Amidation Enzyme Research Articles

Novel components of Tityus serrulatus venom: A transcriptomic approach

Several research groups have studied the components produced by the venom gland of the scorpion Tityus serrulatus, which has one of the most lethal venoms in the world. Various methodologies have been employed to clarify the complex mechanisms of action of these components, especially neurotoxins and enzymes. Transcriptomes and proteomes have provided important information for pharmacological, biochemical, and immunological research. Next-generation sequencing (NGS) has allowed the description of new transcripts and completion of partial sequence descriptions for peptides, especially those with low expression levels. In the present work, after NGS sequencing, we searched for new putative venom components. We present a total of nine new transcripts with neurotoxic potential (Ts33–41) and describe the sequences of one hyaluronidase (TsHyal_4); three enzymes involved in amidation (peptidyl-glycine alpha-amidating monooxygenase A, peptidyl-alpha-hydroxyglycine alpha-amidating lyase, and peptidylglycine alpha-hydroxylating monooxygenase), which increases the lethal potential of neurotoxins; and also the enzyme Ts_Chitinase1, which may be involved in the venom's digestive action. In addition, we determined the level of transcription of five groups: toxins, metalloproteases, hyaluronidases, chitinases and amidation enzymes, including new components found in this study. Toxins are the predominant group with an expression level of 91.945%, followed by metalloproteases with only 7.790% and other groups representing 0.265%.

Mass spectrometric evidence for neuropeptide-amidating enzymes in Caenorhabditis elegans

Neuropeptides constitute a vast and functionally diverse family of neurochemical signaling molecules and are widely involved in the regulation of various physiological processes. The nematode Caenorhabditis elegans is well-suited for the study of neuropeptide biochemistry and function, as neuropeptide biosynthesis enzymes are not essential for C. elegans viability. This permits the study of neuropeptide biosynthesis in mutants lacking certain neuropeptide-processing enzymes. Mass spectrometry has been used to study the effects of proprotein convertase and carboxypeptidase mutations on proteolytic processing of neuropeptide precursors and on the peptidome in C. elegans However, the enzymes required for the last step in the production of many bioactive peptides, the carboxyl-terminal amidation reaction, have not been characterized in this manner. Here, we describe three genes that encode homologs of neuropeptide amidation enzymes in C. elegans and used tandem LC-MS to compare neuropeptides in WT animals with those in newly generated mutants for these putative amidation enzymes. We report that mutants lacking both a functional peptidylglycine α-hydroxylating monooxygenase and a peptidylglycine α-amidating monooxygenase had a severely altered neuropeptide profile and also a decreased number of offspring. Interestingly, single mutants of the amidation enzymes still expressed some fully processed amidated neuropeptides, indicating the existence of a redundant amidation mechanism in C. elegans All MS data are available via ProteomeXchange with the identifier PXD008942. In summary, the key steps in neuropeptide processing in C. elegans seem to be executed by redundant enzymes, and loss of these enzymes severely affects brood size, supporting the need of amidated peptides for C. elegans reproduction.

Bile acid‐CoA ligase deficiency—a new inborn error of bile acid metabolism

Born at 27 weeks gestation, a child of consanguineous parents of Pakistani origin required prolonged parenteral nutrition. She developed jaundice, with extensive fibrosis and architectural distortion at liver biopsy; jaundice resolved with supportive care. Serum γ-glutamyl transpeptidase values were within normal ranges. The bile acids in her plasma and urine were >85% unconjugated (non-amidated). Two genes encoding bile-acid amidation enzymes were sequenced. No mutations were found in BAAT, encoding bile acid-CoA : aminoacid N-acyl transferase. The patient was homozygous for the missense mutation c.1012C > T in SLC27A5, predicted to alter a highly conserved amino-acid residue (p.H338Y) in bile acid-CoA ligase (BACL). She also was homozygous for the missense mutation c.1772A > G in ABCB11, predicted to alter a highly conserved amino-acid residue (p.N591S) in bile salt export pump (BSEP). BACL is essential for reconjugation of bile acids deconjugated by gut bacteria, and BSEP is essential for hepatocyte-canaliculus export of conjugated bile acids. A female sibling born at term had the same bile-acid phenotype and SLC27A5 genotype, without clinical liver disease. She was heterozygous for the c.1772A > G ABCB11 mutation. This is the first report of a mutation in SLC27A5. The amidation defect may have contributed to cholestatic liver disease in the setting of prematurity, parenteral nutrition, and homozygosity for an ABCB11 mutation.

Molecular Organization of Drosophila Neuroendocrine Cells by Dimmed

In Drosophila, the basic-helix-loop-helix protein DIMM coordinates the molecular and cellular properties of all major neuroendocrine cells, irrespective of the secretory peptides they produce. When expressed by nonneuroendocrine neurons, DIMM confers the major properties of the regulated secretory pathway and converts such cells away from fast neurotransmission and toward a neuroendocrine state. We first identified 134 transcripts upregulated by DIMM in embryos and then evaluated them systematically using diverse assays (including embryo insitu hybridization, invivo chromatin immunoprecipitation, and cell-based transactivation assays). We conclude that of eleven strong candidates, six are strongly and directly controlled by DIMM invivo. The six targets include several large dense-core vesicle (LDCV) proteins, but also proteins in non-LDCV compartments such as the RNA-associated protein Maelstrom. In addition, a functional invivo assay, combining transgenic RNA interference with MS-based peptidomics, revealed that three DIMM targets are especially critical for its action. These include two well-established LDCV proteins, the amidation enzyme PHM and the ascorbate-regenerating electron transporter cytochrome b(561-1). The third key DIMM target, CAT-4 (CG13248), has not previously been associated with peptide neurosecretion-it encodes a putative cationic amino acid transporter, closely related to the Slimfast arginine transporter. Finally, we compared transcripts upregulated by DIMM with those normally enriched in DIMM neurons of the adult brain and found an intersection of 18 DIMM-regulated genes, which included all six direct DIMM targets. The results provide a rigorous molecular framework with which to describe the fundamental regulatory organization of diverse neuroendocrine cells.

Isolation, identification and biological activity of gastrin-releasing peptide 1–46 (oGRP 1–46), the primary GRP gene-derived peptide product of the pregnant ovine endometrium

We have previously demonstrated that pregnant ovine endometrium expresses the gastrin-releasing peptide (GRP) gene at a high level following conceptus implantation. Here we report the isolation, characterization and biological activity of ovine GRP1–46, the primary product of this gene in the pregnant endometrium. Full thickness 125–140-day pregnant sheep uterus (term is 145 day) was homogenized in 80% acetonitrile/2% trifluoroacetic acid (1:7 ACN/TFA), concentrated on reverse-phase C18 cartridges and chromatographed successively on gel filtration (Sephadex G-50) and reverse-phase HPLC (C18 μBondapak). Purification was monitored by RIA. Purified GRP peptide was analysed by mass spectrometry giving a major mass ion at 4963 which corresponds exactly to GRP1–46. Other mass ions from pro-GRP did not contain a biologically active N-terminus or antigenic determinant. Proteolytic cleavage of pro-GRP to give rise to GRP 1–46 would require preferential cleavage at the Glu–Glu bond by a Glu-C2-like enzyme, rather than the trypsin-like and C-terminal amidation enzymes (PAM) that produce GRP 18–27 and GRP 1–27 in other tissues. GRP1–46 was synthesized and receptor binding and biological activity tested on a range of rodent and human cell lines that express GRP-related receptors GRPR, NMBR and BRS3. GRP1–46 bound GRPR and NMBR with low affinity, and mobilized inositol phosphate in cell lines expressing the GRPR and NMBR, but not BRS-3. This study describes a new processed product of the GRP gene, GRP1–46, which is highly expressed in the pregnant sheep endometrium and which acts as a weak agonist at the GRPR and NMBR.

Structure-function relationships of tachyplesins and their analogues.

Haemocytes of the horseshoe crab (Limulus) contain a new family of arthropodous peptide antibiotics, termed the tachyplesin family. These cationic peptides are composed of 17-18 amino acid residues with a C-terminal arginine alpha-amide. Tachyplesin I takes on a fairly rigid conformation constrained by two disulphide bridges and adopts a conformation consisting of an antiparallel beta-sheet connected by a beta-turn. Isopeptides of tachyplesin I with amino acid replacements, tachyplesins II and III, and polyphemusins I and II have also been found in the haemocytes of the South-East Asian species and Limulus polyphemus. These peptides are present in abundance in the small granules of the haemocytes and inhibit strongly the growth of not only Gram-negative and Gram-positive bacteria but also fungi such as Candida albicans. Tachyplesin exists in the prepro form consisting of 77 residues; this precursor is probably processed by intracellular proteases and an amidation enzyme before incorporation into the small granules of the haemocytes. We examined the mode of action of tachyplesin I on biomembranes, comparing it with that of gramicidin S. Tachyplesin caused an efflux of K+ from Staphylococcus aureus and Escherichia coli cells similar to that caused by gramicidin S. Another antimicrobial substance, anti-LPS factor, has been isolated from haemocytes.

Adrenomedullin amidation enzyme activities in hypertensive patients.

Adrenomedullin (AM) is a potent vasodilating peptide secreted from the vasculature of various organs. It is biologically active when its C-terminus is amidated. Recently, an RIA method was developed for measurement of the active form of AM, or mature AM. We here employed this method to investigate the significance of amidation of AM in controlling cardiovascular function. Thirty-six patients under hemodialysis were recruited and divided into hypertensive (n = 25; 157/86 mmHg) and normotensive (n= 11; 116/66 mmHg) groups. Mature AM, immature AM and blood pressure were monitored during hemodialysis in all patients. There was a significant reduction in blood pressure during hemodialysis in both groups, although after hemodialysis blood pressure was still higher in hypertensives than in normotensives (139 +/-14.8/76 +/- 2.5 mmHg vs. 110 +/- 5.1/66.7 +/- 3.1 mmHg). Mature AM before hemodialysis were lower in hypertensives than normotensives and it decreased in both groups. Although mature AM decreased more in normotensives than in hypertensives (-27 +/- 8% vs. -17 +/- 5%), at the end point, its level was still higher in normotensives. The ratio of mature AM/immature AM decreased only in normotensives (-11.4 8.7%), whereas it remained stable in hypertensives (0.2 +/- 5.6%). Both groups showed similar changes in ANP, endothelin, catecholamines, cGMP, and NOx. The low level in mature AM level in hypertensives may have contributed to the higher blood pressure in this group. The attenuation of AM amidation in normotensives indicates that an unspecified amidative enzyme of AM was regulated in order to normalize blood pressure.

An efficient cellular system for mutational analysis of prohormone processing.

A novel system for heterologous expression of prohormones based on transient transfection of the HIT beta-cell line was established using human progastrin as a model. Progastrin was expressed at high levels compared to other gene transfer systems in endocrine cells, and the processing pattern was similar to that of normal antral gastrin cells. Thus, gastrin was partially tyrosine O-sulfated and carboxyamidated. Cell extracts contained mainly gastrin-17 and gastrin-34 and the corresponding glycine-extended forms. In contrast, the media contained more incompletely processed gastrin forms. This suggests that gastrin was directed to the regulated secretory pathway but that some progastrin products were constitutively secreted. Glucose increased both the level of gastrin gene expression and maturation to carboxyamidated peptides, indicating that glucose influences the activity of the amidation enzyme complex, peptidylglycine alpha-amidating mono-oxygenase (PAM), in insulin cells. Mutational analysis of tyrosine sulfation of gastrin demonstrated that substitution of the uncharged residue carboxy-terminal to the tyrosine with an acidic residue does not increase sulfation in contrast to previous results, where the amino-terminal residue was replaced with an acidic residue. The mutant peptides displayed sulfation-dependent processing, supporting our recent suggestion that tyrosine sulfation increases the proteolytic processing of prohormones.

Production of bioactive gastrin from the non-endocrine cell lines CHO and COS-7

We made a mutated progastrin cDNA construct that contains a cleavage site (-Arg −4-Arg −3-Lys −2 -Arg −1) specific for the Kex2-like endoprotease furin, located ahead of the bioactive gastrin. For expressing the mutated progastrin cDNA, we used two non-endocrine cell lines, CHO and COS-7. CHO cells exhibit amidating enzyme activity and levels of amidation enzyme mRNA as high as those in the pituitary-derived endocrine cell line GH 3, whereas COS-7 cells have far less amidating activity and lower amounts of mRNA. Mutant progastrin-expressing CHO cells produced mostly amidated gastrin. Gel filtration showed the size of this gastrin corresponded to that of the synthetic human gastrin-17. In contrast, COS-7 cells produced glycine-extended gastrin and only a small amount of amidated gastrin. The difference in the amount of amidated gastrin products produced by the two non-endocrine cell lines is due to differing amounts of the amidation enzyme contained in each cell line.

Comparison of peptidyl-glycine α-amidation activity in medullary thyroid carcinoma cells, pheochromocytomas, and serum

With an assay based on the radioimmunological detection of the formation of the C-terminal amide function on a neuropeptide Y-like substrate, amidation enzyme activity with apparent M r of 56,000 and 38, 000 was found in pheochromocytoma extracts. The larger molecular form of amidating enzyme was also expressed and secreted from medullary thyroid carcinoma cells in a dexamethasone-suppressible way. Serum contained high levels of amidating enzyme activity with no difference between normal subjects and patients with pheochromocytomas. However, the majority of the amidating activity in serum was of much larger size, M r between 80 and 105,000, compared to that released from the endocrine cells. No major difference was found between the molecular forms of amidation enzyme from tissues and from serum either in respect of enzyme kinetics or in respect of requirements for the cofactors copper and ascorbate. The major serum forms of enzyme were relatively independent of exogenous copper; however, they could still be quenched by cobber chelating agents. It is concluded that the molecular weight forms of the amidating enzyme circulating in serum are much larger than the soluble enzyme stored and secreted from most endocrine tissues.

Ontogeny of peptidylglycine alpha-amidating monooxygenase activity in rapidly mineralizing bone from neonatal mouse.

Peptidylglycine alpha-amidating monooxygenase is hormonally, developmentally, and nutritionally regulated. In several tissues concomitant changes in enzyme activity and the level of expression of a known amidated peptide have been demonstrated. We report that neonatal mouse calvarium, a rapidly mineralizing bone, has detectable amidation enzyme activity. The level of activity varied 3-10-fold during the first 9 d of life. Production of one or more amidated peptides by bone may be coordinately regulated.

Glucocorticoid regulation of amidating enzyme in a neoplastic C-cell line

Posttranslationar carboxyl-terminal amidation of many peptides is accomplished by peptidylglycine α-amidating monooxygenase. We have previously demonstrated that glucocorticoids stimulate production of amidated products by the CA-77 rat medullary thyroid carcinoma cell line. The present investigation was undertaken to determine whether amidation enzyme activity changes in parallel. Enzyme activity, similar to that found in other tissues, was readily detected in cell extracts and conditioned cultured medium. Stimulation with the calcitonin secretagogue calcium increased secretion of enzyme activity and lowered cell extract activity. Treatment of cultures with dexamethasone, but no other steroid, decreased by 50–70% the basal amidation enzyme activity secreted. There was no associated change in cellular activity. The decrease in medium activity was partially reversible and steroid-dose dependent. The glucocorticoid-induced change in medium activity was due to a decreased V m . These experiments demonstrate that the a-amidating activity of the CA-77 cells can be hormonally regulated.

Glycine-extended progastrin processing intermediates: accumulation and cosecretion with gastrin.

Glycine-extended intermediates of peptide processing serve as substrates for carboxyl-terminal amidation, hence activation, of many brain-gut peptides. To explore the dynamics of accumulation and secretion of these important intermediates we utilized primary cultures of canine antral mucosal G-cells as a model system. Glycine-extended progastrin processing intermediates (G-Gly) accumulated rapidly in G-cells cultured in ascorbate-deficient media, exhibiting a fourfold increase over a 51-h culture period, while gastrin content fell to less than half of the initial level. In contrast, G-cells cultured in ascorbate-supplemented media accumulated G-Gly at a relatively low rate, while gastrin was preserved at a higher level. Under either condition, G-Gly and gastrin were progressively released into the culture media. The release of both immunoreactivities could be stimulated by bombesin and inhibited by somatostatin in similar fashion. By electron microscopy, the cultured G-cells exhibited no ultrastructural alterations. These data suggest that 1) the cellular homeostasis of G-Gly is regulated by the activity of an ascorbate-dependent amidation enzyme similar to one previously described in pituitary tissues, 2) carboxyl-terminal amidation is not an obligatory step for secretion of gastrin, and 3) the proportions of gastrin and G-Gly cosecreted from G-cells reflect their proportional accumulation within G-cell secretory granules. The physiological relevance of the released G-Gly has yet to be determined.

Ascorbic acid increases the thyrotropin-releasing hormone content of hypothalamic cell cultures

Thyrotropin-releasing hormone (TRH) is one of many COOH-terminal alpha-amidated neuropeptides. Recent work with the intermediate pituitary has indicated that ascorbate is a required cofactor for the COOH-terminal alpha-amidation of alpha-melanotropin. This is consistent with the ascorbate requirement of an enzyme found in pituitary and hypothalamus capable of converting peptides with a COOH-terminal glycine (-X-Gly) to alpha-amidated molecules (-H-NH2). Thus, it has been proposed that COOH-terminal glycine-extended TRH (TRH-Gly) may be the direct precursor to TRH. In the present study, primary hypothalamic cultures supplemented with ascorbate for 7 d contained two- to threefold more TRH immunoactivity (amide-specific) than cultures maintained without ascorbate. A dose-response experiment indicated that 20 microM ascorbate was capable of producing 50% of the maximum observable increase in culture TRH immunoactivity; this concentration is similar to the Km value for ascorbate uptake obtained in adrenal chromaffin and pituitary cells. A stereoisomer of ascorbate, D-isoascorbate, was also capable of producing an increase in TRH immunoactivity, but oxidized ascorbate was not. Recent studies have shown that the amidation enzyme from pituitary is capable of utilizing both L-ascorbate and D-isoascorbate but is incapable of utilizing oxidized ascorbate. The culture extracts were analyzed further by reversed-phase high-performance liquid chromatography; the increased TRH immunoactivity observed in extracts of cultures maintained in ascorbate comigrated with standard synthetic TRH.(ABSTRACT TRUNCATED AT 250 WORDS)