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PubMed1990

Labeling of epsilon-lysine crosslinking sites in proteins with peptide substrates of factor XIIIa and transglutaminase.

Peptides patterned on the N-terminal sequence of fibronectin were synthesized and tested for amine acceptor qualities in reactions with dansylcadaverine catalyzed either by coagulation factor XIIIa or intracellular transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13). On the basis of inverse half-saturations of the enzymes, the order of acceptor substrate affinity for factor XIIIa was pEAQQIV much greater than Boc-AQQIV greater than Boc-QQIV, and for transglutaminase, Boc-QQIV greater than Boc-AQQIV greater than pEAQQIV (amino acid residues are shown in one-letter code; pE, pyroglutamic acid; Boc, tert-butyloxycarbonyl). Sequence analysis of dansylcadaverine-substituted pEAQQIV indicated that the first of the two adjacent glutamine residues was the target of enzymatic modification. Boc-QIV showed no substrate activity with either enzyme. Crosslinking of crystallins in Ca2(+)-treated rabbit lens homogenate was readily inhibited by Boc-QQIV, Boc-AQQIV, and pEAQQIV, as was the formation of alpha-chain polymers in human fibrin by pEAQQIV in the presence of human factor XIIIa. SDS/PAGE analysis suggested that the inhibitory peptides selectively blocked the electron donor functionalities in these enzymatic crosslinking reactions.

Amino Acid Sequence↗

PubMed1994

The complete amino acid sequence and disulphide bond arrangement of oat alcohol-soluble avenin-3.

We have elucidated the complete amino acid sequence of one of the avenin components, avenin-3, isolated from oat (Avena sativa L.), variety Narymsky 943. The sequence of the protein was determined by sequencing of CNBr and trypsin-generated peptides in combination with mass spectrometry. The protein is a single polypeptide chain, consisting of 201 amino acid residues with M(r) 23,252.8. The N-terminal amino acid residue of the protein is blocked with 5-oxoproline (pyroglutamic acid). All eight cysteine residues in avenin-3 are involved in disulphide bonds. The positions of these bonds were established by identification of a CNBr cleavage product of the intact avenin containing all the disulfide bonds (S-S core). Subsequent subdigestion of this S-S core allowed isolation of disulphide bonded peptides detected by differential reverse-phase HPLC before and after reduction. As a result, all four disulphides Cys50 Cys183, Cys58 Cys77, Cys84 Cys85 and Cys97 Cys191 were identified. Comparison of avenins with other prolamins demonstrates a high degree of similarity, which is especially pronounced around the cysteine residues. Avenins differ slightly from other prolamins in having unique N-terminal sequences and some differences in the repeated sequence motifs.

Amino Acid Sequence↗

PubMed1989

Conformational properties of 2,3-methanopyroglutamic acid in peptides: NMR and x-ray diffraction studies.

The effects of replacing L-pyroglutamic acid with the cyclopropane analogue 2,3-methanopyroglutamic acid (2,3-MeGlp) on conformation and enzymatic stability have been investigated in 2,3-MeGlp-NHMe and the novel thyrotropin releasing hormone (TRH) analogue [2,3-MeGlp1]-TRH by x-ray diffraction and nmr. While 2,3-MeGlp-NHMe adopts a folded conformation (small psi angle) in the solid state, several conformations are available to the molecule in solution. 1H-nmr of the diastereomeric mixture [(+/- )-2,3-MeGlp1]-TRH indicates a close orientation of the pyrrolidone and imidazole rings. The 2,3-MeGlp-His amide bond is considerably more stable to pyroglutamate aminopeptidase than the Glp-His bond in TRH.

Magnetic Resonance Spectroscopy↗

PubMed1986

Metabolism of thyrotropin-releasing hormone in human cerebrospinal fluid. Isolation and characterization of pyroglutamate aminopeptidase activity.

Pyroglutamate aminopeptidase, which catalyzes metabolism of thyrotropin-releasing hormone (TRH) to cyclo(His-Pro), is the major enzyme of TRH metabolism in human CSF. The partially purified CSF pyroglutamate aminopeptidase has a pH optimum between 6.0 and 7.4, and a Km of 15.9 +/- 3.1 microM. A number of potential competitive inhibitors of the enzymatic activity were examined, of which luteinizing hormone-releasing hormone and bombesin were the most effective. An examination of the structure of various peptides that inhibit pyroglutamate aminopeptidase activity indicated that the enzyme generally prefers a substrate having amino-terminal pyroglutamic acid (pGlu) and a COOH-terminal that is either blocked or distant from amino-terminal pGlu. Heavy metals, EDTA and reducing agents inactivated the enyzme, whereas benzamidine, phenylmethylsulfonylfluoride, trypsin inhibitor and alkylating agents had little or no effect on the enzymatic activity. Thiol-oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid), however, considerally inhibited the enzymatic activity. We hypothesize that CSF pyroglutamate aminopeptidase may play a role in the biologic actions of TRH.

Aminopeptidases↗

PubMed2006

Purification and characterization of cassiicolin, the toxin produced by Corynespora cassiicola, causal agent of the leaf fall disease of rubber tree.

Cassiicolin, a phytotoxin produced by the necrotrophic fungus Corynespora cassiicola, was purified to homogeneity from a rubber tree isolate. The optimized protocol involves reverse phase chromatography followed by size exclusion chromatography, with monitoring of the toxicity on detached rubber tree leaves. Cassiicolin appeared to be a peptide composed of 27 amino acids, glycosylated on the second residue, with a N-terminal pyroglutamic acid and 6 cysteines involved in disulfide bonds. Its molecular mass was estimated to be 2885 Da. No significant sequence homology with other proteins could be found. The availability of pure toxin in sufficient amount is a prerequisite for its structure determination, which is a key step in the understanding of the aggression mechanism.

Amino Acid Sequence↗

PubMed2004

Tandem functionalization of nonactivated alkenes and alkynes in intramolecular N-acyloxyiminium ion carbocyclization. Synthesis of 6-substituted hydroindole 2-carboxylic acids.

[reaction: see text] Five-membered N-Boc acyliminium ions derived from L-pyroglutamic acid harboring 4-butenyl and 4-butynyl tethers undergo Lewis acid-mediated halo and tandem Friedel-Crafts carbocyclization within minutes at -78 degrees C to give stereodefined 6-substituted octahydroindole and hexahydroindole 2-carboxylic acid methyl esters, respectively. The cyclic vinyl bromides are excellent substrates for Pd-catalyzed Suzuki-Miyaura, Heck, and Stille couplings. The method also provides access to enantiopure sp2- and sp3-arylated azabicyclics that are novel and versatile scaffolds for chemical diversification.

Journal Article↗

PubMed1992

Cloning and sequencing of the xynA gene encoding xylanase A of Aspergillus kawachii.

We have cloned the xynA gene coding for xylanase A, a major component of the xylanase family, from Aspergillus kawachii. The cDNA was isolated from an A. kawachii cDNA library by immunoscreening using antibody raised against the purified xylanase A protein. Nucleotide sequence analysis of the cDNA showed a 981-bp open reading frame that encoded a protein of 327 amino acid residues. The signal peptide was composed of 25 amino acid residues and the N-terminus of the mature protein was pyroglutamic acid. The transformed yeast with a cloned cDNA produced xylanase. The genomic DNA was arranged as ten exons and nine introns.

Amino Acid Sequence↗

PubMed2005

Mass spectral signature for insect adipokinetic hormones.

Insect adipokinetic hormones (AKHs) are structurally similar. They consist of 8 to 10 amino acid residues, and are post-translationally modified at the N-terminus (pyroglutamic acid) and at the C-terminus (carboxyamide). They contain aromatic amino acids at position 4 (mostly Phe, in a few cases Tyr) and at position 8 (Trp). Position 9 is always Gly which is used in the octapeptides for the amidation, and the majority of the peptides have no charge. AKHs exhibit a characteristic ion signature both in matrix-assisted laser desorption/ionization (ion pair [M+Na](+)/[M+K]+) and in electrospray mass spectrometry ([M+H+K]2+, [M-17+H]+, [M+H]+, [M+Na]+, [M+K]+). Their high affinity for Na+ and K+ alkali cations is observed even after reversed-phase purification. AKHs rarely form doubly charged ions with protons or sodium while the [M+H+K]2+ ion is often abundant suggesting a special conformation of the larger metal ion complex possibly related to its size. Here, we present analyses of several AKHs of different insect species and discuss their ionization behavior with respect to their sequence. The mass spectral signature observed is useful for AKH detection from mixtures and so an unassigned 990.7 Da molecule was found in dragonfly which is currently under investigation.

Amino Acid Sequence↗

PubMed1990

Isolation and characterization of human reg protein produced in Saccharomyces cerevisiae.

reg was originally identified as a gene expressed during the regeneration of insulin-producing pancreatic beta-cells of the rat. We built an expression vector containing human reg cDNA to drive Saccharomyces cerevisiae to synthesize the reg protein, and purified it from the culture medium. The 144-amino acid sequence of the recombinant protein was consistent with that deduced from the cDNA and genomic DNA sequence except that the signal sequence of 22 amino acids was eliminated, and the amino-terminal residue of the protein was pyroglutamic acid. The secondary structure of the reg protein was predicted by determination of the intramolecular cystine linkage and of alpha-helix and beta-sheet contents.

Amino Acid Sequence↗

PubMed1998

Enantioselective multidimensional gas chromatography-mass spectrometry in the analysis of urinary organic acids.

Enantioselective multidimensional gas chromatography-mass spectrometry is a valuable tool for the enantioselective analysis of compounds from complex matrices. Samples are separated initially on a precolumn and selected substances then transferred on-line to a main-column coated with suitable chiral stationary phase for enantioselective analysis with subsequent mass selective detection. In this paper the method is used as an adjunct to urinary organic acid analysis to provide information in patients with suspected inborn errors of metabolism. Lactic acid, alpha-hydroxyisocaproic acid, 3-phenyllactic acid and 3-(4-hydroxyphenyl)-lactic acid are separated in a single run. In addition, the enantioselective analysis of pyroglutamic acid is presented. D-Enantiomers of amino acids and alpha-hydroxycarboxylic acids derived from amino acids, point to a bacterial origin whereas the L-enantiomer is predominantly of endogenous origin. Therefore the enantiomeric ratio of chiral metabolites is an important parameter for the understanding of metabolic processes.

Carboxylic Acids↗

PubMed2005

Total synthesis of cyclic tetrapeptide FR235222, a potent immunosuppressant that inhibits mammalian histone deacetylases.

[structure: see text] The total synthesis of FR235222, a potent immunosuppressant with in vivo activities, has been achieved. The key steps include assembling its (2S,9R)-2-amino-9-hydroxy-8-oxodecanoic acid residue via an olefin cross-metathesis of a methyl (R)-lactate-derived homoallyl ketone with protected allyl amino acid and constructing its trans-(2R,4S)-4-methylproline unit from protected (R)-pyroglutamic acid in seven steps.

Acremonium↗

PubMed2003

Determination of the post-translational modifications of salivary acidic proline-rich proteins.

Human salivary acidic proline-rich proteins were analyzed by electrospray-ion trap mass spectrometry and by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. All acidic-PRP isoforms share a common N-terminal region, which contains a pyroglutamic acid residue at the N-terminus, and two phosphorylation sites on Ser 8 and 22. At the same time, HPLC-MS spectra revealed isoforms of PRP-1 and PRP-3 having a different number of phosphoserine residues, namely, a mono-phosphorylated form of PRP-1 and PRP-3 and a tri-phosphorylated form of PRP-1. The analysis of the masses of tryptic digests suggested that the third phosphate residue should be located on Ser 17. Another protein with a mass of 30,923 amu was detected along the HPLC pattern and MS data of its tryptic digest suggested that it corresponds to the dimer of Pa, the isoform of PRP-1 with a substitution Arg-Cys at 103 position. Finally, structural identification is pending for another post-translational modification of acidic-PRP that provides an increase of 111-114 amu.

Amino Acid Sequence↗

PubMedDate not supplied

Preventing the spontaneous modification of an HLA-A2-restricted peptide at an N-terminal glutamine or an internal cysteine residue enhances peptide antigenicity.

The p68-derived peptide, QIVDVCHDV, was identified by a reverse immunology approach as capable of reconstituting an epitope recognized by the melanoma-reactive cytotoxic T lymphocyte (CTL) line VMM5. The peptide has not been demonstrated definitively on the cell surface by mass spectrometry; thus, it is not yet considered appropriate for use in human melanoma vaccines. Interestingly, however, the antigenicity of this peptide was affected by spontaneous modifications at two distinct residues. Spontaneous modification of the QIVDVCHDV peptide can occur at the cysteine residue at position 6 or at the N-terminal glutamine residue, and both modifications dramatically affect CTL recognition. Avoidance of an acidic environment prevents the conversion of the N-terminal glutamine residue to pyroglutamic acid, a conversion that inhibits binding of the peptide to HLA-A2 and diminishes recognition by CTLs. Substitution of asparagine for the N-terminal glutamine and substitution of serine for the cysteine were shown to enhance the binding of the peptide to HLA-A2 and to enhance the recognition of the peptide by CTLs. These findings suggest general strategies for enhancing the antigenicity of other peptides containing similar amino acids in their sequence.

Antigens, Neoplasm↗

PubMed1988

The amino acid sequence of antistasin. A potent inhibitor of factor Xa reveals a repeated internal structure.

Antistasin is a 15-kDa protein from the salivary glands of the Mexican leech, Haementeria officinalis, which manifests anticoagulant activity by inhibiting factor Xa. Previous work demonstrating the presence of this activity in salivary gland extracts and its partial purification has been reported (Tuszynski, G. P., Gasic, T. B, and Gasic, G.J. (1987) J. Biol. Chem. 262, 9718-9723). The present study includes further purification to homogeneity of antistasin and its subsequent fragmentation and complete amino acid sequence determination. The protein, which possesses 119 amino acid residues, is blocked at its amino terminus by the presence of a pyroglutamic acid residue and has an unusually high cysteine content, with 20 cysteine residues. The primary structure of antistasin shows no homology to hirudin, a 65-residue anticoagulant protein from the medicinal leech, Hirudo medicinalis. Of great interest is the finding of significant internal homology within antistasin where a 2-fold internal repeated structure is observed. At least four isoforms of antistasin have been identified in leech salivary gland extracts by high performance liquid chromatography analysis, and partial amino acid sequence analysis of these isoforms indicates they differ by 1 or 2 amino acid residues.

Amino Acid Sequence↗

PubMed1995

Purification, characterization and specificity of chondroitin lyases and glycuronidase from Flavobacterium heparinum.

The chondroitin lyases from Flavobacterium heparinum (Cytophaga heparinia) have been widely used in depolymerization of glycosaminoglycan and proteoglycan chondroitin sulphates. Oligosaccharide products derived from chondroitin sulphate can be further degraded by glycuronidases and sulphatases obtained from the same organism. There has been no reported purification of these enzymes to homogeneity nor is there any information on their physical and kinetic characteristics. The absence of pure enzymes has resulted in a lack of understanding of the optimal conditions for their catalytic activity and their substrate specificity. This has limited the use of these enzymes as reagents for preparation of oligosaccharides for structure and activity studies. Reproducible schemes to purify a chondroitin AC lyase, a glycuronidase and chondroitin B lyase from Flavobacterium heparinum to apparent homogeneity are described. Chondroitin AC lyase (chondroitinase AC, EC 4.2.2.5), glycuronidase [chondro-(1-->3)-glycuronidase, no EC number] and chondroitin B lyase (chondroitinase B, no EC number) have M(r) values (assessed by SDS/PAGE) of 74,000, 41,800 and 55,200 respectively, and isoelectric points (determined by isoelectric focusing) of 8.85, 9.28 and 9.05 respectively. Chondroitin lyase AC and B contain pyroglutamic acid at their N-termini precluding their analysis by Edman degradation. Deblocking with pyroglutamate aminopeptidase facilitated the determination of their N-terminal sequences. The kinetic properties of these enzymes have been determined as well as the optimum conditions for their catalytic activity. The specificity of the glycouronidase, determined using 17 different disaccharide substrates, shows that it only acts on unsulphated or 6-O-sulphated 1-->3 linkages. The chondroitin lyases are both endolytic enzymes, and oligosaccharide mapping shows their expected specificity towards the chondroitin and dermatan sulphate polymers.

Amino Acids↗

PubMed1994

Microidentification of N-terminal-blocked amino acid residues of proteins and peptides.

Proteins or peptides having an N-terminal-blocked amino acid were successively digested by pronase E, proteinase K, and carboxypeptidase Y. The N-blocked amino acids released from proteins or peptides were derivatized with 9-anthryldiazomethane (ADAM) to the corresponding esters followed by addition of formic acid to remove the remaining ADAM which interfered with further analysis. The anthryl esters were analyzed by high-performance liquid chromatography equipped with a fluorimetric detector. Twelve N-acetylamino acids (Asn, Gln, Ser, Thr, Gly, Ala, Tyr, Pro, Met, Val, Ile, and Leu), pyroglutamic acid, N-formylMet, and N-myristoylGly could be separated from each other and identified on the same chromatographic run. As examples of applied experiments to proteins and peptides, N-acetyl derivatives of Ser, Ala, Met, Gly, Tyr, and Pro as well as N-myristoylGly could be satisfactorily identified using 100 pmol each of seven proteins and peptides. The method reported here is an improved one that was reported in the previous paper based on the same principles.

Amino Acids↗

PubMed1990

Amino acid sequence of the acidic Kunitz-type trypsin inhibitor from winged-bean seed [Psophocarpus tetragonolobus (L.) DC].

The primary sequence of trypsin inhibitor-2 (WBTI-2) from Psophocarpus tetragonolobus (L.) DC seeds was determined. This inhibitor consists of a single polypeptide chain of 182 amino acids, including four half-cystine residues, and an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2 showed 57% identity to the basic trypsin inhibitor (WBTI-3) and 50% identity to the chymotrypsin inhibitor (WBCI) of winged bean, and 54% identity to the trypsin inhibitor DE-3 from Erythrina latissima seed. The similarity to the soybean Kunitz trypsin inhibitor (40%) and the other Kunitz-type inhibitors from Adenanthera pavonina (30%) and wheat (26%) was much lower. Sequence comparisons indicate that the Psophocarpus and Erythrina inhibitors are more closely related to each other than to other members of the Kunitz inhibitor family.

Amino Acid Sequence↗

PubMed1981

5-oxoprolinuria due to hereditary 5-oxoprolinase deficiency in two brothers--a new inborn error of the gamma-glutamyl cycle.

Two brothers, aged 16 and 11 years, had recurrent episodes of vomiting, diarrhoea and abdominal pain, starting in infancy. In spite of extensive investigations no cause of their enterocolitis could be established. After several years symptomatic treatment was discontinued without any recurrence of symptoms. Their father and several paternal relatives have had kidney stones. Both boys developed urolithiasis and an oxalate-containing stone was removed from the elder brother's kidney. He had no hypercalciuria. His glomerular and tubular function tests were normal. Gas chromatography of urine from both brothers revealed massive excretion of L-5-oxoproline (pyroglutamic acid). Glutathione levels in erythrocytes of both patients were normal. The activities of enzymes of the gamma-glutamyl cycle were analysed in erythrocytes, leukocytes and cultured skin fibroblasts. The level of glutathione synthetase was normal, as was the affinity of this enzyme for its substrate gamma-glutamyl-cysteine. Feedback inhibition of gamma-glutamyl-cysteine synthetase by glutathione was also normal. Both patients had a specific deficiency of 5-oxoprolinase, the activity of which was 2-4% of that of control subjects. Their parents had intermediate 5-oxoprolinase activities in fibroblasts, indicating a recessive mode of inheritance. Thus, 5-oxoprolinuria in these two patients was due to a lack of 5-oxoprolinase, i.e., a new inborn error in the gamma-glutamyl cycle.

Adolescent↗