Generation of a cysteine sulfinic acid analog for incorporation in peptides using solid phase peptide synthesis

The sulfinic acid analog of aspartic acid, cysteine sulfinic acid, introduces a sulfur atom that perturbs the acidity and oxidation properties of aspartic acid. Cysteine sulfinic acids are often introduced in peptides and proteins by oxidation of cysteine, but this method is limited as all cysteine residues are oxidized and cysteine residues are often oxidized to sulfonic acids. To provide the foundation for the specific incorporation of cysteine sulfinic acids in peptides and proteins, we synthesized a 9-fluorenylmethyloxycarbonyl (Fmoc) benzothiazole sulfone analog. Oxidation conditions to generate the sulfone were examined and oxidation of the Fmoc-protected sulfide (3) with NbC in hydrogen peroxide provided the corresponding sulfone (4) in the highest yield and purity. Reduction with sodium borohydride generated the cysteine sulfinic acid (5) suggesting this approach may be an efficient method to incorporate a cysteine sulfinic acid in biomolecules. A model tripeptide bearing a cysteine sulfinic acid was synthesized using this approach. Future studies are aimed at using this method to incorporate cysteine sulfinic acids in peptide hormones and proteins for use in the study of biological function.     

S-acylated residues of the acyl-carrier protein subunit of Klebsiella, aerogenes citrate lyase

Oxidation of the isolated deacetyl acyl-carrier protein subunit of citrate lyase from $\text{Klebsiella}\text{,}\text{aerogenes}$ with Cu²⁺-$\text{o}$-phenanthroline complex leads exclusively to intrapeptide disulfide bridge formation indicating that the cysteamine and the cysteine residues are located in close proximity. The S-acetylation of the cysteine residue in deacetyl acyl-carrier protein subunit is catalysed by a citrate lyase ligase preparation in presence of acetate and ATP. Reaction-inactivation of citrate lyase results in deacetylation of the S-acetyl cysteamine residue of the prosthetic group but not of the S-acylated cysteine residue in the acyl-carrier protein.     

Structural and functional roles of cysteine residues of Bacillus polymyxa beta-amylase.

Bacillus polymyxa beta-amylase contains three cysteine residues at positions 83, 91, and 323, which can react with sulfhydryl reagents. To determine the role of cysteine residues in the catalytic reaction, cysteine residues were mutated to construct four mutant enzymes, C83S, C91V, C323S, and C-free. Wild-type and mutant forms of the enzyme were expressed in, and purified to homogeneity from, Bacillus subtilis. A disulfide bond between Cys83 and Cys91 was identified by isolation of tryptic peptides bearing a fluorescent label, IAEDANS, from wild-type and C91 V enzymes followed by amino acid sequencing. Therefore, only Cys323 contains a free SH group. Replacement of cysteine residues with serine or valine residues resulted in a significant decrease in the kcat/Km value of the enzyme. C323S, containing no free SH group, however, retained a high specific activity, approximately 20% of the wild-type enzyme. None of the cysteine residues participate directly in the catalytic reaction.     

Covalent chromatography — The isolation of tryptophanyl containing peptides by novel polymeric reagents

Specific polymeric reagents for reversible covalent binding of tryptophan residues have been developed. Polymers bearing Aryl-SxCl groups (x=2–3) were prepared by binding thioaryl groups to cross-linked polyacrylamide, and subsequently reacting the products with an excess of S₂Cl₂. The resulting polymers were allowed to react with various mixtures of amino acids and peptides (excluding cysteine and its peptides) in acidic media. It was found that tryptophan as well as tryptophan-containing peptides were selectively bound to the polymer. Upon reduction with thiols (e.g. dithiothreitol), 2-thiotryptophan or its peptide derivatives were cleaved from the polymeric matrix. The proposed method is used for a one step isolation of tryptophanyl-containing peptides from peptide mixtures as well as for introducing thiol groups at the tryptophanyl residues.     

Alkylation of cysteine-containing peptides to mimic palmitoylation.

Numerous proteins that are involved in cell signaling and viral replication require post-translational modification by palmitoylation to function properly. The molecular details by which this palmitoyl modification affects protein function remain poorly understood. To facilitate in vitro biochemical and structural studies of the role of palmitoylation on protein function, a method was developed for alkylating peptides with saturated C16 groups at cysteine residues and demonstrated using peptides derived from the palmitoylated region of Sindbis virus E2 glycoprotein. The synthetic approach takes advantage of disulfide chemistry to specifically modify only the cysteine residues within peptides and covalently links C16 groups via disulfide bridges using a new thioalkylating reagent, hexyldexyldithiopyridine. The chemistry presented here takes place in solution under mild conditions without the need for protection of the peptide functional groups. A method for purifying these modified peptides is also described. This protocol can be of general use to investigators studying the role of palmitoylation in biological systems.     

Configuration and racemization determination of cysteine residues in peptides by chiral derivatization and HPLC: application to oxytocin peptides.

An improved RP-HPLC method was developed for the determination of the configuration and stereochemical purity of cysteine residues in peptides. The method consists of oxidation of cysteine and cystine residues to cysteic acid, followed by hydrolysis and pre-column chiral derivatization with Val-Marfey's reagent.     

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.     

Probes of the catalytic site of cysteine dioxygenase

The first major step of cysteine catabolism, the oxidation of cysteine to cysteine sulfinic acid, is catalyzed by cysteine dioxygenase (CDO). In the present work, we utilize recombinant rat liver CDO and cysteine derivatives to elucidate structural parameters involved in substrate recognition and x-ray absorption spectroscopy to probe the interaction of the active site iron center with cysteine. Kinetic studies using cysteine structural analogs show that most are inhibitors and that a terminal functional group bearing a negative charge (e.g. a carboxylate) is required for binding. The substrate-binding site has no stringent restrictions with respect to the size of the amino acid. Lack of the amino or carboxyl groups at the alpha-carbon does not prevent the molecules from interacting with the active site. In fact, cysteamine is shown to be a potent activator of the enzyme without being a substrate. CDO was also rendered inactive upon complexation with the metal-binding inhibitors azide and cyanide. Unlike many non-heme iron dioxygenases that employ alpha-keto acids as cofactors, CDO was shown to be the only dioxygenase known to be inhibited by alpha-ketoglutarate.     

Determination of dehydroalanine residues in proteins and peptides: An improved method

Dehydroalanine residues in peptides and proteins react with 4-pyridoethanethiol in alkaline solution to form 4-pyridoethyl cysteine residues, which after acid hydrolysis, give the corresponding amino acid. An experimental protocol has been developed, and with simple dehydroalanine derivatives, conversions of 96–99% are achieved. Tests with peptides and proteins show that serine and cysteine/cystine residues do not interfere with this analytical procedure. A sample of wool keratin contained 57 µmol dehydroalanine/g.     

Treatment of YAC128 mice and their wild-type littermates with cystamine does not lead to its accumulation in plasma or brain: implications for the treatment of Huntington disease

Cystamine is beneficial to Huntington disease (HD) transgenic mice. To elucidate the mechanism, cystamine metabolites were determined in brain and plasma of cystamine-treated mice. A major route for cystamine metabolism is thought to be: cystamine --> cysteamine --> hypotaurine --> taurine. Here we describe an HPLC system with coulometric detection that can rapidly measure underivatized cystamine, cysteamine and hypotaurine, as well as cysteine and glutathione in the same deproteinized tissue sample. A method is also described for the coulometric estimation of taurine as its isoindole-sulfonate derivative. Using this new methodology we showed that cystamine and cysteamine are undetectable (< or = 0.2 nmol/100 mg protein) in the brains of 3-month-old HD transgenic (YAC128) mice (or their wild-type littermates) treated daily for 2 weeks with cystamine (225 mg/kg) in their drinking water. No significant changes were observed in brain glutathione and taurine but significant increases were observed in brain cysteine. Cystamine and cysteamine were not detected in the plasma of YAC128 mice treated daily with cystamine between the ages of 4 and 12 or 7 and 12 months. These findings suggest that cystamine is not directly involved in mitigating HD but that increased brain cysteine or uncharacterized sulfur metabolites may be responsible.     

Synthesis of d-labeled N-alkylmaleimides and application to quantitative peptide analysis by isotope differential mass spectrometry

d-Labeled N-alkylmaleimides have been prepared for specific modification of the terminal SH groups of cysteine residues in proteins or peptides. These reagents are useful tools for quantitative analysis of peptides by stable isotope differential mass spectrometry.     

Redox regulation of enzymatic activity and proteolytic susceptibility of ribulose-1,5-bisphosphate carboxylase/oxygenase fromEuglena gracilis

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase fromEuglena gracilis decays steadily when exposed to agents that induce oxidative modification of cysteine residues (Cu²⁺, benzofuroxan, disulfides, arsenite, oxidized ascorbate). Inactivation takes place with a concomitant loss of cysteine sulfhydryl groups and dimerization of large subunits of the enzyme. 40% activity loss induced by the vicinal thiol-reagent arsenite is caused by modification of a few neighbor residues while the almost complete inactivation achieved with disulfides is due to extensive oxidation leading to formation of mixed disulfides with critical cysteines of the protein. In most cases oxidative inactivation is also accompanied by an increased sensitivity to proteolysis by trypsin, chymotrypsin or proteinase K. Both enzymatic activity and resistance to proteolysis can be restored through treatment with several thiols (cysteamine, cysteine, dithiothreitol and, more slowly, reduced glutathione). Redox effectors which are thought to regulate the chloroplast activity (NADPH, ferredoxin and thioredoxin) do not reactivate the oxidized enzyme. When ribulose-1,5-bisphoshate carboxylase/oxygenase is incubated with cystamine/cysteamine mixtures having different disulfide/thiol ratio (r), inactivation takes place around r=1.5 while proteolytic sensitization occurs under more oxidative conditions (r=4). It is suggested that oxidative modification may happen in vivo under exceptional circumstances, such as senescence, bleaching or different kinds of stress, leading to enzyme inactivation and triggering the selective degradation of the carboxylase that has been repeatedly observed during these processes.     

Synthesis of D-labeled naphthyliodoacetamide and application to quantitative peptide analysis by isotope differential mass spectrometry

D-labeled and -unlabeled N-beta-naphthyliodoacetamides have been synthesized for specific modification of the sulfhydryl groups of cysteine residues in proteins or peptides, and have been applied to quantitative analysis of several peptides. A combination of these reagents, coupled with mass spectrometry, is anticipated to serve as a useful tool for quantitative analysis of peptides and hence proteins.     

NADPH-cytochrome P-450 reductase (pig liver). Studies on the sequence of the cyanogen bromide peptides from the catalytic domain and on the reactivity of the thiol groups

The reactivity of the cysteine residues in the non-denatured catalytic domain of the NADPH-cytochrome P-450 reductase (pig liver) was studied using the -SH reagent monobromobimane. Prerequisite was the characterization of the cysteine residues by their surrounding amino-acid sequences. In pursuit of these aims the CNBr fragments obtained from the catalytic domain were sequenced. The cysteine residues are distributed on six CNBr fragments of the catalytic domain [Vogel and Lumper (1984) Hoppe-Seyler's Z. Physiol. Chem. 365, 1074]. Only the 11-kDa CNBr peptides with the N-terminal sequences Val-Gly-Pro-Thr- and Ala-Ser-Ser-Ser-, respectively, contain two cysteine residues each. The cysteine residues of the catalytic domain accessible to monobromobimane were localized on three CNBr peptides with the N-terminal sequences Val-Gly-Pro-Thr-, Ala-Ser-Ser-Ser- and Ala-Arg-Asp-Val-, respectively. Inactivation of the trypsin-solubilized enzyme by -SH-directed reagents is caused by the modification of the accessible cysteine residue (which can be protected by NADPH) in the 11-kDa CNBr fragment (N-terminal sequence: Val-Gly-Pro-Thr-). The cosubstrate NADPH protected a second cysteine residue localized in the 11-kDa CNBr peptide with the N-terminal sequence Ala-Ser-Ser-Ser-, which is however modified at a distinctly slower rate than the critical cysteine residue characterized by the sequence -Gly-Glu-Thr-Leu-Leu-Tyr-Tyr-Gly-Cys-Arg-Arg. Five non-reacting thiol groups were localized on CNBr fragments with the N-terminal sequences Val-Gly-Pro-Thr-, Ala-Ser-Ser-Ser-, Ser-Leu-Asn-Asn-, Gly-Lys-Tyr-Val-Asp- and Ala-Ala-Asp-Pro-.     

Effects of thiol compounds on in vitro maturation of canine oocytes collected from different reproductive stages

Various thiol compounds are known to improve cytoplasmic and/or nuclear maturation of oocytes in vitro. The present study examined the effects of two thiol compounds, cysteine (0.1, 0.5, and 1.0 mM) and cysteamine (50, 100, and 200 microM), on cytoplasmic and nuclear maturation of canine oocytes. Oocytes collected from different reproductive stages were cultured in TCM-199 supplemented with 10% fetal bovine serum, 2.2 mg/ml sodium carbonate, 2.0 microg/ml estrogen, 0.5 microg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin-streptomycin solution for 72 h. Data were analyzed by two-way ANOVA after arcscine transformation and protected by Bonferroni post hoc test. The effects of cysteine and cysteamine on canine IVM were varied depending on the reproductive stage of oocyte donor bitches. In the follicular stage, significantly more oocytes reached the metaphase II (M II) stage when cultured with 0.5 or 1.0 mM cysteine (16.7% and 16.9%, respectively) compared to the control (6.2%). In the follicular stage, cysteamine increased oocyte maturation rate upto the M II stage (15.1% to 17.0%) compared to the control (4.4%). Both the 0.5 mM cysteine and 100 microM cysteamine, alone or together, increased the intracellular GSH level of canine oocytes compared to the control. Irrespective of reproductive stage, no further beneficial effects on nuclear or cytoplasmic maturation were observed when 0.5 mM cysteine and 100 microM cysteamine were supplemented together. In conclusion, addition of 0.5 mM cysteine and 100 microM cysteamine to the maturation medium improved IVM of canine oocytes.     

Synthesis of 13C-labeled iodoacetanilide and application to quantitative peptide analysis by isotope differential mass spectrometry

13C-Labeled and unlabeled iodoacetanilides have been synthesized for covalent modification of the sulfhydryl groups of cysteine residues in proteins or peptides. A combination of these reagents, coupled with mass spectrometry, is a powerful tool for quantitative analysis of peptides and hence proteins.     

Isotope-coded, iodoacetamide-based reagent to determine individual cysteine pK(a) values by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Cysteine reactivity in enzymes is imparted to a large extent by the stabilization of the deprotonated form of the reduced cysteine (i.e., the thiolate) within the active site. Although this is likely to be an important chemical attribute of many thiol-based enzymes, including cysteine-dependent peroxidases (peroxiredoxins) and proteases, only relatively few pK(a) values have been determined experimentally. Presented here is a new technique for determining the pK(a) value of cysteine residues through quantitative mass spectrometry following chemical modification with an iodoacetamide-based reagent over a range of pH buffers. This isotope-coded reagent, N-phenyl iodoacetamide (iodoacetanilide), is readily prepared in deuterated (d(5)) and protiated (d(0)) versions and is more reactive toward free cysteine than is iodoacetamide. Using this approach, the pK(a) values for the two cysteine residues in Escherichia coli thioredoxin were determined to be 6.5 and greater than 10.0, in good agreement with previous reports using chemical modification approaches. This technique allows the pK(a) of specific cysteine residues to be determined in a clear, fast, and simple manner and, because cysteine residues on separate tryptic peptides are measured separately, is not complicated by the presence of multiple cysteines within the protein of interest.     

A new method for the selective isolation of cysteine-containing peptides. Specific labelling of the thiol group with a hydrophobic chromophore.

A new method for the selective isolation of cysteine-containing peptides was designed. The method is based on the specific labelling of thiol groups with a hydrophobic chromophore followed by enzymic fragmentation of the labelled protein and reversed-phase high-pressure liquid-chromatographic separation of the peptide mixture. This new method has several distinct advantages: (1) the hydrophobic-chromophore-labelled cysteine-containing peptides are easily separated from non-cysteine-containing peptides by reversed-phase high-pressure liquid chromatography; (2) only cysteine-containing peptides are detected in the visible region with sensitivity at the low picomole level; this high sensitivity allows isolation of nanogram amounts of pure cysteine-containing peptide; (3) during sequence determination of the chromophore-labelled cysteine-containing peptides, the cysteine residues are released as coloured anilinothiazolinone derivatives and can be detected directly in the picomole range; (4) with proteins bearing several disulphide groups, each disulphide group may undergo a different degree of reduction, and therefore the recovery of individual cysteine-containing peptides may be used to deduce the disulphide linkages present in the native protein. Two thiol-specific reagents, 4-dimethylaminoazobenzene-4'-iodoacetamide and 4-dimethylaminoazobenzene-4'-N-maleimide, were synthesized and characterized. The method was successfully used to isolate five cysteine-containing peptides from a completely reduced monoclonal-antibody kappa-light chain raised against the azobenzenearsonate determinant and six cysteine-containing peptides from a kappa-light chain raised against streptococcal group A polysaccharide. The principle of this method is applicable to the isolation of any peptide containing amino acid residues that can be specifically labelled with a hydrophobic chromophore.     

Thiols as myeloperoxidase-oxidase substrates.

Nine low-Mr thiols were compared with regard to their ability to function as myeloperoxidase-oxidase substrates under conditions where no auto-oxidation of the thiols could be observed. The methyl and ethyl esters of cysteine were found to be about twice as active as cysteamine at pH 7.0, in terms of increased O2 consumption. Cysteine itself was poorly active, whereas glutathione, N-acetylcysteine and penicillamine were completely inactive as myeloperoxidase-oxidase substrates under these conditions. The structure-activity relationships indicated that both a free thiol and free amino group were required for peroxidase-oxidase activity, and also that a free carboxy group abolished activity. In analogy with cysteamine, the activities of both cysteine esters were inhibited by superoxide dismutase (less than 5 micrograms/ml) and by catalase and not by the hydroxyl-radical scavenger mannitol. In contrast with cysteamine, the activities of both cysteine esters were stimulated more than 2-fold by high concentrations (greater than 5 micrograms/ml) of superoxide dismutase. The activities of both cysteine esters exhibited broad pH optima at pH 7. A mechanism for the myeloperoxidase-oxidase oxidation of the cysteine esters is proposed, which is partly different from that previously proposed for cysteamine.     

Disulfide bond formation in NGR fiber-modified adenovirus is essential for retargeting to aminopeptidase N

The peptide motif NGR (asparagine-glycine-arginine) is known to bind to aminopeptidase N (APN). We have constructed five adenoviruses (Ads) bearing NGR in the HI loop of the adenoviral fiber protein. We compared the targeting properties of the NGR peptide within different amino acid environments and showed that their cellular receptor(s) were not identical. Ads containing NGR within potentially cyclic sequences flanked by cysteines retargeted viruses mainly to APN, while Ads containing NGR within linear sequences not containing cysteines retargeted Ads mainly to alpha(v)beta(3) integrin, albeit with a lower affinity. Finally, we show evidence that disulfide bond formation within an Ad bearing the CDCNGRCFC sequence is essential for retargeting to APN, suggesting that this sequence does indeed assume a cyclic structure which facilitates NGR binding to APN. Therefore, our study underscores the importance of cysteine residues flanking targeting peptides for not only affinity but also specificity of the retargeted Ad.