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PubMed1995

Chemical shift differences between free and Fab-bound peptide correlate with a two-stage selection of peptide sequences from a random phage display library to delineate critical and non-critical residues for antibody recognition.

Epitope libraries provide a method to identify peptide ligands for antibodies, receptors or other binding proteins. As such, they provide a powerful tool to rapidly identify lead ligands in the drug discovery process. In an attempt to correlate structural information with the results from peptide screening, we have used NMR spectroscopy of peptide/antibody complexes to demonstrate that core residues identified through a two-stage selection process undergo a larger structural change upon binding antibody than do positions in the peptide amenable to a variety of side chains. The model system used was the M2 monoclonal antibody/Flag octapeptide epitope system. We have analyzed two peptides: Ac-Asp-Tyr-Lys-Leu-Gly-Asp-Asp-Leu-NH2 (peptide 1), which contains several non-core positions randomized, and Ac-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Leu-NH2 (peptide 2), which closely corresponds to the original Flag sequence. Enrichment of the peptides with 15N facilitated the investigation by permitting spectral editing of the peptide resonances in the presence of antibody. For peptide 1 the absolute shifts for the free vs. Fab-bound peptide were found to be largest for the amide groups of Asp-1 and Asp-6, in agreement with classification of these residues as critical by the phage display library selection process. For peptide 2 the largest absolute shifts were observed for Asp-1 and Asp-4, with the other aspartic acid residues also showing significant but smaller changes.

Amino Acid Sequence↗
PubMed1982

Signal peptide digestion in Escherichia coli. Effect of protease inhibitors on hydrolysis of the cleaved signal peptide of the major outer-membrane lipoprotein.

Upon incubation of the envelope fraction of Escherichia coli a precursor of the major outer membrane lipoprotein that accumulates in the cytoplasmic membrane of the globomycin-treated cell is processed to the mature form [Hussain, M., Ichihara, S., and Mizushima, S. (1980) J. Biol. Chem. 255, 3707-3712; (1982) J. Biol. Chem. 257, 5177-5182]. When this precursor-containing envelope fraction was incubated in the presence of protease inhibitors such as antipain, leupeptin, chymostatin and elastatinal, a new peptide appeared on a polyacrylamide gel at the position where the signal peptide was expected to appear. This was proved to be the signal peptide of the lipoprotein from the following facts: (a) its appearance is in proportion to the appearance of the lipoprotein and disappearance of the precursor; (b) when the cleavage of the signal peptide from the precursor was inhibited by globomycin, the peptide did not appear on the gel; and (c) the results of labeling of the peptide with [3H]leucine, [35S]methionine and [3H]arginine were consistent with the amino acid composition of the signal peptide. The signal peptide thus accumulated in the envelope fraction was hydrolyzed by an enzyme named 'signal peptide peptidase' when the envelope fraction was washed to remove the inhibitors. The hydrolysis was inhibited by re-addition of these inhibitors. The signal peptide peptidase hydrolyzed the signal peptide only after its cleavage from the lipoprotein precursor.

Bacterial Proteins↗
PubMed1990

Crystal structures of an antibody to a peptide and its complex with peptide antigen at 2.8 A.

The three-dimensional structures of an antibody to a peptide and its complex with the peptide antigen have been determined at 2.8 A resolution. The antigen is a synthetic 19-amino acid peptide homolog of the C helix of myohemerythrin (Mhr). The unliganded Fab' crystals are orthorhombic with two molecules per asymmetric unit, whereas the complex crystals are hexagonal with one molecule per asymmetric unit. The Fab' and the Fab'-peptide complex structures have been solved independently by molecular replacement methods and have crystallographic R factors of 0.197 and 0.215, respectively, with no water molecules included. The amino-terminal portion of the peptide sequence (NH2-Glu-Val-Val-Pro-His-Lys-Lys) is clearly interpretable in the electron density map of the Fab'-peptide complex and adopts a well-defined type II beta-turn in the concave antigen binding pocket. This same peptide amino acid sequence in native Mhr is alpha-helical. The peptide conformation when bound to the Fab' is inconsistent with binding of the Fab' to native Mhr, and suggests that binding of the Fab' to conformationally altered forms of the native Mhr or to apo-Mhr. Immunological mapping previously identified this sequence as the peptide epitope, and its fine specificity correlates well with the structural analysis. The binding pocket includes a large percentage of hydrophobic residues. The buried surfaces of the peptide and the antibody are complementary in shape and cover 460 A2 and 540 A2, respectively. These two structures now enable a comparison of a specific monoclonal Fab' both in its free and antigen complexed state. While no major changes in the antibody were observed when peptide was bound, there were some small but significant side chain and main chain rearrangements.

Amino Acid Sequence↗
PubMed1998

Identification of an erythrocyte binding peptide from the erythrocyte binding antigen, EBA-175, which blocks parasite multiplication and induces peptide-blocking antibodies.

A biotinylated peptide covering a sequence of 21 amino acids (aa) from the erythrocyte binding antigen (EBA-175) of Plasmodium falciparum bound to human glycophorin A, an erythrocyte receptor for merozoites, as demonstrated by enzyme-linked immunosorbent assay (ELISA) and to erythrocytes as demonstrated by flow cytometry analysis. The peptide, EBA(aa1076-96), also bound to desialylated glycophorin A and glycophorin B when tested by ELISA. The peptide blocked parasite multiplication in vitro. The glycophorin A binding sequence was further delineated to a 12-aa sequence, EBA(aa1085-96), by testing the binding of a range of truncated peptides to immobilized glycophorin A. Our data indicate that EBA(aa1085-96) is part of a ligand on the merozoite for binding to erythrocyte receptors. This binding suggests that the EBA(aa1085-96) peptide is involved in a second binding step, independent of sialic acid. Antibody recognition of this peptide sequence may protect against merozoite invasion, but only a small proportion of sera from adults from different areas of malaria transmission showed antibody reactivities to the EBA(aa1076-96) peptide, indicating that this sequence is only weakly immunogenic during P. falciparum infections in humans. However, Tanzanian children with acute clinical malaria showed high immunoglobulin G reactivity to the EBA(aa1076-96) peptide compared to children with asymptomatic P. falciparum infections. The EBA(aa1076-96) peptide sequence from EBA-175 induced antibody formation in mice after conjugation of the peptide with purified protein derivative. These murine sera inhibited EBA(aa1076-96) peptide binding to glycophorin A.

Adolescent↗
PubMed2004

Major histocompatibility complex class I peptide presentation after Salmonella enterica serovar typhimurium infection assessed via stable isotope tagging of the B27-presented peptide repertoire.

Reactive arthritis (ReA) induced by infection with several gram-negative bacteria is strongly associated with expression of the major histocompatibility complex class I molecule HLA-B27. It is thought that due to the intracellular lifestyle of ReA-inducing bacteria, bacterial fragments can be presented by HLA-B27. Cytotoxic T cells recognizing such bacterial peptides or other induced host peptides could cross-react with self peptides presented in the joints, giving rise to disease. Studies to analyze the B27 peptide repertoire in relation to infection were severely hampered, as complex peptide profiles obtained from separate infected and noninfected cell preparations had to be compared. For this study, we applied a new approach to examine the effect of Salmonella enterica serovar Typhimurium infection on the B27 peptide repertoire presented by the HLA-B*2704 subtype associated with disease. Firstly, we showed that both host cell and S. enterica serovar Typhimurium proteins can be tagged metabolically with stable-isotope-labeled arginine. We then designed experiments so that either the tagged endogenous or tagged bacterial B*2704-presented peptide repertoires from infected cells could be analyzed by mass spectrometry from single peptide preparations that included uninfected controls. Using this new approach, we found no evidence for significant changes in endogenous B*2704 peptide presentation after infection or for any S. enterica serovar Typhimurium-derived B27-bound peptide. In conclusion, the hypothesis that S. enterica serovar Typhimurium induces changes in B27 peptide presentation could not be supported.

Amino Acid Sequence↗
PubMed2006

Peptide mimotopes selected from a random peptide library for diagnosis of Epstein-Barr virus infection.

Epstein-Barr virus (EBV) is a ubiquitous, worldwide infectious agent that causes infectious mononucleosis, affecting >90% of the world's population. Currently, enzyme-linked immunosorbent assay, mostly with purified preparations of EBV cell extracts to capture immunoglobulin M (IgM) antibodies in patients' serum, is used for primary diagnosis. Our objective was to determine whether a small set of peptides could contain sufficient immunogenic information to replace solid-phase antigens in EBV diagnostics. Using monoclonal antibodies, we selected four peptides that mimic different epitopes of EBV from a phage-displayed random peptide library. To assess their diagnostic value, we screened a panel of 62 individual EBV IgM sera for their reactivities with the peptides alone. For all peptides, there was a clear distinction between the EBV-positive and the EBV-negative samples, resulting in 100% specificity. The sensitivities were 88%, 85%, 71%, and 54% for peptides F1, A3, gp125, and A2, respectively. Any combination of peptides increased the sensitivity, indicating that individual peptides react with different subsets of antibodies. Furthermore, when the F1 and the gp125 peptides were coupled to bovine serum albumin and screened against 216 serum samples, there were dramatic improvements in sensitivities (95% and 92%, respectively) and little cross-reactivity with the other peptides encountered during acute viral infections, including rheumatoid factor. This study shows the potential for the use of peptide mimotopes as alternatives to the complex antigens used in current serodiagnostics for EBV infection.

Amino Acid Sequence↗
PubMed1974

The responses of rat intestinal brush border and cytosol peptide hydrolase activities to variation in dietary protein content: dietary regulation of intestinal peptide hydrolases.

The effects of variation in dietary protein content on small intestinal brush border and cytosol peptide hydrolase activities have been investigated. One group of rats was fed a high protein diet (55% casein) and another group was fed a low protein diet (10% casein). After 1 wk, brush border peptide hydrolase activity (L-leucyl-beta-naphthylamide as substrate) and cytosol peptide hydrolase activity (L-prolyl-L-leucine as substrate) were determined in mucosae taken from the proximal, middle, and distal small intestine. As judged by several parameters, brush border peptide hydrolase activity was significantly greater in rats fed the high protein diet when data for corresponding segments were compared. In contrast, no significant difference was seen in cytosol peptide hydrolase activity. IN A SECOND STUDY, BRUSH BORDER AND CYTOSOL PEPTIDE HYDROLASE ACTIVITIES WERE DETERMINED IN THE PROXIMAL INTESTINE BY UTILIZING AN ADDITIONAL THREE PEPTIDE SUBSTRATES: L-leucyl-L-alanine, L-phenylalanylglycine, and glycyl-L-phenylalanine. Sucrase, maltase, and alkaline phosphatase activities were also determined. As before, brush border peptide hydrolase activities were significantly greater in rats fed the high protein diet. However, activities of the nonproteolytic brush border enzymes did not vary significantly with diet. In contrast to the results obtained with L-prolyl-L-leucine as substrate for the cytosol enzymes, cytosol activity against the three additional peptide substrates was greater in rats fed the high protein diet. It is suggested that the brush border peptide hydrolase response to variation in dietary protein content represents a functional adaptation analogous to the regulation of intestinal disaccharidases by dietary carbohydrates. The implication of the differential responses of the cytosol peptide hydrolases is uncertain, since little is known of the functional role of these nonorgan-specific enzymes.

Animals↗
PubMed1995

Kaliuretic peptide: the most potent inhibitor of Na(+)-K+ ATPase of the atrial natriuretic peptides.

The present investigation was designed to determine whether atrial natriuretic peptides consisting of amino acids 1-30 (i.e. long-acting natriuretic peptide), 31-67 (vessel dilator), 79-98 (kaliuretic peptide), and 99-126 [atrial natriuretic factor (ANF)] of the 126 amino acid ANF prohormone inhibit sodium-potassium-ATPase as part of their mechanism(s) of action for producing a natriuresis and/or kaliuresis. Kaliuretic peptide, long-acting natriuretic peptide, vessel dilator and ANF at their 10(-11) M concentrations inhibited Na(+)-K(+)-ATPase 39.5%, 27.8%, 19.2%, and 4% respectively, in bovine renal medulla, whereas their inhibition in renal cortical membranes was 37.5%, 27.5%, 20%, and 0%, respectively. Ouabain (0.5 mM) inhibited kidney medullary Na(+)-K(+)-ATPase 45% and in the cortex, 38%. There was no additive effect of any of these peptides with ouabain suggesting that they are interacting with the same site on the Na(+)-K(+)-ATPase as ouabain. To help elucidate the mechanism of these peptides' interaction with Na(+)-K(+)-ATPase, naproxen (0.5 mM), an inhibitor of prostaglandin synthesis, and direct measurement of prostaglandin E2 by RIA were used. Naproxen completely blocked the inhibition of Na(+)-K(+)-ATPase by kaliuretic peptide, long-acting natriuretic peptide, and vessel dilator suggesting that their inhibition of Na(+)-K(+)-ATPase in both the kidney medulla and cortex are mediated by prostaglandins. Direct measurement of prostaglandin E2 revealed that kaliuretic peptide > long-acting natriuretic peptide > vessel dilator increased prostaglandin E2 synthesis, whereas ANF did not have any effect. Of interest, angiotensin II and ouabain inhibition of Na(+)-K(+)-ATPase were also completely blocked by naproxen.

Analysis of Variance↗
PubMed1997

Quantification of circulating peptides and assessment of peptide uptake across the gastrointestinal tract of sheep.

Gastrointestinal absorption of peptides was examined in sheep fed a forage-based diet. Peptide concentrations were determined in arterial, portal, and mesenteric blood and plasma by quantification of amino acid concentrations before and after acid hydrolysis of samples that had been first deproteinized then subjected to Sephadex G-15 gel-filtration to remove residual protein. In contrast to other studies of ruminants, peptide concentrations for individual amino acids were lower than for the corresponding free amino acids with peptide (expressed as a proportion of total nonprotein amino acid) representing not more than .25 to .3 of total amino acid. Peptide concentrations in arterial, mesenteric, and portal blood and plasma were similar, indicating that on this diet there was no net uptake of peptides from the small intestine (mesenteric-drained viscera, MDV) or the whole tract (portal-drained viscera, PDV). Increasing the intake of alfalfa pellets from 800 to 1,200 g/d, while increasing the absorption and net flux across the MDV and PDV of free amino acids, had no effect on peptide absorption. Preparation of blood and plasma samples for peptide analysis with methods used in studies in which substantial peptide absorption has been reported indicated no net MDV or PDV flux of peptide. Such conflicting data on the extent of gastrointestinal peptide flux are discussed in the context of methodological differences and the importance of diet and physiological state of the animal.

Amino Acids↗
PubMed1994

Dituftsin and polytuftsin induce an anti-peptide IgG response to non-immunogenic peptides in mice.

The effect of covalently attaching multiple forms of the immunomodulating tetrapeptide tuftsin to normally non-immunogenic peptides was studied in BALB/c and C57Bl/6 mice. The peptides: (NANP)3 from the Plasmodium falciparum circumsporozoite protein and peptides 136-152 and 205-213 derived from the capsid protein of foot-and-mouth disease virus were coupled to polytuftsin or dituftsin. Anti-peptide IgG titers were determined after two immunizations. All of these three non-immunogenic peptides coupled to polytuftsin or dituftsin induced anti-peptide antibody production in mice while peptides alone did not elicit IgG. In addition, a conjugate of (NANP)3 and polytuftsin with built-in glycopeptide adjuvant elicited an anti-peptide response comparable in magnitude with that of a peptide-KLH conjugate. The data suggest that when non-immunogenic peptides are synthesized in tandem with dituftsin or conjugated to polytuftsin a significant immune response to the peptides may be elicited. This approach may be employed in synthetic vaccine design.

Amino Acid Sequence↗
PubMed2003

Anti-HER2/neu peptide producing vector system for biologic therapy - is it possible to mass-produce the peptide?

A humanized monoclonal antibody against HER2 has been using in a clinical setting and has been shown to possess therapeutic properties. A mimetic peptide against HER2 was also reported to bind to the HER2 receptor with some therapeutic potential. Based on a previous report and the sequence of Herceptin, we designed oligonucleotides of anti-HER2 mimetic peptides, named V2 and V3 peptides, in order to develop a peptide- producing vector system for biologic therapy against HER2- overexpressing cancers. We also adopted the sequence of a previously reported mimetic peptide, V1 (Park BW et al. Nat. Biotechnol, 2000, 18:194-198), as a reference peptide. We examined the effects of the V2 and V3 peptides against the HER2-overexpressing cell lines, SK-BR-3 and T6-17. Transient transfection of the construct expressing V1 and V2 inhibited cell proliferation in HER2-overexpressing cell lines by 20 - 30%, but had no effect on the HER2-negative NIH3T3 cells. The proliferation inhibition shown by V2 was slightly better than that shown by V1. Recombinant peptides V2 and V3 were produced on a large scale in an E. coli system, and the V2 peptide showed anti-HER2-specific tumor cell proliferation inhibition of 10% to 30%. Current results suggest that anti-HER2 mimetic peptides, overexpressed by a constitutive promoter or produced in an E. coli system, could specifically inhibit the proliferation of HER2-expressing cancer cells. Further efforts to augment the biologic specificity and efficacy and to develop new technologies for the purification of the peptide from the E coli system are needed.

Amino Acid Sequence↗
PubMed2004

Selection of a peptide mimicking neutralization epitope of hepatitis E virus with phage peptide display technology.

AIM: To select the peptide mimicking the neutralization epitope of hepatitis E virus which bound to non-type-specific and conformational monoclonal antibodies (mAbs) 8C11 and 8H3 fromed 7-peptide phage display library, and expressed the peptide recombinant with HBcAg in E.coli, and to observe whether the recombinant HBcAg could still form virus like particle (VLP) and to test the activation of the recombinant polyprotein and chemo-synthesized peptide that was selected by mAb 8H3. METHODS: 8C11 and 8H3 were used to screen for binding peptides through a 7-peptide phage display library. After 4 rounds of panning, monoclonal phages were selected and sequenced. The obtained dominant peptide coding sequences was then synthesized and inserted into amino acid 78 to 83 of hepatitis B core antigen (HBcAg), and then expressed in E.coli. Activity of the recombinant proteins was detected by Western blotting, VLPs of the recombinant polyproteins were tested by transmission electron microscopy and binding activity of the chemo-synthesized peptide was confirmed by BIAcore biosensor. RESULTS: Twenty-one positive monoclonal phages (10 for 8C11, and 11 for 8H3) were selected and the inserted fragments were sequenced. The DNA sequence coding for the obtained dominant peptides 8C11 (N'-His-Pro-Thr-Leu-Leu-Arg-Ile-C', named 8C11A) and 8H3 (N'-Ser-Ile-Leu-Pro- Tyr-Pro-Tyr-C', named 8H3A) were then synthesized and cloned to the HBcAg vector, then expressed in E.coli. The recombinant proteins aggregated into homodimer or polymer on SDS-PAGE, and could bind to mAb 8C11 and 8H3 in Western blotting. At the same time, the recombinant polyprotein could form virus like particles (VLPs), which could be visualized on electron micrograph. The dominant peptide 8H3A selected by mAb 8H3 was further chemo-synthesized, and its binding to mAb 8H3 could be detected by BIAcore biosensor. CONCLUSION: These results implicate that conformational neutralizing epitope can be partially modeled by a short peptide, which provides a feasible route for subunit vaccine development.

Amino Acid Sequence↗
PubMed2001

Thermal stability of MHC class I-beta 2-microglobulin peptide complexes in the endoplasmic reticulum is determined by the peptide occupancy of the transporter associated with antigen processing complex.

Once MHC class I heavy chain binds beta(2)-microglobulin (beta(2)m) within the endoplasmic reticulum, an assembly complex comprising the class I heterodimer, TAP, TAPasin, calreticulin, and possibly Erp57 is formed before the binding of high affinity peptide. TAP-dependent delivery of high affinity peptide to in vitro translated K(b)beta(2)m complexes within microsomes (TAP(+)/TAPasin(+)) was studied to determine at which point peptide binding becomes resistant to thermal denaturation. It was determined that the thermal stability of K(b)-beta(2)m-peptide complexes depends on the timing of peptide binding to K(b)beta(2)m relative to TAP binding high affinity peptide. Premature exposure of the TAP complex to high affinity peptide before its association with class I heavy chain results in K(b)beta(2)m-peptide-TAP complexes that lose peptide upon exposure to elevated temperature after solubilization away from microsome-associated proteins. These findings suggest that the order in which class I heavy chain associates with endoplasmic reticulum-resident chaperones and peptide determines the stability of K(b)beta(2)m-peptide complexes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

In vivo evidence that peptide vaccination can induce HLA-DR-restricted CD4+ T cells reactive to a class I tumor peptide.

Vaccination with class I tumor peptides has been performed to induce tumor-reactive CD8(+) T cells in vivo. However, the kinds of immune responses that vaccination might elicit in patients are not fully understood. In this study we tried to elucidate the mechanisms by which vaccination of class I binding tumor peptides into an HLA-A2(+) lung cancer patient elicited dramatic amounts of IgG1 and IgG2 specific to a nonamer peptide, ubiquitin-conjugated enzyme variant Kua (UBE2V)(43-51). The UBE2V(43-51) peptide contains cysteine at the sixth position. HLA-DR-restricted and UBE2V(43-51) peptide-recognizing CD4(+) T cells were induced from postvaccination, but not from prevaccination, PBMCs of the cancer patient. In addition, a CD4(+) T cell line (UB-2) and its clone (UB-2.3), both of which recognize the UBE2V(43-51) peptide in the context of HLA-DRB1*0403 molecules, were successfully established from postvaccination PBMCs. The peptide vaccination increased the frequency of peptide-specific T cells, especially CD4(+) T cells. In contrast, mass spectrometric analysis revealed that the vaccinated UBE2V(43-51) peptide contained both monomeric and dimeric forms. Both forms, fractionated by reverse phase HPLC, were recognized by UB-2 and UB-2.3 cells. Recognition by these CD4(+) T cells was observed despite the addition of a reduction reagent or the fixation of APC. Overall, these results indicate that vaccination with class I tumor peptides can induce HLA-DR-restricted CD4(+) T cells in vivo and elicit humoral immune responses, and that a cysteine-containing peptide can be recognized by CD4(+) T cells not only as a monomer, but also as a dimer.

Aged↗
PubMed1992

Role of hapten-anchoring peptides in defining hapten-epitopes for MHC-restricted cytotoxic T cells. Cross-reactive TNP-determinants on different peptides.

Synthetic hapten-peptide conjugates selectively modify cell-bound MHC class I molecules in a haplotype-specific way. We investigated the contribution of the carrier peptides to the structural specificity of T cell-antigenic TNP epitopes, using different H-2Kb-binding TNP-peptides and a collection of TNP/Kb-specific CTL clones. Adjustment of peptide sequences to the proposed Kb-specific "motif" (octamers with F or Y and L in positions 5 and 8, respectively) enhanced Kb-binding and antigenicity by many orders of magnitude. Moreover, several clones reacted to peptides, containing the "motif" and TNP-lysine in position 4 but were otherwise unrelated by sequence. TNP in other positions was not recognized by these cells, but other CTL reacted to TNP in position 7. This points to the positioning of hapten determinants within the MHC binding groove as a major role of the anchoring peptide. However, determination of the limiting amounts of TNP peptides that elicit antigenicity or inhibit other Kb-restricted CTL reactions revealed that TCR also recognize variations in the sequences of carrier peptides. This contribution is low for TNP in position 4 but high in position 7, indicating lysine in position 4 as a particularly dominant and cross-reactive hapten-anchoring site in Kb-associated peptides. This implies that cell modification with lysine-reactive TNP reagents results in immunodominant, highly repetitive TNP epitopes, which may explain the strong antigenicity and the allergenic properties of TNP, as well as the restricted TCR repertoire directed against this hapten. Our data further recommend hapten peptides for general studies of TCR-Ag interactions because in contrast to pure protein Ag, hapten epitopes tolerate substantial structural variations in the MHC-anchoring peptide, and can be located by hapten-specific antibodies.

Amino Acid Sequence↗
PubMed1986

Design of biologically active peptides with non-peptidic structural elements. Biological and physical properties of a synthetic analogue of beta-endorphin with unnatural amino acids in the region 6-12.

Modelling studies with beta-endorphin have clearly demonstrated that an amphiphilic secondary structural segment is a salient feature of the biologically active conformation of this 31-residue opioid peptide hormone. Here, we have initiated the synthesis of peptide models using unnatural building blocks by designing a beta-endorphin analogue (peptide 6) in which the hydrophilic linker region between the NH2-terminal enkephalin (residues 1-5) and the COOH-terminal helix (residues 10-28, sequence identical to that of peptide 3 in region 13-31, Fig. 1) consists of four units of gamma-amino-gamma-hydroxymethylbutyric acid connected by isopeptidic linkages. Peptide 6 has physical properties similar to that of peptide 3, as shown by surface monolayer and circular dichroism studies. The binding affinities of the two peptides to delta- and mu-receptors are also similar. In rat vas deferens assays, the present model is equipotent to peptide 3. The most striking result of all is the potent analgesic activity displayed by peptide 6 when injected intracerebroventricularly into mice. The potencies of peptides 6 and 3 are comparable in these assays. These studies clearly illustrate that one can use unusual building blocks to construct structural regions of synthetic analogues and still preserve the biological activity of peptide hormones.

Analgesics↗
PubMed1987

Evidence that arginyl-glycyl-aspartate peptides and fibrinogen gamma chain peptides share a common binding site on platelets.

Synthetic peptides corresponding to the carboxyl terminus of the fibrinogen gamma chain inhibit the binding of fibrinogen, fibronectin, and von Willebrand factor to platelets, yet the active decapeptide sequence has only been found in fibrinogen to date. In contrast, all three proteins contain Arg-Gly-Asp sequences, and peptides containing Arg-Gly-Asp are potent inhibitors of their binding to activated platelets. We have analyzed the relationship between these peptide sets by direct binding assays. H12 (gamma 400-411) inhibited the binding of an Arg-Gly-Asp-containing peptide to platelets with similar dose response to inhibition of fibronectin binding. We have previously reported that GPIIb-IIIa binds to immobilized Arg-Gly-Asp peptides and can be eluted by Arg-Gly-Asp-containing peptides in solution. Both H12 and L10 (gamma 402-411) completely eluted GPIIb-IIIa bound to immobilized Arg-Gly-Asp peptides. Conversely, when GPIIb-IIIa was bound to immobilized L10, either L10 or an Arg-Gly-Asp peptide could elute it. Peptide specificity was established by the failure of Gly-Arg-Gly-Glu-Ser-Pro or acetylated L10 to elute GPIIb-IIIa from the immobilized peptides. These results indicate that the two peptide sets interact with the same receptor which contains GPIIb-IIIa.

Amino Acid Sequence↗
PubMed1994

Binding of apolipoprotein A-I model peptides to lipid bilayers. Measurement of binding isotherms and peptide-lipid headgroup interactions.

Amphiphatic alpha-helices are the lipid-binding motif in many apolipoproteins. Two model peptides, namely Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe (18A) and Lys-Trp-Leu-Asp-Ala-Phe-Tyr-Lys-Asp-Val-Ala-Lys-Glu-Leu-Glu-Lys-Ala- Phe (18R), have been synthesized previously to mimic the structural and functional properties of apolipoprotein A-1. Here a quantitative thermodynamic analysis of the binding process of 18A and 18R to neutral and negatively charged lipid membranes is provided. Peptide 18A has a higher lipid affinity than 18R, and both peptides bind better to mixed 1-palmitoyl-2-oleoyl-3-sn-glycero-phosphocholine-1- palmitoyl-2-oleoyl-3-sn-glycero-phosphoglycerol (POPC/POPG) bilayers than to pure POPC bilayers. At lipid-to-peptide ratios > 100, the binding of 18A and 18R to phospholipid bilayers can be described by an apparent surface partition equilibrium with binding constants in the range of 40-900 M-1. At high peptide concentrations, the membrane affinity of 18A and 18R increases dramatically. NMR studies provide evidence that peptide-peptide interactions make additional contributions to the binding energy. A cooperative binding model is developed to describe the binding process over the whole concentration range. The cooperativity parameter sigma is identical for 18A and 18R yielding a peptide-peptide interaction energy of about -2.4 kcal/mol. The free energy of membrane insertion is about -6.5 kcal/mol for 18A and -5.5 kcal/mol for 18R. The binding reaction is driven by the hydrophobic surface energy which is partially balanced by the loss in translational and rotational degrees of freedom. A molecular analysis of the free energy of binding predicts a 40-60% insertion of the peptides into the hydrophobic membrane environment. Deuterium and phosphorus solid state NMR were used to monitor the influence of 18A and 18R on the long range and short range order of the phospholipids. The spectra are characteristic of fluid-like lipid bilayers and provide no evidence for the formation of discoidal particles. However, both peptides change the conformation of the phosphocholine dipoles, moving the N+ end of the latter toward the water phase. The rotation of the -P-N+ dipoles is due to the interaction of the phospholipids with the positive charges on 18A and 18R, with 18A being more effective than 18R. For 18R the NMR data predict a pK shift and a partial charge neutralization of the carboxylate groups located at the edge of the polar/nonpolar interface.

Amino Acid Sequence↗