Specific labeling of the essential cysteine residue of L-methionine gamma-lyase with a cofactor analogue, N-(bromoacetyl)pyridoxamine phosphate

L-Methionine gamma-lyase from Pseudomonas putida is composed of four identical polypeptide chains and contains four cysteinyl residues per subunit. We have found one of them catalytically essential by its specific cyanylation with 2-nitro-5-thiocyanobenzoic acid. We have shown its essentiality also with N-(bromoacetyl)pyridoxamine 5'-phosphate (BAPMP), which is a cofactor analogue and also an affinity-labeling agent. The kinetic data show that the apoenzyme forms a binary complex with BAPMP prior to covalent binding. The stoichiometry of inactivation was 1 mol of BAPMP per subunit. We have shown that the cysteine residue modified with BAPMP is identical with that labeled specifically with [14C]iodoacetic acid. The amino acid sequences of the peptides containing the essential cysteine residue and the lysine residue to which pyridoxal 5'-phosphate is bound were determined by automated Edman degradation.     

Identification of koningic acid (heptelidic acid)-modified site in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase

The sesquiterpene antibiotic koningic acid (heptelidic acid) has been previously demonstrated to modify glyceraldehyde-3-phosphate dehydrogenase in specific manner, probably by binding to the sulfhydryl residue at the active site of the enzyme (Sakai, K., Hasumi, K. and Endo, A. (1988) Biochim. Biophys. Acta 952, 297-303). Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase labeled with [3H]koningic acid was digested with trypsin. Reverse-phase HPLC revealed that the label is associated exclusively with a tryptic peptide having 17 amino acid residues. Microsequencing and fast atom bombardment mass spectrometry demonstrated that the peptide has the sequence Ile-Var-Ser-Asn-Ala-Ser-Cys-Thr-Thr-Asn-Cys-Leu-Ala-Pro-Leu-Ala-Lys. In comparison to the amino acid sequence of glyceraldehyde-3-phosphate dehydrogenase from other species, this peptide is in a highly conserved region and is part of the active site of the enzyme. The cysteine residue corresponding to the Cys-149 in the pig muscle enzyme, which has been shown to be an essential residue for the enzyme activity, was shown to be the site modified by koningic acid. Structural analyses of the reaction product of koningic acid and L-cysteine suggested that the epoxide of koningic acid reacts with the sulfhydryl group of cysteine residue, resulting in a thioether.     

Differential reactivity of maleimide and bromoacetyl functions with thiols: application to the preparation of liposomal diepitope constructs

The comparative reactivity of maleimide and bromoacetyl groups with thiols (2-mercaptoethanol, free cysteine, and cysteine residues present at the N-terminus of peptides) was investigated in aqueous media. These studies were performed (i) with water-soluble functionalized model molecules, i.e., polyoxyethylene-based spacer arms that could also be coupled to lipophilic anchors destined to be incorporated into liposomes, and (ii) with small unilamellar liposomes carrying at their surface these thiol-reactive functions. Our results indicate that an important kinetic discrimination (2-3 orders of magnitude in terms of rate constants) can be achieved between the maleimide and bromoacetyl functions when the reactions with thiols are performed at pH 6.5. The bromoacetyl function which reacts at higher pH values (e.g., pH 9.0) retained a high chemoselectivity; i.e., under conditions where it reacted appreciably with the thiols of, e.g., HS-peptides, it did react with other nucleophilic functions such as alpha- and epsilon-amino groups or imidazole, which could also be present in peptides. This differential reactivity was applied to design chemically defined and highly immunogenic liposomal diepitope constructs as synthetic vaccines, i.e., vesicles carrying at their surface two different peptides conjugated each to a specific amphiphilic anchor. This was realized by coupling sequentially at pH 6.5 and 9.0 two HS-peptides to preformed vesicles containing lipophilic anchors functionalized with maleimide and bromoacetyl groups [Boeckler, C., et al. (1999) Eur. J. Immunol. 29, 2297-2308].     

Synthesis of novel protected Nalpha(omega-thioalkyl) amino acid building units and their incorporation in backbone cyclic disulfide and thioetheric bridged peptides.

General methods for the preparation of protected Nalpha(omega-thioalkyl) amino acids building units for backbone cyclization using reductive alkylation and on-resin preparation are described. The synthesis of non-Gly Fmoc-protected S-functionalized N-alkylated amino acids is based on the reaction of readily prepared protected omega-thio aldehyde with the appropriate amino acid. Preparation of Fmoc-protected S-functionalized N-alkylated Gly building units was carried out using two methods: reaction of glyoxylic acid with Acm-thioalkylamine and an on-resin reaction of bromoacetyl resin with Trt-thioalkylamines. Three model peptides were prepared using these building units. The GlyS2 building unit was incorporated into a backbone cyclic analog of somatostatin that contains a disulfide bridge. Formation of the disulfide bridge was performed by on-resin oxidation using 12 or Tl(CF3COO-)3. Both methods resulted in the desired product in a high degree of purity in the crude. The AspS3 building unit was also successfully incorporated into a model peptide. In addition, the in situ generation of sulfur containing Gly building units was demonstrated on a Substance P backbone cyclic analog containing a thioether bridge.     

Amino acid and peptide ratio in protein hydrolysates for parenteral feeding

Using the method of amino acid analysis and routine methods of protein biochemistry, the ratio of amino acids and peptides in acid and enzyme protein hydrolyzates was determined. Depending on the production procedure, the hydrolyzates under study contained various amounts of free amino acids and peptides in which the number of amino acid residues varied from 2 to 7. Additional hydrolysis of these preparations by leucine aminopeptidase led to a decrease in the peptide content and to an increase in the amino acid content. This may have a beneficial effect on the quality of protein hydrolyzates.     

Synthesis of sulfhydryl cross-linking poly(ethylene glycol)-peptides and glycopeptides as carriers for gene delivery

Sulfhydryl cross-linking poly(ethylene glycol) (PEG)-peptides and glycopeptides were prepared and tested for spontaneous polymerization by disulfide bond formation when bound to plasmid DNA, resulting in stable PEG-peptide and glycopeptide DNA condensates. A 20 amino acid synthetic peptide possessing a single sulfhydryl group on the N-terminal cysteine, with two or five internal acetamidomethyl (Acm)-protected cysteine residues, was reacted with either PEG vinyl sulfone or iodoacetamide tyrosinamide triantennary N-glycan. Following RP-HPLC purification, Acm groups were removed by silver tetrafluoroborate to generate sulfhydryl cross-linking PEG-peptides and glycopeptide that were characterized by either (1)H NMR or LC-MS. Sulfhydryl cross-linking PEG-peptides and glycopeptides were found to bind to plasmid DNA and undergo disulfide cross-linking resulting in stable DNA condensates with potential utility for in vivo gene delivery.     

The use of cysteinyl peptides to effect portage transport of sulfhydryl-containing compounds in Escherichia coli

We describe a method by which sulfhydryl compounds may be transported into Escherichia coli as the mixed disulfides with a cysteine residue of a di- or tripeptide. Transport occurs through the di- or oligopeptide transport systems, and it is suggested that subsequent release of the sulfhydryl compound occurs as a result of a disulfide exchange reaction with components of the sulfhydryl-rich cytoplasm. The free sulfhydryl compounds used here (2-mercaptopyridine and 4-[N-(2-mercaptoethyl)]aminopyridine-2,6-dicarboxylic acid) show weak growth-inhibitory properties in their own right, but disulfide linkage to a cysteinyl peptide results in a considerable enhancement (up to 2 orders of magnitude). This is the first example of the use of the peptide transport systems of E. coli to effect portage transport of a poorly permeant molecule by using attachment to the side chain of one of the amino acid residues of a peptide; all previous examples have involved the incorporation of amino acid analogues into the peptide backbone. The synthesis of cysteinyl peptides containing disulfide-linked 2-mercaptopyridine is described. Displacement of the 2-mercaptopyridine by sulfhydryl compounds of interest proceeds rapidly and quantitatively in aqueous alkaline solution to provide the required peptide disulfides.     

A novel cyclic octapeptide as an analogue of cyclic peptide in triostin A

This paper reported an ongoing study of cyclic peptides as carriers of potential anti-tumor agents. In an effort to carry out anti-cancer drug design, we synthesized another novel cyclic peptide as the analogue of the cyclic peptide in Triostin A. The linear peptide chains were synthesized by coupling protected amino acid residues according to Pfp/DCC methods (Pfp: Pentafluorophenol, DCC: N,N'-Dicyclohexyl-carbodiimide) in solution. After deblocking the Boc- group of the linear octapeptide chain, the cyclic product was achieved by employing diphenylphosphoryl azide (DPPA) as cyclic agent at low temperature in DMF. Further study on cyclic octapeptide-drug conjugates is in progress.     

Novel strategy for the synthesis of template-assembled analogues of rat relaxin.

The 'template-assembled synthetic protein' (TASP) concept provides a simple and elegant approach for the preparation of analogues that retain key structural elements. We have synthesized TASP molecules containing the putative active site of relaxin, a peptide that has similar structural features to insulin but a markedly different biological role. Two types of chemoselective thiol ligation strategies (thioether and thiazolidine) were used and compared. The synthetic pendant peptides contain an essential region for bioactivity that is located in the alpha-helical region of the relaxin B-chain. Depending on whether the thioether or the thiazolidine chemistry was used to attach the peptides to the template, the reacting amino acid was placed either at the C-terminus or N-terminus, respectively, thus allowing the choice of orientation relative to the carrier molecule. The template molecule consists of a decapeptide with two proline-glycine turns and four evenly spaced lysine residues that were functionalized with the appropriate chemical moiety. This allowed reaction with the appropriately derivatized peptides in solution. To improve the template ligation step using the thioether approach, a pendant peptide C-terminal cysteamine residue was used to reduce potential steric hindrance during conjugation. The design of the peptides as well as the synthetic strategy resulted in the acquisition of mimetics showing weak non-competitive and weak competitive antagonist properties.     

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.     

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.     

Conjugation of epitope peptides with SH group to branched chain polymeric polypeptides via Cys(Npys)

Since bioconjugates may play an important role as therapeutics in the future, the development of new and effective conjugation strategies is necessary. For the attachment of peptide-like molecules to carriers, there are two main coupling methods involving amide or disulfide bonds. Conjugation through an amide bond can be achieved in several well-defined ways known from peptide chemistry. However, the formation of disulfide bridges between cysteine-containing peptides and carrier molecules still has some problems. In this paper, we describe a novel approach in which the carrier polypeptide is modified by 3-nitro-2-pyridinesulfenyl (Npys)-protected cysteine and this derivative has been applied for conjugation of Cys-containing epitope peptides with poly(L-lysine)-based branched polypeptides. Considering the stability of Npys group in the presence of pentafluorophenol, Boc-Cys(Npys)-OPfp dervivative was selected for introduction to the N-terminal of branches of polypeptides backbone. The branches of the polymers were built up from oligo(DL-alanine) (poly[Lys(DL-Ala(m))], AK) and elongated by an optically active amino acid [poly[Lys(X(i)-DL-Ala(m))], XAK]. We found that the nature of X (Glu, Ser, Thr) has great influence on the incorporation of the protected cysteine residue. Herpes simplex virus and adenovirus epitope peptides were conjugated to Boc-Cys(Npys)-modified polypeptides. Results indicate that the incorporation of epitope peptides depends on the number of Npys group on the polymers as well as on the presence/absence of Boc-protecting group on the Cys residue. This new class of Cys(Npys)-derivatized branched polypeptides is stable for a couple of months and suitable for effective preparation of epitope peptide conjugates possessing increased water solubility.     

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.     

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.     

Automated synthesis and use of N-chloroacetyl-modified peptides for the preparation of synthetic peptide polymers and peptide-protein immunogens

A method to incorporate N-chloroacetyl moieties at the amino termini of synthetic peptides using a standard program with an automated peptide synthesizer has been developed. The N-chloroacetyl-modified peptides react well with sulfhydryl containing proteins such as 4-mercaptobutyrimide-modified bovine serum albumin to form stable protein-peptide conjugates. By incorporating cysteine into the synthetic peptide, autopolymerization or cyclization of the synthetic peptide occurs by reaction of the free sulfhydryl with the chloroacetyl group. N-Chloroacetyl-derivatized peptides may be useful as reagents for potential peptide immunogens and vaccines.     

Determination of the Cysteine and Cystine Content of Proteins by Amino Acid Analysis: Application to the Characterization of Disulfide-Coupled Folding Intermediates

A method has been developed for the simultaneous detection of cysteine and cystine in proteins by amino acid analysis. In this method, the sulfhydryl groups of the cysteine residues are first blocked with 2-aminoethyl methanethiosulfonate (AEMTS). This reagent converts all free sulfhydryl groups to mixed disulfides with 2-aminoethanethiol (AET). The isolated blocked protein is subjected to oxidation with performic acid prior to hydrolysis and amino acid analysis. This procedure quantitatively converts the 2-aminoethanethiol blocking groups into taurine, and all cysteine residues (including those involved in disulfide bonds) into cysteic acid. Both of these derivatives are stable and can be recovered quantitatively by amino acid analysis. The speed and specificity with which AEMTS reacts with thiols make this method particularly effective for the characterization of disulfide-coupled folding intermediates.     

Rotavirus VP4 and VP7-Derived Synthetic Peptides as Potential Substrates of Protein Disulfide Isomerase Lead to Inhibition of Rotavirus Infection

Rotavirus infection of MA104 cells has been shown to be inhibited by cell membrane-impermeant thiol/disulfide exchange inhibitors and anti-PDI antibodies. To characterise the amino acid sequences of rotavirus structural proteins potentially mediating cell surface PDI?Csubstrate interactions, rotavirus-derived peptides from VP4 and VP7 (RRV) and VP7 (Wa), and their modified versions containing serine instead of cysteine were synthesized. Cysteine-containing VP7 peptides corresponding to residues 189?C210 or 243?C263 caused an infectivity inhibitory effect of about 64 and 85?%, respectively, when added to cells. Changing cysteine to serine significantly decreased the inhibitory effect. A cysteine-containing peptide corresponding to VP4 residues 200?C219 and its scrambled version reduced infectivity by 92 and 80?%, respectively. A cysteine to serine change in the original VP4 200?C219 peptide did not affect its inhibitory effect. Non-rotavirus related sequences containing cysteine residues efficiently inhibited rotavirus infectivity. Antibodies against VP7 residues 189?C210 or 243?C263 significantly inhibited rotavirus infectivity only after virus attachment to cells had occurred, whereas those against VP4 200?C219 peptide inhibited infectivity irrespective of whether virus or cell-attached virus was antibody-treated. A direct PDI?Cpeptide interaction was shown by ELISA for cysteine-containing VP7 and VP4 peptides. Virus?Ccell attachment was unaffected by the peptides inhibiting virus infectivity. The results showed that even though cysteine residues in the peptides tested are important in both virus infectivity inhibition and in vitro PDI?Cpeptide interaction, the accompanying amino acid sequence also plays some role. As a whole, our findings further support our hypothesis that cell surface PDI from MA104 cells might be contributing to rotavirus entry at a post-attachment step.     

Optimized conditions to couple two water-soluble biomolecules through alkylamine thiolation and thioetherification

A simple method for introducing, in buffered saline, a reactive sulfhydryl group on water-soluble molecules bearing an alkyl-amino group is described. This method is based on the use of two water-soluble reagents: 2-iminothiolane and 6,6'-dithiodinicotinic acid. The first one is open upon reaction with an amino group, and the generated thiol group is immediately protected by action of the second reagent. The optimal conditions were determined by taking into account the stability and the reactivity of both reagents with regards to pH and temperature. This method was validated through two applications, the substitution of bovine serum albumin with a bromoacetyl peptide and the substitution of an amino link at the 5' end of an oligonucleotide by reaction with either a fluorescent tag, iodoacetamidofluorescein, or a bromoacetyl peptide, upon reduction of the protected disulfide bridge with a third water-soluble reagent, namely tris(2-carboxyethyl)phosphine.     

Preparation of 'side-chain-to-side-chain' cyclic peptides by Allyl and Alloc strategy: potential for library synthesis.

Automated and manual deprotection methods for allyl/allyloxycarbonyl (Allyl/Alloc) were evaluated for the preparation of side-chain-to-side-chain cyclic peptides. Using a standard Allyl/Alloc deprotection method, a small library of cyclic peptides with lactam bridges (with seven amino acids) was prepared on an automatic peptide synthesizer. We demonstrate that the Guibe method for removing Allyl/Alloc protecting groups under specific neutral conditions [Pd(PPh3)4/PhSiH3)/DCM] can be a useful, efficient and reliable method for preparing long cyclic peptides on a resin. We have also manually synthesized a cyclic glucagon analogue containing 24 amino acid residues. These results demonstrated that properly controlled palladium-mediated deprotection of Allyl/Alloc protecting groups can be used to prepare cyclic peptides on the resin using an automated peptide synthesizer and cyclic peptides with a long chain.     

Solid phase synthesis and biological activity of mast cell degranulating (MCD) peptide: a component of bee venom

Mast cell degranulating (MCD) peptide, a 22 amino acid residue basic peptide from bee venom, was synthesized by stepwise solid phase synthesis on a benzhydrylamine resin support. N alpha-t-butyloxycarbonyl and benzyl type side chain protection was used. The two disulfide bridges were formed selectively by using S-acetamidomethyl protection for the cysteine residues in position 5 and 19 and S-methylbenzyl protection for the cysteine residues in positions 3 and 15. Crude synthetic MCD peptide was obtained following deprotection and cleavage from the resin by the low/high HF method. The peptide was isolated in pure form by ion exchange chromatography and gel filtration. The final product has physical, chemical, and biological properties identical with those reported for the natural product. The synthetic strategy utilized for MCD peptide will facilitate the availability of structurally similar analogs for evaluating antihistaminic and anti-inflammatory activities.