Gamma-carboxylation Of Glutamic Acid Research Articles

Cysteine-free cone snail venom peptides: Classification of precursor proteins and identification of mature peptides.

The cysteine-free acyclic peptides present in marine cone snail venom have been much less investigated than their disulfide bonded counterparts. Precursor protein sequences derived from transcriptomic data, together with mass spectrometric fragmentation patterns for peptides present in venom duct tissue extracts, permit the identification of mature peptides. Twelve distinct gene superfamiles have been identified with precursor lengths between 64 and 158 residues. In the case of Conus monile, three distinct mature peptides have been identified, arising from two distinct protein precursors. Mature acyclic peptides are often post-translationally modified, with C-terminus amidation, a feature characteristic of neuropeptides. In the present study, 20 acyclic peptides from Conus monile and Conus betulinus were identified. The common modifications of C-terminus amidation, gamma carboxylation of glutamic acid (E to ϒ), N-terminus conversion of Gln (Q) to a pyroglutamyl residue (Z), and hydroxylation of Pro (P) to Hyp (O) are observed in one or more peptides identified in this study. Proteolytic trimming of sequences by cleavage at the C-terminus of Asn (N) residues is established. The presence of an asparagine endopeptidase is strengthened by the identification of legumain-like sequences in the transcriptome assemblies from diverse Conus species. Such sequences may be expected to have a cleavage specificity at Asn-Xxx peptide bonds.

Cone Snail Glutaminyl Cyclase Sequences from Transcriptomic Analysis and Mass Spectrometric Characterization of Two Pyroglutamyl Conotoxins.

The post-translational modification of N-terminal glutamine (Q) to a pyroglutamyl (Z) residue is observed in the conotoxins produced by marine cone snails. This conversion requires the action of the enzyme glutaminyl cyclase (QC). Four complete QC sequences from the species C. araneosus, C. frigidus, C. litteratus, and C. monile and two partial sequences from C. amadis and C. miles have been obtained by analysis of transcriptomic data. Comparisons with mammalian enzyme sequences establish a high level of identity and complete conservation of functional active site residues, including a cluster of hydrogen-bonded acidic side chains. Mass spectrometric analysis of crude venom samples coupled to conotoxin precursor protein sequences obtained from transcriptomic data establishes the presence of pyroglutamyl conotoxins in the venom of C. frigidus and C. amadis. The C. frigidus peptide belongs to the M superfamily, with cysteine framework III, whereas the C. amadis peptide belongs to the divergent superfamily with cysteine framework VI/VII. Additionally, gamma carboxylation of glutamic acid and hydroxylation of proline are observed in the C. frigidus peptide. Mass spectral data are available via ProteomeXchange with identifier PXD009006.

Mass spectrometric identification of bromotryptophan containing conotoxin sequences from the venom of C. amadis

Four 30 residue conotoxin have been identified from the venom of C. amadis. MS/MS analysis of crude venom subjected to global reduction/alkylation yielded fragmentation patterns, which permitted searching and matching with a database of putative mature toxin sequences obtained from transcriptomic analysis. Of the four sequences identified, Am3408(Am6.1b), Am3452(Am6.1c), Am3136(Am6.2a) and Am3214(Am6.2b), three contain bromotryptophan residues, while an additional post translational modification, gamma carboxylation of glutamic acid, is present in Am3408(Am6.1b)/3452(Am6.1c). The conotoxins belong to the O1/O2 gene superfamily and possess cysteine framework VI/VII. While, the cysteine patterns show a similarity to omega conotoxins, the three C. amadis peptides are highly negatively charged and possess a significant content of hydrophobic residues.

Contryphan Genes and Mature Peptides in the Venom of Nine Cone Snail Species by Transcriptomic and Mass Spectrometric Analysis.

The occurrence of contryphans, a class of single-disulfide-bond-containing peptides, is demonstrated by the analysis of the venom of nine species of cone snails. Ten full gene sequences and two partial gene sequences coding for contryphan precursor proteins have been identified by next-generation sequencing and compared with available sequences. The occurrence of mature peptides in isolated venom has been demonstrated by LC-ESI-MS/MS analysis. De novo sequencing of reduced, alkylated contryphans from C. frigidus and C. araneosus provides evidence of sequence variation and post-translational modification, notably gamma carboxylation of glutamic acid. The characterization of Fr965 (C. frigidus) provides a rare example of a sequence lacking Pro at position 5 in the disulfide loop. The widespread occurrence of contryphan genes and mature peptides in the venom of diverse cone snails is suggestive of their potential biological significance.

Long-term propylthiouracil use and acute myeloid leukemia: A case report and review of the literature

Dear Editor, We report a 31-year-old female who had been administered propylthiouracil for 15 years and shortly after delivery developed acute myeloid leukemia with complex karyotype, including a rare t(2;21) translocation. We discuss here the potential mechanisms by which this drug could have contributed to leukemia and review the literature for propylthiouracil-related hematological disorders. Acute Myeloid Leukemia (AML) has very few welldefined chemical triggers. Chronic drug use has been associated with increased AML risk, but no definitive evidence is available yet. Many drugs have in vitro leukemogenicity, and for some of them, literature exists reporting causal association with AML in patients. Thionamide-type thyroid peroxidase inhibitors are commonly used to treat primary hyperthyroidism. Despite the fact that methimazole (MMI) is the drug of choice, many patients develop side effects such as hepatitis, cholestatic jaundice, splenomegaly, and lupus-like syndrome, in addition to mild and common side effects like granulocytopenia, pruritus, urticaria, and maculopapular or papular eruption [1], so that they are then shifted to the older drug of this class, 6-n-propylthiouracil (PTU), originally used as a measure of taste genetics. In addition, in comparison with MMI, PTU has a shorter duration of action and has significantly less transfer across the placenta and into breast milk, making PTU the preferred antithyroid agent for pregnant women. PTU can induce antigranulocyte cytoplasm antibodies [2, 3], as well as antinuclear antibodies [4]. Since its approval by the Food and Drug Administration in 1947, PTU has been associated with many hematological adverse events [5], including granulocytopenia, agranulocytosis [6], and eosinophilia [7]. Thionamides resemble the methyltetrazole-thiol leaving group of certain cephalosporin antibiotics, and both thionamides and cephalosporins have been found to inhibit the vitamin-K-dependent gamma-carboxylation of glutamic acid in vitro [8]: actually, PTU has been associated with hypoprothrombinemia [9], disseminated intravascular coagulation [10], and acute thrombocytopenic purpura [11]. Most importantly, PTU has been associated with bone marrow aplasia [12, 13] and AML [14, 15]. AML itself is favored by immunosuppression. We report here on a patient who had been on PTU for 15 years and who developed AML shortly after delivery. A previously healthy, 31-year-old female was admitted at our division because of sudden onset of ecchymoses and a complete blood count showing leukocytosis and thrombocytopenia. A peripheral blood smear suggestive for AML was confirmed by bone marrow aspiration and trephine biopsies. Immunohistochemistry and immunophenotyping showed acute myelomonoblastic leukemia (FAB M4). Cytogenetics on bone marrow leukocytes showed a complex karyotype with two abnormal clones, one with t(2;21) and another with both t(3;16) and del8p. At past medical history, she reported only having been diagnosed with primary hyperthyroidism at age 16, which was initially treated with MMI. Because of drug intolerance, she was then shifted to PTU 25 mg daily po, a drug that she took for 15 years before AML diagnosis. At that time, she had just been pregnant, having an uncomplicated delivery 4 months before the onset of AML. Ann Hematol (2008) 87:233–235 DOI 10.1007/s00277-007-0369-4

Prevention of vitamin K deficiency bleeding in newborns.

Newborn babies are born vitamin K deficient; however, the deficiency is not sufficiently severe to cause a vitamin K deficiency coagulopathy and haemorrhagic disease of the newborn (HDN). Severe vitamin K deficiency can develop quickly in breast-fed newborns and can result in the appearance of classic HDN during the first week of life or late HDN during the first 2 months of life. Both forms of the disease can be severe, causing brain damage and death. Classic and late HDN are prevented by the intramuscular administration of vitamin K at birth. Oral prophylaxis prevents classic HDN but is ineffective in preventing late HDN. Despite proven effectiveness of intramuscular vitamin K prophylaxis there have been concerns about the need for, and safety of, this therapy. This review provides evidence that there is need for intramuscular vitamin K prophylaxis for all babies in order to eradicate haemorrhagic disease of the newborn and concludes that there is no evidence that this therapy is harmful.

Rate limitations in posttranslational processing by the mammary gland of transgenic animals.

Our studies in transgenic animal bioreactors sought to determine the rate limitations in posttranslational processing of recombinant human protein C (rhPC) made in mammary gland of mice and pigs. Human protein C (hPC) is a complex plasma protein containing nine gamma-carboxylated glutamic acid (gla) residues that bind calcium at about 1 to 3 mM. Gamma carboxylation is a vitamin K-dependent posttranslational modification. The effect of rhPC synthesis rate on the extent of gamma-carboxylation of glutamic acid was studied. We have perturbed the biosynthesis of rhPC by using two different transgenes to direct mammary gland-specific expression. Promoter elements of the murine whey acid protein (mWAP) gene were used to drive the expression of hPC-cDNA and hPC-genomic transgenes. Transgenic mice with hPC-cDNA and hPC-genomic sequences gave expression levels of 11 +/- 4 micrograms rhPC/ml of milk and 895 +/- 21 micrograms rhPC/ml of milk, respectively. Transgenic pigs with hPC-cDNA and hPC-genomic sequences gave expression levels of 100 to 500 micrograms rhPC/ml of milk and 800 to 2000 micrograms rhPC/ml of milk, respectively. A monoclonal antibody (7D7B10-mAb) that binds an epitope in the gla domain of hPC in the absence of calcium was used to study the conformational behavior of immunopurified rhPC. Immunopurified rhPC from lower expressing mice and pigs gave a calcium-dependent binding inhibition by 7D7B10-mAb similar to that of hPC. Immunopurified rhPC from higher expressing mice and pigs gave a less calcium-dependent response. This study suggests that a rate limitation in gamma-carboxylation by the mammary gland occurs at expression levels about > 20 micrograms/ml in mice and > 500 micrograms/ml in pigs.

MECHANISM OF THIOAMIDE ANTITHYROID DRUG ASSOCIATED HYPOPROTHROMBINEMIA

The thioamide class of antithyroid drugs has been associated with the development of hypoprothrombinemia. Two drugs in this class, propylthiouracil and methimazole, resemble the methyltetrazole-thiol leaving group of certain cephalosporin antibiotics. Both were found in vitro to inhibit the vitamin K dependent step in clotting factor synthesis, the gamma-carboxylation of glutamic acid with 50 per cent inhibitory concentrations of 2 mM for propylthiouracil and 0.1 mM for methimazole. Methimazole was also found to inhibit vitamin K epoxide reductase, an enzyme related to the carboxylation reaction, with a 50 per cent inhibitory concentration of 25 uM. In vivo methimazole, administered twice at a dose of 500 mg/kg to rats on a vitamin K deficient diet produced hypoprothrombinemia. These results suggest that the mechanism of hypoprothrombinemia associated with thioamide antithyroid drugs may be similar to the mechanism of hypoprothrombinemia associated with cephalosporins which contain the methyltetrazole-thiol leaving group.

Production of hypoprothrombinaemia by cefazolin and 2-methyl-1,3,4-thiadiazole-5-thiol in the rat.

Recent findings have suggested that the 1-methyltetrazole-5-thiol (MTT) group contained in several beta-lactam antibiotics may be responsible for the hypoprothrombinaemia associated with these drugs. In order to determine if the hypoprothrombinaemia associated with cefazolin is due to the presence of the structurally related 2-methyl-1,3,4,-thiadiazole-5-thiol (MTD) group which it possesses, the ability of MTD and cefazolin to produce hypoprothrombinaemia in rats was examined. Female rats maintained on a vitamin K-deficient diet for ten days developed hypoprothrombinaemia after the intravenous administration of cefazolin for two subsequent days. Hypoprothrombinaemia was also produced by the oral administration to vitamin K-deficient rats of either cefazolin or MTD, while the oral administration of cefotaxime, which does not contain a thiol group, had no effect. In a rat liver microsomal system, MTD was found to be a much more potent inhibitor than cefazolin of the vitamin K-dependent step in clotting factor synthesis, the gamma-carboxylation of glutamic acid. These results suggest that the hypoprothrombinaemia associated with cefazolin may be due to the MTD group.

N-METHYL-THIO-TETRAZOLE INHIBITION OF THE GAMMA CARBOXYLATION OF GLUTAMIC ACID: POSSIBLE MECHANISM FOR ANTIBIOTIC-ASSOCIATED HYPOPROTHROMBINAEMIA

Several new antibiotics which contain an N-methyl-thio-tetrazole side chain have been associated with the development of hypoprothrombinaemia. This side chain was found to be capable of the in-vitro inhibition of a necessary reaction in the synthesis of prothrombin, the gamma carboxylation of glutamic acid. The 50% inhibitory concentration was 1·1 mmol/l. The intact antibiotics latamoxef, cephamandole, and cefoperazone, which contain the side chain, did not inhibit the reaction at concentrations up to 2 mmol/l. These results suggest that hypoprothrombinaemia associated with antibiotics containing N-methyl-thio-tetrazole side chains may be a consequence of the in-vivo degradation of these antibiotics and the subsequent inhibition of the gamma carboxylation of glutamic acid by the side chain.