Peptide research最新文献

An 18-residue-long fragment of vasoactive intestinal polypeptide [VIP(11-28)-NH2] that is known to be analgesic was synthesized by solid-phase t-Boc methodology on a 4-methylbenzhydrylamine resin. Circular dichroism spectroscopy gave evidence that the peptid acquires about 60% helical structure in 50/50 methanol/phosphate buffer, pH 6.0, and 65% (+/-5%) helicity in 80/20 methanol/phosphate buffer pH 7.0, A 2.0 mM solution of VIP (11-28) NH2 in 80% methanol, 20% phosphate buffer pH 7.0 was subjected to 2-dimensional nuclear magnetic resonance (NMR) studies The NMR results suggested formation of an extended helical structure extending from residue 11 to 27 essentially the same region found to be helical in a VIP(1-28)-NH2 and log. This finding suggests that the sequence required for analgesia assumes a helical structure at the receptor.

We have developed a functional assay to verify the delivery into the cytoplasm of a 14-amino acid hydrophilic peptide, modified by the incorporation of a palmitoyl-lysine residue into the N- or C-terminal end. This assay is based on the use of a pseudo-substrate sequence for the protein kinase C (PKC) isoenzymes of alpha and beta for the quantification of PKC-mediated tumor necrosis factor (TNF) secretion after T-cell activation by phorbol ester and anti-CD3 MAb. This cellular assay is simple, and it allows for a rapid and comparative study of several peptides. The lipidic analogues of the pseudo-substrate peptide were able to inhibit TNF secretion in intact-activated Jurkat cells, with an EC50 in the 40-60 microM range, whereas the unmodified peptide was not active. Two control lipopeptides were also inactive, demonstrating that the palmitoyl-lysine group had no effect by itself. This study confirms that the modification of a relatively long peptide by the insertion of a palmitoyl-lysine into the N- or C-terminal end is sufficient to allow entry into intact cells.

To probe the interactions between major histocompatibility class-II molecules and the amide bonds of the antigenic peptide main chain, we synthesized ethylenic and reduced analogues of HEL(52-61), an immunogenic peptide for murine major histocompatibility class-II IA k restricted T-cell clones. The synthesis of the corresponding ethylenic analogue of HEL(52-61) in position 53-54 was performed by coupling the Fmoc-protected tripeptide Asp-Tyr-psi [E, CH = CH]Gly with HEL(55-61). Biological tests showed that the ethylenic peptide was presented by major histocompatibility class-II IA kappa molecule and recognized by HEL(52-61)-specific T-cell clones. The corresponding reduced peptide of HEL(52-61) at position 53-54 neither stimulated T-cell clones nor competed with the natural peptide. These results show that, while reduced pseudopeptides might not be appropriate, ethylenic pseudopeptides may be used as probes to dissect the role of hydrogen bonding between the peptide main chain and MHC residues and also help in the design of more stable immunogenic peptides.

The derivative N alpha-9-fluorenylmethyloxycarbonyl-O-phospho-L-tyrosine [Fmoc-Tyr(PO3H2)-OH] has been used successfully for the solid-phase synthesis of a wide variety of phosphorylated peptides. However, when it is used to incorporate consecutive phosphotyrosine residues, a pyrophosphate linkage can form between the two adjacent tyrosines. Incorporation of unprotected phosphotyrosine during the synthesis of peptides with multiple phosphotyrosine residues has been studied as a function of coupling conditions and the absence or presence of intervening amino acid residues. The pyrophosphate-forming side reaction is more severe with increased coupling times and/or repetitions of coupling and occurs only when the phosphotyrosine residues are directly adjacent to one another.

Intercellular junctions present a formidable challenge for the paracellular delivery of drugs. Cadherins are calcium-dependent cell-cell adhesion molecules, which are responsible for the formation and regulation of these junctions. Anti-E-cadherin monoclonal antibody can bind to E-cadherin (uvomorulin) and inhibit cell-cell adhesion through the inhibition of cadherin-cadherin interactions. The objective of this study was to utilize this monoclonal anti-E-cadherin antibody to map the extracellular domains of E- and N-cadherin. This was accomplished by using two different enzyme-linked immunosorbent assays (ELISAs), a regular indirect ELISA and an immobilized-peptide ELISA. Two peptides from each extracellular domain were recognized by this anti-E-cadherin antibody. By mapping the extracellular domains of cadherins, peptides that have discrete roles in cell-cell adhesion can be identified. This will aid in the design of synthetic peptides that can modulate intercellular junctions to improve drug delivery.

The antigenic sequence Ac-IASRYDQL (gp63-SRYD) of the major surface glycoprotein of Leishmania, gp63, was covalently attached to the Lys-N epsilon H2 groups of a new sequential oligopeptide carrier (SOCn), namely, (Lys-Aib-Gly)n (n = 5.6), in order to obtain potent immunogens and site-specific antibodies. It was shown, using 1H-NMR spectroscopy, that the gp63-SRYD octapeptides bound to the SOCn retain their original structural profile outlined by an ionic interaction between R and D side chains and a type 1 beta-turn involving the QNH-->RCO hydrogen bonding. Also, the gp63-SRYD octapeptides linked to the carrier do not experience conformational restrictions, probably because of the favorable conformation of the SOCn. Immunizations of outbred rabbits with the peptide carriers designed resulted in high-titered antibody response to the gp63-SRYD octapeptide and the gp63 cognate protein. Thus, this chemically defined model may be used for incorporating "protective" Leishmania epitopes and ultimately for the design of a multivalent synthetic vaccine against leishmaniosis.

The original trimethylsilyl bromide (TMSBr) method of peptide resin deprotection and cleavage has been modified for convenience and applicability to larger peptides. Equal amounts of a 66-residue test peptide resin were cleaved by the standard hydrogen fluoride (HF) procedure, the original TMSBr method and the modified TMSBr method. The peptide profile from the original TMSBr cleavage procedure showed multiple products and a lower overall yield. In contrast, the modified TMSBr procedure gave high yields of crude products comparable in purity to those obtained by HF cleavage.

This papers addresses many of the optimization parameters necessary to convert from high resolution capillary electrophoresis (CE) analytical separation parameters to automated, micropreparative multiple fraction collection using software-controlled, interrupted applied voltage. Optimization of two parameters are crucial: 1) preparative sample loading and 2) the determination of peak collection windows. Factors affecting sample loading volume are discussed, such as capillary inner diameters, sample temperatures and sample injection times. Peak collection windows have been determined experimentally and offer an advantage to windows calculated using a linear mobility relationship, especially for long run times, high current levels, and multiple voltage ramping required for multiple fraction collection. Reinjection analysis of both non-glycopeptides and glycopeptides are examined, and clearly indicate peak mobility can be employed for identifying the collected peptides. Difficulties associated with quantitation of the collected peaks by CE are described and appear to be predominantly associated with sample matrix effects.

The N alpha-9-fluorenylmethyloxycarbonyl (Fmoc), N omega-9H-xanthen-9-yl (Xan), N omega-2-methoxy-9H-xanthen-9-yl (2-Moxan) or N omega-3-methoxy-9H-xanthen-9-yl (3-Moxan) derivatives of asparagine and glutamine were prepared conveniently by acid-catalyzed reactions of appropriate xanthydrols with Fmoc-Asn-OH and Fmoc-Gln-OH. The Xan and 2-Moxan protected derivatives have been used in Fmoc solid-phase syntheses of several challenging peptides: a modified Riniker's peptide to probe tryptophanalkylation side reactions, Briand's peptide to assess deblocking, at the N-terminus and Marshall's ACP (65-74) to test difficult couplings. Removal of the Asn and Gln side-chain protection occurred concomitantly with release of peptide from the support, under the conditions for acidolytic cleavage of the tris(alkoxy)benzylamide (PAL) anchoring linkage by use of trifluoroacetic acid/scavenger mixtures. For each of the model peptides, the products obtained by the new protection schemes were purer than those obtained with N omega-2,4,6-trimethoxybenzyl (Tmob) or N omega-triphenylmethyl (Trt) protection for Asn and Gln.

The binding specificity of a monoclonal antibody (MAb 12) known to recognize the surface antigen of hepatitis B virus (HBsAg) was studied using a positional scanning synthetic combinatorial library. A hexapeptide library totaling more than 30 x 10(6) sequences, made up of 120 mixtures having a single position defined with individual amino acids and the remaining five positions composed of mixtures of amino acids, was screened by competitive enzyme-linked immunosorbent assay (ELISA). This led to the identification of Ac-STTSMM-NH2 (IC50 = 170 nM), which specifically inhibited the interaction between HBsAg and MAb 12. One of the most active individual mixtures from the library was Ac-XXXPXX-NH2; however, none of the individual peptides synthesized containing proline at the fourth position showed significant activity (IC50 > 100,000 nM). To identify the individual peptide(s) responsible for the activity of Ac-XXXPXX-NH2, a bidirectional iterative synthesis and selection process was carried out. A completely different but active peptide sequence was identified (Ac-SVGPPH-NH2, IC50 = 165 nM). The two different hexapeptide sequences were prepared as linear homo- and heterodimer peptides in an attempt to improve the antigenicity. Of the four different sequences prepared, one heterodimer (Ac-STTSMMGGGSVGPPH-NH2) was found by ELISA to have a 10-fold improvement in activity over the two individual hexapeptides, and was equal to the inhibitory activity of the protein antigen. The equilibrium affinity constant of the MAb 12 toward this heterodimer sequence was 50-fold higher than the protein antigen when measured in a biosensor system. Since motifs from these two hexapeptides, namely, -STTS- and -GP-, were located in the primary sequence of the protein (residues 114-120, subtype ad), overlapping hexapeptides of this region were synthesized and assayed. The most active hexapeptide, namely, Ac-TTSTGP-NH2 (IC50 = 2.3 microM), was 10-fold less active than either hexapeptide found from the library. Extending the specific motif sequences to eleven residues resulted in an analog (Ac-STTSTGPSRTC-NH2) having an equilibrium affinity constant similar to the heterodimer. The combined use of positional scanning libraries and the iterative synthesis and selection process in this study illustrates the power of these methods for the identification of novel peptides that inhibit anti-protein antibodies. These methods can be directly applied to the development of improved immunodiagnostics.