In vivo structure-activity studies on the dark-color-inducing neurohormone of locusts.

In the 11-residue long dark-color-inducing neurohormone (DCIN = [His7]-corazonin), of locusts, from residue 2 to residue 11, one amino acid at each time was substituted by d-phenylalanine (d-Phe). The dark-color-inducing effect of these peptides was investigated in comparison with unaltered DCIN by a bioassay based on nymphs of a DCIN-deficient albino mutant of the migratory locust, Locusta migratoria. Substitution of any single amino acid by d-Phe always reduced the activity, but did not abolish it completely. Maximum inactivation was obtained after substitution of Gln4, Ser6, or Trp9. The latter two residues are within the partial sequence -Ser-Xxx-Gly-Trp- (Xxx = His in the DCIN) that seems to be important for the dark-color-inducing activity, as found also in another study (Insect Biochem. Mol. Biol. 32, 2002, 909). Gln4, however, is outside of this partial sequence. Minimal, although still considerable, inactivation occurred after substitution of Gly8, Phe3, or Asn11, despite the fact that Gly8 is within the -Ser-Xxx-Gly-Trp- partial sequence. In conclusion, no single active core was found, indicating that the whole sequence of the DCIN is necessary to induce maximum darkening effect. No difference was found in the activity of the peptides in which Gly8 was substituted by d-Phe or by l-Phe. Therefore the -Ser-Xxx-Gly-Trp- partial sequence does not seem to be stabilized by a type II beta-turn. Nevertheless, existence of another kind of turn that includes this partial sequence is feasible. A single unsuccessful attempt was made to discover an antagonist to the DCIN.     

Nuclear magnetic resonance (NMR) studies of the dark-color-inducing neurohormone of locusts and corazonin

The dark-color-inducing neurohormone (DCIN) of locusts and corazonin of a cockroach, both 11 residue-long peptides, induce dark coloration in albino nymphs of Locusta migratoria when injected after a nymphal molt. These peptides differ at position 7 (His in DCIN and Arg in corazonin) and elicit an almost identical darkening response. The three-dimensional structures of these peptides, dissolved in dimethylsulfoxide (DMSO), were determined by NMR. Structural elements determined at atomic resolution may provide insight into the biological activity of these two neurohormones. The calculated structures of DCIN and corazonin indicate clear, prevalent conformations with similar secondary features. The generated low-energy solution structures of each show structural elements within residues Phe3 to Trp9 with a turn situated at the core of the peptide from which the sidechains of residue 7 of each peptide protrude. A calculated negative electrostatic potential surface almost completely covers both neurohormones and only the 7th residue sidechains of each peptide emerge in their entirety. Within these residues there is a partial sequence seen in several neurohormones that control various physiological functions in Arthropods: -Ser-X-Gly-Trp- (X=His in DCIN and Arg in corazonin). This partial sequence may play a role in the physiological activity of some Arthropod neurohormones.     

Structure-activity relations of the dark-colour-inducing neurohormone of locusts

The dark-colour-inducing effect of several peptides in comparison to that of the dark-colour-inducing neurohormone (DCIN, [His(7)]-corazonin) of locusts was investigated by a bioassay based on nymphs of a DCIN-deficient albino mutant of Locusta migratoria. The study was aimed at elucidating the active part of the DCIN and to explore the contribution of its amino acids to the activity. Graded doses of all peptides were injected in oil. [Arg(7)]-corazonin and DCIN were equally effective. Certain arthropod neuropeptides having the -SXGW- partial sequence (a part of the DCIN and of [Arg(7)]-corazonin; X=His and X=Arg, respectively) yielded the following findings: Scg-AKH-II (adipokinetic hormone II of Schistocerca gregaria X=Thr), Grb-AKH ( adipokinetic hormone of Gryllus bimaculatus X=Thr) and RPCH (red pigment concentrating hormone of crustaceans X=Pro) evoked a moderate darkening response, but Lom-AKH-II (adipokinetic hormone II of L. migratoria X=Ala) was ineffective. Step by step shortening of the sequence of the DCIN at the N-terminal, from pGlu-3-11DCIN to pGlu-9-11DCIN, resulted in a decreasing activity, but even pGlu-9-11DCIN induced a weak response with high doses. Shortening of the DCIN from the C-terminal revealed a moderate activity of 1-7DCIN-NH(2) and a weak activity of 1-5DCIN-NH(2). An octadecapeptide which induces dark colour in moth larvae, having the pentamer FTPRL-NH(2) at its C-terminal, evoked no darkening in the albino locusts. We conclude that although the -SXGW- partial sequence has some role in induction of darkening, for obtaining maximal effect the whole sequence of the DCIN (or of [Arg(7)]-corazonin) is necessary.     

The Amino Acid Sequence of Monal Pheasant Lysozyme and Its Activity

The amino acid sequence of monal pheasant lysozyme and its activity were analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had one amino acid substitution at position 102 (Arg to Gly) comparing with Indian peafowl lysozyme and four amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), and 121 (Gln to His) with chicken lysozyme. Analysis of the time-courses of reaction using N-acetylglucosamine pentamer as a substrate showed a difference of binding free energy change (-0.4 kcal/mol) at subsites A between monal pheasant and Indian peafowl lysozyme. This was assumed to be caused by the amino acid substitution at subsite A with loss of a positive charge at position 102 (Arg102 to Gly).     

The amino acid sequence of satyr tragopan lysozyme and its activity

The amino acid sequence of satyr tragopan lysozyme and its activity was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had three amino acid substitutions at positions 103 (Asn to Ser), 106 (Ser to Asn), and 121 (His to Gln) comparing with Temminck's tragopan lysozyme and five amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), 101 (Asp to Gly) and 103 (Asn to Ser) with chicken lysozyme. The time course analysis using N-acetylglucosamine pentamer as a substrate showed a decrease of binding free energy change, 1.1 kcal/mol at subsite A and 0.2 kcal/mol at subsite B, between satyr tragopan and chicken lysozymes. This was assumed to be responsible for the amino acid substitutions at subsite A-B at position 101 (Asp to Gly), however another substitution at position 103 (Asn to Ser) considered not to affect the change of the substrate binding affinity by the observation of identical time course of satyr tragopan lysozyme with turkey and Temminck's tragopan lysozymes that carried the identical amino acids with chicken lysozyme at this position. These results indicate that the observed decrease of binding free energy change at subsites A-B of satyr tragopan lysozyme was responsible for the amino acid substitution at position 101 (Asp to Gly).     

Structural requirements for glycosyl-phosphatidylinositol-anchor attachment in the cellular receptor for urokinase plasminogen activator.

The urokinase-plasminogen-activator receptor (u-PAR) is a glycosyl-phosphatidylinositol(glycosyl-PtdIns)-anchored membrane protein. Using site-directed mutagenesis, we have studied features in the u-PAR sequence important for successful glycosyl-PtdIns attachment. Two critical sequence elements were identified. In the sequence Ser282-Gly283-Ala284, simultaneous substitution of all of these residues prevented membrane anchoring. Individual substitution of each of the residues indicated that Gly283 is the more critical residue and the likely attachment site. However, it was unexpectedly found that mutation of this residue gave rise only to a partial impairment of glycosyl-PtdIns attachment. We therefore propose that more than one residue within this sequence can be utilized as glycosyl-PtdIns-attachment site. In the last eight COOH-terminal amino acids encoded in u-PAR cDNA, deletion of this sequence (residues 306-313) completely prevented glycosyl-PtdIns attachment. However, the remaining COOH-terminal region proved still to possess a potential glycosyl-PtdIns signal activity; it could be converted to a new functional glycosyl-PtdIns signal by substitution of a single positively charged residue (Arg304). Substitution of Arg304 by Leu converted this truntaced u-PAR to a glycosyl-PtdIns-anchored protein, indistinguishable from the wild type. Substitution of Arg304 by a negatively charged residue (Glu) led to a partial acquisition of the glycosyl-PtdIns-anchoring ability. These findings show that charged amino acids placed in the COOH-terminus interfere negatively with glycosyl-PtdIns-anchoring, and, furthermore, that this effect is more pronounced for positively charged than for negatively charged amino acid residues.     

Backbone cyclic peptide antagonists, derived from the insect pheromone biosynthesis activating neuropeptide, inhibit sex pheromone biosynthesis in moths.

We describe an application of the backbone cyclization and cycloscan concept for the design and synthesis of pheromone biosynthesis activating neuropeptide (PBAN) antagonists capable of inhibiting sex pheromone biosynthesis in Heliothis peltigera female moths. Two backbone cyclic (BBC) sub-libraries were designed and synthesized. The structure of the first sub-library ([Arg27]PBAN27-33NH2, termed the Ser sub-library) was based on the active C-terminal hexapeptide sequence (Tyr-Phe-Ser-Pro-Arg-Leu-NH2) of PBAN1-33NH2, which was found to comprise its active core. The second sub-library ([Arg27, D-Phe30]PBAN27-33NH2, termed the D-Phe sub-library) was based on the sequence of the lead antagonist Arg-Tyr-Phe-(D)Phe-Pro-Arg-Leu-NH2. In both sub-libraries the Pro residue was replaced by an Nalpha(omega-amino-alkyl)Gly building unit having various lengths of the alkyl chain. All the cyclic peptides in each sub-library had the same primary sequence and the same location of the ring. The members of each library differed from each other by the bridge size and bridge chemistry. Screening of the two libraries for pheromonotropic antagonists resulted in the disclosure of four compounds that fully inhibited sex pheromone biosynthesis at 1 nmol and were devoid of agonistic activity. All antagonistic peptides originated from the D-Phe sub-library. Substitution of the D-Phe30 amino acid with a Ser resulted in a loss of antagonistic activity. Agonistic activities were exhibited by peptides from both sub-libraries.     

Position effect on apparent helical propensities in the C-peptide helix

A search has been made for position effects on apparent helix propensities when another amino acid is substituted for alanine in the C-peptide helix of ribonuclease A. Three internal alanine residues (Ala4, Ala5, Ala6) are used as sites for substitution. Five amino acids, Glu, His, Arg, Lys and Phe, are substituted singly in individual peptides at each of these three positions, and the pH profiles of helix content for the substituted peptides have been determined. The effect of using an acetyl or a succinyl amino-terminal-blocking group has also been determined for each substitution. A strong position effect is found at Ala5: the helix content of the substituted peptide is significantly higher for substitution at position 5 than at positions 4 or 6 in almost all cases. The reason for the position 5 effect is unknown. The results also show that electrostatic interactions often influence substitution experiments, and they provide data on the variability of substitution experiments made with a natural sequence peptide.     

Functional expression and mutational analysis of flavonol synthase from Citrus unshiu.

Flavonols are produced by the desaturation of flavanols catalyzed by flavonol synthase. The enzyme belongs to the class of intermolecular dioxygenases which depend on molecular oxygen and FeII/2-oxoglutarate for activity, and have been in focus of structural studies recently. Flavonol synthase cDNAs were cloned from six plant species, but none of the enzymes had been studied in detail. Therefore, a cDNA from Citrus unshiu (Satsuma mandarin) designated as flavonol synthase was expressed in Escherichia coli, and the purified recombinant enzyme was subjected to kinetic and mutational chacterizations. The integrity of the recombinant synthase was revealed by a molecular ion from MALDI-TOF mass spectrometry at m/z 37888 +/- 40 (as compared to 37899 Da calculated for the translated polypeptide), and by partial N-terminal sequencing. Maximal flavonol synthase activity was observed in the range of pH 5-6 with dihydroquercetin as substrate and a temperature optimum at about 37 degrees C. Km values of 272, 11 and 36 micro m were determined for dihydroquercetin, FeII and 2-oxoglutarate, respectively, with a sixfold higher affinity to dihydrokaempferol (Km 45 micro m). Flavonol synthase polypeptides share an overall sequence similarity of 85% (47% identity), whereas only 30-60% similarity were apparent with other dioxygenases. Like the other dioxygenases of this class, Citrus flavonol synthase cDNA encodes eight strictly conserved amino-acid residues which include two histidines (His221, His277) and one acidic amino acid (Asp223) residue for FeII-coordination, an arginine (Arg287) proposed to bind 2-oxoglutarate, and four amino acids (Gly68, His75, Gly261, Pro207) with no obvious functionality. Replacements of Gly68 and Gly261 by alanine reduced the catalytic activity by 95%, while the exchange of these Gly residues for proline completely abolished the enzyme activity. Alternatively, the substitution of Pro207 by glycine hardly affected the activity. The data suggest that Gly68 and Gly261, at least, are required for proper folding of the flavonol synthase polypeptide.     

Nuclear magnetic resonance characterization of peptide models of collagen-folding diseases

Misfolding of the triple helix has been shown to play a critical role in collagen diseases. The substitution of a single Gly by another amino acid breaks the characteristic repeating (Gly-X-Y)n sequence pattern and results in connective tissue disease such as osteogenesis imperfecta. Nuclear magnetic resonance (NMR) studies of normal and mutated collagen triple-helical peptides offer an opportunity to characterize folding and conformational alterations at the substitution site, as well as at positions upstream and downstream of a Gly mutation. The NMR studies suggest that the local sequences surrounding the substitution site, and the renucleation sequences N-terminal to and adjacent to the substitution site, may be critical in defining the clinical phenotype of osteogenesis imperfecta. These studies may pave the way to understanding the mechanism by which a single Gly substitution in collagen can lead to pathological conditions.     

Systematic optimization of a lead-structure identities for a selective short peptide agonist for the human orphan receptor BRS-3.

The orphan receptor, human bombesin receptor subtype 3 (BRS-3) was assigned to the G-protein coupled bombesin receptor family because of its high sequence homology with the neuromedin B receptor (NMB-R) and gastrin-releasing peptide receptor (GRP-R). Since its pharmacology is stiIl unknown, new highly potent and selective tool-substances are needed, that may be able to elucidate its possible role in obesity and cancer. We have performed structure activity relationship studies on the high affinity peptide agonists [D-Phe6,beta-Ala11,Phe13,Nle14]Bn(6-14) and [D-Phe6,Phe13]Bn(6-13)propylamide, using their ability to mobilize intracellular calcium in BRS-3 transfected CHOGa-16 cells combined with receptor binding studies. It was demonstrated that for [D-Phe,beta-Ala11,Phe13,Nle14]Bn(6-14) the side chains of the residues Trp8 and Phe13, and to a smaller extent beta-Ala11, are the important amino acid side chains for receptor activation and binding, however for [D-Phe6,Phe13]Bn(6-13) propylamide His12 seems to be more important than Phe13. C-and N-terminal deletions and amino acid substitutions allowed further understanding. It was demonstrated that substitution of His 12 by Tyr leads to a high selectivity towards GRP-R. Using the acquired information, a small tetrapeptide library was designed with compounds presenting Trp and Phe at varying stereochemistry and distances, which led to the discovery of the lead-structure H-D-Phe-Gln-D-Trp-Phe-NH2. Systematic SAR revealed the important structural features of this peptide, C-terminal optimization resulted in the highly active and selective BRS-3 agonist H-D-Phe-Gln-D-Trp-1-(2-phenylethyl)amide. In summary, the size of the peptide was reduced from 8 or 9 amino acids to a tripeptide for BRS-3.     

Selective cleavage of an azaGly peptide bond by copper(II). Long‐range effect of histidine residue

Several reports have highlighted the interest of replacing Gly, a frequent amino acid within bioactive peptides, by azaGly (Agly) to improve their stability, activity or for the design of prodrugs. Because metal catalysis is increasingly used for tailoring peptide molecules, we have studied the stability of Agly peptides in the presence of metal ions. In this study, we show that Cu(II), unlike other metal ions such as Fe(II), Fe(III), Pd(II), or Pt(II), induces the cleavage of Agly peptides at room temperature and pH 7.3. The cleavage occurred in the absence of an anchoring His residue within the peptide but it was accelerated when this amino acid was present in the sequence. The influence of His residue on the cleavage rate was minimal when His and Agly were adjacent, whereas large effects were observed for distant His residues. The reaction between Cu(II) and Agly peptides induced the formation of Cu(I) species, which could be detected using bicinchoninic acid as a probe. The nature of products formed in this reaction allowed suggesting a mechanism for the Cu(II)‐induced cleavage of Agly peptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Apolipoprotein A-I-mimetic peptides with antioxidant actions

To augment antioxidant action of apolipoprotein A-I (Apo A-I)-mimetic peptide, the peptide F3,6,14,18 18A (DWFKAFYDKVAEKFKEAF) was modified by incorporating antioxidant amino acid residues. Introduction of His residue at position 2 or 3 at N-terminal of the peptide remarkably enhanced antioxidant action against Cu2+ oxidation of LDL and the capability of sequestering Cu2+. Likewise, the substitution of Ala for Cys residue at position 12 increased antioxidant action against Cu2+ oxidation of LDL. Additionally, the Cys substitution contributed to enhanced capabilities in the removal of hypochlorous acid (HOCl) and 13-hydroperoxyoctadecadienoic acid. Furthermore, the combined incorporation of His and Cys residues enhanced antioxidant actions in preventing Cu2+ oxidation and reducing HOCl and hydroperoxide levels. Separately, in solubilizing phosphatidylcholine, either peptides with His residue at N-terminal position 2 or 3, or those containing Cys residue at position 11 or 12 were equipotent to peptide F3,6,14,18 18A. Further, the lipid-solubilizing ability of those containing both His and Cys residues was comparable to that of peptide F3,6,14,18 18A. In support of this, a similar structural importance was observed with Trp fluorescence study illustrating the penetration of peptides in phosphatidylcholine liposome. Besides, the modified peptides were also comparable to peptide F3,6,14,18 18A in restoring phosphatidylserine-induced loss of PON1 activity. These results indicate that the insertion of His or Cys residue into peptide F3,6,14,18 18A at appropriate positions could lead to enhanced antioxidant action with no significant change of lipid-solubilizing action.     

Effect of substitutions in position 12 of bombesin on antagonist activity

Recent studies show that substitutions for the His in position 12 of bombesin (Bn) are important in determining antagonist activity. The present study was designed to investigate the chemical properties of the substitution in position 12 of Bn that determined antagonist activity and affinity. Nine [Leu14]Bn analogues with a single amino acid substitution and two analogues with multiple substitutions in addition to position 12 were synthesized. Replacing His12 with Phe12 resulted in an agonist with 100-fold decrease in potency and as reported previously, replacement with D-Phe12 resulted in an antagonist, but with a 10,000-fold decrease in affinity. Substitution of D-beta-naphthylalanine (D-Nal12), a larger and more hydrophobic group than D-Phe, produced a complete loss of antagonist activity, whereas substitution of D-pyridylalanine (D-Pal12), a group more hydrophilic and similar in size to D-Phe, converted the analogue to a very weak agonist with 300-fold lower affinity than the D-Phe analogue. Antagonist activity depended on the nature of the aromatic moiety, with a D-Trp12 resulting in an inactive analogue, and with a D-Tyr12 resulting in a weak antagonist being 100-fold less potent than the D-Phe12 substitution. The addition of an electron withdrawing group to the D-Phe substitution (D-Cpa12) resulted in a minimal decrease in antagonist activity, whereas the addition of an electron donating group (p-hydroxy in D-Tyr12) resulted in a 30-fold decrease in antagonist activity. The addition of a basic group (D-Arg12 or D-Pal12) resulted in weak agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced antitumour activity of 15-residue bovine lactoferricin derivatives containing bulky aromatic amino acids and lipophilic N-terminal modifications.

In a structure-antibacterial activity relationship study of a peptide fragment of bovine lactoferricin consisting of FKCRRWQWRMKKLGA (LFB 17-31), it was revealed that the two Trp residues were important for antibacterial activity. It has further been demonstrated that the size, shape and the aromatic character of the side chains were even more important than the Trp itself. In this study the antitumour effect of a series of LFB 17-31 derivatives are reported, in which the two Trp residues in position 6 and 8 were replaced with the larger non-coded aromatic amino acids Tbt, Tpc, Bip and Dip. The counterproductive Cys in position 3 was also substituted with these larger aromatic residues. In addition, the effect of introducing lipophilic groups of different size and shape in the N-terminal of the LFB 17-31 sequence was addressed. The resulting peptide derivatives were tested for activity against three human tumour cell lines and against normal human umbilical vein endothelial cells and fibroblasts. High antitumour activity by several of the peptides demonstrated that Trp successfully could be substituted by the bulky aromatic residues, and peptides containing the large and rigid Tbt residue in position 6 and/or 8 in LFB 17-31 were the most active candidates. The antitumour effect was even more increased by the Tbt-modified peptides when the three counterproductive amino acids Cys3, Gln7 and Gly14 were replaced by Ala. Enhanced antitumour activity was also obtained by modifying the N-terminal of LFB 17-31 with either long-chained fatty acids or bulky moieties. Thus, our results revealed that the size and shape of the lipophilic groups and their position in the peptide sequence were important for antitumour activity.     

Collagen model peptides: Sequence dependence of triple-helix stability

The triple helix is a specialized protein motif, found in all collagens as well as in noncollagenous proteins involved in host defense. Peptides will adopt a triple-helical conformation if the sequence contains its characteristic features of Gly as every third residue and a high content of Pro and Hyp residues. Such model peptides have proved amenable to structural studies by x-ray crystallography and NMR spectroscopy, suitable for thermodynamic and kinetic analysis, and a valuable tool in characterizing the binding activities of the collagen triple helix. A systematic approach to understanding the amino acid sequence dependence of the collagen triple helix has been initiated, based on a set of host-guest peptides of the form, (Gly-Pro-Hyp)(3)-Gly-X-Y-(Gly-Pro-Hyp)(4). Comparison of their thermal stabilities has led to a propensity scale for the X and Y positions, and the additivity of contributions of individual residues is now under investigation. The local and global stability of the collagen triple helix is normally modulated by the residues in the X and Y positions, with every third position occupied by Gly in fibril-forming collagens. However, in collagen diseases, such as osteogenesis imperfecta, a single Gly may be substituted by another residue. Host-guest studies where the Gly is replaced by various amino acids suggest that the identity of the residue in the Gly position affects the degree of destabilization and the clinical severity of the disease.     

Identification of the active site residues in dipeptidyl peptidase IV by affinity labeling and site-directed mutagenesis.

The active site of dipeptidyl peptidase IV (DPPIV) was examined by chemical modification and site-directed mutagenesis. Purified DPPIV was covalently modified with [3H]diisopropyl fluorophosphate (DFP). The radiolabeled DPPIV was digested with lysyl endopeptidase, and the peptides were separated by high-performance liquid chromatography. A single 3H-containing peptide was obtained and analyzed for amino acid sequence and radioactivity distribution. A comparison of the determined sequence with the predicted primary structure of DPPIV [Ogata, S., Misumi, Y., & Ikehara, Y. (1989) J. Biol. Chem. 264, 3596-3601] revealed that [3H]DFP was bound to Ser631 within the sequence Gly629-Trp-Ser-Tyr-Gly633, which corresponds to the consensus sequence Gly-X-Ser-X-Gly proposed for serine proteases. To further identify the essential residues in the active-site sequence, we modified the DPPIV cDNA by site-directed mutagenesis to encode its variants. Expression of the mutagenized cDNAs in COS-1 cells demonstrated that any single substitution of Gly629, Ser631, or Gly633 with other residues resulted in the complete loss of the enzyme activity and DFP binding. Although substitution of Trp630----Glu or Tyr632----Phe caused no effect on the enzyme activity, that of Tyr632----Leu or Gly abolished the activity. These results indicate that the sequence Gly-X-Ser-(Tyr)-Gly is essential for the expression of the DPPIV activity.     

Amino Acid Sequence of Porcine Myelin Basic Protein

The myelin basic protein (BP) of pig brain was cleaved into its constituent tryptic peptides and the amino acid composition of each was determined. Those tryptic peptides that had not been sequenced previously were cleaved with dipeptidyl peptidases and the resulting dipeptides were trimethylsilated, separated by gas chromatography, and identified by mass spectrometry. Carboxypeptidases B and Y were used to establish the COOH-terminal sequences of some of the tryptic peptides; one tryptic peptide (sequence 76-92) was cleaved with thermolysin and the thermolytic peptides were analyzed. From the results of the present study together with those reported previously, it has been possible to determine the complete amino acid sequence of the protein. The protein consists of 172 residues and has a theoretical molecular weight of 18,604. Its amino acid sequence is identical with that reported for the homologous bovine protein with the following exceptions: Ser replaces (bovine) Ala2; His-Gly is inserted between Arg9 and Ser10; Ala replaces Ser45; His and Gly replace Gly76 and His77, respectively; Pro replaces Ser131 and Ser135; Ala is inserted between Gly142 and His143; and Gln replaces His143.     

Conservation of primary structure of the pyridoxyl peptide of Escherichia coli and Serratia marcescens tryptophan synthase beta2 protein.

Two labeled peptides were recovered from tryptic digests of the NaB3H4-reduced, performic acid-oxidized beta2 protein of Serratia marcescens tryptophan synthase. These two pyridoxyl peptides were identical except for the presence or absence of an NH2-terminal arginyl residue. Tryptic digestion of nonreduced, performic acid-oxidized protein allowed isolation of the peptides that comprise the two halves of the pyridoxyl peptide. The partial primary structure for this region of the protein was shown to be Arg-Glx-Asx-Ler-Leu-His(Gly,Gly,Ala,His)Lys(Pxy)-Thr-Asx-Glx-Val(Leu,Gly,Glx,Ala,Leu,Leu,Ala)Lys. All the data available indicate that the sequence is identical with the homologous region from the Escherichia coli enzyme.     

A panel of unique HLA-A2 mutant molecules define epitopes recognized by HLA-A2-specific antibodies and cytotoxic T lymphocytes

HLA-A2.1 and HLA-A2.3, which differ from one another at residues 149, 152, and 156, can be distinguished by the mAb CR11-351 and many allogeneic and xenogeneic CTL. Site-directed mutagenesis was used to incorporate several different amino acid substitutions at each of these positions in HLA-A2.1 to evaluate their relative importance to serologic and CTL-defined epitopes. Recognition by mAb CR11-351 was completely lost when Thr but not Pro was substituted for Ala149. A model to explain this result based on the 3-dimensional structure of HLA-A2.1 is presented. In screening eight other mAb, only the substitutions of Pro for Val152 or Gly for Leu156 led to the loss of mAb binding. Because other non-conservative substitutions at these same positions had no effect, these results suggest that the loss of serologic epitopes is in many cases due to a more indirect effect on molecular conformation. Specificity analysis using 28 HLA-A2.1-specific alloreactive and xenoreactive CTL clones showed 19 distinct patterns of recognition. The epitopes recognized by alloreactive CTL clones demonstrated a pronounced effect by all substitutions at residue 152, including the very conservation substitution of Ala for Val. Overall, the most disruptive substitution at amino acid residue 152 was Pro, followed by Glu, Gln, and then Ala. In contrast, substitutions at 156 had little or no effect on allogeneic CTL recognition, and most clones tolerated either Gly, Ser, or Trp at this position. Similar results were seen using a panel of murine HLA-A2.1-specific CTL clones, except that substitutions at position 156 had a greater effect. The most disruptive substitution was Trp, followed by Ser and then Gly. In addition, when assessed on the entire panel of CTL, the effects of Glu and Gln substitutions at position 152 demonstrated that the introduction of a charge difference is no more disruptive than a comparable change in side chain structure that does not alter charge. Taken together, these results indicate that the effect of amino acid replacements at positions 152 and 156 on CTL-defined epitopes depends strongly on the nature of the substitution. Thus, considerable caution must be exercised in evaluating the significance of particular positions on the basis of single mutations. Nonetheless, the more extensive analysis conducted here indicates that there are differences among residues in the class I Ag "binding pocket," with residue 152 playing a relatively more important role in formation of allogeneic CTL-defined epitopes than residue 156.