Eclosion hormone of the silkworm <i>Bombyx mori</i> Expression in <i>Escherichia coli</i> and location of disulfide bonds

Kono, Toshiaki; Nagasawa, Hiromichi; Kataoka, Hiroshi; Isogai, Akira; Fugo, Hajime; Suzuki, Akira

doi:10.1016/0014-5793(90)81413-i

Cited by 33 publications

(22 citation statements)

References 13 publications

(13 reference statements)

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“…The importance of the C-terminus of Bombyx EH for biological activity has been examined using peptide expressed in E. coli [57]. In this study, a molecule lacking the four amino acids at the C-terminus was 100-fold less active than native EH.…”

Section: Discussionmentioning

confidence: 99%

“…Another notable feature of the sequence comparisons with EH peptides from moths is the conservation of the six Cys residues. These form intramolecular disulfide bonds and the positions of these disulfide bonds has been determined for native Manduca EH [58], and for Bombyx EH expressed in E. coli [57]. Formation of the disulfide bonds to fold the molecule into its proper secondary structure is essential for bioactivity, since reduction by 2-mercaptoethanol resulted in loss of biological activity, both for Manduca EH [59], and for recombinant Bombyx EH [57].…”

Section: Discussionmentioning

confidence: 99%

“…These form intramolecular disulfide bonds and the positions of these disulfide bonds has been determined for native Manduca EH [58], and for Bombyx EH expressed in E. coli [57]. Formation of the disulfide bonds to fold the molecule into its proper secondary structure is essential for bioactivity, since reduction by 2-mercaptoethanol resulted in loss of biological activity, both for Manduca EH [59], and for recombinant Bombyx EH [57]. We have begun to examine the structure of EH from other insects using a PCR protocol in which a portion of the DNA encoding EH is amplified using degenerate oligonucleotide primers.…”

Section: Discussionmentioning

confidence: 99%

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Isolation, characterization and expression of the eclosion hormone gene of Drosophila melanogaster

Horodyski

Ewer

Riddiford

et al. 1993

European Journal of Biochemistry

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Eclosion hormone (EH) is a neuropeptide that triggers the performance of ecdysis behaviors at the end of a molt. We have isolated the EH gene from Drosophila melanogasfer, and localized the gene to the right arm of chromosome 3 at band position 90B1-2. The 97-amino-acid translation product contains a signal peptide followed by a 73-amino-acid prohormone. The N-terminus of the prohormone has diverged from lepidopteran EH both in its length and amino acid composition, and contains a potential endoproteolytic cleavage site. The deduced sequence of Drosophila EH is 58% identical (36 of 62 amino acids) to that of Manduca EH. The EH gene is expressed as a 0.8-kb transcript in a single pair of brain neurons which extend their processes the entire length of the central nervous system and also to the corpora cardiaca portion of the ring gland. These cells show massive depletion of immunoreactive EH at ecdysis.Over the last 10 years, work in invertebrates in general and insects in particular has led to identification of a large number of neuropeptides, many of which are expressed in a small number of cells within the central nervous system (CNS) [l, 21. These findings raise a number of important questions such as the role of these peptides in the normal physiology and development of the organism and the mechanisms that restrict peptide expression to a select set of cells within the CNS. To answer both questions, Drosophila melunogaster provides a number of distinct advantages. Mutants in a desired gene can be generated with relative ease and P-element transformation technology allows examination of regulatory regions that control peptide expression [3, 41. Consequently, we have turned to Drosophila to further our analysis of the neuropeptide eclosion hormone (EH), that triggers ecdysis of insects [5].Eclosion hormone was first found in moths [6] and later shown to be a 62-amino-acid peptide [7 -91 produced by two pairs of ventromedial neurosecretory cells in the brain [lO-131. In moths it has marked behavioral and developmental actions. It is known to act directly on the CNS to release the stereotyped motor programs that bring about the shedding of the old cuticle at the end of each molt [14]. Circulating peptide also acts on peripheral tissues to cause a diverse set of effects such as an increase in plasticity of wing cuticle [15], discharge of secretion by dermal glands [16], and the programmed degeneration of ecdysial muscles [17]. Yet the full range of EH actions are not known because it is not possible to block selectively either release or action of EH in moths. It appeared feasible to study EH in Drosophila because CNS extracts of both Calliphora [5] and Drosophila (Kimura and Truman, unpublished data) show EH activity in moth bioassays. Moreover, blood from ecdysing fleshflies, Sarcophaga bullata, stimulates premature ecdysis behavior in pharate (before ecdysis) flies [18]. The recent cloning of the EH gene from two species of moths, Munduca sexfa [ll] and Bombyx mori [13], has provided an avenue to search for t...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Isolation, characterization and expression of the eclosion hormone gene of Drosophila melanogaster

Horodyski

Ewer

Riddiford

et al. 1993

European Journal of Biochemistry

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“…The latter authors also isolated and sequenced the cDNA for Bom-EH. Bom-EH has been expressed in yeast (139) and Escherichia coli (140), the latter used to establish the location of the 3 disulfide bonds in Bom-EH and to show that the 6 residues at the N-terminal end are dispensable whereas the 4 residues at the C-terminal end are required for activity, ^identical location for the disulfide bonds of Mas-EH (Cys -Cys , Cys -Cys , Cys -Cys ) was recently determined (141). Using a nucleotide probe from Mas-EH and PCR methods, Horodyski and co-workers determined a 73-amino-acid sequence for D. melanogaster EH which has ca.…”

Section: Eclosion Hormonesmentioning

confidence: 99%

Insect Neuropeptides

Kelly

Masler

Menn

1993

Natural and Engineered Pest Management Agents

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The physiological processes regulated by insect neuropeptides are extensive and include growth, development, molting, reproduction, diapause, behavior, color change, ion and water balance, and muscle contraction. Nearly 80 novel insect neuropeptides have been sequenced to date, 10 or so gene sequences have been determined, numerous analogs have been synthesized, and neuropeptide genes have been expressed in vector systems. Investigations into sites of synthesis and release, and tissue specificity and action, continue to reveal complexity in the (classically simple) insect neuroendocrine system. The rapidly increasing knowledge in this area suggests that useful prototypes for the design of selective pest control agents will emerge in the forseeable future.The field of insect neuroendocrinology has expanded rapidly, fostering numerous reviews within the past five years (1-21), including one book entirely devoted to insect neurohormones (22) and one to insect neuropeptides (23). These publications discuss the discovery, physiology, history and chemistry of insect neuropeptides and present potential applications to pest control. These applications may include: blocking neuropeptide synthesis through expression of antisense RNA or inhibition of specific processing enzymes; inactivation of circulating neuropeptides by binding with specific antibodies, antipeptides or by degradation with specific peptidases; disruption of target binding or signal transduction by peptidomimetics; and disruptive expression of natural or modified neuropeptides through baculoviruses or other expression vectors. As suspected in 1988 (4), the number of purified and sequenced insect neuropeptides has continued to increase, and many of the corresponding nucleotide sequences have followed in rapid succession. Nearly eighty novel amino acid sequences for insect neuropeptides have been identified, and novel nucleotide sequences approach ten. This review updates the recent progress in identification of insect neuropeptide structures and their genes and other areas relevant to the development of pest control agents, This chapter not subject to U.S.

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“…These may form three pairs of disulfide bonds, because EH was inactivated by reduction with dithiothreitol. The arrangement of the disulfide bonds was estimated by using the synthetic EH(1-62) 18. Thermolysin digestion of the synthetic EH generated 11 cystine-containing peptides, all of which were sequenced.…”

Section: Ectosion Hormone (Eh)mentioning

confidence: 99%