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PubMed2006

Structural requirements for cellular uptake and antisense activity of peptide nucleic acids conjugated with various peptides.

Peptide nucleic acids (PNAs) have shown great promise as potential antisense drugs; however, poor cellular delivery limits their applications. Improved delivery into mammalian cells and enhanced biological activity of PNAs have been achieved by coupling to cell-penetrating peptides (CPPs). Structural requirements for the shuttling ability of these peptides as well as structural properties of the conjugates such as the linker type and peptide position remained controversial, so far. In the present study an 18mer PNA targeted to the cryptic splice site of a mutated beta-globin intron 2, which had been inserted into a luciferase reporter gene coding sequence, was coupled to various peptides. As the peptide lead we used the cell-penetrating alpha-helical amphipathic peptide KLAL KLAL KAL KAAL KLA-NH2 [model amphipathic peptide (MAP)] which was varied with respect to charge and structure-forming properties. Furthermore, the linkage and the localization of the attached peptide (C- vs N-terminal) were modified. Positive charge as well as helicity and amphipathicity of the KLA peptide was all required for efficient dose-dependent correction of aberrant splicing. The highest antisense effect was reached within 4 h without any transfection agent. Stably linked conjugates were also efficient in correction of aberrant splicing, suggesting that a cleavable disulfide bond between CPP and PNA is clearly not essential. Moreover, the placement of the attached peptide turned out to be crucial for attaining antisense activity. Coadministration of endosome disrupting agents such as chloroquine or Ca2+ significantly increased the splicing correction efficiency of some conjugates, indicating the predominant portion to be sequestered in vesicular compartments.

Antisense Elements (Genetics)↗
PubMed2000

Analysis of peptide length preference of the rat MHC class Ia molecule RT1-A(u), by a modified random peptide library approach.

Using random peptide libraries we have previously shown that both mouse and rat class I molecules can exhibit different peptide length preferences. Such studies required expression of the particular class I molecules in RMA-S, a cell line deficient in the transporter associated with antigen presentation (TAP). For another rat class I molecule called RT1-A(u), however, we found that expression in RMA-S was poor and could not be increased sufficiently by incubation at 26 degrees C. To circumvent this problem we performed our studies on C58, a rat cell line that expresses RT1-A(u) naturally in the presence of a functional TAP transporter. Using C58 cells, cell-surface-expressed class I molecules were 'stripped' of peptides and beta(2)-microglobulin by washing the cells with an acidic citrate buffer (pH 3.3). Peptide stabilization assays, assessed by FACS analysis, were then performed using either specific peptides or synthetic random peptide libraries of different lengths (7-15 amino acids), supplemented with recombinant rat beta(2)-microglobulin. As a positive control an RT1-A(u)-specific nonamer peptide was designed using the previously determined peptide binding motif and this was found to bind to RT1-A(u) at nanomolar concentrations. Both length preference and importance of free N- and C-termini were tested using free base, formylated and acetylated peptide libraries. Results showed that RT1-A(u) was not able to accommodate N- or C-terminally blocked peptides but displayed a preference for peptides of 9-12 amino acids, similar to the preference observed for the RT1-A1(c) allotype, the other rat TAP-B-associated molecule tested thus far. These results suggest that length preference remains a consideration to explain the allelic class I-TAP associations of the RT1-A region.

Animals↗
PubMed1985

Peptide specificity for stimulation of corticotropin secretion: activation of overlapping pathways by the vasoactive intestinal peptide family and corticotropin-releasing factor.

The hypothalamic peptide vasoactive intestinal peptide (VIP) stimulates ACTH and endorphin secretion by the AtT20/D16 clonal strain of mouse pituitary tumor cells. The dose dependence for VIP stimulation of hormone release is biphasic, indicating that VIP is able to activate at least two classes of receptors in D16 cells (ED50 = 1.6 and 160 nM). We show that at high concentrations (ED50 greater than or equal to 150 nM), other natural peptides with primary structures homologous to that of VIP also increased ACTH secretion by D16 cells, whereas structurally unrelated peptides did not. The stimulatory actions of GH-releasing factor (GRF) and porcine heptacosapeptide with amino-terminal histidine and carboxy-terminal isoleucine amide (PHI) were mediated by high affinity VIP receptors because their effects were not additive with that of 10 nM VIP. In addition, GRF and PHI behaved as antagonists at low affinity VIP receptors; both peptides inhibited stimulation by 1 microM VIP. In contrast, glucagon and gastric inhibitory polypeptide appeared to stimulate ACTH release via low affinity VIP receptors because their effects were additive with that of 10 nM, but not 1 microM, VIP. Since all of the VIP-like peptides increased ACTH secretion only at high concentrations, they were unlikely to represent a physiological ligand for the receptor activated by high concentrations of VIP. Therefore, we determined whether cross-reactivity occurred between VIP-like peptides and corticotropin-releasing factor (CRF), a potent stimulator of ACTH secretion both in vitro and in vivo. The dose-response curve for CRF stimulation of ACTH secretion by D16 cells extended over more than a 1000-fold range of concentrations and was biphasic (ED50 = 2.6 and greater than 300 nM), indicating that CRF interacted with multiple receptor types in D16 cells. However, since the effect of 10 nM CRF was additive with that of 1 microM VIP, the CRF receptor was not the site at which high concentrations of VIP stimulated ACTH release. In contrast, the effect of 1 microM CRF was not additive with that of 1 microM VIP or other VIP-like peptides. Therefore, high concentrations of CRF and the previously recognized VIP-like peptides stimulated ACTH secretion by overlapping pathways. Comparison of the amino acid sequence of CRF with those of the VIP-like peptides showed that 18 of the 41 amino acids in CRF match a corresponding amino acid in at least 1 member of the VIP peptide family.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenocorticotropic Hormone↗
PubMedDate not supplied

Screening of a small set of random peptides: a new strategy to identify synthetic peptides that mimic epitopes.

Small diversity libraries, composed of 4550 synthetic dodecapeptides and 8000 synthetic tripeptides, have been used to identify sequences homologous to small linear and non-linear parts of epitopes. Here we report that synthetic peptides identified through alignment of dodecapeptides and tripeptides derived from these small libraries have, in direct ELISA and/or competitive ELISA, activities similar to that of peptides covering the native epitope and similar to that of peptides derived from large expression libraries composed of 10(6)-10(7) random peptides. This result was obtained with the monoclonal antibodies 6A.A6 and M2. Mab 6A.A6 binds the transmissible gastroenteritis virus (TGEV) and mAb M2 binds the FLAG-peptide, an affinity tag. It was also found that the antibody binding activity of peptides, derived from small or large libraries, can strongly depend on the way in which the peptide is presented to the antibody, i.e. high antibody titers were obtained when these peptides were synthesized on pins or coated onto microtiter plates, whereas low IC50s were obtained with these peptides in solution. We postulate that small peptide libraries may be a powerful tool to quickly identify new peptides that can be used as sensitive markers for mAbs of interest.

Animals↗
PubMed1992

Neuropeptides of the vasoactive intestinal peptide/helodermin/pituitary adenylate cyclase activating peptide family elevate plasma cAMP in mice: comparison with a range of other regulatory peptides.

A number of regulatory peptides were investigated for their ability to elevate plasma cAMP. Pituitary adenylate cyclase activating peptide (PACAP)-27, PACAP-38, helodermin, helospectin I and II, vasoactive intestinal peptide (VIP), glucagon, parathyroid hormone (PTH), calcitonin and calcitonin gene-related peptide were among the peptides that were highly effective in raising plasma cAMP when given intravenously in equimolar doses to conscious mice. PACAP-27 and -38 were more effective than any of the other peptides. PACAP 16-38, secretin, gastrin-17, galanin, somatostatin, cholecystokinin-8s, pancreatic polypeptide, substance P, peptide YY and neuropeptide Y were inactive and also did not interfere with the PACAP-27-evoked rise in plasma cAMP levels. Repeated injections of PACAP-27 every 30 min caused a progressive reduction in the plasma cAMP response (measured 5 min after each injection). Forskolin, an activator of adenylate cyclase, dose-dependently raised the plasma concentration of cAMP and displayed a synergistic effect when given in a low dose concurrently with PTH or PACAP-38. The phosphodiesterase inhibitor rolipram dose-dependently raised the plasma concentration of cAMP. Combined treatment with PACAP-27 and a threshold dose of rolipram resulted in an exaggerated plasma cAMP response. Kidney hilus ligation suppressed the responses to PACAP-38, PTH, helodermin, helospectin, VIP, glucagon and calcitonin. Hepatectomy suppressed the response to glucagon but was without effect on the response to the other peptides. Pancreatectomy and spleenectomy reduced the response to VIP, but was without effect on the response to the other peptides. PACAP-27 stimulated cAMP efflux from the isolated rat tail vein. Hence, it cannot be excluded that blood vessels contribute to the peptide evoked plasma cAMP response in vivo.

Animals↗
PubMed1995

Insulin secretion and plasma levels of glucose-dependent insulinotropic peptide and glucagon-like peptide 1 [7-36 amide] after oral glucose in cirrhosis.

A blunted initial insulin secretory response may contribute to oral glucose intolerance in cirrhosis. Oral glucose is a better stimulant to insulin secretion than intravenous (IV) glucose in part because of release of gut peptides, notably glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide 1 [7-36 amide] (GLP-1 [7-36 amide]). Because impaired release of or resistance to these gut peptides could explain impaired insulin secretion after oral glucose, we measured insulin secretion, plasma GIP, and GLP-1 [7-36 amide] levels, basally and after 75 g oral glucose, in 10 cirrhotics and 10 controls. Insulin secretion was calculated from a two-compartment analysis of serum C-peptide levels using kinetic parameters derived from IV injection of recombinant human C-peptide. C-peptide metabolic clearance rate, and the fractional rate constants for C-peptide (using the two-compartment model) were not significantly different, but the volume of the central compartment was 15% greater in cirrhotics (P < .01). Fasting blood glucose levels were similar (cirrhotics, 4.9 +/- 0.2; controls, 4.6 +/- 0.1 mmol/L) but serum insulin was six times higher in cirrhotics (P < .001). Cirrhotics had higher fasting GIP (215 +/- 72 vs. 42 +/- 18 pmol/L) and GLP-1 [7-36 amide] levels (25 +/- 3 vs. 16 +/- 1 pmol/L) (both P < .05). After oral glucose, blood glucose levels were significantly higher in cirrhotics. The timing of the gut peptide response to oral glucose was similar in the two groups, but peak levels of both peptides were approximately x2 higher in the cirrhotics.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1987

Identification of a uveitogenic cyanogen bromide peptide of bovine retinal S-antigen and preparation of a uveitogenic, peptide-specific T cell line.

Peptide fragments of bovine retinal S-antigen produced by cyanogen bromide (CB) digestion have been purified and tested for their ability to induce experimental autoimmune uveoretinitis (EAU) and pinealitis (EAP) in Lewis rats. Following immunization with the various peptides in complete Freund's adjuvant, one of the peptides, CB123, was found to be potently uveitogenic. A CB123-specific, class II restricted T helper lymphocyte line (R208) prepared from one of the CB123 peptide-immunized animals by repeated in vitro selection with purified CB123 was able to transfer severe EAU and EAP to naive rats. Peptides CB36, CB46, CB51 and CB66 were immunogenic as assessed by the presence of antibodies detected in the enzyme-linked immunosorbent assay and sensitized lymphocytes found in [3H]thymidine incorporation assays using lymphocytes from the peptide-immunized rats, but they did not induce significant EAU or EAP. Antibodies induced by immunization with the peptides also bound intact S-antigen, unlike the lymphocytes which were only weakly responsive to S-antigen. Cross-reactivity of antibodies and lymphocytes from CB51 and CB66 immune animals suggested that these peptides overlap and contain a common epitope. No measurable immunity to any CB peptide or intact S-antigen was found in the animals immunized with peptide CB26. Although a T cell line (R17) raised to human S-antigen was also able to transfer EAU, it was weakly responsive to the CB123 peptide; also, the R208 line did not respond well to human S-antigen. Since both human and bovine S-antigen are uveitogenic, these results suggest that the uveitogenic epitope in CB123, which is of bovine origin, is not the same as the uveitogenic epitope of the human antigen.

Animals↗
PubMed2004

Isotope-coded N-terminal sulfonation of peptides allows quantitative proteomic analysis with increased de novo peptide sequencing capability.

Recently various methods for the N-terminal sulfonation of peptides have been developed for the mass spectrometric analyses of proteomic samples to facilitate de novo sequencing of the peptides produced. This paper describes the isotope-coded N-terminal sulfonation (ICenS) of peptides; this procedure allows both de novo peptide sequencing and quantitative proteomics to be studied simultaneously. As N-terminal sulfonation reagents, 13C-labeled 4-sulfophenyl[13C6]isothiocyanate (13C-SPITC) and unlabeled 4-sulfophenyl isothiocyanate (12C-SPITC) were synthesized. The experimental and reference peptide mixtures were derivatized independently using 13C-SPITC and 12C-SPITC and then combined to generate an isotopically labeled peptide mixture in which each isotopic pair differs in mass by 6 Da. Capillary reverse-phase liquid chromatography/tandem mass spectrometry experiments on the resulting peptide mixtures revealed several immediate advantages of ICenS in addition to the de novo sequencing capability of N-terminal sulfonation, namely, differentiation between N-terminal sulfonated peptides and unmodified peptides in mass spectra, differentiation between N- and C-terminal fragments in tandem mass spectra of multiply protonated peptides by comparing fragmentations of the isotopic pairs, and relative peptide quantification between proteome samples. We demonstrate that the combination of N-terminal sulfonation and isotope coding in the mass spectrometric analysis of proteomic samples is a viable method that overcomes many problems associated with current N-terminal sulfonation methods.

Amino Acid Sequence↗
PubMed2000

Two peptides from CD23, including the inverse RGD sequence and its related peptide, interact with the MHC class II molecule.

The human CD23 molecule (low affinity receptor for IgE) has a C-type lectin domain, a reversed Arg-Gly-Asp (RGD) sequence near the C-terminus, and an "RGD-binding inhibitory peptide" at the root of the N-sugar chain. Three peptides were synthesized to determine their functions, i.e., #1, including an inverse RGD sequence near the C-terminus; #2, RGD-binding inhibitory peptides in the gpIIIa chain of platelet integrin gpIIb/IIIa; and #3, the inverse sequence located at the root of the N-sugar chain of CD23 which has homology to peptide 2. Among the three peptide, only peptide 3 inhibited aggregation of L-KT9 cells. Isotope-labeled peptides 1 and 3 bound to MHC class II molecules but peptide 1 did not bind to CD23 molecules. Peptide 3 showed a higher affinity to MHC class II than did peptide 1. Both peptides in CD23, therefore, seem to have interesting and important functions in relation to MHC class II molecules and also to CD23 molecules when CD23 on EBV-transformed B cells acts as a lectin in homotypic cell aggregation. The physiological function of CD23 was discussed from an evolutional point of view.

Amino Acid Sequence↗
PubMed1989

Co-localisation of substance P-, bombesin- and peptide histidine isoleucine (PHI)-like peptides in gut endocrine cells of the dogfish Scyliorhinus stellaris.

The gastrointestinal tract of cartilaginous fishes, like that of higher vertebrates, is known to contain endocrine cells and nerves immunoreactive for a wide variety of peptides, some of which have been structurally characterised. Since we have found that substance P-, bombesin- and peptide histidine isoleucine-like immunoreactivities are similarly distributed in the endocrine cells of the dogfish pyloric stomach, we have tried to establish whether any of these peptides are co-localised. The cells were compared in thin serial sections with both light- and electron microscopical immunocytochemistry. Double immunolabelling was also used to show two immunoreactive peptides in the same tissue section. Further characterisation of the immunoreactivity was attempted by preabsorbing the antibodies with various peptides or synthetic fragments of peptide molecules. Immunoreactivity for all three peptides was frequently present in the same cells, whereas antibodies to other peptides such as gastrin and somatostatin marked different cells. Electron microscopy indicated that all the secretory granules in three morphologically different cell types reacted with antibodies to all three peptides. Dual localisation of unrelated peptides in endocrine cells or nerves is established in many cases, but triple localisation is as yet unusual. The immunoreaction for bombesin-like peptides is different in endocrine cells and nerves, indicating that dogfish bombesin may be present in two forms, in agreement with biochemical evidence.

Animals↗
PubMed1989

Studies on adenosine triphosphate transphosphorylases. XVIII. Synthesis and preparation of peptides and peptide fragments of rabbit muscle ATP-AMP transphosphorylase (adenylate kinase) and their nucleotide-binding properties.

Two peptide fragments, derived from the head and tail of rabbit muscle myokinase, were found to possess remarkable and specific ligand-binding properties (Hamada et al., 1979). By initiating systematic syntheses and measurements of equilibrium substrate-binding properties of these two sets of peptides, or portions thereof, which encompass the binding sites for (a) the magnesium complexes of the nucleotide substrates (MgATP2- and MgADP-) and (b) the uncomplexed nucleotide substrates (ADP3- and AMP2-) of rabbit muscle myokinase, some of the requirements for binding of the substrates to ATP-AMP transphosphorylase are being deduced and chemically outlined. One requirement for tight nucleotide binding appears to be a minimum peptide length of 15-25 residues. In addition, Lys-172 and/or Lys-194 may be involved in the binding of epsilon AMP. The syntheses are described as a set of peptides corresponding to residues 31-45, 20-45, 5-45, and 1-45, and a set of peptides corresponding to residues 178-192, 178-194, and 172-194 of rabbit muscle adenylate kinase. The ligand-binding properties of the first set of synthetic peptides to the fluorescent ligands: epsilon MgATP/epsilon ATP and epsilon MgADP/epsilon ADP are quantitatively presented in terms of their intrinsic dissociation constants (K'd) and values of N (maximal number of moles bound per mole of peptide); and compared with the peptide fragment MT-I (1-44) obtained from rabbit muscle myokinase (Kuby et al., 1984) and with the native enzyme (Hamada et al., 1979). In addition, the values of N and K'd are given for the second set of synthetic peptides to the fluorescent ligands epsilon AMP and epsilon ADP as well as for the peptide fragments MT-XII(172-194) and CB-VI(126-194) (Kuby et al., 1984) and, in turn, compared with the native enzyme. A few miscellaneous dissociation constants which had been derived kinetically are also given for comparison (e.g., the Ki for epsilon AMP and the value of KMg epsilon ATP obtained for the native enzyme) (Hamada and Kuby, 1978), and the K'd measured for Cr3+ ATP [corrected] and the synthetic peptide I1-45 (Fry et al., 1985b).

Adenosine Diphosphate↗
PubMed2000

The use of a rapid ELISPOT assay to analyze peptide-specific immune responses in carcinoma patients to peptide vs. recombinant poxvirus vaccines.

An enzyme-linked immunosorbent spot (ELISPOT) assay for interferon gamma production has been used to analyze specific T cell responses to a Flu 9-mer peptide, and a 9-mer peptide of carcinoembryonic antigen (CEA). Assays were performed on peripheral blood mononuclear cells (PBMC) of HLA-A2-positive patients with CEA-expressing carcinomas, both before and after vaccination with CEA-based vaccines, and from HLA-A2-positive healthy blood donors. The ELISPOT assay utilized aliquots of frozen PBMC, and assays were performed after 24 h in culture with peptide to rule out any artifacts due to long-term in vitro stimulation cycles. An internal standard was used for each assay to define reproducibility of the assay, and all samples from a given patient (pre- and post-vaccination, with both the Flu and CEA peptides) were analyzed simultaneously. The results indicated a trend towards healthy blood donors having higher levels of Flu-specific T cell precursors than do colon carcinoma patients, but these results were not statistically significant (P = 0.06). On the other hand, slightly higher CEA-specific T cell responses were observed in cancer patients with CEA-expressing carcinomas than in healthy blood donors. PBMC from two CEA-based vaccine clinical trials were analyzed for T cell responses to the same CEA peptide and to the Flu control peptide. The first trial consisted of three monthly vaccinations of CEA peptide (designated PPP) in adjuvant. The second trial consisted of cohorts receiving three monthly vaccinations of avipox-CEA recombinant (designated AAA) or cohorts receiving a primary vaccination with recombinant vaccinia-CEA followed by two monthly vaccinations with avipox-CEA (designated VAA). Few, if any, CEA-specific T cell responses were seen in the PPP vaccinations, while the majority of patients receiving the poxvirus CEA recombinants demonstrated increases in CEA-specific T cell responses and no increases in Flu-specific responses. CEA-specific IgG responses were also demonstrated in patients following recombinant CEA poxvirus vaccinations. Statistical analyses of the T cell responses to the same CEA peptide demonstrated a P value of 0.028 for the recombinant poxvirus vaccines, as compared with the peptide vaccine. There were no differences seen (P = 0.37) in Flu-specific responses after these two types of CEA vaccination. These results thus provide the first evidence that poxvirus recombinant-based vaccines are more potent in the initiation of tumor-antigen-specific T cell responses than vaccines employing peptide in adjuvant, when assays are conducted in an identical manner, and in defining responses to the same peptide. These results also demonstrate for the first time that an ELISPOT assay, performed over a 24-h period and without in vitro sensitization, can be successfully used to monitor immune responses to a tumor-associated antigen in cancer patients.

Adjuvants, Immunologic↗
PubMed2000

A novel approach for the identification of unique tumor vasculature binding peptides using an E. coli peptide display library.

BACKGROUND: Tumor neovascularization is necessary for continued tumor growth and metastasis. During the process of endothelial cell (EC) recruitment and tumor infiltration, specific molecular markers unique for this interaction are expressed on the EC surface. Targeting these molecular markers would, in effect, allow for specific tumor targeting. Tripeptide sequence motifs have previously been reported that will bind to angiogenic tumor ECs. These sequences were identified from in vivo phage peptide display libraries. The purpose of this study was to use a more simplified bacterial peptide display library in an in vitro system to seek out peptide motifs with unique binding to tumor microvasculature. METHODS: FliTrx is a bacterial peptide display library containing the entire repertoire of possible random dodecapeptides expressed on the flagella tip of E. coli. Two EC populations were used for the screening process, Matrigel invading cells (MAGIC) and tumor-derived endothelial cells (TDEC). MAGIC are obtained from ECs that infiltrate a subcutaneous fibroblast growth factor-containing Matrigel deposit, and TDEC are ECs selectively obtained from tumor vasculature. FliTrx cells were incubated with MAGIC at 4 degrees C to remove any potential clones displaying peptides that will bind to nonspecific EC surface targets. The non-binding cells were then incubated with TDEC, allowing for clones displaying potential binding peptides to bind tumor specific targets on TDECs. The bacterial population was then expanded and this "panning" process was carried out a total of five times. Peptide insert sequences from 100 bacterial colonies were analyzed for potential repetitive peptide motifs. RESULTS: Recurring peptide sequences were detected that were 3-mers (13 sequences) and 4-mers (4 sequences). Of the 3-mers, four repeated 3 times, whereas none of the 4-mers repeated more than twice. All of the repeated sequences were basic in charge, and arginine was the most commonly seen amino acid. A tripeptide basic-basic-nonpolar amino acid arrangement was the most prevalent charge sequence in all repetitive motifs (17 repeat sequences). Two test peptides showed TDEC binding specificity, and both conformed to the basic-basic-nonpolar motif. CONCLUSIONS: We report peptide sequences derived from panning an in vitro system designed to detect tumor-EC specific markers. These putative motifs may serve as molecular determinants for a novel therapeutic modality aimed at specifically targeting tumors through tumor angiogenic vessels.

3T3 Cells↗
PubMed1991

Synthesis of peptide analogues using the multipin peptide synthesis method.

Modification of the multipin peptide synthesis method which allows the simultaneous synthesis of large numbers of different peptide analogues is described. Peptides were assembled on polyethylene pins derivatized with a 4-(beta-alanyloxymethyl)benzoate (beta-Ala-HMB) handle. For comparative purposes, peptides were also assembled on the diketopiperazine-forming handle N epsilon-(beta-alanyl)lysylprolyloxylactate. In model studies it was demonstrated that beta-Ala-HMB-linked peptides were cleaved from polyethylene pins with dilute sodium hydroxide or 4% methylamine/water to yield analogues with beta-Ala-free acid (beta-Ala-CO2H) and beta-Ala-methylamide (beta-Ala-CONHCH3), respectively. To assess the suitability of this approach for T-cell determinant analysis, analogues of a known T-cell determinant were synthesized with the various C-terminal endings. Peptides were characterized by amino acid analysis and fast atom bombardment-mass spectrometry. HPLC of the crude cleaved peptides indicated that 22 of the 24 peptides were greater than 95% pure. These crude peptide solutions were nontoxic in sensitive cell culture assays without further purification. All three cleavage procedures gave comparable activities in T-cell proliferation assays. These results demonstrate the potential of the multipin peptide synthesis method for the production of large numbers of different peptide analogues.

Amino Acid Sequence↗
PubMed1989

Multiple antigen peptide. A novel approach to increase detection sensitivity of synthetic peptides in solid-phase immunoassays.

We describe a novel approach to detect antibodies to synthetic peptide antigens in solid-phase radioimmunoassays, using a multiple antigen peptide (MAP) system. The MAPs consist of multiple copies of peptides that are synthesized as single units on a branching lysyl matrix using a solid-phase peptide synthesis method. The efficacy of the MAP approach in solid-phase immunoassays was compared with the conventional approach using a monomeric peptide of the immunodominant epitope of the circumsporozoite proteins of two species of malaria. Two monomeric peptides with 12 and 17 residues were found to bind poorly to plastic surfaces at a concentration up to 30 micrograms/ml, and showed no immunoreactivity to specific polyclonal or monoclonal antibodies, while the corresponding MAP-containing peptides showed excellent binding capacity and immunoreactivity at a concentration of 0.11 microgram/ml. The immunoreactivity of MAP-containing peptides was also superior to that of monomeric peptides conjugated to a protein carrier. The effects of various arrangements of lysyl branching of MAP on antigenicity were also studied, and the optimal number for lysyl branching of MAP was found to be octameric. Thus, the MAP, by enhancing the coating capacity and the avidity of synthetic peptides, provides increased sensitivity and reliability for the use of synthetic peptide to study antigen-antibody interactions on solid surfaces.

Antibodies↗
PubMed1995

Fine chemical modifications at N- and C-termini enhance peptide presentation to T cells by increasing the lifespan of both free and MHC-complexed peptides.

We investigated the effect of modifying the N- and/or C-termini of the snake toxin peptide 24-36 on its presentation to T cells. Acetylation at the N-terminus as well as amidation at the C-terminus enhanced the capacity of the peptide to activate T cells. Simultaneous modifications further increased the stimulating activity, the peptide becoming approximately 100-fold more potent than the unmodified peptide. Clearly, the introduced modifications increased the lifetime of the peptide free in solution, by decreasing its proteolytic degradation, during the T cell stimulation assays. Paradoxically, however, at similar concentrations of free peptides, the modified ones, especially those having an acetylated N-terminus, were much more active than the unmodified peptide, irrespective of the experimental conditions. These observations suggested that components other than protection from proteolytic degradation should be associated with the higher stimulating activities of the modified peptides. Accordingly, chasing experiments with APC revealed that acetylation at N-terminus caused a higher persistence of the peptides at APC surface. Together, our data indicate that (i) the T cell stimulating capacity of a peptide is associated with its lifespans in the free and MHC II bound states; and (ii) these lifespans can be greatly enhanced by introducing fine chemical modifications at N- and C-termini. These data may have some implications in designing more potent peptidic immunomodulators.

Amino Acid Sequence↗
PubMedDate not supplied

The role of the RGD peptides and the gamma chain peptide of fibrinogen on fibrinogen binding to activated platelets.

The interaction of fibrinogen with platelet membrane glycoprotein (GP) IIb/IIIa complex is inhibited by the RGD peptides and the peptides corresponding to a sequence unique to fibrinogen in the carboxyl-terminal domain of the gamma chain. The present study was designed to examine the effects of these synthetic peptides on fibrinogen binding to thrombin-activated platelets. The order of potency of these peptides was as follows: RGDS = RGDF > GQQHHLGGAKQAGDV (G15). The RGES peptide had no inhibitory activity. The inhibitory effects of the peptides were synergistic when fibrinogen binding was measured in the mixture of the RGD peptide and the gamma chain peptide. By contrast, the inhibitory effect was additive when the binding was measured in the presence of the RGDS peptide and the RGDF peptide. Thrombin-induced platelet aggregation was also inhibited by these synthetic peptides. The order of potency was the same as that of the inhibition binding assay. These results provide evidence that the RGD sequences present at two different sites in the alpha chain and the gamma chain sequence inhibit fibrinogen binding to the GPIIb/IIIa complex, are mutually exclusive, and are in close spatial proximity in the folded molecule. We conclude that the presence of the RGD sequence and the gamma chain sequence involved in fibrinogen binding domains increases the affinity of fibrinogen to activated platelets.

Amino Acid Sequence↗
PubMed1988

Immunostaining for peptides of the egg-laying hormone/bag cell peptide precursor protein in the head ganglia of Aplysia.

Egg-laying hormone and alpha-bag cell peptide are two neuropeptides derived from a common precursor protein in the marine mollusk Aplysia. Previous studies indicate that they are neurotransmitters that co-exist in individual bag cell neurons and most bag cell processes in the abdominal ganglion. In the present investigation we used double-label immunocytochemistry with highly specific antisera to describe their distribution elsewhere in the CNS. We found that a small cluster of cells and their fibers in the pleural ganglion that were previously described as being immunoreactive for egg-laying hormone were also immunoreactive for alpha-bag cell peptide(1-9). A previously described group of small cell bodies in the cerebral ganglion also stained for both peptides. However, the fiber arborizations located near them were immunoreactive for alpha-bag cell peptide(1-9), but not egg-laying hormone. This suggests that there is alternative processing of the precursor protein or differential transport of the peptides from the cell bodies. The specificities of the antibodies indicate that all of the neurons that stain for egg-laying hormone-like peptides in the CNS synthesize peptides derived from the egg-laying hormone/bag cell peptide precursor, rather than peptides derived from other members of the egg-laying hormone gene family. They also suggest that peptides derived from the related A or B precursor proteins are not synthesized in the CNS, or at levels too low to detect. The results are consistent with the proposal that the behavior associated with egg-laying is initiated and controlled by peptide transmitters derived from a single gene and expressed in specific neurons of the CNS.

Animals↗