Identity of a second type of allatostatin from cockroach brains: an octadecapeptide amide with a tyrosine-rich address sequence.

Abstract

An octadecapeptide that inhibits juvenile hormone synthesis has been isolated by HPLC from brainretrocerebral complexes of the cockroach Diploptera punctata. The primary structure of this allatostatin has been elucidated by tandem mass spectrometry: Ala-Tyr-Ser-Tyr-Val-Ser-GluTyr-Lys-Arg-Leu-Pro-Val-Tyr-Asn-Phe-Gly-Leu-NH2 (ASB2). The amidated three-residue C terminus of this type B allatostatin is identical to that of four known type A allatostatins, and the preceding three residues show close structural homology. ASB2 has over twice the activity of the type A tridecapeptide Ala-Pro-Ser-Gly-Ala-Gln-Arg-Leu-Tyr-GlyPhe-Gly-Leu-NH2 (ASA1) in inhibiting juvenile hormone biosynthesis in corpora allata from females in early vitellogenesis (day 2), and its efficacy persists during pregnancy, but it is equally effective as ASA1 on glands from day-10 females (IC50 = 0.31 nM). The octadecapeptide is characterized by a potential dibasic cleavage site, Lys'-Arg"', the integrity of which is needed for high potency. The ASB2-(11-18)-octapeptide amide gives a full response at high concentrations at day 10 (ICso = 48 nM), but the C-truncated (1-9)-, (1-11)-, and (1-17)-amide fragments of ASB2 are inactive. Thus, the endocrine message is located at the C terminus. Na-acetylation of the N-truncated (9-18), (10-18), and (11-18) fragments of ASB2 increases activity relative to the nonacetylated peptides. The site ofaction of type A and type B allatostatins is located before mevalonate kinase in the biosynthetic pathway for juvenile hormone.

Molecular cloning of the gene for the allatostatin family of neuropeptides from the cockroach Diploptera punctata.

“…To isolate the coding sequence of an internal portion of the AST-encoding gene, oligonucleotides were designed to amplify between the ends of the largest AST peptide sequence, the octadecapeptide ASB2 (4). The degenerate sequences are diagrammed in Fig.…”

“…Seven ASTs ranging in size from 8 to 18 amino acids have been purified from the cockroach Diploptera punctata (3)(4)(5). All share the common C-terminal sequence Tyr-Xaa-Phe-GlyLeu-NH2, which has been demonstrated in vitro to be the minimum sequence capable of inhibiting JH biosynthesis (6,7).…”

A Family of Neuropeptides That Inhibit Juvenile Hormone Biosynthesis in the Cricket, Gryllus bimaculatus

“…In the assay on CA from 3-day-old virgin females, the peptides showed 50% inhibition of JH III synthesis at 1 ϫ 10 Ϫ8 to 7 ϫ 10 Ϫ8 M. This suggests that cricket CA are highly sensitive to both types of cricket allatostatic neuropeptides when they are at the height of activity during middle-late vitellogenesis (day 3 after imaginal moult). In D. punctata, an inverse relationship between the activity of the CA and their sensitivity to allatostatins was observed (3,23). Also in crickets, CA with lower basal rates of JH III biosynthesis (e.g.…”

“…The occurrence of Trp at position 2, which is a common feature of all B allatostatins and of Lom-MIP, might indicate the importance of this residue with regard to the biological activity of these peptides. The information about the bioactivity therefore might be encoded not only in the C-terminal region, as shown for the Tyr-Xaa-Phe-Gly-Leu-NH 2 allatostatin family (3,24).…”

“…Inhibition of juvenile hormone (JH) 1 biosynthesis by corpora allata (CA) in vitro has provided the basis for the isolation of a family of allatostatins (Tyr-Xaa-Phe-Gly-Leu-NH 2 ) from brain extracts of various cockroach species (Diploptera punctata, Periplaneta americana, and Blattella germanica) (1)(2)(3)(4)(5)(6). In D. punctata and P. americana, molecular cloning has led to the isolation of cDNA that encodes a precursor polypeptide containing 13 and 14 potential allatostatic sequences, respectively, including those formerly identified through conventional purification techniques (7,8).…”