Ronald J. Nachman

Locustatachykinin I and II, two novel insect neuropeptides with homology to peptides of the vertebrate tachykinin family

Two myotropic peptides termed locustatachykinin I (Gly‐Pro‐Ser‐Gly‐Phe‐Tyr‐Gly‐Val‐Arg‐NH2) and locustatachykinin II (Ala‐Pro‐Leu‐Ser‐Gly‐Phe‐Tyr‐Gly‐Val‐Arg‐NH2) were isolated from brain‐corpora cardiaca‐corpora allata‐suboesophageal ganglion extracts of the locust, Locusta migratoria. Both peptides exhibit sequence homologies with the vertebrate tachykinins. Sequence homology is greater with the fish and amphibian tachykinins (up to 45%) than with the mammalian tachykinins. In addition, the intestinal myotropic activity of the locustatachykinins is analogous to that of vertebrate tachykinins. The peptides discovered in this study may just be the first in a whole series of substances from arthropod species to be identified as tachykinin family peptides. Moreover, both chemical and biological similarities of vertebrate and insect tachykinins substantiate the evidence for a long evolutionary history of the tachykinin peptide family.

Isolation, primary structure and synthesis of leucomyosuppressin, an insect neuropeptide that inhibits spontaneous contractions of the cockroach hindgut☆

Abstract 1. A neuropeptide that inhibits spontaneous contractions of the isolated cockroach hindgut was purified from head extracts of the cockroach, Leucophaea maderae . 2. The inability of aminopeptidase M to degrade the peptide and the presence of glutamic acid in the hydrolysate suggested N -terminal blocking by pyroglutamic acid. The N -terminal p Glu was removed enzymatically and the unblocked fragment was sequenced with an automated peptide sequencer. 3. The structure determined ( p Glu-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-NH 2 ) was synthesized and shown to be both chemically and biologically identical with the natural product. 4. Leucomyosuppressin is the first inhibitory neuropeptide isolated and structurally identified from an insect source.

Leucokinins, a new family of ion transport stimulators and inhibitors in insect Malpighian tubules

Leucokinins are octapeptides isolated from heads of the cockroach Leucophaea maderae. In the cockroach they increase motility of the isolated hindgut. Surprisingly, synthetic leucokinins have biological activity in a different insect and in a different tissue. In isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti, leucokinins depolarize the transepithelial voltage. This effect on voltage is dependent on extracellular Cl. One leucokinin, LK-8, the effects of which were studied further in isolated Malpighian tubules, was found to inhibit transepithelial fluid secretion at low concentrations (10(-11) M threshold), and to stimulate fluid secretion at high concentrations (3.5 x 10(-9) M threshold). Together, the depolarizing effects on voltage and the stimulation of fluid secretion suggest that leucokinins increase the Cl permeability of the tubule wall thereby increasing the availability of Cl for secretion with Na, K and water. Structure-function comparisons of the seven leucokinins studied suggest that the active region of the octapeptide is segregated to the C-terminal pentapeptide. In view of the known effects of leucokinins on hindgut motility in the cockroach, our finding of effects in mosquito Malpighian tubules suggests that leucokinins may be widely distributed in insects where they may have diverse functions in a variety of organs.

Leucosulfakinin-II, a blocked sulfated insect neuropeptide with homology to cholecystokinin and gastrin

A sulfated neuropeptide [pGlu-Ser-Asp-Asp-Tyr(SO3H)-Gly-His-Met-Arg-Phe-NH2], with a blocked N-terminus and related to the undecapeptide leucosulfakinin, has been isolated from head extracts of the cockroach, Leucophaea maderae. It exhibits sequence homology with the hormonally-active portion of vertebrate hormones cholecystokinin, human gastrin II and caerulin. This peptide, termed leucosulfakinin-II, shares a common C-terminal heptapeptide fragment with leucosulfakinin and a comparison of the two sequences provides an assessment of the importance of the constituent amino acids to biological activity. Leucosulfakinin-II shows a greater resemblance to cholecystokinin than does leucosulfakinin. Leucosulfakinin-II and leucosulfakinin are the only two reported invertebrate sulfated neuropeptides. As with leucosulfakinin, the intestinal myotropic activity of leucosulfakinin-II is analogous to that of gastrin and cholecystokinin. The sequence homology between the leucosulfakinins and the vertebrate hormones, as well as their analogous myotropic activity, suggest that gastrin/cholecystokinin-like neuropeptides are not confined to vertebrates, but also occur in invertebrates.

Primary structure and synthesis of a blocked myotropic neuropeptide isolated from the cockroach, Leucophaea maderae☆

A peptide which stimulates the contractile activity of the cockroach hindgut was isolated from head extracts of the cockroach, Leucophaea maderae. The inability of aminopeptidase M to degrade the peptide and the presence of glutamic acid in the hydrolysate suggested N-terminal blocking by pyroglutamic acid. The N-terminal pGlu was removed enzymatically and the unblocked fragment was sequenced with an automated gas-phase peptide sequencer. The structure determined (pGlu-Thr-Ser-Phe-Thr-Pro-Arg-Leu-NH2) was synthesized and shown to be both chemically and biologically identical with the natural product.

Isolation, identification and synthesis of locustamyoinhibiting peptide (LOM-MIP), a novel biologically active neuropeptide from Locusta migratoria.

A novel peptide termed locustamyoinhibiting peptide (LOM-MIP) was isolated from brain-corpora cardiaca-corpora allata-suboesophageal ganglion extracts of the locust, Locusta migratoria. The primary structure of this nonapeptide has been determined Ala-Trp-Gln-Asp-Leu-Asn-Ala-Gly-Trp-NH2. LOM-MIP suppresses the spontaneous contractions of the hindgut and oviduct of Locusta migratoria and of the hindgut of Leucophaea maderae. This novel peptide is, however, structurally different from leucomyosuppressin, a hindgut suppressing peptide isolated from Leucophaea maderae heads. LOM-MIP has a Gly-TrpNH2 carboxy-terminal in common with APGWamide, a penis retractor muscle inhibiting peptide isolated from the snail, Lymnea stagnalis. In addition, it shows carboxy-terminal sequence similarities with locust AKH II which ends in AGWamide. No sequence similarities were found with other vertebrate or invertebrate peptides. Synthetic LOM-MIP showed biological as well as chemical characteristics indistinguishable from those of native LOM-MIP.

The diuretic activity of a series of cephalomyotropic neuropeptides, the achetakinins, on isolated Malpighian tubules of the house cricket, Acheta domesticus

Abstract At 10−9 M achetakinins double the rate of fluid secretion by isolated Malpighian tubules of the house cricket, Acheta domesticus. They appear to act independently of cAMP, and the diuretic responses to a cAMP analogue (8-bromo-cAMP) and achetakinin-I are additive. At 10−9 M achetakinin-I potentiates the diuretic response to exogenous 8-bromo-cAMP. In the presence of 10−5 M 8-bromo-cAMP, achetakinins, I, II, III, IV and V stimulate fluid secretion maximally, and have ED50 values ranging from 1.8 × 10−11 M to 3.2× 10−10 M. The pentapeptide C-terminal sequence FX1X2WG. NH2 (where X1 = H, N, S, Y, and X2 = S or P), common to achetakinins I–V, is all that is required for full biological activity. A possible role for achetakinins as diuretic hormones in vivo is discussed.

Isolation, primary structure and synthesis of two neuropeptides from Leucophaea maderae: members of a new family of cephalomyotropins

Abstract 1. 1. Two peptides which stimulate the contractile activity of the cockroach hindgut were isolated from head extracts of the cockroach, Leucophaea maderae. 2. 2. A series of four high-performance liquid-chromatographie (HPLC) fractionations yielded sufficient quantities of pure peptides for micro amino acid analyses and primary sequence determinations. The sequences obtained for the two peptides were: Asp-Pro-Ala-Phe-Asn-Ser-Trp-Gly-NH2 and Asp-Pro-Gly-Phe-Ser-Ser-Trp-Gly-NH2. The two octapeptides were designated leucokinins I and II (L-I, L-II), respectively. 3. 3. Minimum concentrations of synthetic L-I and L-II required to produce a response from the isolated cockroach hindgut were 2.0 × 10−10 and 1.6 × 10−10M, respectively. These concentrations were virtually identical to the threshold concentrations observed with the naturally occurring peptides.

Peptidomics of CNS-associated neurohemal systems of adult Drosophila melanogaster: A mass spectrometric survey of peptides from individual flies

Neuropeptides are important messenger molecules that influence nearly all physiological processes. In insects, they can be released as neuromodulators within the central nervous system (CNS) or as neurohormones into the hemolymph. We analyzed the peptidome of neurohormonal release sites and associated secretory peptidergic neurons of adult Drosophila melanogaster. MALDI‐TOF mass spectrometric analyzes were performed on single organs or cell cluster from individual flies. This first peptidomic characterization in adult fruit flies revealed 32 different neuropeptides. Peptides not directly predictable from previously cloned or annotated precursor genes were sequenced by tandem mass spectrometry. These peptides turned out to be either intermediate products of neuropeptide processing or shorter versions of known peptides. We found that the peptidome of the CNS‐associated neurohemal organs is tagma‐specific in Drosophila. Abdominal neurohemal organs and their supplying peptidergic neurons contain the capa gene products periviscerokinins and pyrokinin‐1, thoracic neurohemal organs contain FMRFamides, and the neurohemal release sites of the brain contain pyrokinin‐12‐15, pyrokinin‐2, corazonin, myosuppressin, and sNPF as their major putative release products. Our results show that peptidomic approaches are well suited to study differential neuropeptide expression or posttranslational modifications in morphologically defined parts of the nervous system and in a developmental and physiological context in animals as small as Drosophila melanogaster. J. Comp. Neurol. 474:379–392, 2004.

Expression and functional characterization of a Drosophila neuropeptide precursor with homology to mammalian preprotachykinin A.

Peptides structurally related to mammalian tachykinins have recently been isolated from the brain and intestine of several insect species, where they are believed to function as both neuromodulators and hormones. Further evidence for the signaling role of insect tachykinin-related peptides was provided by the cloning and characterization of cDNAs for two tachykinin receptors fromDrosophila melanogaster. However, no endogenous ligand has been isolated for the Drosophila tachykinin receptors to date. Analysis of the Drosophila genome allowed us to identify a putative tachykinin-related peptide prohormone (prepro-DTK) gene. A 1.5-kilobase pair cDNA amplified from aDrosophila head cDNA library contained an 870-base pair open reading frame, which encodes five novel D rosophila tachykinin-related peptides (called DTK peptides) with conserved C-terminal FXGXR-amide motifs common to other insect tachykinin-related peptides. The tachykinin-related peptide prohormone gene (Dtk) is both expressed and post-translationally processed in larval and adult midgut endocrine cells and in the central nervous system, with midgut expression starting at stage 17 of embryogenesis. The predictedDrosophila tachykinin peptides have potent stimulatory effects on the contractions of insect gut. These data provide additional evidence for the conservation of both the structure and function of the tachykinin peptides in the brain and gut during the course of evolution.