Invertebrate neuropeptide processing: Partial characterization of peptidylglycine α‐amidating monooxygenase in the tobacco hornworm, <i>Manduca sexta</i>

Bernasconi, Paul; Li, Jorge P.; Reagan, Jeff D.; Kramer, Steven J.

doi:10.1002/arch.940210207

Cited by 5 publications

(4 citation statements)

References 30 publications

(20 reference statements)

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“…Our results in this regard are similar to those of Rouillt et al (1992a) who found that conversion of vasopressinyl-glycine to mature, amidated vasopressin by neurohypophysial enzymes was also weak. In general, the PAM activity described in this study strongly resembles PAM activities described in amphibians, fish, mammals (Eipper et al, 1992) and in another insect, Manduca sexta (Bernasconi et al, 1992).…”

Section: Discussionsupporting

confidence: 84%

“…during the reaction was measured and used to calculate the inhibition (the quantity produced in the absence of inhibitors represents 0% inhibition). nervous system of an insect, Munducu sextu (Bernasconi et al, 1992). Our failure to produce amidated AKH in vitro may have been due to the absence of the appropriate cofactors in the reaction buffer.…”

Section: Characterization Of Peptidylglycine-a-amidating Activitymentioning

confidence: 96%

“…Restoration of carboxypeptidase activity by zinc and cobalt. Synthetic AKH-Gly-Lys-Arg was incubated for 1 h with corpora cardiaca extract in vitro as described in the Experimental Procedures section and the legend to nervous system of an insect, Munducu sextu (Bernasconi et al, 1992). Our failure to produce amidated AKH in vitro may have been due to the absence of the appropriate cofactors in the reaction buffer.…”

Section: Characterization Of Peptidylglycine-a-amidating Activitymentioning

confidence: 99%

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Reconstitution of adipokinetic hormone biosynthesis in vitro indicates steps in prohormone processing

Rayne

O’Shea

1994

European Journal of Biochemistry

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We have used a complete, synthetic precursor to adipokinetic hormone I (AKH I) and oligopeptides derived from this precursor as substrates for prohormone-processing enzymes extracted from AKH-synthesizing neurosecretory cells to reconstitute the post-translational steps in AKH biosynthesis in vitro. The results demonstrate the existence of endoproteolytic activity which cleaves the precursor only at the appropriate processing site (at the C-terminal side of Argl3). Further proteolytic processing of C-terminally extended AKH I (AKH-Gly-Lys-Arg) by a carboxypeptidase H-like activity removes the basic residues producing AKH-Gly-Lys, followed by AKH-Gly. Finally, a peptidylglycine-a-amidating-monooxygenase activity produces the amidated bioactive product from the glycine-extended peptide in a two-step process, the first of which requires ascorbate and Cu". Our results show that all steps in AKH precursor processing can be reconstituted and studied in vitro, providing a system to characterize the processing enzymes and to investigate the development of enzyme inhibitors for use as potential insecticides.Neuropeptides are produced by specific, limited proteolysis of larger precursor polypeptides, or prohormones (Docherty and Steiner, 1982;Sossin et al., 1989;Darby and Smyth, 1990). Prohormones are cleaved by endoproteases, often at sites adjacent to pairs of basic amino acid residues (e.g. lysine, arginine). This initial step has been attributed in some cases to members of a recently identified superfamily of subtilisin-like endopeptidases referred to as prohormone convertases (Steiner et al., 1992). Endoproteolytic cleavage is followed by removal of basic residues from C-terminally extended processing intermediates by carboxypeptidase H (also referred to as carboxypeptidase E; Fricker, 1988;Skidgel, 1988). For neuropeptides which require C-terminal amidation, glycine is normally the residue which, via the action of the peptidylglycine-a-amidating-monooxygenase (PAM) enzymes, contributes its amino group to form an amidated C-terminus (Eipper et al., 1992).

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Section: Discussionsupporting

confidence: 84%

Section: Characterization Of Peptidylglycine-a-amidating Activitymentioning

confidence: 96%

Section: Characterization Of Peptidylglycine-a-amidating Activitymentioning

confidence: 99%

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Reconstitution of adipokinetic hormone biosynthesis in vitro indicates steps in prohormone processing

Rayne

O’Shea

1994

European Journal of Biochemistry

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“…Since no peptidylglycine α-amidating enzyme (PAM) activity has been found in yeast, an in itro α-amidation reaction using commercially available PAM was chosen to complete the synthesis of biologically active Mas-DH. The C-terminal Gly is required for α-amidation of the peptide by PAM, a bifunctional enzyme found in the brain of M. sexta [3] and many vertebrates [4]. Some peptides less than 100 amino acids in length have been successfully expressed and secreted in yeast-based expression systems ; however, the majority of these peptides are heavily Abbreviations used : DH, diuretic hormone ; Mas-DH, M. sexta diuretic hormone ; CRF, corticotropin-releasing factor ; PAM, peptidylglycine αamidating enzyme ; TFA, trifluoroacetic acid ; BSA, bovine serum albumin ; RPLC, reversed-phase liquid chromatography ; ESI-MS, electrospray ionization mass spectrometry ; MS-saline, M. sexta saline ; IBMX, isobutylmethylxanthine ; DTT, dithiothreitol ; GAPDH, glyceraldehyde phosphate dehydrogenase.…”

Section: Introductionmentioning

confidence: 99%

Expression, processing and secretion of a proteolytically-sensitive insect diuretic hormone by Saccharomyces cerevisiae requires the use of a yeast strain lacking genes encoding the Yap3 and Mkc7 endoproteases found in the secretory pathway

Copley

Alm

Schooley

et al. 1998

Biochemical Journal

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A system is described for the heterologous expression of peptides in Saccharomyces cerevisiae. A synthetic gene encoding a precursor of the 41 amino acid Manduca sexta diuretic hormone (Mas-DH) was expressed at 0.8 mg/l purified peptide. A precursor of a mutant peptide of Mas-DH, Mas-DH[K22Q] was also expressed. The peptides were purified, then treated with peptidylglycine alpha-amidating enzyme to generate the alpha-amidated, mature, form of Mas-DH or Mas-DH[K22Q], which were biologically active. Successful expression of full-length Mas-DH+Gly depended upon the use of a protease-deficient yeast strain. In wild-type strains, Mas-DH+Gly was recovered only as proteolytic fragments, even in the presence of various protease inhibitors. Expression of Mas-DH+Gly in strains deficient in either the Mkc7 or the Yap3 protease reduced proteolysis, while no proteolysis of Mas-DH+Gly was detectable in a strain lacking both proteases. This protease-deficient strain may prove of general utility for expression of peptides. Analysis of recovered proteolytic fragments revealed a complex pattern of cleavage sites. Both the Yap3 and Mkc7 proteases preferred to cleave at a single Glu-Lys downward arrow-Glu-Arg site. Analysis of secondary cleavage sites showed that Yap3 preferred to cleave after either Lys or Arg and Mkc7 after Lys. This paper is the first report on the in vivo activity and specificity of Yap3 and Mkc7 expressed at physiological levels.

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Peptide amidation in an invertebrate: Purification, characterization, and inhibition of peptidylglycine α‐hydroxylating monooxygenase from the heads of honeybees (apis mellifera)

Zabriskie

Klinge

Szymanski

et al. 1994

Arch Insect Biochem Physiol

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Peptidylglycine alpha-hydroxylating monooxygenase (PHM), an enzyme involved in formation of neuropeptides with a C-terminal amide functionality in mammals and amphibians, was isolated from the head of an invertebrate, the honeybee, Apis mellifera, and purified 220-fold in 1% overall yield. The bee PHM has a molecular weight of 71,000, is membrane associated but can be solubilized with a detergent (n-octyl-beta-D-glucopyranoside), and cross-reacts with rabbit antibodies generated toward bacterially expressed rat PHM. In the presence of copper, oxygen, and ascorbic acid, the enzyme hydroxylates model tripeptides such as dansyl-L-Phe-L-Phe-Gly on the methylene carbon of the glycine residue with retention of configuration. Using this tripeptide as substrate, the Km is 1.7 microM and the Vmax is 2.3 nmol.micrograms-1.h-1. Treatment of the insect PHM with D-Phe-L-Phe-D-vinylglycine, a substrate analogue and mechanism-based inactivator of PHM from pig pituitary, results in irreversible loss of activity. The diastereomeric analogue, D-Phe-L-Phe-L-vinylglycine, is only a competitive inhibitor (IC50 = 320 microM).

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Invertebrate neuropeptide processing: Partial characterization of peptidylglycine α‐amidating monooxygenase in the tobacco hornworm, Manduca sexta

Cited by 5 publications

References 30 publications

Reconstitution of adipokinetic hormone biosynthesis in vitro indicates steps in prohormone processing

Reconstitution of adipokinetic hormone biosynthesis in vitro indicates steps in prohormone processing

Expression, processing and secretion of a proteolytically-sensitive insect diuretic hormone by Saccharomyces cerevisiae requires the use of a yeast strain lacking genes encoding the Yap3 and Mkc7 endoproteases found in the secretory pathway

Peptide amidation in an invertebrate: Purification, characterization, and inhibition of peptidylglycine α‐hydroxylating monooxygenase from the heads of honeybees (apis mellifera)

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