Spermosin, a trypsin-like protease from ascidian sperm: cDNA cloning, protein structures and functional analysis.

We have previously reported that two trypsin-like enzymes, acrosin and spermosin, play key roles in sperm penetration through the vitelline coat of the ascidian (Urochordata) Halocynthia roretzi [Sawada et al. (1984), J. Biol. Chem. 259, 2900-2904; Sawada et al. (1984), Dev. Biol. 105, 246-249]. Here, we show the amino-acid sequence of the ascidian preprospermosin, which is deduced from the nucleotide sequence of the isolated cDNA clone. The isolated ascidian preprospermosin cDNA consisted of 1740 nucleotides, and an open reading frame encoding 388 amino acids, which corresponds to a molecular mass of 41 896 Da. By sequence alignment, it was suggested that His178, Asp230 and Ser324 make up a catalytic triad and that ascidian spermosin be classified as a novel trypsin family member. The mRNA of preprospermosin is specifically expressed in ascidian gonads but not in other tissues. Purified spermosin consists of 33- and 40-kDa bands as determined by SDS/PAGE under nonreducing conditions. The 40-kDa spermosin consists of a heavy chain (residues 130-388) and a long light chain designated L1 (residues 23-129), whereas the 33-kDa spermosin includes the same heavy chain and a shorter light chain designated L2 (residues 97-129). The L1 chain contains a proline-rich region, designated L1(DeltaL2) which is lacking in L2. Investigation with the glutathione-S-transferase (GST)-spermosin-light-chain fusion proteins, including GST-L1, GST-L2, and GST-L1(DeltaL2), revealed that the proline-rich region in the L1 chain binds to the vitelline coat of ascidian eggs. Thus, we propose that sperm spermosin is a novel trypsin-like protease that binds to the vitelline coat and also plays a part in penetration of sperm through the vitelline coat during ascidian fertilization.     

Structure and activity of the insect cytokine growth-blocking peptide. Essential regions for mitogenic and hemocyte-stimulating activities are separate.

Growth-blocking peptide (GBP) is a 25-amino acid insect cytokine found in Lepidopteran insects that possesses diverse biological activities such as larval growth regulation, cell proliferation, and stimulation of immune cells (plasmatocytes). The tertiary structure of GBP consists of a structured core that contains a disulfide bridge and a short antiparallel beta-sheet (Tyr(11)-Arg(13) and Cys(19)-Pro(21)) and flexible N and C termini (Glu(1)-Gly(6) and Phe(23)-Gln(25)). In this study, deletion and point mutation analogs of GBP were synthesized to investigate the relationship between the structure of GBP and its mitogenic and plasmatocyte spreading activity. The results indicated that deletion of the N-terminal residue, Glu(1), eliminated all plasmatocyte spreading activity but did not reduce mitogenic activity. In contrast, deletion of Phe(23) along with the remainder of the C terminus destroyed all mitogenic activity but only slightly reduced plasmatocyte spreading activity. Therefore, the minimal structure of GBP containing mitogenic activity is 2-23 GBP, whereas that with plasmatocyte spreading activity is 1-22 GBP. NMR analysis indicated that these N- and C-terminal deletion mutants retained a similar core structure to wild-type GBP. Replacement of Asp(16) with either a Glu, Leu, or Asn residue similarly did not alter the core structure of GBP. However, these mutants had no mitogenic activity, although they retained about 50% of their plasmatocyte spreading activity. We conclude that specific residues in the unstructured and structured domains of GBP differentially affect the biological activities of GBP, which suggests the possibility that multifunctional properties of this peptide may be mediated by different forms of a GBP receptor.     

Carbohydrate recognition of gramicidin S analogues in aqueous medium.

We have designed and synthesized of carbohydrate-binding peptides, gramicidin S analogues. Asn/Asp/Gln and Trp residues in the peptides were employed as the binding sites for carbohydrates by hydrogen-bonding interaction and the creation units for hydrophobic pocket to promote the interaction, respectively. The data of fluorescence spectroscopy and affinity column chromatography indicated that the peptides possessed the binding ability for some carbohydrates in aqueous medium. As a result of 1H NMR study, nuclear Overhauser effects between aromatic side chains of a peptide, [Gln(1,1'),Trp(3,3')]-gramisidin S and mannose were observed, indicating that the interaction of the peptide with the sugar occurred in the hydrophobic environment formed by Trp and Phe residues.     

Study on the interaction between soybean beta-amylase and substrate by the stopped-flow method.

The hydrolysis of substrates (maltoheptaose, maltopentaose, and maltotetraose) catalyzed by soybean beta-amylase [EC 3.2.1.2] at pH 5.4 and 25 degrees C was followed by monitoring small changes in the quenching of fluorescence due to tryptophan residues by the stopped-flow method. By analysis of whole time course, the dissociation constants, KdS, of enzyme-substrate and enzyme-product complexes were reasonably evaluated; and the difference in fluorescence intensities per mol between the enzyme-complex (ES or EP) and the free enzyme, delta F, was determined. The molecular activity, k0, was also determined by a new method of half time analysis. The KdS and k0 values are in good agreement with our kinetic data reported previously. The delta Fs of substrates were of smaller magnitude than those of products (G2 and G3), which means that the higher the binding affinity of the ligand is, the smaller the delta F value is. This indicates that at least two tryptophan residues must be located in the active site if the enzyme is rigid, or that if there is only one, the active site must undergo a structural change caused by the binding of ligand.     

Construction of an expression system of insect lysozyme lacking thermal stability: the effect of selection of signal sequence on level of expression in the Pichia pastoris expression system.

Expression systems of human and silkworm lysozymes were constructed using the methylotrophic yeast Pichia pastoris as a host. The leader sequence and its prepro peptide of alpha-factor (a peptide pheromone derived from yeast) and the native signal sequences of these lysozymes, were used as secretion signals. When the alpha-factor leader is used as the signal sequence, human lysozyme is secreted at a much higher level than is silkworm lysozyme. On the other hand, silkworm lysozyme, when its native signal is used, is secreted more efficiently than human lysozyme. Therefore, we expected that human lysozyme cDNA with a silkworm native signal would be secreted more efficiently than human lysozyme with its native signal. However, its level of expression was not increased. This result indicates that the native signal of silkworm lysozyme does not promote the secretion of the lysozyme, but rather alpha-factor leader inhibits the secretion. Silkworm lysozyme with the alpha-factor leader is so unstable that it could be easily attacked by some proteases and our findings suggest that the level of expression of heterologous protein with signal peptides and its stability are greatly affected by the selection of the appropriate secretion signal sequence.     

Specialized Plant Metabolism Characteristics and Impact on Target Molecule Biotechnological Production

Plant secondary metabolism evolved in the context of highly organized and differentiated cells and tissues, featuring massive chemical complexity operating under tight environmental, developmental and genetic control. Biotechnological demand for natural products has been continuously increasing because of their significant value and new applications, mainly as pharmaceuticals. Aseptic production systems of plant secondary metabolites have improved considerably, constituting an attractive tool for increased, stable and large-scale supply of valuable molecules. Surprisingly, to date, only a few examples including taxol, shikonin, berberine and artemisinin have emerged as success cases of commercial production using this strategy. The present review focuses on the main characteristics of plant specialized metabolism and their implications for current strategies used to produce secondary compounds in axenic cultivation systems. The search for consonance between plant secondary metabolism unique features and various in vitro culture systems, including cell, tissue, organ, and engineered cultures, as well as heterologous expression in microbial platforms, is discussed. Data to date strongly suggest that attaining full potential of these biotechnology production strategies requires being able to take advantage of plant specialized metabolism singularities for improved target molecule yields and for bypassing inherent difficulties in its rational manipulation.     

Gyrodactylus medaka n. sp. (Monogenea: Gyrodactylidae) parasitic on wild and laboratory-reared medaka Oryzias latipes (Beloniformes: Adrianichthyidae) in Japan

Gyrodactylus medaka n. sp. (Monogenea: Gyrodactylidae) is described from the skin, fins, and gills of medaka Oryzias latipes (Beloniformes: Adrianichthyidae) from Japan. This new species was collected from wild medaka in Hiroshima, Aichi, Saga, and Kumamoto prefectures, and laboratory-reared medaka in Chiba and Aichi prefectures. The small marginal hook sickle (≤4?μm) and the length of the marginal hook of the new species are the diagnostic morphological characters differentiated from other gyrodactylids reported from Asia. The pairwise sequence divergences for the interspecific variation in ITS regions and the phylogenetic analysis suggest that the populations of G. medaka n. sp. may have a similar genetic variation as the medaka populations in Japan. Gyrodactylus medaka n. sp. and Dactylogyrus oryziasi (Monogenea: Dactylogyridae) can maintain their populations in laboratory aquaria using medaka as their hosts, and these monogeneans and medaka have the potential as experimental model animals for clarifying various aspects of their host-parasite relationships. In addition, we report the composition of modified ammonium picrate-glycerin (APG) and show it is advantageous for monogenean taxonomy.     

Genome organization of Magnaporthe grisea: integration of genetic maps, clustering of transposable elements and identification of genome duplications and rearrangements

 A high-density genetic map of the rice blast fungus Magnaporthe grisea (Guy11×2539) was constructed by adding 87 cosmid-derived RFLP markers to previously generated maps. The new map consists of 203 markers representing 132 independently segregating loci and spans approximately 900 cM with an average resolution of 4.5 cM. Mapping of 33 cosmid probes from the genetic map generated by Sweigard et al. has allowed the integration of two M. grisea maps. The integrated map showed that the linear order of markers along all seven chromosomes in both maps is in good agreement. Thirty of eighty seven markers were derived from cosmid clones that contained the retrotransposon MAGGY (M. grisea gypsy element). Mapping of single-copy DNA sequences associated with the MAGGY cosmids indicated that MAGGY elements are scattered throughout the fungal genome. In eight cases, the probes associated with MAGGY elements showed abnormal segregation patterns. This suggests that MAGGY may be involved in genomic rearrangements. Two RFLP probes linked to MAGGY elements, and another flanking other repetitive DNA elements, identified sequences that were duplicated in the Guy11 genome. Most of the MAGGY cosmids also contained other classes of repetitive DNA suggesting that repetitive DNA sequences tend to cluster in the M. grisea genome. Received: 17 February 1997 / Accepted: 21 February 1997