chain containing a leucine class endosomal sorting signal, known to be recognised as an autonomous functional unit during targeting of class II complexes to antigen processing compartments. STUDY DESIGN: A screening procedure where a synthetic 15-mer invariant chain peptide was coupled to a methacrylate matrix of high binding capacity was developed, and three rounds of selection were performed from a random 10-mer fUSE5 display library. RESULTS: The peptide display library was successfully enriched for phage clones with affinity for the invariant chain peptide. Furthermore, the binding phage clones were able to distinguish between a functional and a mutated form of the target. These clones therefore displayed possible peptide mimetics of signal recognition sites in the cellular sorting machinery. CONCLUSION: The size of a protein target may be scaled down to peptide size and be recognised by a 10-mer peptide displayed on filamentous phage. This approach may particularly be useful when the peptide target contains a functional unit for recognition.

Amino Acid Sequence↗
PubMed2005

Fluorescence-based peptide labeling and fractionation strategies for analysis of cysteine-containing peptides.

This study demonstrates that 1,5-I-AEDANS (5-({2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid) can be used as a versatile fluorescence-based peptide quantification tool and provides readily interpretable tandem mass spectra for de novo peptide sequencing. Two AEDANS-cysteinyl-peptide fractionation strategies were evaluated. One AEDANS-cysteinyl-peptide fractionation strategy employs immobilized metal affinity chromatography (IMAC) to recover AEDANS-labeled peptides and reduce the complexity of peptide mixtures. In an alternate solid-phase approach, 1,5-I-AEDANS was coupled to an o-nitrobenzyl-based photocleavable resin to produce a resin that can label and isolate thiols and cysteine-containing peptides with a modified-AEDANS label (mAEDANS: 5-((4-amino-4-oxobutanoyl){2-[(iodoacetyl)amino]ethyl}amino)naphthalene-1-sulfonic acid). This fractionation protocol enriches cysteine-containing peptides more specifically than the IMAC strategy. Using micro-LC-ESI-MS with an on-line fluorescence detector and a Q-TOF mass spectrometer, we generated fluorescence-based elution profiles and corresponding positive ion mass spectra of AEDANS-labeled peptides. This study demonstrates that AEDANS-peptides produce positive ion ESI-MS mass spectra with detection limits comparable to those of the unlabeled peptide. Collision-induced dissociation (CID) of fluorescent AEDANS-peptides revealed readily interpretable product ion spectra with the label intact. Similar to the AEDANS-labeled peptide, an mAEDANS-labeled thiol is fluorescent and CID of a mAEDANS-labeled peptide also reveals an interpretable product ion spectrum with the label intact.

Affinity Labels↗
PubMedDate not supplied

Liposomal formulations of synthetic MUC1 peptides: effects of encapsulation versus surface display of peptides on immune responses.

Synthetic human MUC1 peptides are important candidates for therapeutic cancer vaccines. To explore whether a human MUC1 peptide BP25 (STAPPAHGVTSAPDTRPAPGSTAPP) can be rendered immunogenic by incorporation in liposomes, the effects of physical association of the peptide with liposomes on immune responses were investigated. Lipid conjugated and nonconjugated MUC1 peptides were incorporated in liposomes with a composition of distearoylphosphatidylcholine/cholesterol/dimyristoylphosphatidylglyc erol (3:1:0.25, molar ratio) containing monophosphoryl lipid A (1% w/w of the total lipids). Liposomes were characterized for peptide retention by HPLC and for surface peptide display of MUC1 epitopes by flow cytometry. C57BL/6 mice were immunized with lipopeptide alone, peptide mixed with peptide-free liposomes, and peptide associated with liposomes in entrapped or surface-exposed forms. T cell proliferative responses, cytokine patterns, and antibody isotypes were studied. Results showed that immune responses were profoundly influenced by the liposome formulations. Physically associated, either encapsulated or surface-exposed, peptide liposomes elicited strong antigen-specific T cell responses, but not lipopeptide alone or peptide mixed with peptide-free liposomes. Analysis of the cytokines secreted by the proliferating T cells showed a high level of IFN-gamma and undetectable levels of IL-4, indicating a T helper type 1 response. Thus, physical association of the peptide with liposomes was required for T cell proliferative responses, but the mode of association was not critical. On the other hand, the nature of the association significantly affected humoral immune responses. Only the surface-exposed peptide liposomes induced MUC1-specific antibodies. A domination of anti-MUC1 IgG2b over IgG1 (94 versus 6%) was observed. Our results support the hypothesis that different immune pathways are stimulated by different liposome formulations. This study demonstrated that a liposome delivery system could be tailored to induce either a preferential cellular or humoral immune response.

Amino Acid Sequence↗
PubMed2000

Limited plasticity in the recognition of peptide epitope variants by an alloreactive CTL clone correlates directly with conservation of critical residues and inversely with peptide length.

Although self-restricted T cells are peptide-specific and can distinguish among closely related ligands, they have some flexibility in the recognition of sequence variants of their natural peptide epitopes. Alloreactive cytotoxic T lymphocytes (CTL) can recognize specific peptides bound to the allo-major histocompatibility complex (MHC) molecule, but their plasticity in the recognition of related peptide variants has not been properly defined. The anti-B*2705 alloreactive CTL 27S69 specifically recognizes a natural octamer ligand of HLA-B*2705. In this study, we tested the recognition of a nested set of epitope variants by this CTL clone. Although none of these peptides was recognized equally as the natural epitope, two of the peptide variants were recognized with only slightly decreased efficiency. Peptide sensitization assays showed that CTL recognition of epitope variants correlated directly with conservation of two non-anchor residues that were critical for recognition of the natural epitope, and inversely with peptide length. Molecular modeling of the peptide variants complexed with B*2705 provided a rational explanation for their differential recognition. Location of the two critical peptide residues at the right three-dimensional space favored efficient recognition by CTL 27S69. The negative effect of increasing peptide length on recognition was due to the bigger bulging surface between the two critical residues, which precluded for optimal interaction with the specific T-cell receptors (TCR). Our results demonstrate that an alloreactive CTL has a degree of plasticity in the recognition of peptide epitope variants that is comparable to that of peptide-specific self-restricted CTL, and define the structural features determining crossreaction among related peptides.

Cloning, Molecular↗
PubMed2002

High-throughput peptide synthesis and peptide purification strategy at the low micromol-scale using the 96-well format.

The increasing demand for short- and medium-sized peptides in many fields of biological, medical and pharmaceutical research requires optimized and universally applicable high-throughput synthesis and purification techniques at the low-micromol scale. Here, we describe a continuous peptide synthesis/purification approach using the 96-well format. First, a micromol scale peptide synthesis on resin beads was optimized on a novel miniaturized 96-reaction vessel block employing standard Fmoc/tBu-chemistry. Almost 90% of the synthesized peptides contained the target sequence as the main component, as judged from matrix-assisted laser desorption/ionization (MALDI) mass spectra. Impurities were mostly related to partially protected peptides. Second, we tested the applicability of ion pair reversed-phase solid-phase extraction (IP-RP-SPE) to purify individual peptides. Depending on the length and predicted hydrophobicity of the peptides, elution was performed with 25 or 35% aqueous acetonitrile in the presence of 0.1% trifluoroacetic acid (TFA). Thus, scavengers used during TFA cleavage and partially protected peptides carrying very hydrophobic protecting groups were effectively removed. Using a narrow step gradient, the target peptides were even separated from deleted sequences and protected peptides with similar hydrophobicities. Third, we combined the micromol-scale synthesis in the 96-well format with purification by IP-RP-SPE on a 96-well micro-extraction plate format. This simple, fast and parallel approach was tested on 12-mer and 15-mer peptides to map epitopes of T- and B-cell clones, respectively. Approximately 80% of all peptides were obtained at purities > 90% without purification by RP-HPLC. In summary, this novel approach has several advantages: (i) the micromol-scale reduced the cost of peptide synthesis, (ii) large numbers of peptides were purified faster, (iii) the volumes of eluents and waste were significantly reduced, and (iv) the RP-HPLC column was not contaminated with hydrophobic impurities.

Acetonitriles↗
PubMed2003

Biophysical studies and anti-growth activities of a peptide, a certain analog and a fragment peptide derived from alpha-fetoprotein.

A chemically synthesized 34-amino acid peptide, an analog, and a fragment of the peptide have been purified and studied. Biophysical studies were carried out to determine some of the metal ion binding properties of the original peptide and an analog of this parent peptide, in which the two histidine residues were replaced by alanines. As shown by visible absorption spectroscopy, Co (II) forms a complex with the parent peptide, but not with the analog peptide, and one or two histidines in the parent peptide are ligands for Co (II) ion binding. The effects on disulfide bond formation in the peptide by Zn (II) and Co (II) ions were also examined for this analog. Anti-growth assays were performed using the original cysteine-containing peptide with Zn (II) ion complexed to the peptide through the two cysteine residues. These rat uterine growth assays showed that the complexing of Zn (II) ion to the peptide maintained the anti-growth activity of the peptide, while gel-filtration experiments showed the zinc ions maintained the peptide in its anti-growth form indefinitely in solution. A saliently important part of this research was the discovery that a fragment of the peptide consisting of a middle sequence of 14 amino acids was found to have significant anti-growth activity in the rat uterine assay. Its activity suggested that this fragment might be considered a viable candidate for testing in anti-cancer protocols.

Amino Acid Sequence↗
PubMed1995

Stimulation of inositol 1,4,5-trisphosphate production by peptides corresponding to the effector domain of different Rab3 isoforms and cross-linking of an effector domain peptide target.

Rab3 proteins are localized on secretory vesicles and appear to be involved in regulated exocytosis. We have previously shown that a modified peptide corresponding to the effector domain of the small molecular mass GTP-binding protein Rab3A, Rab3AAL, stimulates inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production and amylase release in digitonin-permeabilized pancreatic acini. Experiments using monoclonal antibodies reveal that the Rab3-like protein present in pancreatic acini is not the Rab3A isoform. However, since the putative effector domains of the four as yet known Rab3 proteins (A, B, C and D) differ only in the C-terminal four amino acid residues, Rab3A effector domain peptide could mimic the action of the pancreas-specific Rab3 isoform. In the present study we report that peptides corresponding to the different Rab3 isoforms stimulate both Ins(1,4,5)P3 production and amylase secretion with an order of potency Rab3B/D > Rab3AAL > Rab3A = Rab3C. For Rab3A, B/D and C effector domain peptides the concentrations causing half-maximal response (EC50) were 3, 0.2 and 3 nM for Ins(1,4,5)P3 accumulation and 0.3, 0.02 and 0.3 nM for amylase release, respectively. A Rab1A effector domain peptide, Rab1AAL, and a scrambled peptide of Rab3AAL were less potent by several orders of magnitude in eliciting these responses compared with native Rab3 effector domain peptides. None of the peptides influenced Ins(1,4,5)P3 production and amylase release in intact acini. Cross-linking of 125I-Rab3B/D peptide to pancreatic acinar membranes showed a band at 70 to 75 kDa with maximum intensity at 75 kDa. Radiolabelling of the substrates could be displaced by unlabelled Rab3B/D peptide, and to a lesser extend by Rab3A peptide, whereas the scrambled peptide of Rab3AAL had no effect. These data suggest that phospholipase C and exocytosis might be regulated by Rab3B-or Rab3D-like proteins in pancreatic acinar cells. A 75 kDa protein that preferentially cross-linked to 125I-Rab3B/D effector domain peptide is a potential candidate as an effector protein of Rab3 effector domain peptides.

Amino Acid Sequence↗
PubMed2001

Binding of antigenic peptide to the endoplasmic reticulum-resident protein gp96/GRP94 heat shock chaperone occurs in higher order complexes. Essential role of some aromatic amino acid residues in the peptide-binding site.

Vaccination with heat shock protein gp96-antigenic peptide complexes produces a powerful specific immune response against cancers and infectious diseases in some experimental animal models, and gp96-peptide complexes are now being tested in human clinical trials. gp96 appears to serve as a natural adjuvant for chaperoning antigenic peptides into the immune surveillance pathways. A fundamental issue that needs to be addressed is the mechanism of binding of antigenic peptide to gp96. Here, we show using scanning transmission electron microscopy that recombinant gp96 binds peptide in stable multimeric complexes, which may have biological significance. To open the possibility for genetically engineering gp96 for improved immunogenicity and to understand if molecular recognition plays a role in the binding of antigenic peptide, we mutagenized some specific aromatic amino acids in the presumed peptide-binding pocket. Replacement of Tyr-667 or Tyr-678 to Ala reduced affinity for peptide whereas conversion of Trp-654 to Tyr increased peptide binding. Similarly, changing Trp-621 to Phe or Leu or Ala or Ile negatively affected peptide binding whereas changing Trp-621 to Tyr or Val positively affected peptide binding. Probing the peptide microenvironment in gp96-peptide complexes, suggested that hydrophobic interactions (and perhaps hydrogen bonding/stacking interactions) may play a role in peptide loading by gp96.

Antigens, Neoplasm↗
PubMed2001

Ca2+-dependent dual functions of peptide C. The peptide corresponding to the Glu724-Pro760 region (the so-called determinant of excitation-contraction coupling) of the dihydropyridine receptor alpha 1 subunit II-III loop.

Both in vivo and in vitro studies suggest that the Glu(724)-Pro(760) (peptide C) region of the dihydropyridine receptor alpha1 II-III loop is important for excitation-contraction coupling, although its actual function has not yet been elucidated. According to our recent studies, peptide C inhibits Ca(2+) release induced by T-tubule depolarization or peptide A. Here we report that peptide C has Ca(2+)-dependent dual functions on the skeletal muscle ryanodine receptor. Thus, at above-threshold [Ca(2+)]s (> or =0.1 microm) peptide C blocked peptide A-induced activation of the ryanodine receptor (ryanodine binding and Ca(2+) release); peptide C also blocked T-tubule depolarization-induced Ca(2+) release. However, at sub-threshold [Ca(2+)]s (<0.1 microm), peptide C enhanced ryanodine binding and induced Ca(2+) release. If peptide A was present, together with peptide C, both peptides produced additive activation effects. Neither peptide A nor peptide C produced any appreciable effect on the cardiac muscle ryanodine receptor at both high (1.0 microm) and low (0.01 microm) Ca(2+) concentrations. These results suggest the possibility that the in vivo counterpart of peptide C retains both activating and blocking functions of the skeletal muscle-type excitation-contraction coupling.

Animals↗