Susumu Izumi

Dally regulates Dpp morphogen gradient formation in the Drosophila wing

Decapentaplegic (Dpp), a Drosophila TGFβ/bone morphogenetic protein homolog, functions as a morphogen to specify cell fate along the anteroposterior axis of the wing. Dpp is a heparin-binding protein and Dpp signal transduction is potentiated by Dally, a cell-surface heparan sulfate proteoglycan, during assembly of several adult tissues. However, the molecular mechanism by which the Dpp morphogen gradient is established and maintained is poorly understood. We show evidence that Dally regulates both cellular responses to Dpp and the distribution of Dpp morphogen in tissues. In the developing wing, dally expression in the wing disc is controlled by the same molecular pathways that regulate expression of thickveins, which encodes a Dpp type I receptor. Elevated levels of Dally increase the sensitivity of cells to Dpp in a cell autonomous fashion. In addition, dally affects the shape of the Dpp ligand gradient as well as its activity gradient. We propose that Dally serves as a co-receptor for Dpp and contributes to shaping the Dpp morphogen gradient.

Molecular properties and biosynthesis of major plasma proteins in Bombyx mori

Abstract In the silkworm, Bombyx mori, a group of structurally related proteins referred to as ‘30K proteins’ comprises the major plasma proteins of the last instar larvae. Four protein components consisting of 30K proteins were purified to homogeneity from the larval hemolymph and designated Component 1, 2, 3 and 4, respectively. Close similarity in amino acid composition was noticed between Components 1 and 3, and between Components 2 and 4. Rabbit antibody prepared against Component 4 crossreacted with Component 2 as well as Component 4 but not with Components 1 or 3. In a cell-free translation system, RNA isolated from the fat body of the last instar larvae directed the synthesis of proteins reactive with anti-Component 4 antibody. Developmental change in the hemolymph concentration of 30K proteins well reflected the level of functional mRNA for these proteins in the fat body, indicating that the biosynthesis of 30K proteins is regulated during development at pre-translational level.

Purification and molecular properties of vitellin from the silkworm, Bombyx mori

Abstract Vitellin was purified from the eggs of the silkworm, Bombyx mori by a simple method which included a specific precipitation at pH 6 under low ionic concentration and DEAE-cellulose column chromatography. The final preparation was highly homogeneous as judged by gel electrophoresis, electron microscopy and ultracentrifugation. Vitellin was defined as glycolipoprotein with a sedimentation coefficient (S 20, W ) of 13.5S and a molecular weight of 440,000. The molecule was almost spherical in shape with a diameter of 13 nm. The molecule contained 3% mannose and 7.5% total lipids which comprised triacylglycerol, diacylglycerol, cholesterol, phosphatidylcholine and phosphatidylethanolamine. The amino acid composition displayed a high content of glutamic and aspartic acids and a low content of methionine. The molecule was composed of two non-identical subunits with molecular weights of 180,000 and 42,000, and the native molecule was assumed to be a tetramer composed of two molecules of each of these subunits. Separation of the two subunits was achieved, and mannose was covalently associated only with the heavier subunit. The rabbit anti-egg vitellin antibody cross-reacted with the haemolymph vitellogenin but not with other haemolymph proteins, nor with the vitellogenin from Locusta migratoria . The antibody also reacted with the haemolymph vitellogenin of the silkworm, Philosamia cynthia .

Structure and expression of mRNA for vitellogenin in Bombyx mori

Vitellogenin, a precursor of major yolk protein of the silkworm, Bombyx mori is a tetramer composed of each two molecules of heavy and light subunits. We cloned mRNA sequence for the B. mori vitellogenin and analyzed its structure. Sequence alignment of several overlapping cDNA clones indicated that the vitellogenin mRNA is approx. 5.7 kb, containing an open reading frame for a peptide with 1782 amino acid residues. By comparing the deduced amino acid sequence with the amino-terminal primary structures of vitellogenin subunits, it is suggested that the heavy and light subunits of the B. mori vitellogenin are encoded by a single contiguous mRNA. The primary translation product of the vitellogenin mRNA was detected in the microsomal fraction prepared from the fat body of vitellogenic females. Northern blot analysis of the fat body RNA demonstrated that the biosynthesis of vitellogenin in B. mori is regulated in a tissue-, sex- and stage-specific manner at the level of mRNA. Possible cause for discrepancy between the present results and our previous proposal (Izumi, S. and Tomino, S. (1983) Insect Biochem. 13, 81-85) on the biosynthesis of B. mori vitellogenin is also discussed.

Yolk proteins from insect eggs: Structure, biosynthesis and programmed degradation during embryogenesis

Abstract Since insect vitellogenesis is a heterosynthetic process, yolk protein follows a complicated fate. This includes: hormonally stimulated gene activity, secretion into the blood, specific recognition and uptake by oocytes, packaging into yolk granules, and proteolysis in developing eggs. During the process, proteins are modified co- or post-translationally. Each of these steps has been intensively studied in insects. This offers a basis for more exciting development not only in the field of insect physiology, but also in more basic or fundamental areas of biochemistry and cellular biology. Bombyx acid cysteine proteinase is a case in point. It is now evident that proteolytic reactions play a key role in the control of many physiological functions. Thus the control mechanisms involved in the proteolytic processes require attention and proteinases have been purified and characterized. In many cases, however, it is difficult to analyze the physiological substrates of them, and difficult to know the regulatory mechanism in vivo . Insect yolk protein systems provide a suitable system for studying regulation of cellular proteinases, since yolk proteins (substrate of proteinases) are purified and characterized. This review summarizes recent progress in understanding the structure, biosynthesis and programmed degradation of yolk proteins in insects.

Vitellogenin gene of the silkworm, Bombyx mori: Structure and sex-dependent expression

Vitellogenin of Bombyx mori is a precursor of major yolk protein synthesized in the female fat body at larval—pupal ecdysis. The gene for B. mori vitellogenin is composed of seven exons interspersed by six introns. Developmental profile of the primary transcript of the gene indicated that the biosynthesis of B. mori vitellogenin is regulated transcriptionally in a sex‐ and stage‐dependent manner in the fat body. The Arg‐X‐Arg‐Arg sequence, which conforms to the recognition site of mammalian furin, occurs in a region just upstream of the putative proteolytic cleavage site of B. mori previtellogenin.

Developmental and sex-dependent regulation of storage protein synthesis in the silkworm, Bombyx mori

The mechanism of sex-dependent expression of a major plasma protein, referred to as storage protein 1 (SP-1) was studied during development of the silkworm, Bombyx mori. SP-1 occurred in the hemolymph of the female as well as in the male larvae until the end of the fourth larval instar. In the last instar larvae, the amount of SP-1 in the hemolymph greatly increased in females, but markedly declined in males. The level of fat body mRNA for SP-1 reflected the developmental and sex-dependent changes in the hemolymph concentration of SP-1. The developmental patterns of hemolymph proteins in the third and the fourth instar larvae of sex-mosaic individuals were quite analogous to those observed in normal larvae at the same developmental stages. The hemolymph concentration of SP-1 at the last larval instar of the sex mosaics varied among individuals irrespective of the gonad compositions. In vitro culture of the fat body cells dissected from several locations of a sex-mosaic larva provided evidence that each fat body cell in a common hemolymph milieu synthesizes a high (female type) or a low (male type) level of SP-1 depending on the sex chromosome composition. The amount of vitellogenin in the hemolymph of the sex-mosaic pupae was in proportion to that of SP-1 at the last larval instar. From these results, it is suggested that the sex-dependent expression of SP-1 and vitellogenin in B. mori is genetically determined and developmentally regulated without participation of the reproductive organs or any sex-specific humoral factors.

Structure and expression of mRNA for a pupal cuticle protein of the silkworm, Bombyx mori

Electrophoretic and immunoblot analyses of proteins extracted from the salt-washed integuments of the silkworm Bombyx mori demonstrated that the pupal cuticle contains structural proteins distinct from those present in the larval cuticle. The cDNA clone encoding a pupal cuticle protein was isolated from the cDNA library constructed from epidermal mRNA of pharate pupae. Northern blot hybridization by use of a cDNA probe provided evidence that mRNA for the pupal cuticle protein accumulate in integument during larval-pupal transformation, though temporal rise of the mRNA level was also noticed at the stages of larval molting. Primary structure of the pupal cuticle protein was deduced from the nucleotide sequence of cDNA. The cloned mRNA sequence encodes a 27 kDa protein rich in alanine and proline, containing characteristic repeats of Ala-Pro-Ala-His-Gln-(Asp/Ser)-Trp-Asn sequence in the carboxyl-proximal domain. The sequence (Ile/Val)-(Leu/Ala)-(Asp/Glu)-Thr-Pro-Glu-Val-Ala-(Gln/Ala)-Ala-Arg-Ala-Ala-His-(Leu/Ile)-(Ala/Ser)-Ala-(Leu/His) occurs in three hydrophobic domains of the molecule.

Structure and expression of a cyst specific protein of Acanthamoeba castellanii

The life cycle of Acanthamoeba is divided into a growth-division phase and two distinctive processes of cellular differentiation, termed encystment and excystment. Polyacrylamide gel electrophoresis revealed that a specific protein of 21 kDa in molecular weight occurs in the cyst, but not in the trophozoite stages of A. castellanii Neff strain. This cyst-specific protein, designated as CSP21, was purified from guanidine-HCl extract of cyst wall and anti-CSP21 antibody was produced. Immunoblotting of proteins extracted from a variety of species of Acanthamoeba genus suggested that the antibody is specific for group II amoebae, therefore, providing a useful tool for Acanthamoebae taxonomy. A cDNA clone for A. castellanii CSP21 was isolated by immunoscreening of a cDNA expression library constructed from mRNA of amoebae at encysting stage. The deduced primary structure indicated that CSP21 is a hydrophilic protein showing no significant homology with peptides thus far published. RNA blot analysis showed that the expression of CSP21 mRNA was restricted within early stages of encystment, suggesting that the biosynthesis of CSP21 is regulated at mRNA level.

Molecular cloning and characterization of cDNA for insect biogenic peptide, growth-blocking peptide

Growth‐blocking peptide (GBP) is an insect biogenic peptide that prevents the onset of metamorphosis from larva to pupa. A cDNA coding for GBP is described. Mixed oligonucleotides derived from a GBP peptide sequence were used to generate amplified DNA by the polymerase chain reaction (PCR). Based on the sequence of the amplified DNA, a 41 bases oligonucleotide was designed for screening a cDNA library which was constructed from the armyworm Pseudaletia separata larvae parasitized with the parasitic wasp Cotesia kariyai. The cloned cDNA for GBP was 809 base pairs in length. An open reading frame of 429 base pairs encodes a pre‐pro‐peptide of 143 amino acid residues in which GBP is localized at the C‐terminal region, and other three peptides including a putative signal peptide and appropriate processing sites for endoproteolytic cleavage precede the GBP sequence. Northern blot analyses demonstrate the presence of a 800‐base mRNA transcript in fat body and 2.5‐kilobase transcript in brain and nerve cord, suggesting the possibility that the transcription of GBP gene is regulated in a tissue‐dependent manner. This interpretation was supported by isolating a GBP cDNA fragment from cDNA pool of brain‐nerve cords. GBP mRNA is constantly expressed in both parasitized and non‐parasitized last instar larvae and there is no difference in the levels of the mRNA between both larvae, thus indicating that parasitism may effect on translational or posttranslational level to elevate plasma GBP concentration.