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PubMed1994

Inhibition of experimental autoimmune encephalomyelitis by MHC class II binding competitor peptides depends on the relative MHC binding affinity of the disease-inducing peptide.

Blocking of the Ag presentation function of MHC molecules by competitor peptides has been proposed as a potential immunotherapy for MHC-associated autoimmune diseases. Despite the fact that successful inhibition of experimental autoimmune encephalomyelitis (EAE) by coimmunization with competitor peptides had been achieved, it remained questionable whether the in vivo activity of such peptides was solely the result of MHC blockade. In the peptide MBP72-85-induced EAE model in Lewis rats, we designed a single amino acid-substituted analogue of MBP72-85 with a superior MHC binding capacity, and with the capacity to activate encephalitogenic MBP72-85-specific T cells. Subsequently, two well-defined competitor peptides, one EAE related and one non-EAE related, were studied for their respective efficacies to inhibit the in vitro proliferation of an encephalitogenic T cell clone induced by the original MBP72-85 or the superior MHC binding analogue peptide. It appeared that the response to MBP72-85 was inhibited very efficiently by both competitor peptides, whereas the response to the superior MHC binding analogue peptide was not. Co-immunization of either the related or non-related competitor peptide together with MBP72-85 inhibited EAE induction in a concentration-dependent manner. In such protected rats, polyclonal T cell responses against MBP72-85 were dramatically decreased. However, EAE induced by the stronger MHC binding MBP72-85 analogue could not be inhibited by either of the competitor peptides. Moreover, in these rats, T cell priming for both MBP72-85 and the MBP72-85 analogue was not inhibited. These results show that competition for MHC binding in vivo could lead to inhibition of EAE induction.

Amino Acid Sequence↗

PubMed1993

A peptide variant of an arthritis-related T cell epitope induces T cells that recognize this epitope as a synthetic peptide but not in its naturally processed form.

The immune system has the potential to utilize a diverse T cell repertoire for the recognition of Ag in the context of MHC molecules. Here we describe the analysis of two rat T cell clones, both of which recognize a synthetic peptide comprised of the arthritis-associated 180-188 amino acid sequence of the mycobacterial 65-kDa heatshock protein (hsp65 180-188), but which differ in the recognition of the naturally processed hsp65. The arthritogenic T cell clone A2b, generated by immunization with whole Mycobacterium tuberculosis, recognized the hsp65 180-188 synthetic peptide as well as the processed hsp65, whereas T cell clone ATL11, generated after immunization with a single amino acid substituted peptide analog of hsp65 180-188, recognized peptide hsp65 180-188 but not the processed hsp65. For both T cell clones the minimal stimulatory sequence was hsp65 180-186. However, within this minimal stimulatory sequence marked differences between the clones were found with regard to peptide residues interacting with the TCR. Furthermore, addition of extra residues at the N terminus of the hsp65 180-186 sequence abrogated the recognition by clone ATL11, but not by A2b. These findings demonstrate that, upon in vivo immunization with a synthetic peptide comprised of a single amino acid variant of a T cell epitope, T cells can be triggered that recognize a peptide comprised of the original epitope sequence, but that do not recognize this epitope in its naturally processed protein fragment. The possibility of triggering such T cells by immunization with synthetic peptides, may well have consequences for the design of peptide vaccines or peptide immunomodulatory agents.

Amino Acid Sequence↗

PubMedDate not supplied

[Use of a series of synthetic peptides and purified major histocompatibility complex I (H-2K(b)) molecules for inhibiting T-suppressors, specific for the K(b) molecule, and their induction by peptides in vivo].

Six synthetic peptides of the MHC class 1 molecule corresponding to individual H-2kb participants in amino acid sequences of domains alpha 1 (peptide 1 and 2) and alpha 2 (peptide 3, 4, 5, 6) were selected. Kb-specific suppressor T cells (Ts) were in vivo in mice, then pretreated with a set of peptides and assayed by proliferation decrease in the third-partial mixed lymphocyte culture (MLC). Effector function of Ts was abolished by the complex of the alpha 2-domain peptides (but not by the alpha 1-domain peptides) and decreased by each peptide (4, 5, 6) of the alpha 2-domain. Both alpha 1 and alpha 2 domain peptides, added at high concentrations, decreased the otherwise efficient enrichment of Ts during the absorption-elution procedure on the syngeneic macrophage (MP) monolayers. A similar significant effect was observed the purified Kb molecule (100 mg/ml) on the allogeneic MP monolayer. Interaction between Ts receptors with some MHC peptides indicates effector Ts activation in vivo by induction with peptides 5+6 of the alpha 2 domain. The fine mechanisms of interaction between MHC class I molecule epitopes and T cell receptors (TCR) of each of the T cell subsets separately are under study now.

Amino Acid Sequence↗

PubMed1997

Chimeric peptides: a new approach to enhancing the immunogenicity of peptides with low MHC class I affinity: application in antiviral vaccination.

Recruitment of the CTL repertoire specific for subdominant epitopes that have a low MHC class I-binding affinity could be the way to achieve an efficient protective immunity against spontaneous tumors and viruses with high mutation rate. However, we have reported recently that subdominant peptides of influenza A Puerto Rico/8/34 (flu PR8) nucleoprotein (NP) with low Db affinity are only partially able to protect mice against lethal influenza infection. This seems to be due to their inability to recruit the specific CTL repertoire, and suggests that subdominant peptides could be used for vaccination only if they become highly immunogenic. In this work, we describe an approach that allows enhancement of the immunogenicity of every low affinity peptide presented by the Db molecule. It consists in producing chimeric peptides composed by amino acids from a high Db affinity peptide (NP366) in positions that interact with the MHC, and amino acids from low Db affinity nonimmunogenic influenza NP-derived peptides (NP17, NP97, NP330, and NP469) in positions that are exposed to the TCR. All chimeric peptides tested exhibited a high Db affinity and efficiently recruited the CTL repertoire specific for the corresponding low Db affinity peptide. Furthermore, vaccination with chimeric peptides that corresponded to subdominant NP17 and NP97 peptides induced a very potent anti-flu PR8 protective immunity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Enhancement of tumor outgrowth through CTL tolerization after peptide vaccination is avoided by peptide presentation on dendritic cells.

Synthetic peptide-based vaccines have been shown to induce potent protective and therapeutic T cell-mediated immunity in preclinical animal models and are now being evaluated in clinical phase I/II studies for their efficacy against tumors or infectious diseases. However, such vaccines might also specifically tolerize T cells causing enhanced tumor outgrowth, as shown by vaccination with two CTL epitopes derived from the adenovirus type 5 early region 1 (Ad5E1) oncogenes. We now report that modification of the Ad5E1 peptide vaccine either through incorporation of the peptides into liposomes or by ligation of the peptides to lipid tails, another vaccine formulation being tested in the clinic, fails to convert immunosuppression into effective antitumor vaccination. Inclusion of a helper T cell epitope into the vaccine likewise induces enhanced tumor outgrowth and thus does not diminish the capacity of the peptides to tolerize Ad5E1-specific CTL. In contrast, the Ad5E1-derived peptides evoke a strong tumor-protective CTL response when presented on dendritic cells (DC), indicating that the in vivo CTL-tolerizing potential of these peptides is converted to specific immunostimulation when presented on DC. These findings have important implications for the development of peptide-based immune intervention strategies and emphasize the superior nature of Ag-pulsed DC over other peptide-based vaccination protocols as well as the crucial importance of the mode of peptide-Ag delivery in setting the balance between T cell stimulation and tolerization.

Amino Acid Sequence↗

PubMed2000

Differential effects of calcitonin gene-related peptide and calcitonin gene-related peptide 8-37 upon responses to N-methyl-D-aspartate or (R, S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate in spinal nociceptive neurons with knee joint input in the rat.

Calcitonin gene-related peptide is involved in the spinal processing of nociceptive input from the knee joint and in the generation and maintenance of joint inflammation-evoked hyperexcitability of spinal cord neurons. The present study examined whether this peptide influences the excitation of nociceptive spinal cord neurons by agonists at the N-methyl-D-aspartate and the non-N-methyl-D-aspartate [(R, S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate] receptors, both of which are essential for the excitation and hyperexcitability of spinal cord neurons. In anaesthetized rats extracellular recordings were made from dorsal horn neurons with knee input, and compounds were administered ionophoretically close to the neurons recorded. When calcitonin gene-related peptide was administered the responses of the neurons to the application of both N-methyl-D-aspartate and AMPA were increased. The coadministration of the antagonist calcitonin gene-related peptide 8-37 had no effect on the responses to N-methyl-D-aspartate, but it prevented the enhancement of the responses to N-methyl-D-aspartate by calcitonin gene-related peptide. By contrast, the administration of calcitonin gene-related peptide 8-37 enhanced the responses of the neurons to AMPA, and it did not antagonize but rather increased the effects of calcitonin gene-related peptide on these responses. The data suggest that the facilitatory role of calcitonin gene-related peptide on the development and maintenance of inflammation-evoked hyperexcitability is caused at least in part by the modulation of the activation of the dorsal horn neurons through their N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors. The different effects of calcitonin gene-related peptide 8-37 on the respones to N-methyl-D-aspartate and AMPA suggest that different intracellular pathways may facilitate the activation of N-methyl-D-aspartate and ionotropic non-N-methyl-D-aspartate receptors.

Animals↗

PubMed1995

Extensive contact between Gi2 and N-formyl peptide receptor of human neutrophils: mapping of binding sites using receptor-mimetic peptides.

The N-formyl peptide receptor (FPR) of human neutrophils is a member of the G protein-coupled receptor (GPCR) superfamily. Sites on agonist-occupied FPR involved in binding the Gi2 protein were investigated by competition with synthetic receptor-mimetic peptides. Twenty-three synthetic FPR-mimetic and control peptides were tested for their ability to disrupt functionally active complexes of FPR and Gi2 in octyl glucoside, assayed by changes in sedimentation rates of FPR in detergent-containing sucrose gradients. GPCRs are thought to contain seven transmembrane segments with three cytoplasmic connecting loops and a cytoplasmic tail. Only certain peptides from regions in or adjoining each of the four predicted cytoplasmic domains of the 350 amino acid FPR, including the first cytoplasmic loop, were able to disrupt the reconstituted FPR-Gi2 complex. The IC50s of the peptides that were able to fully disrupt the FPR-Gi2 complex ranged from 20 microM (C2W 122-134) to 1.4 mM (C3A 230-246), a range similar to peptide inhibition of other G protein-coupled receptor-G protein interactions. Detergent concentrations above and below the critical micelle concentration had no effect on the activity of even the most hydrophobic peptide, C3B, and there was no apparent correlation of activity with hydrophobic moment, hydrophilic index, or net charge of the peptides. Control peptides from irrelevant proteins with similar physical properties and FPR extracellular domains did not dissociate the reconstituted FPR-Gi2 complex up to 5 mM, the highest concentration tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMedDate not supplied

A new member of the hypothalamic RF-amide peptide family, LPXRF-amide peptides: structure, localization, and function.

Recently, we identified a novel hypothalamic neuropeptide with a C-terminal LPLRF-amide sequence in the quail brain. This avian neuropeptide was shown to inhibit gonadotropin release from the cultured anterior pituitary. This peptide is the first hypothalamic peptide that inhibited gonadotropin release reported in vertebrates. We, therefore, termed it gonadotropin-inhibitory hormone (GnIH). After this finding, we found that GnIH-related peptides were present in the brains of other vertebrates, such as mammals, amphibians, and fish. These GnIH-related peptides possessed a LPXRF-amide (X=L or Q) motif at their C-termini in all investigated animals. Mass spectrometric analyses combined with immunoaffinity chromatography were powerful techniques for the identification of mature endogenous LPXRF-amide peptides. The identified LPXRF-amide peptides were found to be localized in the hypothalamus and brainstem areas, and to regulate pituitary hormone release. Subsequently, cDNAs that encode LPXRF-amide peptides were characterized in vertebrate brains. In this review, we summarize the identification, localization, and function of a new member of the hypothalamic RF-amide peptide family, LPXRF-amide peptides in vertebrates. Recent studies on the receptors for LPXRF-amide peptides will also be reviewed.

Amides↗

PubMedDate not supplied

Aplysia californica neurons R3-R14: primary structure of the myoactive histidine-rich basic peptide and peptide I.

The R3-R14 neurons of the marine mollusc Aplysia are neuroendocrine cells that express a gene encoding peptides I, II and histidine-rich basic peptide (HRBP), a myoactive peptide that excites Aplysia heart and enhances gut motility in vitro. Peptide II has been chemically characterized (35), but the complete primary structures of peptide I and HRBP have not been established by amino acid sequence analysis. HRBP, peptide I, and the prohormone (proHRBP) were therefore purified from acid extracts of Aplysia californica neural tissue using sequential gel filtration and reverse-phase high-performance liquid chromatography and chemically characterized. Amino acid sequence analysis demonstrated that HRBP was a 43-residue peptide whose sequence was: less than Glu-Val-Ala-Gln-Met-His-Val-Trp-Arg-Ala-Val-Asn-His-Asp-Arg-Asn-His-Gly- Thr-Gly - Ser-Gly-Arg-His-Gly-Arg-Phe-Leu-Ile-Arg-Asn-Arg-Tyr-Arg-Tyr-Gly-Gly-Gly- His-Leu - Ser-Asp-Ala-COOH. Compositional and sequence analyses of peptide I and proHRBP demonstrated that peptide I was a 26-residue peptide with the following sequence: NH2-Glu-Glu-Val-Phe-Asp-Asp-Thr-Asp-Val-Gly-Asp-Glu-Leu-Thr-Asn-Ala- Leu-Glu-Ser-Val-Leu-Thr-Asp-Phe-Lys-Asp-COOH. These results demonstrated that the pro-HRBP sequence predicted by nucleotide sequence analysis of a cDNA clone (24) was in fact synthesized in R3-R14 neurons. Hydrophilicity and hydrophobicity profiles of preproHRBP, combined with charge distribution profiles and predictive secondary structural analysis, showed that cleavage at dibasic sequences was strongly associated with peaks of hydrophilicity in alpha-helical regions of the preprohormone.

Amino Acid Sequence↗

PubMed1995

Integrated light-scattering spectroscopy, a sensitive probe for peptide-vesicle binding: application to the membrane-bound colicin E1 channel peptide.

Integrated light-scattering (ILS) spectroscopy was used to monitor the binding of the colicin E1 channel peptide to POPC:POPG large unilamellar vesicles (LUV; 60:40, mol:mol) at acidic pH (3.5). Binding conditions were chosen such that nearly all of the channel peptide was bound to the vesicles with little free peptide remaining in solution. The increase in vesicle size upon the insertion of the channel peptide was measured by performing a discrete inversion technique on data obtained from an ILS spectrometer. Vesicle size number distributions were determined for five different systems having peptide/vesicle ratios of approximately 0, 77, 154, 206, and 257. The experiment was repeated four times (twice at two different vesicle concentrations) to determine reproducibility. The relative changes in vesicle radius upon peptide binding to the membrane vesicles was remarkably reproducible even though these changes represented only a few nanometers. A comparison of vesicle size number distributions in the absence of bound peptide was made between ILS and dynamic light scattering (DLS) data and showed similar results. However, DLS was incapable of detecting the small changes due to peptide-induced vesicle swelling. The membrane-bound volume of the colicin E1 channel peptide was approximately 177 +/- 22 nm3. These data indicate that in the absence of a membrane potential (closed channel state) the colicin E1 channel peptide inserts into the membrane resulting in a significant displacement of the lipid bilayer as evidenced from the dose-dependent increase in the vesicle radius. These results indicate that ILS spectroscopy is a sensitive sizing technique that is capable of detecting relatively small changes in membrane vesicles and may have a wide application in the determination of peptide binding to membrane vesicles.

Biophysical Phenomena↗

PubMed2001

Umbilical artery N-terminal peptide of proatrial natriuretic peptide in hypertensive pregnancies and fetal acidemia during labor.

OBJECTIVE: To assess the activity of the human fetal atrial natriuretic peptide system in hypertensive pregnancies with and without signs of increased fetal systemic venous pressure and in pregnancies complicated by fetal acidemia during labor. METHODS: Umbilical artery plasma N-terminal peptide of proatrial natriuretic peptide concentrations were measured in neonates by radioimmunoassay. The control group consisted of 50 neonates with uncomplicated gestation and labor. In group 1, there were 22 newborns of hypertensive pregnancies. Doppler ultrasonography showed abnormal umbilical artery blood velocity waveform in five cases and normal nonpulsatile umbilical vein blood velocity profile in every case. Group 2 consisted of five newborns of pregnancies complicated by maternal hypertensive disorder. Atrial pulsations in the umbilical vein and retrograde diastolic blood velocity pattern in the umbilical artery were detected in every case. Group 3 was composed of 27 newborns of uncomplicated pregnancies with fetal acidemia (pH 7.10 or less) during labor. RESULTS: In groups 1-3, N-terminal peptide of proatrial natriuretic peptide concentrations were higher (P <.001) than in the control group. In group 1, neonates with abnormal umbilical artery blood velocity pattern had higher N-terminal peptide of proatrial natriuretic peptide concentrations than neonates with normal umbilical artery Doppler findings (P <.006). N-terminal peptide of proatrial natriuretic peptide concentrations were higher in group 2 (P <.002) than in groups 1 and 3. CONCLUSIONS Maternal hypertensive disorder and fetal acidemia during labor stimulate fetal atrial natriuretic peptide production, which was greatest in fetuses with severe placental insufficiency and signs of congestive heart failure.

Adult↗

PubMed1999

Properties of TCA-insoluble peptides in kimoto (traditional seed mash for sake brewing) and conditions for liberation of the peptides from rice protein.

It was found that a large amount of TCA (trichloroacetic acid)-insoluble peptides were liberated into the supernatant of kimoto on the 7th-10th day of mashing. These TCA-insoluble peptides had five polypeptide groups (12, 21, 31, 38, and 55 kDa) on SDS-PAGE (SDS-polyacrylamide gel electrophoresis), and a large amount of high molecular weight peptides, higher than 10,000, were observed upon gel filtration chromatography using TSKgel G2000swxl (Tosoh Co.). Four of these peptides (12, 21, 31, and 38 kDa on SDS-PAGE) appeared specifically in kimoto, and were not detected at all either in sokujo-moto or in the main mash for sake brewing. These TCA-insoluble peptides were fractionated from the supernatant of kimoto on the 9th day, and it was revealed that free amino acids were produced abundantly from them in the presence of the enzyme of rice-koji. Therefore, it was assumed that the peptides are related to the abundant production of free amino acids in kimoto. For the liberation of these TCA-insoluble peptides from rice protein, the enzyme of rice-koji was indispensable. The enzyme liberating the TCA-insoluble peptides from rice protein was purified from rice-koji, and was presumed to be identical with acid protease (AP) of rice-koji. The presence of a high concentration of glucose (higher than 20%) was also indispensable for the liberation of the TCA-insoluble peptides. Furthermore, it was revealed that the peptides were liberated from rice protein under a limited pH of around 4.5.

Journal Article↗

PubMed2002

Ductus ejaculatorius peptide 99B (DUP99B), a novel Drosophila melanogaster sex-peptide pheromone.

We have characterized a glycosylated, 31 amino-acid peptide of 4932 Da isolated from Drosophila melanogaster males. The mature peptide contains a sugar moiety of 1184 Da at a NDT consensus glycosylation site and a disulfide bond. It is synthesized in the male ejaculatory duct via a 54 amino-acid precursor containing an N-terminal signal peptide and Arg-Lys at the C-terminus which is cleaved off during maturation. The gene contains an intron of 53 bp and is localized in the cytological region 99B of the D. melanogaster genome. The peptide is therefore named DUP99B (for ductus ejaculatorius peptide, cytological localization 99B). The C-terminal parts of mature DUP99B and D. melanogaster sex-peptide (ACP70A) are highly homologous. Injected into virgin females, DUP99B elicits the same postmating responses as sex-peptide (increased oviposition, reduced receptivity). These effects are also induced by de-glycosylated native peptide or synthetic DUP99B lacking the sugar moiety. Presence of the glycosyl group, however, decreases the amount needed to elicit the postmating responses. Homologies in the coding regions of the two exons of DUP99B and sex-peptide, respectively, suggest that the two genes have evolved by gene duplication. Thus, we consider these two genes to be members of the new sex-peptide gene family.

Amino Acid Sequence↗

PubMed1996

Covalent modification of protein kinase C isozymes by the inactivating peptide substrate analog N-biotinyl-Arg-Arg-Arg-Cys-Leu-Arg-Arg-Leu. Evidence that the biotinylated peptide is an active-site affinity label.

We recently reported that the peptide substrate analog Arg-Lys-Arg-Cys-Leu-Arg-Arg-Leu (RKRCLRRL) irreversibly inactivates the protein kinase C (PKC) isozymes alpha, beta, and gamma in a dithiothreitol-sensitive manner by an active site-directed mechanism. We hypothesized that the inactivation mechanism entailed covalent complex formation between the PKC isozyme and the inactivator peptide. In this report, N-biotinylated analogs of RKRCLRRL that inactivate Ca2+-dependent PKC activity were designed and tested for their ability to covalently label PKC isozymes. A purified PKC isozyme mixture (alpha, beta, gamma, epsilon, zeta) was incubated with the N-biotinylated peptides and then subjected to denaturing gel electrophoresis, transferred to nitrocellulose, and probed for avidin-reactive species. The Ca2+-dependent PKC subfamily members PKC-alpha, -beta, and -gamma comigrated at 82 kDa and were distinguished by isozyme-specific immunoprecipitation. N-Biotinyl-RRRCLRRL covalently labeled all of the isozymes examined. When the isozymes were denatured prior to incubation with the N-biotinylated peptides, no labeling was observed. Inactivation of the Ca2+-dependent PKC subfamily by the N-biotinylated peptides was associated with covalent labeling of the 82-kDa PKC subspecies. The concentration dependence curves observed with N-biotinyl-RRRCLRRL were similar for inactivation and covalent labeling. The rank order of potency of three N-biotinylated peptides was the same for the inactivation and covalent labeling. Both the inactivation and covalent labeling were dithiothreitol-sensitive, and they were each subject to protection by MgATP and a peptide substrate analog. The covalent label was mapped to the catalytic domain of PKC by limited proteolysis of the modified enzyme. These results provide evidence that the N-biotinylated inactivator peptides are active-site affinity labels of PKC. The inactivator peptides most likely function by S-thiolating the active-site Cys residue conserved in PKC. This is the first report to demonstrate covalent labeling of PKC by a peptide substrate analog.

Animals↗

PubMed1997

Structural properties of chimeric peptides containing a T-cell epitope linked to a fusion peptide and their importance for in vivo induction of cytotoxic T-cell responses.

We have previously shown that when administered to mice without adjuvant, a chimeric peptide consisting of the fusion peptide F from measles virus protein linked at the C-terminus of a cytotoxic T-cell epitope from the M2 protein of respiratory syncytial virus efficiently primes for an major histocompatibility complex (MHC) class-I restricted cytotoxic T lymphocyte (CTL) response. In this report, we demonstrated by microspectrofluorometry that the fusion-peptide moiety bound to the plasma membrane of living cells. When the fusion peptide was linked to the C-terminus of the CTL epitope, the chimeric peptide (M2-F) adopted a marked beta-sheet conformation. In contrast, when the fusion peptide was linked to the N-terminus of the T-cell epitope (F-M2), the chimeric peptide adopted an alpha-helical conformation in the presence of trifluoroethanol. The immunogenicity of the two chimeric peptides for class-I restricted CTL was also significantly different, the one adopting the alpha-helical conformation being more immunogenic. Probably due to its obvious conversion to an alpha-helical conformation, the F-M2 peptide could have a higher propensity to insert into membranes, as shown by microspectrofluorometry, with a resultant better immunogenicity than the M2-F peptide.

Amino Acid Sequence↗

PubMed1990

Peptides derived from the CDR3-homologous domain of the CD4 molecule are specific inhibitors of HIV-1 and SIV infection, virus-induced cell fusion, and postinfection viral transmission in vitro. Implications for the design of small peptide anti-HIV therapeutic agents.

Peptides 12-25 amino acids in length from the V1J1 region of the CD4 molecule (residues 1-120) were synthesized as randomly derivatized, deliberately derivatized, or pure peptide products, and tested for their ability to inhibit HIV-1-induced cell fusion, HIV-1 and SIV infection of CD4-positive human cells, HIV-1 envelope glycoprotein binding to the CD4 molecule, CD4-neutralizing antibody binding to the CD4 holoreceptor, and CD4-dependent cellular immune function in the mixed lymphocyte and cytotoxic T-cell bioassays. Only peptides derived from the complementarity-determining region 3 (CDR3)-homologous domain of CD4, in particular CD4(81-92) and CD4(81-101), were effective antiviral agents. Within the CD4(81-92) series, R-group derivatization of selective amino acid residues was an absolute requirement for biological activity. The prototype compound T1C4E5-tribenzyl-K10-acetyl-TYICEVEDQKEE inhibited HIV-1-induced cell fusion at 32 microM, HIV-1 infection of CEM-SS cells at 10 microM, SIV infection of CEM-174 cells at less than 125 microM, gp120/CD4 binding at 60 microM, and postinfection cell-mediated viral transmission at 10-15 microM. Compounds of identical structure and derivatization, but of altered primary sequence, were substantially less active, or without activity, in these assays. These data indicate that the effect of amino acid derivatization of the CD4(81-92) peptide was most likely restriction of the flexible underivatized peptide backbone to a conformation closely approximating that of the CDR3-homologous gp120 binding site of the native CD4 molecule. Peptide antiviral activity was specific, as judged by lack of cytotoxicity, lack of inhibition of HTLV-1-induced cell fusion, and lack of inhibition of CD4-dependent cellular immune function in vitro. Further derivatization of the prototype compound involving the production of cyclic congeners yielded peptides with submicromolar potency to block HIV-1 infection, strengthening the hypothesis that previous peptide derivations accomplished partial restriction of the conformation of CD4(81-92) to one favorable for interaction with gp120. Concentrations of the original prototype compound T1C4E5-tribenzyl-CD4(81-92) that inhibited infection in vitro more than 50% could be achieved for several hours by intravenous infusion in primates and were well-tolerated at these levels. The peptide was not efficacious to inhibit establishment of viral infection at these doses; however, peptide treatment did lower average viral antigenemia and delay the cumulative time to morbidity relative to the control group.

Acquired Immunodeficiency Syndrome↗

PubMed1998

Intranasal immunization with cytotoxic T-lymphocyte epitope peptide and mucosal adjuvant cholera toxin: selective augmentation of peptide-presenting dendritic cells in nasal mucosa-associated lymphoid tissue.

We previously reported that cholera toxin (CT) was required as a mucosal adjuvant for the induction of peptide-specific cytotoxic T lymphocytes (CTL) following intranasal immunization with CTL epitope peptides (A. Porgador et al., J. Immunol. 158:834-841, 1997). The present study was performed to identify the site and the antigen-presenting cell (APC) population responsible for the presentation of intranasally administered CTL epitope peptide immunogens and to determine whether CT directly affects antigen presentation by these APCs. For these experiments, C57BL/6 mice were intranasally immunized with the ovalbumin H-2Kb-restricted CTL epitope SIINFEKL with or without CT. Cells were then isolated from the cervical lymph nodes (CLN) and the nasal mucosa-associated lymphoid tissue (NALT) and tested for the ability to stimulate the B3Z T-cell hybridoma, which recognizes SIINFEKL in association with H-2Kb. Dendritic cell (DC)-enriched CLN cells from mice immunized with peptide and CT or peptide only could stimulate B3Z cells, while DC-depleted CLN cells from either group were unable to stimulate B3Z cells. NALT cells of mice immunized with peptide and CT, but not with peptide alone, were able to efficiently stimulate B3Z hybridomas. Depletion of N418-positive DC from these NALT cells resulted in significant reduction of B3Z activation. Our results indicate that DC are the APC responsible for the presentation of CTL epitope peptides following intranasal immunization and that CT augments the ability of dendritic cells in the NALT, but not in the draining CLN, to present CLT epitope peptides. This finding suggests that CT acts locally as a mucosal adjuvant and that NALT DC are the predominant APC involved with the induction of immunity after intranasal immunization with peptide immunogens and CT.

Adjuvants, Immunologic↗

PubMed1993

Structural requirements of Bacillus subtilis alpha-amylase signal peptide for efficient processing: in vivo pulse-chase experiments with mutant signal peptides.

The Bacillus subtilis alpha-amylase signal peptide consists of 33 amino acids from its translation initiation site. To analyze the structural requirements for efficient processing of the signal peptide, single and repeated Ala-X-Ala sequences and their modifications were introduced into B. subtilis alpha-amylase signal peptides of different lengths and the mature thermostable alpha-amylase. Then the cleavage positions and processing rates of the signal peptides were analyzed by the NH2-terminal amino acid sequences of the exported thermostable alpha-amylases and by in vivo pulse-chase experiments. In B. subtilis, the most efficient cleavage site was located at the peptide bond between Ala-33 and amino acid X at position 34, even though Val-X-Ala and six repeating Ala-X-Ala sequences were present around the cleavage site. However, the cleavage site was shifted to the peptide bond between Ala-31 and amino acid X when Ala-33 was deleted, and it was also shifted to Ala-35 and X when Ala-33 was replaced with Val-33. The shorter signal peptide consisting of 31 amino acids reduced the processing rate and alpha-amylase production. In contrast, those signal peptides were cleaved preferentially at the peptide bond between Ala-31 and amino acid X in Escherichia coli. In addition to the presence of an Ala residue at the -1 amino acid position, the length of the signal peptide was another important requirement for efficient processing.

Alanine↗