Effect of Modification of the N-Terminal Region of Semax on the Expression of Nootropic Effect of Semax Analogs

A comparative study of nootropic activity of semax (MEHFPGP), an analog of ACTH_4–10, and some of its derivatives, in which the N-terminal methionine was modified or substituted with other amino acid residues, was performed. The effect of these peptides on learning of albino rats in tests with positive (food) and negative (pain) reinforcement was studied. In the case of modification of methionine by attachment of the gluconic-acid residue or substitution of methionine with lysine, the nootropic effect of the peptide was retained. The substitution of methionine with tryptophan or serine resulted in a decrease in the nootropic activity. The substitution of methionine with glycine, threonine, or alanine caused a complete loss of the nootropic activity of the peptide. Therefore, the amino acid residue located at position 1 of the heptapeptide analog semax, plays a key role in retaining the nootropic effects of the peptide and determines the degree of their expression.__________Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 4, 2005, pp. 460–466.Original Russian Text Copyright © 2005 by Glazova, Sebentsova, Levitskaya, Andreeva, Alfeeva, Kamenskii, Myasoedov.     

Convergent solid-phase peptide synthesis. VIII. Synthesis, using a photolabile resin, and purification of a methionine-containing protected peptide

The solid-phase synthesis, photolytic detachment from the solid support and purification in solution, of a fully-protected octapeptide containing a methionine residue (protected as the sulphoxide) is described. Protection of methionine in this manner avoids problems associated with the oxidation of this residue during the photolysis. The peptide has been purified by medium pressure liquid chromatography using solvent mixtures containing a high proportion of dimethylformamide in order to avoid precipitation of the peptide on the column.     

Inactivation of plasminogen activator inhibitor by oxidants

The rapidly acting plasminogen activator inhibitor (PAI) purified from cultured bovine aortic endothelial cells (BAEs) was inactivated during iodination with chloramine T and other oxidizing iodination systems. Inactivation was observed in the absence of iodine, suggesting that the loss of activity resulted from the oxidizing conditions employed. In an attempt to further study the nature of this inactivation, the PAI was treated with chloramine T under conditions that specifically oxidize methionine and cysteine residues. Both PAI inhibitory activity and the ability of the PAI to form complexes with tissue-type PA were decreased in a dose-dependent manner by such treatment. The PAI was more sensitive to oxidative inactivation than urokinase, elastase, and alpha 1-protease inhibitor. Incubation of the chloramine T inactivated PAI with methionine sulfoxide peptide reductase in the presence of dithiothreitol (DTT) restored more than 90% of the PAI activity. The reductase is a DTT-dependent enzyme that specifically converts methionine sulfoxide to methionine. Little activity was restored by either the reductase or DTT alone. These results indicate that the oxidation of at least one critical methionine residue is responsible for the loss of PAI activity upon iodination. In this respect, the BAE PAI resembles alpha 1-protease inhibitor, a well-characterized elastase inhibitor that also is inactivated by oxidants. Both inhibitors are members of the serine protease inhibitor superfamily (Serpins), and both have a methionine residue in their reactive center.     

Neurotoxicity and oxidative stress in D1M-substituted Alzheimer's A beta(1-42): relevance to N-terminal methionine chemistry in small model peptides

Small model peptides containing N-terminal methionine are reported to form sulfur-centered-free radicals that are stabilized by the terminal N atom. To test whether a similar chemistry would apply to a disease-relevant longer peptide, Alzheimer's disease (AD)-associated amyloid beta-peptide 1-42 was employed. Methionine at residue 35 of this 42-mer has been shown to be a key amino acid residue involved in amyloid beta-peptide 1-42 [A beta1-42]-mediated toxicity and therefore, the pathogenesis of AD. Previous studies have shown that mutation of the methionine residue to norleucine abrogates the oxidative stress and neurotoxic properties of A beta(1-42). In the current study, we examined if the position of methionine at residue 35 is a criterion for toxicity. In doing so, we tested the effects of moving methionine to the N-terminus of the peptide in a synthetic peptide, A beta(1-42)D1M, in which methionine was substituted for aspartic acid at the N-terminus of the peptide and all subsequent residues from D1 to L34 were shifted one position towards the carboxy-terminus. A beta(1-42)D1M exhibited oxidative stress and neurotoxicity properties similar to those of the native peptide, A beta(1-42), all of which are inhibited by the free radical scavenger Vitamin E, suggesting that reactive oxygen species may play a role in the A beta-mediated toxicity. Additionally, substitution of methionine at the N-terminus by norleucine, A beta(1-42)D1Nle, completely abrogated the oxidative stress and neurotoxicity associated with the A beta(1-42)D1M peptide. The results of this study validate the chemistry reported for short peptides with N-terminal methionines in a disease-relevant peptide.     

Synthesis of Escherichia coli heat-stable enterotoxin STp as a pre-pro form and role of the pro sequence in secretion.

Escherichia coli heat-stable enterotoxin STp is presumed from its DNA sequence to be synthesized in vivo as a 72-amino-acid residue precursor that is cleaved to generate mature STp consisting of the 18 carboxy-terminal amino acid residues. There are two methionine residues in the inferred STp sequence in addition to the methionine residue at position 1. In order to confirm production of the STp 72-amino-acid residue precursor, we substituted the additional methionine residues by oligonucleotide-directed site-specific mutagenesis. Since these substitutions did not cause a significant change in STp production, it can be concluded that STp is normally synthesized as the 72-amino-acid residue precursor. The length of the STp precursor indicated the existence of a pro sequence between the signal peptide and the mature protein. In order to identify the pro sequence and determine its role in protein secretion, deletion and fusion proteins were made. A deletion mutant in which the gene fragment encoding amino acid residues 22 to 53 of STp was removed was made. STp activity was found in the culture supernatant of cells. Amino acid sequence analysis of the purified STp deletion mutant revealed that the pro sequence encompasses amino acid residues 20 to 54. A hybrid protein consisting of STp amino acids 1 to 53 fused in frame from residue 53 to nuclease A was not secreted into the culture supernatant. These results indicate that the pro sequence does not function to guide periplasmic protein into the extracellular milieu.     

High level expression and purification of peptide methionine sulfoxide reductase in Escherichia coli.

The enzyme peptide methionine sulfoxide reductase catalyzes the conversion of methionine sulfoxide residues in proteins to methionine. The 636 nucleotide coding region of the peptide methionine sulfoxide reductase gene has been amplified from a genomic clone using the polymerase chain reaction and the product was subcloned into plasmid pGEX-2T downstream of the glutathione S-transferase gene under control of the tac promoter. Escherichia coli XL1-Blue cells transformed with this plasmid and induced with isopropylthio-beta-galactoside expressed high levels of the fusion protein. The protein was soluble and was purified to homogeneity by affinity binding to a glutathione-agarose resin followed by cleavage of the fusion protein with thrombin. Both the fusion protein and the purified peptide methionine sulfoxide reductase protein showed high peptide methionine sulfoxide reductase activity.     

Chemotactic factor inactivation by stimulated human neutrophils mediated by myeloperoxidase-catalyzed methionine oxidation

Leukocyte chemoattractants were inactivated when exposed to human neutrophils and either ingestible particles or phorbol esters. Loss of biologic activity was time- and temperature-dependent, required physiologic concentrations of viable neutrophils and a halide, and was inhibited by azide or catalase. Neutrophils from patients with either hereditary myeloperoxidase deficiency or chronic granulomatous disease failed to inactivate the chemoattractants unless purified myeloperoxidase or H2O2, respectively, was added. Susceptibility to inactivation by neutrophils correlated with the presence of methionine in the attractant. Loss of chemotactic activity was blocked by low concentrations of methionine and by higher concentrations of other reducing agents, but was unaffected by oxidized methionine. Paper chromatography demonstrated that exposure of a formyl-methionyl peptide chemotactic factor to either the cellfree myeloperoxidase system or stimulated neutrophils resulted in its conversion to a molecular species whose location in the chromatographs was identical to that of the peptide containing oxidized methionine. Thus, stimulated human neutrophils inactivate peptide chemoattractants by secretion of myeloperoxidase and H2O2, which combine with halides to form oxidants that react with a critical methionine residue. We suggest that myeloperoxidase-catalyzed oxidation of thioethers may constitute an inflammatory control mechanism as well as a general means of modifying the functional properties of biologic mediators.     

Substance P sulfoxide: separation from substance P by high-pressure liquid chromatography, biological and immunological activities, and chemical reduction

Substance P (SP), a biologically active peptide, contains a carboxy-terminal methionine residue that can oxidize to methionine sulfoxide in dilute solutions exposed to air. The oxidized form of SP, SP sulfoxide, is readily separable from SP by reverse-phase high-pressure liquid chromatography (hplc). Oxidation of SP occurs during some conventional extraction and purification procedures. In 2 m acetic acid extracts of rat brain, SP oxidation was largely prevented by addition of 0.01 m 2-mercaptoethanol. SP sulfoxide can be reduced to SP in high yield by 4.2 m 2-mercaptoethanol at 80°C in 1.5 h. The activity of SP sulfoxide in some bio- and immunoassays for SP was examined. SP sulfoxide had 36% of the sialagogie and 40% of the hypotensive activity of SP. Relative to SP, the immunoreactivity of the sulfoxide with two different antisera against SP was 80 and 40%, respectively. Thus, spontaneous oxidation of substance P can affect estimates of its tissue concentration by such assays and must be taken into account when using reverse-phase hplc to identify or purify this peptide.     

The presence of N-terminal methionine on nascent protein of rat liver and rabbit reticulocytes and its cleavage during polypeptide-chain elongation

1. The size of nascent globin peptides from which the N-terminal methionine residue is cleaved has been investigated by comparing the proportion of N-terminal methionine and valine in short and long chains. Nascent chains were labelled in rabbit reticulocyte lysates, fractionated according to length by chromatography on Sephadex G-50, and analysed by the Edman degradation of selected pooled fractions. It was found that different peptide fractions contained either methionine or valine, but not both, as the N-terminal residue. Methionine was present at the N-terminus of globin chains containing up to approx. 50 amino acids whereas valine was found to be the N-terminal amino acid of longer peptides. 2. In similar experiments with nascent proteins of rat liver, labelled either in vivo or in a cell-free system containing microsomal material and cell sap, evidence was obtained for the presence of methionine at the N-terminus of nascent chains up to approx. 65 amino acid residues long. Thus protein synthesis in liver appears to be initiated also by methionine, but in this case cleavage takes place somewhat later during peptide elongation than in globin synthesis.     

Amino acid sequences around 1, 2-epoxy-3-(p-nitrophenoxy)propane-reactive residues in rhizopus chinensis acid protease: homology with pepsin and rennin.

Two different peptides containing an aspartyl residue reactive with 1, 2-epoxy-3-(p-nitrophenoxy)propane (EPNP) in the acid protease from Rhizopus chinensis were isolated from a peptic digest of the EPNP-modified enzyme. One of the peptides was sequenced as Asp-Thr-Gly-Ser-Asp. The amino acid sequence had very high homology with those around the EPNP-reactive aspartyl residues in rennin (chymosin) [EC 3.4.23.4] and pepsin [EC 3.4.23.1]. The other peptide contained no methionine residue and gave the sequence: Asp-Thr-Gly-Thr-Thr-Leu. The N-terminal aspartyl residue of each peptide was deduced to be the EPNP-reactive site.     

Larval and pupal induction and N-terminal amino acid sequence of lysozyme from Heliothis virescens

Fifth instar larvae and prepupae of Heliothis virescens (tobacco budworm) were injected with live Enterobacter cloacae and bled at different times after vaccination. Immune pupal hemolymph showed a 54 times increase in lysozyme activity when compared with normal larval hemolymph, and an 11 times increase of lysozyme activity when compared with immune larval hemolymph. Lysozyme activity of the normal pupal hemolymph increased as greatly as did lysozyme activity of the immune larval hemolymph after metamorphosis. The pupal immune response with regard to lysozyme was much greater than the larval immune response in H. virescens. Lysozyme was purified by heat treatment at 100 degrees C and a chromatography series that included reverse-phase HPLC. The molecular mass of H. virescens lysozyme was approximately 16 kDa by SDS-PAGE which is greater than other insect lysozymes and chicken lysozyme. Amino acid sequence of the N-terminus showed that H. virescens lysozyme is 82% homologous with lysozyme of Manduca sexta and Galleria mellonella. CNBr cleavage of H. virescens lysozyme produced 11 and 6 kDa peptide fragments indicating that one methionine was present, which was also supported by amino acid analysis. However, methionine was located at the carboxyl terminal side rather than the N-terminal side as judged by the N-terminal sequences of each peptide fragment. The residue 22 in most lepidopteran lysozymes is methionine, whereas H. virescens lysozyme had a leucine at residue 22. There was an amino acid deletion near the carboxyl terminal side of H. virescens lysozyme as also found in Trichoplusia ni.     

Site-specific modification of Candida antarctica lipase B via residue-specific incorporation of a non-canonical amino acid

In order to modify proteins in a controlled way, new functionalities need to be introduced in a defined manner. One way to accomplish this is by the incorporation of a non-natural amino acid of which the side chain can selectively be reacted to other molecules. We have investigated whether the relatively simple method of residue-specific replacement of methionine by azidohomoalanine can be used to achieve monofunctionalization of the model enzyme Candida antarctica lipase B. A protein variant was engineered with one additional methionine residue. Due to the high hydrophobicity and low abundance of methionine, this was the only residue out of five that was exposed to the solvent. The use of the Cu (I)-catalyzed [3 + 2] cycloaddition under native conditions resulted in a monofunctionalized enzyme which retained hydrolytic activity. The strategy can be considered a convenient tool to modify proteins at a single position as long as one solvent-exposed methionine is available.     

Neuropeptide Y in guinea pig, rabbit, rat and man. Identical amino acid sequence and oxidation of methionine-17

Neuropeptide Y (NPY) was isolated and characterised from acid-ethanol extracts of rabbit and guinea pig brain. In both instances the chromatographic purification was a two-step procedure of gel filtration followed by reverse-phase high-performance liquid chromatography. The amino acid sequence of rabbit and guinea pig NPY was found to be identical to human and rat NPY as deduced from the cDNA structures. With the exception of the porcine peptide, all mammalian NPYs characterised to date have a methionine residue in position 17. This methionine residue is readily oxidized as indicated by the high degree of spontaneous oxidation of peptides found in the rabbit and guinea pig brain extracts and in NPY extracted from a rat phaeochromocytoma cell line. It is concluded that NPY is among the most highly conserved peptides and that NPYs containing methionine in position 17 are prone to oxidation.     

Hypochlorous acid generated by myeloperoxidase modifies adjacent tryptophan and glycine residues in the catalytic domain of matrix metalloproteinase-7 (matrilysin): an oxidative mechanism for restraining proteolytic activity during inflammation

Dysregulation of matrix metalloproteinase (MMP) activity is implicated in tissue destruction under inflammatory conditions. An important mechanism controlling enzymatic activity might involve reactive oxygen species generated by phagocytes. Myeloperoxidase, a heme protein secreted by neutrophils, monocytes, and macrophages, uses hydrogen peroxide to generate hypochlorous acid (HOCl). We demonstrate that HOCl inhibits the activity of human matrilysin (MMP-7) in vitro, suggesting that it might limit proteolytic activity during inflammation. When MMP-7 was exposed to HOCl generated by myeloperoxidase, the proteinase lost activity. High performance liquid chromatographic analysis of the tryptic digest of the HOCl-treated proteinase demonstrated the absence of two peptides that were present in the untreated enzyme. Tandem mass spectrometric analysis revealed that both of the lost peptides contained methionine and tryptophan-glycine residues. The methionine residue of one of the peptides had been oxidized to methionine sulfoxide. In contrast, the major product from the other peptide was 4 atomic mass units smaller than its precursor (WG-4). This novel oxidation product was derived though modification of adjacent tryptophan and glycine residues in the catalytic domain of the enzyme. Loss of proteolytic activity was associated with conversion of the precursor peptide to WG-4 but not with methionine oxidation. In contrast, hydrogen peroxide failed to oxidize MMP-7 or to inactivate the enzyme. Thus, HOCl inactivates MMP-7, perhaps by site-specific conversion of tryptophan-glycine to WG-4. This inactivation mechanism is distinct from the well studied mechanisms involving tissue inhibitors of metalloproteinases. Our findings suggest that local pericellular production of HOCl by phagocytes is a physiological mechanism for governing MMP activity during inflammation.     

八肽胆囊收缩素的结构与活性关系

本文采用液相多肽合成法制备了八肽胆囊收缩素(CCK_8)的六种类似物,并测定了它们诱导离体的豚鼠胆囊收缩的生物学活性。发现CCK_8的N-端乙酰化不改变其生物活性,脱去N-端氨基的CCK_8类似物即Suc-CCK_7与母体CCK_8相比,其活性明显增加。在Boc-CCK_7中,Met被NIe取代活性可完全保留,Gly~(29)被D-Ala取代后仍保留相当的活性,但Gly~(29)被β-Ala取代后则失去胆囊收缩活性;在Met~(28)-Gly~(29)区域引入刚性的r-内酰胺环作为构象限制,也导致活性完全丧失。     

Yeast methionine aminopeptidase I. Alteration of substrate specificity by site-directed mutagenesis

In eukaryotes, two isozymes (I and II) of methionine aminopeptidase (MetAP) catalyze the removal of the initiator methionine if the penultimate residue has a small radius of gyration (glycine, alanine, serine, threonine, proline, valine, and cysteine). Using site-directed mutagenesis, recombinant yeast MetAP I derivatives that are able to cleave N-terminal methionine from substrates that have larger penultimate residues have been expressed. A Met to Ala change at 329 (Met206 in Escherichia coli enzyme) produces an average catalytic efficiency 1.5-fold higher than the native enzyme on normal substrates and cleaves substrates containing penultimate asparagine, glutamine, isoleucine, leucine, methionine, and phenylalanine. Interestingly, the native enzyme also has significant activity with the asparagine peptide not previously identified as a substrate. Mutation of Gln356 (Gln233 in E. coli MetAP) to alanine results in a catalytic efficiency about one-third that of native with normal substrates but which can cleave methionine from substrates with penultimate histidine, asparagine, glutamine, leucine, methionine, phenylalanine, and tryptophan. Mutation of Ser195 to alanine had no effect on substrate specificity. None of the altered enzymes produced cleaved substrates with a fully charged residue (lysine, arginine, aspartic acid, or glutamic acid) or tyrosine in the penultimate position.     

Role of calpains and kininogens in inflammation.

Neutrophil chemotactic activity was found in the autodigest of calcium dependent cysteine proteinase (calpain) I purified from human erythrocytes, an active peptide was isolated, and its structure was determined. It was an N-acetyl nonapeptide with the sequence: N-acetyl Ser-Glu-Glu-Ile-Ile-Thr-Pro-Val-Tyr. This peptide was identical with the N-terminal amino acid sequence of the large subunit of calpain I deduced from cDNA sequence, except that the peptide was lacking a methionine residue and was acetylated at the N-terminus. A number of N-acetyl peptides with N-terminal amino acid sequences of large and small subunits of calpains I and II were synthesized and their chemotactic activity was estimated. In addition to the N-acetyl nonapeptide from calpain I large subunit, several peptides of different lengths from the small subunit showed dose-dependent migrations of neutrophils. They include N-acetyl tetra, hepta, octa, nona and larger size peptides. Further, it was also revealed that when calpain was incubated with high molecular weight (HMW) or low molecular weight (LMW) kininogen, kinin liberation occurred with simultaneous inhibition of calpains by kininogens. These data suggest that chemical mediators generated from the calpain-kininogen system may participate in migration and accumulation of neutrophils to the inflammatory locus.     

Characterization of N alpha-acetyl methionyl human growth hormone formed during expression in Saccharomyces cerevisiae with liquid chromatography and mass spectrometry

We found a new variant of human growth hormone (hGH) from the recombinant hGH expression process in Saccharomyces cerevisiae. The variant was identified as N(alpha)-acetyl methionyl hGH which may be formed by N(alpha)-acetylation of met-hGH during the intracellular expression of hGH in S. cerevisiae. The variant was isolated from manufacturing process of LG Life Sciences' hGH product. The variant was subjected to trypsin digestion and RP-HPLC analysis, resulting in a delayed retention time and an increased mass (173 Da) of T1 tryptic peptide. The amino acid composition and amino acid sequence of the peptide showed the same result with T1 peptide of met-hGH except the N-terminal modification on methionine in the variant peptide. With collision induced dissociation (CID) experiments of the variant T1 tryptic peptide, we found the sequence and the a(1) fragment of N-terminal residue matched with those of acetyl-methionyl hGH. Within our production process, we produce the methionyl hGH first and then use the aminopeptidase to cut the N-terminal methionine. So the acetylation may inhibit the aminopeptidase to remove methionine and produces N(alpha)-acetyl methionyl hGH. And the biological activity of the variant was comparable to one of the unmodified hGH when tested by rat weight gain bioassay.     

Engineering increased stability in the antimicrobial peptide pediocin PA-1

Pediocin PA-1 is a food grade antimicrobial peptide that has been used as a food preservative. Upon storage at 4 degrees C or room temperature, pediocin PA-1 looses activity, and there is a concomitant 16-Da increase in the molecular mass. It is shown that the loss of activity follows first-order kinetics and that the instability can be prevented by replacing the single methionine residue (Met31) in pediocin PA-1. Replacing Met by Ala, Ile, or Leu protected the peptide from oxidation and had only minor effects on bacteriocin activity (for most indicator strains 100% activity was maintained). Replacement of Met by Asp was highly deleterious for bacteriocin activity.     

Calcium dependent cysteine proteinase is a precursor of a chemotactic factor for neutrophils

A new chemotactic factor for neutrophils is generated from calcium dependent cysteine proteinase (calpain) I by autodigestion. An active peptide was isolated from the autodigest and its structure was determined to be an acetylated nonapeptide with the sequence: N-acetyl Ser-Glu-Glu-Ile-Ile-Thr-Pro-Val-Tyr. Compared with the entire sequence of human calpain I, the peptide was identical with the N-terminal amino acid sequence of the large subunit deduced from the cDNA sequence, except that the peptide was devoid of a methionine residue and acetylated at the N-terminus. The acetyl nonapeptide was synthesized and its chemotactic activity was reconfirmed. The biological significance and possible role of this calpain derived chemotactic factor in inflammation are discussed.