Eiji Kotani

Cloning and expression of the gene of hemocytin, an insect humoral lectin which is homologous with the mammalian von Willebrand factor

Invertebrate lectins play an important role in a non-specific self-defense mechanism, as invertebrates do not synthesize specific antibodies. We report the cloning of several overlapping cDNAs encoding the entire silkworm (Bombyx mori) lectin, which we propose to call hemocytin. The sequence (10477 bp) encoded 3133 amino acids. The characteristics features of the carbohydrate-recognition domain of C-type animal lectin were revealed at C-terminal sequence of hemocytin. When cDNA encoding this region was introduced into baculovirus vector, hemagglutinating activities were detected in the culture fluid of a recombinant virus-infected cells. These activities were inhibited by D-mannose, N-acetyl-D-galactosamine, and D-maltose which are haptenic saccharides of authentic hemocytin. Analysis of dot and Northern blot hybridization revealed that hemocytin gene was transcribed in hemocytes of the silkworm at larval-pupal metamorphosis and/or after the injection of Escherichia coli and lipopolysaccharide. After silkworm larvae were injected with C-terminal portion of hemocytin, aggregation of hemocytes was observed in the hemolymph. Hemocytin has significant homology with mammalian von Willebrand factor which involves in platelet adhesion to subendothelium. Also, hemocytin has a homologous region with coagulation factor V and VIII. These results suggest that hemocytin molecule is an adhesive protein and relates to hemostasis or encapsulation of foreign substances for self-defense.

Characterization of a Bombyx mori cDNA encoding a novel member of the attacin family of insect antibacterial proteins

A Bombyx mori cDNA was cloned that hybridized with Hyalophora cecropia attacin probe and its nucleotide sequence was determined. This cDNA consisted of 846 nucleotides and the deduced amino acid sequence showed that the cDNA encodes an attacin precursor protein. The putative mature protein of B. mori attacin had 70.4, 68.3 and 18.8% identity in amino acid sequences with that of H. cecropia acidic and basic attacins and Sarcophaga peregrina sarcotoxin IIA, respectively. B. mori and H. cecropia attacins and S. peregrina sarcotoxin IIA had two subdomains in each G domain, suggesting that common amino acid residues in the subdomains are conserved during evolution and plays an important role in the activity of the antibacterial proteins. Expression of B. mori attacin gene was rapidly induced by the injection of Escherichia coli cells into B. mori larvae and continued at least for 48 h mainly in fat bodies and hemocytes.

Structure-based targeting of bioactive proteins into cypovirus polyhedra and application to immobilized cytokines for mammalian cell culture.

Certain insect viruses produce stable infectious micro-crystals called polyhedra which function to protect the virus after the death of infected larvae. Polyhedra form within infected cells and contain numerous virus particles embedded in a crystalline lattice of the viral protein polyhedrin. We have previously demonstrated that the N-terminal 75 amino acids of the Bombx mori cypovirus (BmCPV) turret protein (VP3) can function as a polyhedrin recognition signal leading to the incorporation of foreign proteins into polyhedra. Foreign proteins tagged with the VP3 polyhedrin recognition signal were incorporated into polyhedra by co-expression with polyhedrin in insect cells. We have used this method to encapsulate a wide variety of foreign proteins into polyhedra. The atomic structure of BmCPV polyhedrin showed that the N-terminal H1 alpha-helix of polyhedrin plays a significant role in cross-linking and stabilizing polyhedra. Here we show that the polyhedrin H1-helix can also function as a polyhedrin recognition signal and can be used like the VP3 N-terminal sequence to target foreign proteins into polyhedra. In addition, the two targeting methods can be used together to produce polyhedra containing both EGFP and Discosoma sp. Red Fluorescent Protein (DsRed). The modified polyhedra were imaged using dual-wavelength confocal microscopy showing that the two foreign proteins are uniformly incorporated into polyhedra at similar levels. We have investigated the biological and physiological properties of fibroblast growth factor-2 (FGF-2), FGF-7 and epidermal growth factor (EGF) immobilized on polyhedra with either the H1 or the VP3 tag. Growth factors produced by both methods were functional, inducing the growth of fibroblast cells and keratinocytes. The results demonstrate the utility and flexibility of modified polyhedra for encapsulating and stabilizing bioactive proteins.

The use of leukemia inhibitory factor immobilized on virus-derived polyhedra to support the proliferation of mouse embryonic and induced pluripotent stem cells

Human leukemia inhibitory factor (LIF) was immobilized into insect virus-derived microcrystals (polyhedra) to generate LIF polyhedra (LIF-PH) that can slowly release LIF into embryonic stem (ES) cell culture media and thus maintain ES cells in an undifferentiated state. Assays of the biological activities of LIF-PH indicated that a single addition of LIF-PH to the ES cell culture medium can support the proliferation of mouse ES and induced pluripotent stem (iPS) cells continuously for 14 days, and suggest that LIF-PH can be successfully used in the place of a periodic addition of recombinant LIF to the media every 2-3 days. The release of LIF protein from LIF-PH was determined by enzyme-linked immunosorbent assay (ELISA). Maintenance of undifferentiated state of mouse ES and iPS cells cultured with LIF-PH was determined by the detection of pluripotency-related biomarkers Oct3/4 and stage-specific embryonic antigen-1 (SSEA-1) through immunostaining and measurement of alkaline phosphatase activity. In this paper, we propose a closed culture system for mass production of ES and iPS cells that utilize a slow-releasing agent of LIF.

Bone regeneration by polyhedral microcrystals from silkworm virus.

Bombyx mori cypovirus is a major pathogen which causes significant losses in silkworm cocoon harvests because the virus particles are embedded in micrometer-sized protein crystals called polyhedra and can remain infectious in harsh environmental conditions for years. But the remarkable stability of polyhedra can be applied on slow-release carriers of cytokines for tissue engineering. Here we show the complete healing in critical-sized bone defects by bone morphogenetic protein-2 (BMP-2) encapsulated polyhedra. Although absorbable collagen sponge (ACS) safely and effectively delivers recombinant human BMP-2 (rhBMP-2) into healing tissue, the current therapeutic regimens release rhBMP-2 at an initially high rate after which the rate declines rapidly. ACS impregnated with BMP-2 polyhedra had enough osteogenic activity to promote complete healing in critical-sized bone defects, but ACS with a high dose of rhBMP-2 showed incomplete bone healing, indicating that polyhedral microcrystals containing BMP-2 promise to advance the state of the art of bone healing.

Isolation and characterization of a C-type lectin cDNA specifically expressed in the tip of mouthparts of the flesh fly Sarcophaga peregrina.

We have isolated a novel gene, CLEM 36, of the flesh fly Sarcophaga peregrina, which shows significant homology to the C‐type lectin family. CLEM 36 mRNA was transcribed excessively from the second day after eclosion only in the tip of mouthparts. Whole mount in situ hybridization showed that CLEM 36 mRNA was expressed in the C‐type lectin‐producing tissue (CLPT) located at the entrance of the food canal and between the labellum and haustellum. Immunoblot analysis showed that the mature form of CLEM 36 protein was synthesized in the CLPT, then secreted into saliva. Our results indicate that CLEM 36 protein may play an important role in biological defence against pathogens during the food intake of this insect.

De novo gene expression and antisense inhibition in cultured cells of BmTRN-1, cloned from the midgut of the silkworm, Bombyx mori, which is homologous with mammalian TIA-1/R

A cDNA encoding a 388 amino acid TIA-1-type RNA-binding protein (BmTRN-1) was isolated from midgut cDNAs of the silkworm, Bombyx mori, via homologous cloning, in order to characterize its function. The deduced amino acid sequence, most likely encoded by a single copy gene, has significant homology with human TIA-1 and TIAR known as apoptotic regulators and recently reported to function as important factors for either splicing or translation. RT-PCR analysis showed that the BmTRN-1 gene was vigorously transcribed in the midgut at the gut purge stage, indicating a possible relation to the tissue-decomposing process in larval-pupal metamorphosis. We also show that inhibition of the expression of BmTRN-1 by a transfected oligonucleotide encoding the antisense sequence caused a remarkable rise in protein expression from artificially constructed cDNAs encoded by plasmid vectors in Bombyx cells, depending on the constructed ORF sequences of the introduced cDNAs. Furthermore, it was shown that the transcripts from the cDNAs introduced into the cells increased under the antisense-inhibition of BmTRN-1 when the protein levels of these cDNAs also rose, demonstrating that BmTRN-1 could act as a regulator especially of the mechanism eliminating transcripts with possible targets for BmTRN-1 recognition in the authentic post-transcription process.

Control of angiogenesis by VEGF and endostatin-encapsulated protein microcrystals and inhibition of tumor angiogenesis

Encapsulation of cytokines within protein microcrystals (polyhedra) is a promising approach for the stabilization and delivery of therapeutic proteins. Here, we investigate the influence of vascular endothelial growth factor (VEGF) microcrystals and endostatin microcrystals on angiogenesis. VEGF was successfully encapsulated into microcrystals derived from insect cypovirus with overexpression of protein disulfide bond isomerase. VEGF microcrystals were observed to increase the phosphorylation of p42/p44 MAP kinase and to stimulate the proliferation, migration, and network and tube formation of human umbilical vein endothelial cells (HUVECs). Endostatin was also successfully encapsulated into microcrystals. Endostatin microcrystals showed antiangiogenesis activities and inhibited the migration, and network and tube formation of HUVECs. Local administration of endostatin microcrystals in mice inhibited both angiogenesis and tumor growth with clear significant differences between treatment and control groups. Endostatin microcrystals only affected angiogenesis, but had no significant effect on lymphangiogenesis compared to controls. Local therapy using endostatin microcrystals offers a potential approach to achieve sustained therapeutic release of antiangiogenic molecules for cancer treatment.

Expression of freeze-responsive proteins, Fr10 and Li16, from freeze-tolerant frogs enhances freezing survival of BmN insect cells

To date, two novel freeze‐responsive proteins, Fr10 and Li16, have been discovered in the wood frog, Rana sylvatica, and likely support freezing survival. Although previous studies have established tissue distribution of each protein, there have been no studies that explore their functional consequences in intolerant cells. To assess the ability of Fr10 and Li16 to confer freeze tolerance, we transfected each protein into a freeze‐intolerant silkworm cell line (BmN). Selected controls were the transfection of an unrelated protein (CAT) and a no‐transfection sample. Li16 and Fr10 showed 1.8 ± 0.1‐ and 1.7 ± 0.2‐fold, respectively, greater survival after freezing at –6°C for 1 h than did transfection controls. To investigate how these novel proteins protect cells from freezing damage, protein structures were predicted from primary amino acid sequences. Analysis of the structures indicated that Fr10 is a secreted protein and may be a new type IV antifreeze protein, whereas Li16 may have intracellular membrane associated functions. This study shows that freezing protection can be provided to intolerant cells by the overexpression of transfected Li16 and Fr10 frog proteins. Results from this study will provide new insights into adapting intolerant cells for medical organ cryoprotection using a natural vertebrate model of tolerance.—Biggar, K. K., Kotani, E., Furusawa, T., Storey, K. B., Expression of freeze‐responsive proteins, Fr10 and Li16, from freeze‐tolerant frogs enhances freezing survival of BmN insect cells. FASEB J. 27, 3376–3383 (2013). www.fasebj.org

Cell proliferation by silk gut incorporating FGF-2 protein microcrystals

Silk gut processed from the silk glands of the silkworm could be an ideal biodegradable carrier for cell growth factors. We previously demonstrated that polyhedra, microcrystals of Cypovirus 1 polyhedrin, can serve as versatile carrier proteins. Here, we report the generation of a transgenic silkworm that expresses polyhedrin together with human basic fibroblast growth factor (FGF-2) in its posterior silk glands to utilize silk gut as a proteinaceous carrier to protect and slowly release active cell growth factors. In the posterior silk glands, polyhedrin formed polyhedral microcrystals, and FGF-2 became encapsulated within the polyhedra due to a polyhedron-immobilization signal. Silk gut powder prepared from posterior silk glands containing polyhedron-encapsulated FGF-2 stimulated the phosphorylation of p44/p42 MAP kinase and induced the proliferation of serum-starved NIH3T3 cells by releasing bioactive FGF-2. Even after a one-week incubation at 25 °C, significantly higher biological activity of FGF-2 was observed for silk gut powder incorporating polyhedron-encapsulated FGF-2 relative to silk gut powder with non-encapsulated FGF-2. Our results demonstrate that posterior silk glands incorporating polyhedron-encapsulated FGF-2 are applicable to the preparation of biodegradable silk gut, which can protect and release FGF-2 that is produced in a virus- and serum-free expression system with significant application potential.