The action of fish peptide Orpotrin analogs on microcirculation

In order to investigate the relationship between the primary structure of Orpotrin, a vasoactive peptide previously isolated from the freshwater stingray Potamotrygon gr. orbignyi, and its microcirculatory effects, three Orpotrin analogs were synthesized. The analogs have a truncated N‐terminal with a His residue deletion and two substituted amino acid residues, where one Nle is substituted for one internal Lys residue and the third analog has a substitution of a Pro for an Ala (Orp‐desH¹, Orp‐Nle and Orp‐Pro/Ala, respectively). Only Orp‐desH¹ could induce a lower vasoconstriction effect compared with the natural Orpotrin, indicating that besides the N‐terminal, the positive charge of Lys and the Pro residues located at the center of the amino acid chain is crucial for this vasoconstriction effect. Importantly, the suggestions made with bioactive peptides were based on the molecular modeling and dynamics of peptides, the presence of key amino acids and shared activity in microcirculation, characterized by intravital microscopy. Moreover, this study has demonstrated that even subtle changes in the primary structure of Orpotrin alter the biological effects of this native peptide significantly, which could be of interest for biotechnological applications. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Relaxin-like gonad-stimulating peptide is highly conserved in starfish Asterina pectinifera

Relaxin-like gonad-stimulating peptide (RGP) in starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to gonadotropins in vertebrates. Recently, RGP was purified from the radial nerves of starfish Asterina pectinifera, which belongs to the Order Valvatida in the Class Asteroidea. A. pectinifera is an endemic Japanese species, inhabiting rocky shores from northern to southern Japanese waters. This study examined whether genetic variation or polymorphism is found in RGP. Comparing cDNA sequences of RGP in A. pectinifera from 10 local populations in Japanese waters, we found that the coding DNA sequences (CDSs) were exactly the same. This result indicated that RGP is a highly conserved peptide in A. pectinifera. Furthermore, the CDS of RGP identified in Certonardoa semiregularis, which also belongs to Order Valvatida, was completely consistent with that of A. pectinifera. Thus, this also suggested that the chemical structure of A. pectinifera RGP is conserved among starfish of the Order Valvatida beyond species.     

Identification and bioactivity evaluation of a novel bradykinin inhibitory peptide from the skin secretion of Chinese large odorous frog,Odorrana livida

A novel peptide was isolated from the skin secretion of Chinese large odorous frog, Odorrana livida, and was named as Rana‐BI. The cDNA sequencing was obtained by ‘shotgun’ cloning. The amino acid sequence of the mature peptide was identified as Gly‐Leu‐Leu‐Ser‐Gly‐Lys‐Ser‐Val‐Lys‐Gly‐Ser‐Ile‐OH by automated Edman degradation, and the molecular weight of the peptide was confirmed to be 1144.68 Da by MALDI‐TOF and liquid chromatography/MS. Subsequently, the bioactivity of synthetic peptide was evaluated by smooth muscle assay using isolated rat bladder preparation. It was demonstrated that Rana‐BI inhibited the contraction of rat bladder induced by bradykinin. Comparing with other peptides by searching from database, the primary structure of Rana‐BI showed high similarity with that of an antimicrobial peptide of Rana family (12/12 residues). These data revealed a novel biological function of this peptide. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis,conformational study,and spectroscopic characterization of the cyclic Cα,α‐disubstituted glycine 9‐amino‐9‐fluorenecarboxylic acid

A series of terminally blocked peptides (to the pentamer level) from l ‐Ala and the cyclic C^α,α‐disubstituted Gly residue Afc and one Gly/Afc dipeptide have been synthesized by solution method and fully characterized. The molecular structure of the amino acid derivative Boc‐Afc‐OMe and the dipeptide Boc‐Afc‐Gly‐OMe were determined in the crystal state by X‐ray diffraction. In addition, the preferred conformation of all of the model peptides was assessed in deuterochloroform solution by FT‐IR absorption and ¹H‐NMR. The experimental data favour the conclusion that the Afc residue tends to adopt either the fully‐extended (C₅) or a folded/helical structure. In particular, the former conformation is highly populated in solution and is also that found in the crystal state in the two compounds investigated. A comparison with the structural propensities of the strictly related C^α,α‐disubstituted Gly residues Ac₅c and Dϕg is made and the implications for the use of the Afc residue in conformationally constrained analogues of bioactive peptides are briefly examined. A spectroscopic (UV absorption, fluorescence, CD) characterization of this novel aromatic C^α,α‐disubstituted Gly residue is also reported. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Dragonfly Erythemis simplicicollis contains a novel adipokinetic neuropeptide

We have isolated a novel member of the adipokinetic hormone family of peptides from a methanolic extract of corpora cardiaca of the libellulid dragonfly Erythemis simplicicollis by using a single‐step reversed‐phase high performance liquid chromatography method and monitoring biological activity in various heterologous bioassays and a homologous one. The sequence, as determined by Edman degradation and mass spectrometry, was of an uncharged blocked octapeptide: pGlu‐Leu‐Asn‐Phe‐Thr‐Pro‐Ser‐Trp amide. The structure was confirmed by chemical synthesis. The synthetic peptide increased hemolymph lipids in the dragonfly and was active in another libellulid (Orthetrum julia‐falsum) as well, but to a lesser extent than the conspecific peptide Lia‐AKH, which is an isoform of the novel peptide differing by a Val (instead of Leu) at position 2. Since lipids are apparently used as substrate for muscle contraction during flight of Erythemis simplicicollis and the native peptide induces lipid mobilization, this novel peptide is denoted Ers‐AKH. Arch. Insect Biochem. Physiol. 40:99–106, 1999. © 1999 Wiley‐Liss, Inc.     

Examination of the active secondary structure of the peptide 101.10, an allosteric modulator of the interleukin‐1 receptor,by positional scanning using β‐amino γ‐lactams

The relationship between the conformation and biological activity of the peptide allosteric modulator of the interleukin‐1 receptor 101.10 (D ‐Arg‐D ‐Tyr‐D ‐Thr‐D ‐Val‐D ‐Glu‐D ‐Leu‐D ‐Ala‐NH₂) has been studied using (R)‐ and (S)‐Bgl residues. Twelve Bgl peptides were synthesized using (R)‐ and (S)‐cyclic sulfamidate reagents derived from L ‐ and D ‐aspartic acid in an optimized Fmoc‐compatible protocol for efficient lactam installment onto the supported peptide resin. Examination of these (R)‐ and (S)‐Bgl 101.10 analogs for their potential to inhibit IL‐1β‐induced thymocyte cell proliferation using a novel fluorescence assay revealed that certain analogs exhibited retained and improved potency relative to the parent peptide 101.10. In light of previous reports that Bgl residues may stabilize type II′β‐turn‐like conformations in peptides, CD spectroscopy was performed on selected compounds to identify secondary structure necessary for peptide biological activity. Results indicate that the presence of a fold about the central residues of the parent peptide may be important for activity. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

A gonad-stimulating peptide of the crown-of-thorns starfish,Acanthaster planci

The crown-of thorns starfish, Acanthaster planci, has been blamed for coral mortality in a large number of coral reef systems in the Indo-Pacific region. Because population outbreaks of A. planci are closely related to reproduction, it is important to examine the mechanism of reproductive control in this starfish. Previously, a relaxin-like gonad-stimulating peptide (RGP) in starfish Asterina pectinifera has been identified as the gonadotropin responsible for final gamete maturation. On the basis of homology research on RGP cDNAs from several species, this study was carried out to identify gonadotropin in A. planci. The cDNA sequence of RGP was determined using a RACE product of mRNA from the radial nerves of A. planci. The coding DNA sequence consisted of 351 base pairs with an open reading frame encoding a peptide of 116 amino acids (aa), including a signal peptide (29 aa), B-chain (19 aa), C-peptide (44 aa), and A-chain (24 aa). The chemical structure of A. planci RGP was exactly the same as that of A. pectinifera RGP. Furthermore, synthetic RGP could induce gamete spawning and oocyte maturation in the ovarian fragments of A. planci. This strongly suggested that the RGP is a gonadotropin in A. planci.     

Applying Proteolytic Enzymes on Soybean

The soybean cold-insoluble protein was hydrolyzed with pepsin and the hydrolyzate was dialyzed. The diffusate was submitted to gel permeation chromatography of Sephadex G-10 and thin layer chromatography of silica gel G. A ninhydrin-negative bitter peptide was detected by chlorine-starch-iodine test and was purified by paper electrophoresis and by rechromatography on the silica gel. Azeotropic HCl hydrolysis of the peptide gave equal molar ratio of Ala, Glu, Gly, Ile, Leu, Phe, Ser and Val. N-Terminal residue was composed of pyrrolidone carboxylic acid, which was tentatively identified in comparison with the authentic sample by paper electrophoresis and thin layer chromatography. Hydrazinolysis of the peptide, followed by the 2,4-dinitrophenylation, produced α,α,γ-tri-DNP-glutamic acid-α,γ-dihydrazide and DNP-pyrrolidone carboxylic acid hydrazide, also supporting the N-terminal structure. Hydrolysis of the peptide by carboxypeptidase A gave Leu, Val, Phe, Ile, Ala, etc. in the order of the liberation rate. As the peptide fragments remaining in this hydrolyzate were detected pyrrolidone carboxyl-Gly·OH, pyrrolidone carboxyl-Gly-Ser·OH, pyrrolidone carboxyl-Gly-Ser-Ala·OH, etc. In conclusion, the structure of this peptide was proposed as: pyrrolidone carboxyl-Gly-Ser-Ala-Ile-Phe-Val-Leu·OH. Quantitative information about the total amount of pyrrolidone carboxyl residue contained in the peptic hydrolyzate of the soybean protein and sensory study on the bitterness of the peptide as referred by standard solution of phenylthiourea, were demonstrated.     

Peptide actions on oviduct contractions in the large pine weevil,Hylobius abietis

Abstract The peptides proctolin, crustacean cardioactive peptide (CCAP) and FMRFamide, which are known to modulate insect muscle contractions, were assayed for their action on oviduct contractions in Hylobius abietis. A video microscopy technique and computer‐based method of data acquisition and analysis were used to investigate the effects of theses peptides on spontaneous contractions of continuously perfused oviducts. All three peptides tested stimulate spontaneous contraction activity of the pine weevil oviduct, increasing the frequency and amplitude of phasic contractions in a dose‐dependent manner. Proctolin is more potent as a stimulator than CCAP. For proctolin a threshold response of oviduct muscles is at concentration of peptide 10^?11–10^?10 mol/L and for CCAP at concentration range 10^?10–10^?9 mol/L. FMRFamide exerts a weak stimulatory effect on the oviduct, and at higher concentrations of the peptide (above 10^?8 mol/L). The peptides exert different responses on oviduct contractions and they may play a role as functional regulators in such processes as egg movement and oviposition.     

Synthesis and receptor binding of opioid peptide analogues containing β3‐homo‐amino acids

β‐Amino acids containing hybrid peptides and β‐peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the µ‐ and δ‐opioid receptors of β‐peptides, analogues of Leu‐enkephalin, deltorphin I, dermorphin and α,β‐hybrides, analogues of deltorphin I. Substitution of α‐amino acid residues with β³‐homo‐amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation β³h‐D ‐Ala in position 2 or β³hPhe in position 3 of deltorphin I resulted in potent and selective ligand for δ‐opioid receptor. The NMR studies of β‐deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

New pemetrexed‐peptide conjugates: synthesis,characterization and in vitro cytostatic effect on non‐small cell lung carcinoma (NCI‐H358) and human leukemia (HL‐60) cells

Pemetrexed (Pem) is a novel antimetabolite type of anticancer drug that demonstrated promising clinical activity in a wide variety of solid tumors, including non‐small cell lung carcinoma and malignant pleural mesothelioma. It inhibits enzymes involved in the folate pathway, for which the presence of its free carboxylic groups is necessary. The heteroaromatic ring system of Pem has a modifiable amino group, which opens a possibility to apply a new strategy to conjugate Pem to carrier molecules. Considering this as well as the necessity of untouched carboxylic groups of Pem in the new conjugates, we developed a new synthesis strategy. Here, we describe the synthesis and the characterization of new Pem‐peptide conjugates in which cell‐penetrating octaarginine or/and lung‐targeting H‐Ile‐Glu‐Leu‐Leu‐Gln‐Ala‐Arg‐NH₂ peptide is attached to the drug by thioether bond. The conjugates characterized by RP‐HPLC and MS exhibited cytostatic effect in vitro on non‐small cell lung carcinoma as well as on human leukemia cell lines. The IC₅₀ values of the conjugates were similar, but the conjugates with H‐Ile‐Glu‐Leu‐Leu‐Gln‐Ala‐Arg‐NH₂ sequence were slightly more effective. Our data show that the in vitro cytostatic effect of the free Pem was essentially maintained after conjugation with cell‐penetrating or cell‐targeting peptides. Thus, the conjugation strategy reported could lead to the development of a new generation of active Pem conjugates. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

De novo design,synthesis and solution conformational study of two didehydroundecapeptides: effect of nature and number of amino acids interspersed between Phe residues

De novo design of peptides and proteins has recently surfaced as an approach for investigating protein structure and function. This approach vitally tests our knowledge of protein folding and function, while also laying the groundwork for the fabrication of proteins with properties not precedented in nature. The success relies heavily on the ability to design relatively short peptides that can espouse stable secondary structures. To this end, substitution with α,β‐didehydroamino acids, especially α,β‐didehydrophenylalanine (Δ^zPhe), comes in use for spawning well‐defined structural motifs. Introduction of ΔPhe induces β‐bends in small and 3₁₀‐helices in longer peptide sequences. The present work aims to investigate the effect of nature and the number of amino acids interspersed between two ΔPhe residues in two model undecapeptides, Ac‐Gly‐Ala‐ΔPhe‐Ile‐Val‐ΔPhe‐Ile‐Val‐ΔPhe‐Ala‐Gly‐NH₂ (I) and Boc‐Val‐ΔPhe‐Phe‐Ala‐Phe‐ΔPhe‐Phe‐Leu‐Ala‐ΔPhe‐Gly‐OMe (II). Peptide I was synthesized using solid‐phase chemistry and characterized using circular dichroism spectroscopy. Peptide II was synthesized using solution‐phase chemistry and characterized using circular dichroism and nuclear magnetic resonance spectroscopy. Peptide I was designed to examine the effect of incorporating β‐strand‐favoring residues like valine and isoleucine as spacers between two ΔPhe residues on the final conformation of the resulting peptide. Circular dichroism studies on this peptide have shown the existence of a 3₁₀‐helical conformation. Peptide II possesses three amino acids as spacers between ΔPhe residues and has been reported to adopt a mixed 3₁₀/α‐helical conformation using circular dichroism and nuclear magnetic resonance spectroscopy studies. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Improved antimicrobial activity of h‐lysozyme (107–115) by rational Ala substitution

The most challenging target in the design of new antimicrobial agents is the development of antibiotic resistance. Antimicrobial peptides are good candidates as lead compounds for the development of novel anti‐infective drugs. Here we propose the sequential substitution of each Ala residue present in a lead peptide with known antimicrobial activity by specific amino acids, rationally chosen, that could enhance the activity of the resultant peptide. Taking the fragment 107–115 of the human lysozyme as lead, two‐round screening by sequentially replacing both Ala residues (108 and 111) by distinct amino acids resulted in a novel peptide with 4‐ and 20‐fold increased antimicrobial activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, respectively. These results reinforce the strategy proposed, which, in combination with simple and easy screening tools, will contribute to the rapid development of new therapeutic peptides required by the market. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

METHIONINE-ACTIVATING ENZYME IN STARFISH FOLLICLE CELLS *

In starfish follicle cells 1-methyladenine is produced under the influence of a gonad-stimulating hormonal peptide (GSS). Since such production of the substance is enhanced by the addition of L-methionine or S-adenosylmethionine in vitro, the presence of methionine-activating enzyme in the follicle cells of the starfish, Asterina pectinifera, was investigated. To detect enzyme activity, the enzyme was partially purified from the supernatant of the follicle-cell homogenate by precipitation with ammonium sulfate followed by gel-filtration on a Sephadex G-150 column. Using such a preparation of the enzyme, the production of S-adenosylmethionine from L-methionine and adenosine triphosphate was clearly demonstrated by thin-layer chromatography. GSS was found to exert no effect on the activity of the methionine-activating enzyme. The hormonal peptide, GSS, is therefore considered to take part in some reaction other than this step in the formation of 1-methyladenine.     

Structure–activity study of macropin,a novel antimicrobial peptide from the venom of solitary bee Macropis fulvipes (Hymenoptera: Melittidae)

A novel antimicrobial peptide, designated macropin (MAC‐1) with sequence Gly‐Phe‐Gly‐Met‐Ala‐Leu‐Lys‐Leu‐Leu‐Lys‐Lys‐Val‐Leu‐NH₂, was isolated from the venom of the solitary bee Macropis fulvipes. MAC‐1 exhibited antimicrobial activity against both Gram‐positive and Gram‐negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l ‐ or d ‐lysine in selected positions. Furthermore, all‐d analog and analogs with d ‐amino acid residues introduced at the N‐terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α‐helical secondary structure in the presence of trifluoroethanol or membrane‐mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure–activity relationship for the effect of d ‐amino acid substitutions in MAC‐1 using NMR spectroscopy. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Terminal effect of chiral residue on helical screw sense in achiral peptides

To understand the terminal effect of chiral residue for determining a helical screw sense, we adopted five kinds of peptides I – V containing N‐ and/or C‐terminal chiral Leu residue(s): Boc–L ‐Leu–(Aib–ΔPhe)₂–Aib–OMe ( I ), Boc–(Aib–ΔPhe)₂–L ‐Leu–OMe ( II ), Boc–L ‐Leu–(Aib–ΔPhe)₂–L ‐Leu–OMe ( III ), Boc–D ‐Leu–(Aib–ΔPhe)₂–L ‐Leu–OMe ( IV ), and Boc–D ‐Leu–(Aib–ΔPhe)₂–Aib–OMe ( V ). The segment –(Aib–ΔPhe)₂– was used for a backbone composed of two “enantiomeric” (left‐/right‐handed) helices. Actually, this could be confirmed by ¹H‐nmr [nuclear Overhauser effect (NOE) and solvent accessibility of NH resonances] and CD spectroscopy on Boc–(Aib–ΔPhe)₂–Aib–OMe, which took a left‐/right‐handed 3₁₀‐helix. Peptides I – V were also found to take 3₁₀‐type helical conformations in CDCl₃, from difference NOE measurement and solvent accessibility of NH resonances. Chloroform, acetonitrile, methanol, and tetrahydrofuran were used for CD measurement. The CD spectra of peptides I – III in all solvents showed marked exciton couplets with a positive peak at longer wavelengths, indicating that their main chains prefer a left‐handed screw sense over a right‐handed one. Peptide V in all solvents showed exciton couplets with a negative peak at longer wavelengths, indicating it prefers a right‐handed screw sense. Peptide IV in chloroform showed a nonsplit type CD pattern having only a small negative signal around 280 nm, meaning that left‐ and right‐handed helices should exist with almost the same content. In the other solvents, peptide IV showed exciton couplets with a negative peak at longer wavelengths, corresponding to a right‐handed screw sense. From conformational energy calculation and the above ¹H‐nmr studies, an N‐ or C‐terminal L ‐Leu residue in the lowest energy left‐handed 3₁₀‐helical conformation was found to take an irregular conformation that deviates from a left‐handed helix. The positional effect of the L ‐residue on helical screw sense was discussed based on CD data of peptides I – V and of Boc–(L ‐Leu–ΔPhe)_n–L ‐Leu–OMe (n = 2 and 3). © 1999 John Wiley & Sons, Inc. Biopoly 49: 551–564, 1999     

Total synthesis,structural, and biological evaluation of stylissatin A and related analogs

The natural product cyclic peptide stylissatin A ( 1a ) was reported to inhibit nitric oxide production in LPS‐stimulated murine macrophage RAW 264.7 cells. In the current study, solid‐phase total synthesis of stylissatin A was performed by using a safety‐catch linker and yielded the peptide with a trans‐Phe⁷‐Pro⁶ linkage, whereas the natural product is the cis rotamer at this position as evidenced by a marked difference in NMR chemical shifts. In order to preclude the possibility of 1b being an epimer of the natural product, we repeated the synthesis using d ‐allo‐Ile in place of l ‐Ile and a different site for macrocyclization. The resulting product (d ‐allo‐Ile²)‐stylissatin A ( 1c ) was also found to have the trans‐Phe⁷‐Pro⁶ peptide conformations like rotamer 1b . Applying the second route to the synthesis of stylissatin A itself, we obtained stylissatin A natural rotamer 1a accompanied by rotamer 1b as the major product. Rotamers 1a , 1b , and the epimer 1c were separable by HPLC, and 1a was found to match the natural product in structure and biological activity. Six related analogs 2–7 of stylissatin A were synthesized on Wang resin and characterized by spectral analysis. The natural product ( 1a ), the rotamer ( 1b ), and (d ‐allo‐Ile²)‐stylissatin A ( 1c ) exhibited significant inhibition of NO^.. Further investigations were focused on 1b , which also inhibited proliferation of T‐cells and inflammatory cytokine IL‐2 production. The analogs 2–7 weakly inhibited NO^. production, but strongly inhibited IL‐2 cytokine production compared with synthetic peptide 1b . All analogs inhibited the proliferation of T‐cells, with analog 7 having the strongest effect. In the analogs, the Pro⁶ residue was replaced by Glu/Ala, and the SAR indicates that the nature of this residue plays a role in the biological function of these peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

Alloreactive cytotoxic T-lymphocyte-defined HLA-B7 subtypes differ in peptide antigen presentation

We investigated T-cell-defined HLA-B7 subtypes using cDNA sequencing, analysis of bound peptides, and reactivity with a panel of alloreactive cytotoxic T-lymphocyte (CTL) clones. Three subtypes (HLA-B*0702, HLA-B*0703, and HLA-B*0705) differ in nucleotide and predicted amino acid sequence. CTL reactivity and pooled peptide sequencing show that these three HLA-B7 subtypes bind distinct but overlapping sets of peptides. In particular B*0702 expresses D pocket residue Asp 114 and binds peptides with P3 Arg, whereas B*0705 expresses D pocket residue Asn 114 and binds peptides with P3 Ala, Leu, and Met. Consistent with different peptide-binding specificities, three alloreactive CTL differentiate between cells expressing B*0702, B*0703, and B*0705 by detecting specific peptide/HLA-B7 complexes. In contrast, three other T-cell-defined HLA-B7 subtypes are identical to HLA-B*0702. The B*0702-expressing cell lines are differentiated by two of ten CTL clones. One CTL clone differentiates B*0702-expressing cells by their ability to present peptide antigen. Thus differences in peptide presentation can explain differential CTL recognition of cell lines expressing structurally identical and variant HLA-B7.