Hironori Ishizaki

Amino-Terminal Amino Acid Sequence of the Silkworm Prothoracicotropic Hormone: Homology with Insulin

Three molecular forms of prothoracicotropic hormone were isolated from the head of the adult silkworm, Bombyx mori, and the amino acid sequence of 19 amino acid residues in the amino terminus of these prothoracicotropic hormones was determined. These residues exhibit significant homology with insulin and insulin-like growth factors.

Bombyxin, an Insulin-Related Peptide of Insects, Reduces the Major Storage Carbohydrates in the Silkworm Bombyx mori

The effects of an insect insulin-related peptide, bombyxin, on carbohydrate metabolism were investigated in the silkworm Bombyx mori. Bombyxin lowered the concentration of the major hemolymph sugar, trehalose, in a dose-dependent manner when injected into neck-ligated larvae. Bombyxin also caused elevated trehalase activity in the midgut and muscle, suggesting that bombyxin induces hypotrehalosemia by promoting the hydrolysis of hemolymph trehalose to glucose and thereby facilitating its transport into tissues. In addition, bombyxin reduced the glycogen content in the fat body and concurrently raised the percentage of active glycogen phosphorylase in this tissue. Because hemolymph trehalose is also a major storage form of carbohydrate in insects, our results indicate that bombyxin reduces the amount of both principal storage carbohydrates in B. mori larvae. It is therefore suggested that although bombyxin is involved in the control of carbohydrate metabolism like insulin, the physiological role of bombyxin in insects is different from that of insulin in mammals.

Immunohistochemical Localization of Prothoracicotropic Hormone‐Producing Neurosecretory Cells in the Brain of Bombyx mori

A monoclonal antibody that recognized the Bombyx prothoracicotropic hormone (PTTH) was produced by immunizing mice with a synthetic pentadecapeptide corresponding to the amino‐terminal portion of Bombyx PTTH. The antibody recognized both intact and reduced forms of PTTH. Immunohistochemistry with this antibody has demonstrated that PTTH is produced by two pairs of dorso‐lateral neurosecretory cells of the brain and transported to the corpora allata by axons running through the contralateral hemisphere of the brain. Immunoreactive axon terminals in the corpora allata were localized between the glandular cells, suggesting that PTTH is released at the inner part of this organ.

Isolation and some characterization of the prothoracicotropic hormone from Bombyx mori.

The prothoracicotropic hormone (PTTH) was isolated from adult heads of Bombyx mori. Fifty micrograms of pure PTTH was obtained from 648,000 heads through a 15-step purification procedure with a 2 X 10(6)-fold purification and an 8% recovery. Chemical analyses of this PTTH have shown that it is a single-chain peptide consisting of 40-43 amino acid residues (MW, 4330-4740), the N-terminus of which is glycine. As little as 0.1 ng of PTTH elicited adult development in a debrained pupa of Samia cynthia ricini. Five picograms of PTTH directly stimulated the prothoracic glands in vitro so as to enhance ecdysone release. The hemolymph ecdysteroids of brainless Samia pupae that were developed by PTTH injection increased with essentially the same pattern as in developing normal pupae.

Cloning of a Gene Encoding Bombyxin, an Insulin‐Like Brain Secretory Peptide of the Silkmoth Bombyx mori with Prothoracicotropic Activity

A genomic DNA encoding bombyxin, a 5kD brain peptide of the silkmoth Bornbyx mori with prothoracicotropic hormone activity, has been isolated. The nucleotide sequence coding for bombyxin shows high homology with insulin‐gene family members and the overall organization of the preprobombyxin gene is the same as in preproinsulin genes, indicating that bombyxin shares a common ancestral molecule with insulin‐family peptides. The bombyxin gene has no intron contrasting to other members of insulin‐gene family.

Species Specificity of the Insect Prothoracicotropic Hormone (PITH): the Presence of Bombyx‐and Samia‐Specific PTTHs in the Brain of Bombyx mori

Crude extracts of Bombyx mori brains can provoke adult development when injected into brain‐removed dormant pupae of Bombyx mori and Samia Cynthia ricini. From this fact the prothoracicotropic hormone (PTTH) of Bombyx has long been thought to be species‐nonspecifically active on Samia. Chemical fractionation of Bombyx brain or head extracts by fractional precipitation with acetone, Sephadex G‐50 gel‐filtration, and DEAE‐Sepharose CL‐6B chromatography, however, separated the fractions which activated Bombyx brainless pupae from those which activated Samia. Those results reveal the existence of two species‐specific PTTHs.

INSECT PROTHORACICOTROPIC HORMONE : A NEW MEMBER OF THE VERTEBRATE GROWTH FACTOR SUPERFAMILY

Prothoracicotropic hormone (PTTH) is a brain neurosecretory protein that controls insect development. PTTH of the silkmoth Bombyx mori is a homodimeric protein, the subunit of which consists of 109 amino acids. Clear‐cut sequence similarity to any other proteins has not been observed. By disulfide‐bond pattern analysis and modeling of the PTTH structure based on the known three‐dimensional (3D) structures of growth factor family with cystine‐knot motif, we propose that the PTTH protomer adopts the fold unique to the structural superfamily of the growth factors, β‐nerve growth factor (β‐NGF), transforming growth factor‐β2 (TGF‐β2), and platelet‐derived growth factor‐BB (PDGF‐BB). The insect neurohormone PTTH appears to be a member of the growth factor superfamily, sharing a common ancestral gene with the three vertebrate growth factors, β‐NGF, TGF‐β2 and PDGF‐BB.

Synthesis of bombyxin-IV, an insulin superfamily peptide from the silkworm, Bombyx mori, by stepwise and selective formation of three disulfide bridges.

We report the synthesis of bombyxin-IV, a disulfide-linked, heterodimeric, insulin superfamily peptide from the silkworm,Bombyx mori. The two chains (A- and B-chains) were synthesized separately by the solid-phase method using fluoren-9-ylmethoxycarbonyl (Fmoc) group as a protecting group for α-amino group. Three disulfide bonds were bridged step by step (A6–A11, A20–B22, and A7–B10) in a good yield. Synthetic bombyxin-IV was identical with natural one with regard to the retention time on a reversed-phase column and the molecular weight measured by mass spectrometry. Circular dichroism (CD) spectrum of the synthetic bombyxin-IV was very similar to that of the natural one. The specific activity of synthetic bombyxin-IV is equal to that of natural one (0.1 ng/Samia unit). These results suggest that the synthetic bombyxin-IV has the tertiary structure identical with the natural peptide. Our method developed for synthesis of bombyxin-IV would be generally applicable to the synthesis of insulin-like heterodimeric peptides.

Changes in the titer of bombyxin-immunoreactive material in hemolymph during the postembryonic development of the silkmoth Bombyx mori

Monoclonal antibodies were raised against pure, native bombyxin‐II (bombyxin‐II antibody) and against a synthetic nonapeptide corresponding to the amino‐terminus of the C‐peptide of the bombyxin precursor protein (C‐peptide antibody). The bombyxin‐II antibody recognized both bombyxin and probombyxin. A radioimmunoassay for bombyxin using the bombyxin‐II antibody was developed, and developmental change in the titer of bombyxin immunoreactivity in the Bombyx hemolymph was investigated. The titer was low and almost constant during the fourth and early fifth instars. In the male, the titer rose abruptly 3 days after the beginning of wandering. One day after pupation it rose again steeply to reach the maximal level which lasted until the middle of the developing adult stage. The titer decreased thereafter and increased again at adult emergence. In the female, the pattern of titer fluctuation was similar to that in the male, but the female titers during pupal‐adult development were 2–3 times higher than the male titers.

Structure and expression of bombyxin-related peptide genes of the moth Samia cynthia ricini.

From the genomic DNA of the moth Samia cynthia ricini, we cloned and characterized six clustered genes that encode precursor molecules for peptides structurally related to bombyxin, a Bombyx mori brain secretory peptide that is structurally like insulin and functionally like the prothoracicotropic hormone. The precursor molecules deduced from these genes have the domain organization of signal peptide/B-chain/C-peptide/A-chain, as in preprobombyxins and preproinsulins. The Samia bombyxin-related peptide (SBRP) genes are classified into families A and B according to their sequence homology. Two genes belonging to different families are arranged close to each other to form a pair with opposite transcriptional orientations (A-1/B-1, A-2/B-2, and A-3/B-3). None of these genes have introns, and gene B-3 has an in-frame stop codon representing a pseudogene. Four genes, A-1, A-3, B-1, and B-2, are expressed in Samia brain. Genomic Southern hybridization suggests that the Samia genome contains many other SBRP genes.