Amoebapores, a family of membranolytic peptides from cytoplasmic granules of Entamoeba histolytica: isolation, primary structure, and pore bacterial cytoplasmic membranes

Three peptides with pore-forming activity were isolated from the cytoplasmic granules of pathogenic Entamoeba histolytica by acidic extraction, gel filtration and reversed-phase high-performance liquid chromatography. Partial amino acid sequence analysis of the three active peptides revealed that the most abundant of them was amoebapore and the other two were isoforms thereof. Cloning and sequencing of genomic DNA resolved the amino acid sequence of the two newly recognized peptides. The three peptides designated amoebapores A, B and C were found to have the same molecular size but to differ markedly in their primary structure, although all six cysteine residues are conserved. Despite sequence divergence, structural implications predict for the three peptides a similar amphipathic α-helical conformation stabilized by disulphide bonds. All three isoforms exhibit pore-forming activity toward lipid vesicles, but they differ in their kinetics. They also are capable of perturbing the integrity of bacterial cytoplasmic membranes and thereby kill Gram-positive bacteria. The amoebapores represent a distinct family of membrane-active peptides that may function intracellularly as antimicrobial agents but may also confer cytolytic activity on the parasite.     

The primary structure of the cytotoxin alpha-sarcin

The primary structure of the cytotoxin alpha-sarcin was determined. Eighteen of the 19 tryptic peptides were purified; the other peptide has arginine only. The complete sequence of 17 of the peptides was determined; the sequence of the remaining peptide was determined in part. The sequence of the 39 NH2-terminal residues was obtained by automated Edman degradation. The carboxyl-terminal amino acids were identified after carboxypeptidase treatment. The assignment of the amino acids in the tryptic peptides was confirmed and their alignment established from the sequence of the secondary tryptic peptides obtained after cleavage of citraconylated alpha-sarcin, from the sequence of a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine peptide, from the sequence of a chymotryptic peptide, and from the sequence of a peptide obtained with Staphylococcus aureus V8 protease. alpha-Sarcin contains 150 amino acid residues; the molecular weight is 16,987. There are disulfide bridges between cysteine residues at positions 6 and 148 and between residues 76 and 132.     

Nitration and chlorination of folic acid by peroxynitrite and hypochlorous acid,and the selective binding of 10-nitro-folate to folate receptor beta

Peptides pore-forming in cell membrane have been identified from a wide range of animals. A putative pore-forming peptide deduced from a cDNA clone of Clonorchis sinensis (clonorin) was predicted to consist of four amphipathic alpha-helices. Clonorin contained six invariably conserved cysteine residues, identified to form three disulfide bonds. These predicted structural features are highly homologous with pore-forming peptides, the amoebapores. Recombinant clonorin showed hemolytic activity toward rabbit erythrocytes. The hemolytic activity of C. sinensis extract increased dose-dependently and was inhibited by anti-clonorin immune sera. The clonorin was expressed developmentally in juvenile and adult flukes and localized in the intestinal epithelium of adult flukes. It is proposed that, through lysing host cellular components, clonorin could enhance proteolytic digestion in the intestine of C. sinensis.     

Determination of the disulfide array in the human defensin HNP-2. A covalently cyclized peptide

HNP-2 is a 29-residue peptide present in human neutrophils and is a member of the defensin family of antimicrobial peptides. All defensins contain an invariant disulfide infrastructure comprised of 6 half-cystine residues. The disulfide structure of HNP-2 was determined using a novel method to identify the cross-links involving the amino- and carboxyl-terminal cysteine residues. A derivative of HNP-2 was synthesized by covalent modification of the terminal cysteine residues. This derivative was purified, characterized, and subjected to exhaustive proteolytic digestion. Characterization of purified proteolytic fragments by amino acid analysis and/or sequence analysis identified an oligopeptide containing all 6 cystine residues. This oligopeptide was subjected to a single cycle of Edman degradation to cleave the peptide bond linking 2 adjacent cysteines. Purification and characterization of the Edman reaction products allowed for assignment of the disulfide array in HNP-2, revealing a cystine motif unique to the defensin peptide family. Further, the covalent structure of HNP-2 was found to be cyclic as one disulfide links the amino- and carboxyl-terminal cysteine residues. HNP-2 is the only polypeptide known to possess such a configuration.     

The cysteine bonding in TSH receptor and it function in autoantibodies recognition

The majority of epitopes for TSH receptor (TSHR) stimulating autoantibodies are clustered around the Nterminal region of the TSH receptor. The characteristic feature of this region is the presence of four cysteine residues. It was proposed that cysteines in positions 29 and 41 in the receptor are connected by disulfide bonds and they are the target for receptor stimulating antibodies. The present study was aimed to check this possibility. The synthetic peptides: peptide corresponding to the part of TSHR containing the above 29-41 cysteine bond, the peptide similar to this peptide but without disulfide bond and the control peptide, containing sequence absent in the receptor were used for rabbit immunization. The thyroid status of all immunized rabbits was the same. Rabbits immunized with peptides related to TSHR generated antisera reactive with TSHR in immunoenzymatic assay. To check specificity of this reaction the influence of the peptides and the antisera on TSH binding to the receptor in competitive assay (TRAK) and their influence on adenylate cyclase activity were studied. It was found that neither synthetic peptides nor antiserum from any rabbit influenced TSH binding to the receptor in TRAK. In contrast low, but significant adenylate cyclase stimulating activity was noticed for antisera from two of six rabbit immunized by peptide containing the disulfide bond. We concluded that such a bond between cysteine residues 29 and 41 are present in TSHR in the site of stimulating antibodies epitope.     

三叶肽：从结构到功能

三叶结构域是一段由38－39个氨基酸组成的多肽序列,其中包含6个高度保守的半胱氨酸残基,这6个半胱氨酸残基以1－5,2－4,3－6的交联方式形成三对二硫,窝囊鑫肽链折叠成特征性的三叶结构。已发现的哺乳动物三叶肽有三种：pS1、SP及ITF。三叶肽通常位于消化道腔面的粘膜层,具有保护和修复功能,在维持粘膜的完整性中发挥着重要作用。     

Profile of the disulfide bonds in acetylcholinesterase

The inter- and intrasubunit disulfide bridges for the 11 S form of acetylcholinesterase isolated from Torpedo californica have been identified. Localized within the basal lamina of the synapse, the dimensionally asymmetric forms of acetylcholinesterase contain either two (13 S) or three (17 S) sets of catalytic subunits linked to collagenous and noncollagenous structural subunits. Limited proteolysis of these molecules yields a tetramer of catalytic subunits that sediments at 11 S. Each catalytic subunit contains 8 cysteine residues which were identified following tryptic digestion of the reduced, 14C-carboxymethylated protein. The tryptic peptides were purified by gel filtration followed by reverse-phase high performance liquid chromatography (HPLC) and then sequenced. The disulfide bonding profile was determined by treating the native, nonreduced 11 S form of acetylcholinesterase with a fluorescent, sulfhydryl-specific reagent, monobromobimane, prior to tryptic digestion. Peptides again were resolved by gel filtration and reverse-phase HPLC. One fluorescent cysteine-containing peptide was identified, indicating that a single sulfhydryl residue, Cys231, was present in its reduced form. Three pairs of disulfide-bonded peptides were identified. These were localized in the polypeptide chain based on the cDNA-deduced sequence of the protein and were identified as Cys67-Cys94, Cys254-Cys265, and Cys402-Cys521. Hence, three loops are found in the secondary structure. Cys572, located in the carboxyl-terminal tryptic peptide, was disulfide-bonded to an identical peptide and most likely forms an intersubunit cross-link. Since the 6 cysteine residues in acetylcholinesterase that are involved in intrachain disulfide bonds are conserved in the sequence of the homologous protein thyroglobulin, it is likely that both proteins share a common folding pattern in their respective tertiary structures. Cys231 and the carboxyl-terminal cysteine residue Cys572 are not conserved in thyroglobulin.     

A new antifungal peptide from the seeds of Phytolacca americana: characterization, amino acid sequence and cDNA cloning

An antifungal peptide from seeds of Phytolacca americana, designated PAFP-s, has been isolated. The peptide is highly basic and consists of 38 residues with three disulfide bridges. Its molecular mass of 3929.0 was determined by mass spectrometry. The complete amino acid sequence was obtained from automated Edman degradation, and cDNA cloning was successfully performed by 3'-RACE. The deduced amino acid sequence of a partial cDNA corresponded to the amino acid sequence from chemical sequencing. PAFP-s exhibited a broad spectrum of antifungal activity, and its activities differed among various fungi. PAFP-s displayed no inhibitory activity towards Escherichia coli. PAFP-s shows significant sequence similarities and the same cysteine motif with Mj-AMPs, antimicrobial peptides from seeds of Mirabilis jalapa belonging to the knottin-type antimicrobial peptide.     

Determination of the positions of the disulfide bonds in aqualysin I (a thermophilic alkaline serine protease) of Thermus aquaticus YT-1

Aqualysin I is a heat-stable alkaline serine protease produced by Thermus aquaticus YT-1. Aqualysin I comprises 281 amino acid residues and contains four cysteine residues. The cysteine residues seemed to form disulfide bonds in the molecule. Thus, the positions of the disulfide bonds were investigated. Disulfide bond-containing peptides were identified by peptide mapping with HPLC before and after carboxymethylation of chymotryptic peptides of aqualysin I. The disulfide bond-containing peptides were isolated and then carboxymethylated. Carboxymethylcysteine-containing peptides were purified, and their amino acid compositions and sequences were determined. Based on the data obtained and the primary structure of aqualysin I, it was concluded that two disulfide bonds were formed between Cys67 and Cys99, and between Cys163 and Cys194.     

Analysis and separation of residues important for the chemoattractant and antimicrobial activities of beta-defensin 3

beta-Defensins are important in mammalian immunity displaying both antimicrobial and chemoattractant activities. Three canonical disulfide intramolecular bonds are believed to be dispensable for antimicrobial activity but essential for chemoattractant ability. However, here we show that HBD3 (human beta-defensin 3) alkylated with iodoactemide and devoid of any disulfide bonds is still a potent chemoattractant. Furthermore, when the canonical six cysteine residues are replaced with alanine, the peptide is no longer active as a chemoattractant. These findings are replicated by the murine ortholog Defb14. We restore the chemoattractant activity of Defb14 and HBD3 by introduction of a single cysteine in the fifth position (Cys V) of the beta-defensin six cysteine motif. In contrast, a peptide with a single cysteine at the first position (Cys I) is inactive. Moreover, a range of overlapping linear fragments of Defb14 do not act as chemoattractants, suggesting that the chemotactic activity of this peptide is not dependent solely on an epitope surrounding Cys V. Full-length peptides either with alkylated cysteine residues or with cysteine residues replaced with alanine are still strongly antimicrobial. Defb14 peptide fragments were also tested for antimicrobial activity, and peptides derived from the N-terminal region display potent antimicrobial activity. Thus, the chemoattractant and antimicrobial activities of beta-defensins can be separated, and both of these functions are independent of intramolecular disulfide bonds. These findings are important for further understanding of the mechanism of action of defensins and for therapeutic design.     

Sequential comparison of peptides containing half-cystine residues from ovalbumins of six avian species

Hen ovalbumin contains one cystine disulfide (Cys73-Cys120) and four cysteine sulfhydryl groups (Cys11, Cys30, Cys367, and Cys382) in a single polypeptide chain of 385 amino acid residues. To investigate whether or not such a structure is shared by related avian species, the contents of disulfide-involved half-cystine residues and their positions in the primary structure of ovalbumins from five species were compared with those of hen ovalbumin. Ovalbumins were alkylated with a fluorescent dye, IAEDANS, under disulfide-reduced and disulfide-intact conditions and digested with a number of proteolytic enzymes. The sequences were deduced from peptides containing half-cystine residues labeled with the fluorescent dye. The results showed that the number of free cysteine sulfhydryl groups of ovalbumins was different among the species, three for guinea fowl and turkey (Cys11, Cys367, and Cys382); and two for Pekin duck, mallard duck, and Emden goose (Cys11 and Cys331). On the other hand, a single intrachain disulfide bond could be identified from ovalbumins of five species using a combination of peptide mapping and N-terminal amino acid sequencing analysis under reduced and non-reduced conditions, in which the intrachain disulfide bond was like that of hen ovalbumin (Cys73-Cys120). The results also indicated that the variations in amino acid sequences on these peptides containing half-cystine residues bear a close relationship with the phylogeny of the six species.     

The thiol proteinases from the latex of Carica papaya L. III. The primary structure of chymopapain

The amino-acid sequence of chymopapain is presented. It was isolated from the latex of the fruits from the tropical species Carica papaya L. and is, besides papain and papaya proteinase omega, the third thiol proteinase from this source. The primary structure contains 218 amino-acid residues. It was deduced from sequence analysis of the native enzyme and of peptides obtained by tryptic, chymotryptic, peptic, thermolysinolytic and mild acidic hydrolysis. Out of a total of eight cysteine residues, six are involved in the formation of three disulfide bonds, the location of which has been established with the help of peptic and thermolysinolytic peptides and fragments, obtained by mild acidic hydrolysis. Chymopapain shares 126 identical amino-acid residues (58%) with papain and 141 (65%) with papaya proteinase omega, including the three disulfide bridges and the free cysteine in position 25, required for activity. Except some amino-acid residues in the substrate-binding site, all residues involved in the catalytic mechanism are conserved. The homology between papaya proteinases is discussed.     

Synthesis and characterization of the human CC chemokine HCC-2.

Human CC chemokine 2 (HCC-2) is a novel member of the chemokine peptide family that induces chemotaxis of monocytes, T lymphocytes and eosinophils via activation of the CCR-1 and CCR-3 receptors. Fmoc chemistry was optimized and used to synthesize the biologically active 66-residue peptide HCC-2-(48-113). Introduction of the three disulfide bonds was achieved by oxidative folding in the presence of the redox system cysteine/cystine. Alternatively, a semiselective approach utilizing a mixed Acm/Trt protection scheme for disulfide formation was applied. It was found that, without participation of the two HCC-2-specific cysteine residues in positions 64 and 104, the two typical chemokine disulfides are formed predominantly during oxidative folding. In addition, the mutant [Ala64,104]HCC-2-(48-113) lacking the third disulfide bond that discriminates HCC-2 from most other chemokines was synthesized. For disulfide bond formation, oxidative folding was compared with the use of Acm/Trt protection. HCC-2-(48-113) and the mutant [Ala64,104]HCC-2-(48-113) were further analyzed by CD and one-dimensional 1H NMR-spectroscopy. Both peptides adopt a similar stable secondary and tertiary structure in solution.     

C-type natriuretic peptide (CNP): a new member of natriuretic peptide family identified in porcine brain

Two types of natriuretic peptide, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), very similar to each other in structure and in pharmacological effect, are known to be present in mammalian heart and brain. In our present survey for unidentified peptides in porcine brain extracts, we found a new peptide of 22 amino acid residues, eliciting a potent relaxant activity on chick rectum. The amino acid sequence determined for the peptide shows remarkable similarity to those of ANP and BNP, especially in the 17-residue sequences flanked by two cysteine residues. The peptide shows a pharmacological spectrum similar to ANP and BNP. Thus, the peptide was designated "C-type natriuretic peptide (CNP)", the third member to join the natriuretic peptide family. In contrast to ANP and BNP, CNP terminates in the second cysteine residue, lacking a further C-terminal extension.     

Solution structure of cryptdin-4, a mouse paneth cell alpha-defensin

Mammalian defensins are abundant antimicrobial peptides that contribute to host defense. They are characterized by several conserved amino acids, including six invariant cysteine residues which form three intramolecular disulfide bonds and stabilize the tertiary structure. Cryptdin-4 (Crp4), a mouse alpha-defensin with potent in vitro bactericidal activity, has a primary structure distinct from all known alpha-defensins in that its polypeptide backbone uniquely lacks three residues between Cys(IV) and Cys(V). NMR diffusion experiments showed that Crp4 is monomeric in solution, and its three-dimensional solution structure, determined by two-dimensional proton NMR, consists of a triple-stranded antiparallel beta-sheet with the beta-strands joined to each other by a series of tight turns and a beta-hairpin. However, the overall beta-sheet content in Crp4 is lower than that of other alpha-defensin structures, while the shape and orientation of the Crp4 beta-hairpin also differ from those of other alpha-defensin structures. These structural characteristics combined with the high overall cationicity of Crp4 may contribute to its broad bactericidal spectrum and membrane disruptive activity.     

The structure of a novel insect peptide explains its Ca2+ channel blocking and antifungal activities

Diapause-specific peptide (DSP), derived from the leaf beetle, inhibits Ca2+ channels and has antifungal activity. DSP acts on chromaffin cells of the adrenal medulla in a fashion similar to that of omega-conotoxin GVIA, a well-known neurotoxic peptide, and blocks N-type voltage-dependent Ca2+ channels. However, the amino acid sequence of DSP has little homology with any other known Ca2+ channel blockers or antifungal peptides. In this paper, we analyzed the solution structure of DSP by using two-dimensional 1H nuclear magnetic resonance and determined the pairing of half-cystine residues forming disulfide bonds. The arrangement of the three disulfide bridges in DSP was distinct from that of other antifungal peptides and conotoxins. The overall structure of DSP is compact due in part to the three disulfide bridges and, interestingly, is very similar to those of the insect- and plant-derived antifungal peptides. On the other hand, the disulfide arrangement and the three-dimensional structure of DSP and GVIA are not similar. Nevertheless, some surface residues of DSP superimpose on the key functional residues of GVIA. This homologous distribution of hydrophobic and charged side chains may result in the functional similarity between DSP and GVIA. Thus, we propose here that the three-dimensional structure of DSP can explain its dual function as a Ca2+ channel blocker and antifungal peptide.     

Analogs of pancreatic polypeptide and peptide YY with a locked PP-fold structure are biologically active

Pancreatic polypeptide (PP), peptide YY (PYY) and neuropeptide Y (NPY), members of the PP-fold family share a high degree of sequence homology. Nuclear magnetic resonance (NMR) and X-ray crystallography studies have shown these peptides can adopt a tightly organized tertiary structure called the PP-fold, which has long been assumed to be the active structure of this family of peptides. To date, however, no studies have been completed with PYY and PP which confirm if the PP-fold structure is important for their physiological actions. The aim of the study was to test if PYY and PP locked into the PP-fold maintained biological activity. Therefore, we designed and produced analogs of PP and PYY in a cyclic conformation with two cysteine amino acid substitutions at the N-terminus and at position 27. These were oxidized to form a cysteine disulfide bond locking the peptides into the PP-fold structure. Studies demonstrate that the cyclic analogs have both similar in vivo activity to their parent molecules, and affinity for the Y2 and Y4 receptors. Results suggest that the proposed PP and PYY-fold is likely to be their biologically active conformation.     

Amino acid sequence of the Anthopleura xanthogrammica heart stimulant, anthopleurin A.

A highly potent heart stimulant, anthopleurin A, from Anthopleura xanthogrammica was shown to exist as a single polypeptide chain consisting of 49 amino acid residues. The sequence of the peptide was shown to be: Gly-Val-Ser-Cys-Leu-Cys-Asp-Ser-Asp-Gly-Pro-Ser-Val-Arg-Gly-Asn-Thr-Leu-Ser-Gly-Thr-Leu-Trp-Leu-Tyr-Pro-Ser-Gly-Cys-Pro-Ser-Gly-Trp-His-Asn-Cys-Lys-Ala-His-Gly-Pro-Thr-Ile-Gly-Trp-Cys-Cys-Lys-Gln as judged by Edman degradation of the carboxymethylcysteine derivative and the tryptic peptides obtained from the derivative. Although six carboxymethylcysteine residues were present in the polypeptide, no cysteine residues were detectable in the native protein, indicating that there are three cystine residues in anthopleurin A.     

Specific inhibition of procollagen C-endopeptidase activity by synthetic peptide with conservative sequence found in chordin

Procollagen C-endopeptidase (BMP-1) and N-endopeptidase (ADAMTS-2) are key enzymes for correct and efficient conversion of fibrillar procollagens to their self assembling monomers. Thus, they have an essential role in building and controlling the quality of extracellular matrices (ECMs). Here, we tested inhibition of activity of the largest variant of BMP-1, a recombinant mammalian tolloid (mTld), in vitro by three synthetic peptides with conservative amino-acid sequences found in chordin using procollagen type I as a substrate. We also verified the specific action of best inhibitory 16 amino-acid peptide in the procollagen type I cleavage assay with the use of ADAMTS-2 (procollagen N-endopeptidase). Subsequently, we determined the critical residues and minimal sequence of six amino acids in the original 16 amino-acid peptide required to maintain the inhibitory potential. Studies on the interactions of 6 and 16 amino acid long peptides with the enzyme revealed their binding to non-catalytic, regulatory domains of mTld; the inhibitory activity was not due to the competition of peptides with the substrate for the enzyme active center, because mTld did not cleave the peptides. However, in the presence of mTld both peptides underwent cyclization by disulfide bond formation. Concluding, we have shown that procollagen C-endopeptidase may be specifically blocked via its non-catalytic domains by synthetic peptide consisting of 6 amino acids in the sequence found in highly conservative region of chordin. Thus, we hypothesize that the 6 amino-acid peptide could be a good candidate for anti-fibrotic drug development.