Styelin D, an extensively modified antimicrobial peptide from ascidian hemocytes

We isolated styelin D, a 32-residue, C-terminally amidated antimicrobial peptide, from the blood cells (hemocytes) of the solitary ascidian, Styela clava. Styelin D had remarkably extensive post-translational modifications, containing two novel amino acids, dihydroxyarginine and dihydroxylysine, and two distinctly unusual ones, 6-bromotryptophan and 3,4-dihydroxyphenylalanine. In addition, the peptide exhibited microheterogeneity because of differential mono- and dihydroxylation of several lysine residues. The primary sequence of one variant was: GW(*)LR(**)K(**)AAK(**)SVGK(**)FY(*)Y(*)K(**)HK(*)Y(*) Y(*)IK(*)AAWQIG KHAL-NH(2), where W(*) is 6-bromotryptophan, R(**) is dihydroxyarginine, Y(*) is 3,4-dihydroxyphenylalanine, K(*) is 5-hydroxylysine, and K(**) is dihydroxylysine. Styelin D exhibited activity against Gram-negative and Gram-positive bacteria, and this activity was retained in 200 mm NaCl. The role of the extensive modifications may be to preserve activity at low pH and/or high salinity because, under these conditions, the native peptide was considerably more active against the Gram-positive bacterial strains than its unmodified synthetic analogue. The peptide was also hemolytic and quite cytotoxic to eukaryotic cells. These broad ranging activities, combined with its relative abundance in ascidian hemocytes, suggest that styelin D plays a significant role in the innate immune mechanisms of S. clava.     

Structural and kinetic characterization of native laccases from Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii

A comparative study has been performed on five native laccases purified from the three basidiomycete fungi Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii to relate their different catalytic capacities to their structural properties. Spectroscopic absorption features and EPR spectra at various pH values of the five enzymes are very similar and typical of the blue oxidases. The analysis of the dependence of kinetic parameters on pH suggested that a histidine residue is involved in the binding of nonphenolic substrates, whereas both a histidine and an acidic residue may be involved in the binding of phenolic compounds. His and an Asp residue are indeed found at the bottom of a cavity which may be regarded as a suitable substrate channel for approaching to type 1 copper in the 3D homology models of the two laccases from Pleuorotus ostreatus (POXC and POXA1b) whose sequences are known.     

Control of Seed Protein Accumulation in Field Bean (Vicia faba L.)

A study was made of the changes during development in the totalamino acid and 3, 4 dihydroxyphenylalanine (DOPA) content ofbean pod phloem sap, employing EDTA to aid phloem exudation.Two field bean lines, Dacre B and D, selected for their lowand high seed protein content respectively, were compared. Throughoutdevelopment, the sap samples of Dacre D had a greater aminoacid concentration than those from Dacre B. The sap of DacreB contained a higher proportion of DOPA than that of Dacre D.These two lines of Dacre were also studied with respect to accumulationof protein and uncombined amino acid in cotyledons grown bothin vitro and in vivo. Dacre D accumulated more total proteinthan Dacre B but contained a similar amount of uncombined aminoacids when grown in vivo. However, the amount of total proteinaccumulated was similar when the cotyledons were grown in vitro.The data suggest that the supply of nutrients to the pod maybe the basis of the different protein concentrations in themature seed of these lines. Vicia faba L., field bean, phloem sap, cotyledon culture, amino acids, DOPA, protein     

Antimicrobial activity of histidine-rich peptides is dependent on acidic conditions

Synthetic peptides composed of multiples of the consensus heparin-binding Cardin and Weintraub sequences AKKARA and ARKKAAKA are antimicrobial. Replacement of lysine and arginine by histidine in these peptides completely abrogates their antimicrobial and heparin-binding activities at neutral pH. However, the antibacterial activity against Gram-negative (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) as well as the fungus Candida albicans, was restored at acidic conditions (pH 5.5). Fluorescence microscopy and FACS analysis showed that the binding of the histidine-rich peptides to E. coli and Candida was significantly enhanced at pH 5.5. Likewise, fluorescence studies for assessment of membrane permeation as well as electron microscopy analysis of peptide-treated bacteria, paired with studies of peptide effects on liposomes, demonstrated that the peptides induce membrane lysis only at acidic pH. No discernible hemolysis was noted for the histidine-rich peptides. Similar pH-dependent antimicrobial activities were demonstrated for peptides derived from histidine-rich and heparin-binding regions of human kininogen and histidine-rich glycoprotein. The results demonstrate that the presence of an acidic environment is an important regulator of the activity of histidine-rich antimicrobial peptides.     

Hydrogen Peroxide-Dependent Oxidation of 3,4-Dihydroxyphenylalanine in Vacuoles of Mesophyll Cells of Vicia faba L. Participation of Peroxidase in the Oxidation

Peroxidase activity and 3,4-dihydroxyphenylalanine (DOPA) werefound in vacuoles isolated from mesophyll protoplasts of Viciafaba L. A peroxidase isozyme localized in vacuoles migratedto the cathode during electrophoresis at pH 8.7, indicatingthat the vacuole peroxidase was a basic isozyme. When isolatedvacuoles were treated with 2 mM H₂O₂, dopachrome, a productof oxidation of DOPA, was formed in a reaction that was inhibitedby KCN and NaN₃. These results suggest that DOPA can serve asa donor of electrons to the peroxidase in vacuoles. (Received December 25, 1989; Accepted March 22, 1990)     

PPEF: A bisbenzimdazole potent antimicrobial agent interacts at acidic triad of catalytic domain of E. coli topoisomerase IA

BackgroundTopoisomerase is a well known target to develop effective antibacterial agents. In pursuance of searching novel antibacterial agents, we have established a novel bisbenzimidazole (PPEF) as potent E. coli topoisomerase IA poison inhibitor.MethodsIn order to gain insights into the mechanism of action of PPEF and understanding protein-ligand interactions, we have produced wild type EcTopo 67 N-terminal domain (catalytic domain) and its six mutant proteins at acidic triad (D111, D113, E115). The DDE motif is replaced by alanine (A) to create three single mutants: D111A, D113A, E115A and three double mutants: D111A-D113A, D113A-E115A and D111A-E115A.ResultsCalorimetric study of PPEF with single mutants showed 10 fold lower affinity than that of wild type EcTopo 67 (7.32 × 10⁶ M⁻¹for wild type, 0.89 × 10⁶ M⁻¹for D111A) and 100 fold lower binding with double mutant D113A-E115A (0.02 × 10⁶ M⁻¹) was observed. The mutated proteins showed different CD signature as compared to wild type protein. CD and fluorescence titrations were done to study the interaction between EcTopo 67 and ligands. Molecular docking study validated that PPEF has decreased binding affinity towards mutated enzymes as compared to wild type.ConclusionThe overall study reveals that PPEF binds to D113 and E115 of acidic triad of EcTopo 67. Point mutations decrease binding affinity of PPEF towards DDE motif of topoisomerase.General significanceThis study concludes PPEF as poison inhibitor of E. coli Topoisomerase IA, which binds to acidic triad of topoisomerase IA, responsible for its function. PPEF can be considered as therapeutic agent against bacteria.     

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.     

Halocyntin and papillosin,two new antimicrobial peptides isolated from hemocytes of the solitary tunicate,Halocynthia papillosa

We report here the screening of five marine invertebrate species from two taxa (tunicates and echinoderms) for the presence of cationic antimicrobial peptides (AMP) in defence cells (hemocytes). Antimicrobial activities were detected only in the two tunicates Microcosmus sabatieri and Halocynthia papillosa. In addition, we report the isolation and characterization of two novel peptides from H. papillosa hemocytes. These molecules display antibacterial activity against Gram‐positive and Gram‐negative bacteria. Complete peptide characterization was obtained by a combination of Edman degradation and mass spectrometry. The mature molecules, named halocyntin and papillosin, comprise 26 and 34 amino acid residues, respectively. Their primary structure display no significant similarities with previously described AMP. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

两种金小蜂毒液对菜粉蝶蛹血细胞延展、存活及包囊作用的影响

活体微注射测定结果表明,将0.5毒囊当量（venom reservoir equivalent, VRE）的蝶蛹金小蜂毒液注射于其寄主菜粉蝶蛹体内,注射后4～24 h寄主浆血细胞和颗粒血细胞的延展、存活和对Sephadex A50微珠的包囊能力显著下降;以0.002～0.02 VRE/μL的该蜂毒液处理其离体寄主血细胞均能产生同样的生理效应。该毒液抑制90%菜粉蝶蛹浆血细胞和颗粒血细胞延展的浓度各为0.00076 VRE/μL和0.00804 VRE/μL。可见,蝶蛹金小蜂毒液能显著抑制其寄主血细胞的延展和包囊作用,并导致血细胞的死亡。然而,同样条件下丽蝇蛹集金小蜂毒液对其非自然寄主菜粉蝶蛹的血细胞延展、存活和包囊作用则无任何效应。可见,寄生蜂毒液的生理作用具有明显的寄主特异性。     

A new Dol-P-Man:protein O-D-mannosyltransferase activity from Saccharomyces cerevisiae

The deletion of the protein mannosyltransferase 1 gene (PMT1)of Saccharomyces cerevisiae results in viable cells. O-Mannosylationof proteins is reduced to about half of the value in comparisonto wild-type cells. In order to distinguish between the thePMT1 gene product (= Pmt1p) and residual transferase activity,an in vitro assay to measure Dol-P-Man:protein mannosyltransferaseactivity in cells deleted for PMT1 has been developed. The transferaseactivity of these cells exhibits a pH optimum of 6.5 as comparedto pH 7.5 for Pmt1p. The K_$$$ value of the residual enzyme activityfor the hexapeptide YNPTSV is 7 times higher than that of Pmt1pand shows a clear preference for the seryl/residue. Differencesin substrate affinities as well as in seryl/threonyl preferencebetween the two enzymes, however, depend on the specific sequenceof the peptides used in the enzyme assay. The new enzyme activityshows a significantly lower thermal stability as compared toPmt1p. glycoprotein O-glycosylation mannosyltranferase Saccharomyces cerevisiae     

Neuropepride Regulators of Insect Corpora Allata

SYNOPSIS. Neuropeptides of the insect brain that regulate juvenilehormone synthesis by the corpora allata include allatotropins,stimulatory modulators, and allatostatins, inhibitory modulators.A radiochemical assay for juvenile hormone synthesis by corporaallata in vitro was utilized in the high pressure liquid chromatographicisolation of brain neuropeptides leading to the determinationof their primary structure. Identified are an allatotropin andan allatostatin from a Lepidopteran, Manduca sexta, and a familyof five allatostatins from a Dictyopteran, Diploptera punctata.These neuropeptides are all unique, effective at low concentration(10^–10 to 10^–8 M), act quickly (within hrs) andappear to be effective only within the same order of insectsas that from which the peptides were isolated. The physiologicalstate of the corpora allata conditions the effectiveness ofthe allatostatins of D. punctata. These neuropeptide regulatorsof corpora allatal function may have multiple regulatory roles.This is indicated for D. punctata allatostatin I by specificreceptors in brain and fat body as well as in corpora allatalmembrane preparations, and also by immunocytochemical localizationof allatostatin I in medial nerve cells that terminate withinthe brain as well as in the lateral neurosecretory cells thatterminate on corpus allatum cells.     

The long‐term immunological effects of alloferon and its analogues in the mealworm Tenebrio molitor

The subject of this article is a search for the long‐term immunological effects of alloferon and 3 structural analogues of alloferon, which were earlier characterized by the highest pro‐apoptotic activity in Tenebrio molitor. The differences in the actions of these peptides on immune response were observed. Alloferon increased nodulation and significantly phenoloxidase activity in the hemolymph of experimentally infected T. molitor. However, [Phe(p‐NH₂)¹]‐ and [Phe(p‐OMe)¹]‐alloferon strongly inhibited cellular and humoral defense of the mealworm against Staphylococcus aureus infection. One day after injection of these peptides, the specific biochemical and morphological hallmarks of apoptosis in bacteria‐challenged hemocytes were visible; in contrast, 3 days after peptides injection in all hemocytes, caspase activation was not observed. However, these new, circulating hemocytes differed from the control and the peptide‐untreated bacteria‐challenged hemocytes. They had an increased adhesion that led to a separation of viable, anucleated fragments of hemocytes that retain the ability to adhere and to form long filopodia. The peptide‐induced separation of hemocyte fragments may resemble the formation of platelets in mammals and perhaps play a role in sealing wounds in insects. The results of in vivo studies may suggest a long half‐life of studied peptides in the hemolymph of mealworm. Moreover, we showed the importance of the N‐terminal histidine residues at position one of the alloferon molecule for its immunological properties in insects. The results obtained here show that alloferon plays pleiotropic functions in insects.     

Two Goose-Type Lysozymes in Mytilus galloprovincialis: Possible Function Diversification and Adaptive Evolution

Two goose-type lysozymes (designated as MGgLYZ1 and MGgLYZ2) were identified from the mussel Mytilus galloprovincialis. MGgLYZ1 mRNA was widely expressed in the examined tissues and responded sensitively to bacterial challenge in hemocytes, while MGgLYZ2 mRNA was predominately expressed and performed its functions in hepatopancreas. However, immunolocalization analysis showed that both these lysozymes were expressed in all examined tissues with the exception of adductor muscle. Recombinant MGgLYZ1 and MGgLYZ2 could inhibit the growth of several Gram-positive and Gram-negative bacteria, and they both showed the highest activity against Pseudomonas putida with the minimum inhibitory concentration (MIC) of 0.95–1.91 µM and 1.20–2.40 µM, respectively. Protein sequences analysis revealed that MGgLYZ2 had lower isoelectric point and less protease cutting sites than MGgLYZ1. Recombinant MGgLYZ2 exhibited relative high activity at acidic pH of 4–5, while MGgLYZ1 have an optimum pH of 6. These results indicated MGgLYZ2 adapted to acidic environment and perhaps play an important role in digestion. Genomic structure analysis suggested that both MGgLYZ1 and MGgLYZ2 genes are composed of six exons with same length and five introns, indicating these genes were conserved and might originate from gene duplication during the evolution. Selection pressure analysis showed that MGgLYZ1 was under nearly neutral selection while MGgLYZ2 evolved under positive selection pressure with three positively selected amino acid residues (Y¹⁰², L²⁰⁰ and S²⁰²) detected in the mature peptide. All these findings suggested MGgLYZ2 perhaps served as a digestive lysozyme under positive selection pressure during the evolution while MGgLYZ1 was mainly involved in innate immune responses.     

pH-dependent membrane fusion and vesiculation of phospholipid large unilamellar vesicles induced by amphiphilic anionic and cationic peptides.

We studied fusion induced by a 20-amino acid peptide derived from the amino-terminal segment of hemagglutinin of influenza virus A/PR/8/34 [Murata, M., Sugahara, Y., Takahashi, S., & Ohnishi, S. (1987) J. Biochem. (Tokyo) 102, 957-962]. To extend the study, we have prepared several water-soluble amphiphilic peptides derived from the HA peptide; the anionic peptides D4, E5, and E5L contain four and five acidic residues and the cationic peptide K5 has five Lys residues in place of the five Glu residues in E5. Fusion of egg phosphatidylcholine large unilamellar vesicles induced by these peptides is assayed by two different fluorescence methods, lipid mixing and internal content mixing. Fusion is rapid in the initial stage (12-15% within 20 s) and remains nearly the same or slightly increasing afterward. The anionic peptides cause fusion at acidic pH lower than 6.0-6.5, and the cationic peptide causes fusion at alkaline pH higher than 9.0. Leakage and vesiculation of vesicles are also measured. These peptides are bound and associated with vesicles as shown by Ficoll discontinuous gradients and by the blue shift of tryptophan fluorescence. They take an alpha-helical structure in the presence of vesicles. They become more hydrophobic in the pH regions for fusion. When the suspension is made acidic or alkaline, the vesicles aggregate, as shown by the increase in light scattering. The fusion mechanism suggests that the amphiphilic peptides become more hydrophobic by neutralization due to protonation of the carboxyl groups or deprotonation of the lysyl amino groups, aggregate the vesicles together, and interact strongly with lipid bilayers to cause fusion. At higher peptide concentrations, E5 and E5L cause fusion transiently at acidic pH followed by vesiculation.     

An Experimental Study of Calcium Acquisition and its Effects on the Calcifuge Moss Pleurozium schreberi

In a field experiment to investigate the sources and effectson growth of Ca in the calcifuge moss Pleurozium schreberi,significant quantities of Ca reached the growing shoot apicesfrom a CaCO₃ layer placed on the mineral soil surface Top applicationsof 0.5 and 5 mol m^–3 CaCl₂ raised the exchangeable andintracellular Ca concentrations and displaced natural exchangeableK and Mg The 5 mol m^–3 CaCl₂ treatment also caused a significantreduction in intracellular Mg indicating that Mg uptake is dependenton an initial exchange step No growth differences were notedbetween treatments, possibly because ionic changes had not reacheda detrimental level within the 28 weeks of the experiment ina second experiment, shoot apices of Pleurozium schreberi, Pseudoscleropodiumpurum and Calliergon cuspidatum were grown on nylon gauze underintermittent distilled-water mist At weekly intervals the shootswere saturated with CaCl₂ solutions providing factorial combinationsof Ca and pH Growth of C cuspidatum and P purum from chalk soilwas reduced at high (0.01) Ca concentration whereas Pleuroziumschreberi and Pseudoscleropodium purum from acidic clay wereunaffected The pH treatments did not significantly affect mossgrowth Initial tissue levels of K and Mg were lower in the mossesfrom chalk and it is suggested that the CaCl₂ treatments causednutrient deficiencies in these plants Mosses from acidic soilcontained less exchangeable Ca than the chalk plants and grewpoorly in the absence of CaCl₂, perhaps due to the developmentof Ca deficiency Bryophyte growth, calcium uptake, pH, mineral nutrition, Pleurozium schreberi, Pseudoscleropodium purum, Calliergon cuspidatum     

影响叶螨磷酸酯酶活性的四因子数学模型

应用二次回归通用旋转组合设计,组建了影响叶螨磷酸酯酶（酸性和碱性）活性的四因子（缓冲液Ph值X₁、温浴时间X₂、反应温度X₃、底物浓度X₄）数学模型: Y_酸性=0.456380+0.107889X₂+0.069027X₃-0.026836X₁²-0.030794X₃², F=24.98,P<0.01;Y_碱性=0.267286-0.200736X₁+0.049541X₂+0.030930X₃-.049063X₁X₂+0.053585X₁²-0.049665X₂^2, F=57.68,P<0.01。结果表明,温浴时间是影响叶螨酸性磷酸酯酶活性的关键因子,在缓冲液pH 4.4、底物浓度8.5×10^-3 mol/L、42℃温浴40 min测得该酶活性最强。影响碱性磷酸酯酶活性的关键因子则是缓冲液pH值,pH 9.0、37℃恒温30 min、底物7.5×10^-3 mol/L的条件下,光密度值最大。两种酶的最大吸收峰波长为405 nm。     

Formation of filamentous aerobic granules: role of pH and mechanism

Filamentous overgrowth in aerobic granular sludge processes can cause reactor failure. In this work, aerobic granules were cultivated in five identical sequencing batch reactors with acetate or glucose as the carbon source with various values of influent pH (4.5–8). Microscopic observations revealed that acidic pH, rather than the species of carbon source, epistatically controls the aerobic granules with filamentous structure. An acidic pH shifted the structure of the microbial community in the granules, such that the fungus Geotrichum fragrans was the predominant filamentous microorganism therein. The acidic pH reduced the intracellular cyclic diguanylate (c-di-GMP) content for increasing the motility of the bacteria to washout and increase the growth rate of G. fragrans on glucose or acetate, together causing overgrowth of the fungus. Maintaining the suspension under alkaline condition is proposed as an effective way to suppress filamentous overgrowth and maintain granule stability.     

Fibril formation of hsp10 homologue proteins and determination of fibril core regions: differences in fibril core regions dependent on subtle differences in amino acid sequence

Heat shock protein 10 (hsp10) is a member of the molecular chaperones and works with hsp60 in mediating various protein folding reactions. GroES is a representative protein of hsp10 from Escherichia coli. Recently, we found that GroES formed a typical amyloid fibril from a guanidine hydrochloride (Gdn-HCl) unfolded state at neutral pH. Here, we report that other hsp10 homologues, such as human hsp10 (Hhsp10), rat mitochondrial hsp10 (Rhsp10), Gp31 from T4 phage, and hsp10 from the hyperthermophilic bacteria Thermotoga maritima, also form amyloid fibrils from an unfolded state. Interestingly, whereas GroES formed fibrils from either the Gdn-HCl unfolded state (at neutral pH) or the acidic unfolded state (at pH 2.0-3.0), Hhsp10, Rhsp10, and Gp31 formed fibrils from only the acidic unfolded state. Core peptide regions of these protein fibrils were determined by proteolysis treatment followed by a combination of Edman degradation and mass spectroscopy analyses of the protease-resistant peptides. The core peptides of GroES fibrils were identical for fibrils formed from the Gdn-HCl unfolded state and those formed from the acidic unfolded state. However, a peptide with a different sequence was isolated from fibrils of Hhsp10 and Rhsp10. With the use of synthesized peptides of the determined core regions, it was also confirmed that the identified regions were capable of fibril formation. These findings suggested that GroES homologues formed typical amyloid fibrils under acidic unfolding conditions but that the fibril core structures were different, perhaps owing to differences in local amino acid sequences.     

Modifications on amphiphilicity and cationicity of unnatural amino acid containing peptides for the improvement of antimicrobial activity against pathogenic bacteria

The widespread natural sources‐derived cationic peptides have been reported to reveal bacterial killing and/or growth‐inhibiting properties. Correspondingly, a number of artificial peptides have been designed to understand antibacterial mechanism of the cationic peptides. These peptides are expected to be an alternative antibiotic against drug‐resistant pathogenic bacteria because major antimicrobial mechanism of cationic peptides involves bacterial membrane disorder, although those availabilities have not been well evaluated. In this study, cationic peptides containing Aib were prepared to evaluate the availability as an antimicrobial agent, especially against representative pathogenic bacteria. Among them, BRBA20, consisting of five repeated Aib‐Arg‐Aib‐Ala sequences, showed strong antibacterial activity against both Gram‐negative and Gram‐positive bacteria, including methicillin‐resistant Staphylococcus aureus. Additionally, growth of Serratia marcescens and multidrug‐resistant Pseudomonas aeruginosa, known as proteases‐secreting pathogenic bacteria, were also completely inhibited by BRBA20 under 20 µg/ml peptide concentrations. Our results suggested availabilities of Aib‐derived amphiphilicity and protease resistance in the design of artificial antimicrobial peptides. Comparing BRBA20 with BKBA20, it was also concluded that Arg residue is the preferred cationic source than Lys for antimicrobial action of amphiphilic helices. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Crucial role of N-terminal residue of binding peptides in recognition of the monoclonal antibody specific for the peptide-HLA-B5, -B35 complex

 The monoclonal antibody (mAb) 4D12 specific for the HLA-B5, -B35 cross-reacting group (CREG) bound to a fraction of HLA-B^*3501 and HLA-B^*5101 molecules carrying self-peptides. Analysis of the binding of mAb 4D12 to HLA-B^*3501 and -B^*5101 molecules pulsed with chemically synthesized peptides revealed that this mAb recognizes a restricted number of peptides and that P1 of the bound peptides critically influences its binding. The 4D12 mAb bound only to HLA-B^*3501 molecules carrying peptides with Asn, Asp, Glu, Ser, and Val at P1. Analysis using an HLA-B^*3501 crystallographic model suggested that 4D12 may recognize the side chain of the P1 residue that is pointing to the solvent. On the other hand, 4D12 bound only to HLA-B^*5101 molecules carrying peptides with Asn or Asp at P1, suggesting that the 4D12 epitope formed by Glu, Ser, or Val at P1 and the A-pocket was changed by the substitution of His for Tyr at residue 171 of HLA-B^*3501 molecules. This was confirmed by testing the binding of mAb 4D12 to HLA-B^*3501 mutant molecules at residue 171 carrying these peptides. These results together suggest that the conformation of the A-pocket and its hydrogen bound network with the P1 residue is also critical for the binding of mAb 4D12. The present study shows the molecular basis of the specificity of 4D12 for the peptide-HLA class I complex. Received: 19 June 1997 / Revised: 27 August 1997