Storage Protein 2 Research Articles

The jasmonoyl-isoleucine receptor CORONATINE INSENSITIVE1 suppresses defense gene expression in Arabidopsis roots independently of its ligand.

The F-box protein CORONANTINE INSENSITIVE1 (COI1) serves as the receptor for the plant hormone jasmonoyl-isoleucine (JA-Ile). COI1, its co-receptors of the JASMONATE ZIM-domain (JAZ) protein family, and JA-Ile form a functional unit that regulates growth or defense mechanisms in response to various stress cues. Strikingly, COI1, but not JA-Ile, is required for susceptibility of Arabidopsis thaliana towards the soil-borne vascular pathogen Verticillium longisporum. In order to obtain marker genes for further analysis of this JA-Ile-independent COI1 function, transcriptome analysis of roots of coi1 and allene oxide synthase (aos) plants (impaired in JA biosynthesis) was performed. Intriguingly, nearly all of the genes that are differentially expressed in coi1 versus aos and wild type are constitutively more highly expressed in coi1. To support our notion that COI1 acts independently of its known downstream signaling components, coi1 plants were complemented with a COI1 variant (COI1AA ) that is compromised in its interaction with JAZs. As expected, these plants showed only weak induction of the expression of the JA-Ile marker gene VEGETATIVE STORAGE PROTEIN2 after wounding and remained sterile. On the other hand, genes affected by COI1 but not by JA-Ile were still strongly repressed by COI1AA . We suggest that COI1 has a potential moonlighting function that serves to repress gene expression in a JA-Ile- and JAZ-independent manner.

Depletion of Arabidopsis ACYL-COA-BINDING PROTEIN3 Affects Fatty Acid Composition in the Phloem.

Oxylipins are crucial components in plant wound responses that are mobilised via the plant vasculature. Previous studies have shown that the overexpression of an Arabidopsis acyl-CoA-binding protein, AtACBP3, led to an accumulation of oxylipin-containing galactolipids, and AtACBP3pro::BETA-GLUCURONIDASE (GUS) was expressed in the phloem of transgenic Arabidopsis. To investigate the role of AtACBP3 in the phloem, reverse transcription-polymerase chain reaction and western blot analysis of phloem exudates from the acbp3 mutant and wild type revealed that the AtACBP3 protein, but not its mRNA, was detected in the phloem sap. Furthermore, micrografting demonstrated that AtACBP3 expressed from the 35S promoter was translocated from shoot to root. Subsequently, AtACBP3 was localised to the companion cells, sieve elements and the apoplastic space of phloem tissue by immunogold electron microscopy using anti-AtACBP3 antibodies. AtACBP3pro::GUS was induced locally in Arabidopsis leaves upon wounding, and the expression of wound-responsive jasmonic acid marker genes (JASMONATE ZIM-DOMAIN10, VEGETATIVE STORAGE PROTEIN2, and LIPOXYGENASE2) increased more significantly in both locally wounded and systemic leaves of the wild type in comparison to acbp3 and AtACBP3-RNAi. Oxylipin-related fatty acid (FA) (C18:2-FA, C18:3-FA and methyl jasmonate) content was observed to be lower in acbp3 and AtACBP3-RNAi than wild-type phloem exudates using gas chromatography-mass spectrometry. Experiments using recombinant AtACBP3 in isothermal titration calorimetry analysis showed that medium- and long-chain acyl-CoA esters bind (His)6-AtACBP3 with KD values in the micromolar range. Taken together, these results suggest that AtACBP3 is likely to be a phloem-mobile protein that affects the FA pool and jasmonate content in the phloem, possibly by its binding to acyl-CoA esters.

CHO 세포에서 누에 혈림프 유래 Storage-protein 2의 세포응집 및 세포사멸 억제 효과

Chinese hamster ovary (CHO) cells have been widely used for production of various recombinant proteins such as cytokines and monoclonal antibodies. The cell aggregation and cell death in CHO cell culture directly affect cell viability, and productivity and quality of products. In this study, we investigated preventing effects of storage-protein 2 (SP2) derived from silkworm hemolymph on cell aggregation and cell death in CHO cell culture producing albuminerythropoietin (Alb-EPO). The viable cell density in the culture supplemented with 2 mg/mL SP2 was 1.71-fold higher than that in control culture. Increased titer of Alb-EPO was also found in the culture with SP2. Morphology of CHO cells in SP2 supplemented cultures did not differ from that of control. In addition, the cell aggregation rate of the SP2 cultures was reduced 20% compared to the control. Finally, we confirmed that the apoptosis was strongly suppressed by addition of SP2 in the cultures. These results clearly demonstrate that SP2 can be served as an effective supplement for enhancing titer of Alb-EPO via reducing cell aggregation and cell death.

Paired Hierarchical Organization of 13-Lipoxygenases in Arabidopsis.

Embryophyte genomes typically encode multiple 13-lipoxygenases (13-LOXs) that initiate the synthesis of wound-inducible mediators called jasmonates. Little is known about how the activities of these different LOX genes are coordinated. We found that the four 13-LOX genes in Arabidopsis thaliana have different basal expression patterns. LOX2 expression was strong in soft aerial tissues, but was excluded both within and proximal to maturing veins. LOX3 was expressed most strongly in circumfasicular parenchyma. LOX4 was expressed in phloem-associated cells, in contrast to LOX6, which is expressed in xylem contact cells. To investigate how the activities of these genes are coordinated after wounding, we carried out gene expression analyses in 13-lox mutants. This revealed a two-tiered, paired hierarchy in which LOX6, and to a lesser extent LOX2, control most of the early-phase of jasmonate response gene expression. Jasmonates precursors produced by these two LOXs in wounded leaves are converted to active jasmonates that regulate LOX3 and LOX4 gene expression. Together with LOX2 and LOX6, and working downstream of them, LOX3 and LOX4 contribute to jasmonate synthesis that leads to the expression of the defense gene VEGETATIVE STORAGE PROTEIN2 (VSP2). LOX3 and LOX4 were also found to contribute to defense against the generalist herbivore Spodoptera littoralis. Our results reveal that 13-LOX genes are organised in a regulatory network, and the data herein raise the possibility that other genomes may encode LOXs that act as pairs.

Efficient cryopreservation of human mesenchymal stem cells using silkworm hemolymph-derived proteins

Cryopreservation methods for human mesenchymal stem cells (hMSCs) typically depend on the presence of fetal bovine serum (FBS) with dimethyl sulphoxide (DMSO), which is not appropriate for therapeutic applications. In our previous study, we found that storage protein 2 (SP2), a natural material derived from silkworm hemolymph, has an inhibitory effect on the generation of reactive oxygen species (ROS). In this study, we used SP2 as an alternative to establish an effective, low-DMSO and FBS-free cryopreservation agent for the cryostorage of hMSCs. We investigated the cell viability and stem cell characterization of umbilical cord-derived MSCs in different freezing media through the freezing and thawing process. We also evaluated the efficacy of cryostorage using these media over 1 week and 1 year. When the cell characteristics (cell viability and stemness) were analysed after thawing, those obtained using 5 mg/ml SP2 were comparable to those obtained using a freezing medium with FBS. The stable cell viability and characteristics were shown even after 1 year of cryopreservation. In addition, when the cells were differentiated into adipocytes and osteocytes, we confirmed that the differentiation behaviours of the thawed cells were well maintained. The positive results could be also obtained when SP2 was applied to other MSCs. The results clearly indicate that SP2 could be used as an alternative to FBS for a freezing medium with reduced DMSO. Copyright © 2016 John Wiley & Sons, Ltd.

Whitening Effect of Storage Protein 2 from Silkworm Hemolymph

This study investigated the effects of silkworm hemolymph-derived storage protein 2 (SP2) on the whitening process in mouse B16F1 melanoma cells. After the cells were treated with various concentrations of SP2 (0.1 - 1.0 mg/mL), cytotoxicity, melanin contents, and differences in mRNA and protein expression associated with melanogenesis were observed. No cytotoxicity was observed when cells were treated with SP2, even with increased SP2 concentrations of up to 2.0 mg/mL. When treated with various SP2 concentrations in the cells, the protein and mRNA expression of tyrosinase were dose-dependently decreased, respectively, and inhibition of tyrosinase was further increased by 50.0% with increasing SP2 concentration of 1.0 mg/mL. Expression mRNAs coding tyrosinase related protein-1 and protein-2 (TRP-1 and TRP-2) was also significantly decreased in a dose dependent manner. When measuring the melanin content in melanoma cells, SP2 at 1 mg/mL inhibited melanin synthesis by 73.5% compared with non-treated cells. The inhibitory effect was 2.8-fold higher than that obtained using arbutin as a positive control. This study demonstrates that SP2, as a whitening material, is capable of suppressing melanin synthesis through the downregulation of proteins and genes in the melanin biosynthesis pathway.

Expression and Functional Analysis of Storage Protein 2 in the Silkworm,Bombyx mori

Storage protein 2 (SP2) not only is an important source of energy for the growth and development of silkworm but also has inhibitory effects on cell apoptosis. Endothelial cell (EC) apoptosis is an important contributing factor in the development of atherosclerosis; therefore, study of the antiapoptotic activity of SP2 on ECs provides information related to the treatment of atherosclerosis and other cardiovascular diseases. In this study, the sp2 gene was cloned and expressed in Escherichia coli to produce a 6xHis-tagged fusion protein, which was then used to generate a polyclonal antibody. Western blot results revealed that SP2 levels were higher in the pupal stage and hemolymph of fifth-instar larvae but low in the egg and adult stages. Subcellular localization results showed that SP2 is located mainly on the cell membrane. In addition, a Bac-to-Bac system was used to construct a recombinant baculovirus for SP2 expression. The purified SP2 was then added to a culture medium for human umbilical vein ECs (HUVECs), which were exposed to staurosporine. A cell viability assay demonstrated that SP2 could significantly enhance the viability of HUVEC. Furthermore, both ELISA and flow cytometry results indicated that SP2 has anti-apoptotic effects on staurosporine-induced HUVEC apoptosis.

The Lateral Organ Boundaries Domain Transcription Factor LBD20 Functions in Fusarium Wilt Susceptibility and Jasmonate Signaling in Arabidopsis

The LATERAL ORGAN BOUNDARIES (LOB) DOMAIN (LBD) gene family encodes plant-specific transcriptional regulators functioning in organ development. In a screen of Arabidopsis (Arabidopsis thaliana) sequence-indexed transferred DNA insertion mutants, we found disruption of the LOB DOMAIN-CONTAINING PROTEIN20 (LBD20) gene led to increased resistance to the root-infecting vascular wilt pathogen Fusarium oxysporum. In wild-type plants, LBD20 transcripts were barely detectable in leaves but abundant in roots, where they were further induced after F. oxysporum inoculation or methyl jasmonate treatment. Induction of LBD20 expression in roots was abolished in coronatine insensitive1 (coi1) and myc2 (allelic to jasmonate insensitive1) mutants, suggesting LBD20 may function in jasmonate (JA) signaling. Consistent with this, expression of the JA-regulated THIONIN2.1 (Thi2.1) and VEGETATIVE STORAGE PROTEIN2 (VSP2) genes were up-regulated in shoots of lbd20 following treatment of roots with F. oxysporum or methyl jasmonate. However, PLANT DEFENSIN1.2 expression was unaltered, indicating a repressor role for LBD20 in a branch of the JA-signaling pathway. Plants overexpressing LBD20 (LBD20-OX) had reduced Thi2.1 and VSP2 expression. There was a significant correlation between increased LBD20 expression in the LBD20-OX lines with both Thi2.1 and VSP2 repression, and reduced survival following F. oxysporum infection. Chlorosis resulting from application of F. oxysporum culture filtrate was also reduced in lbd20 leaves relative to the wild type. Taken together, LBD20 is a F. oxysporum susceptibility gene that appears to regulate components of JA signaling downstream of COI1 and MYC2 that are required for full elicitation of F. oxysporum- and JA-dependent responses. To our knowledge, this is the first demonstration of a role for a LBD gene family member in either biotic stress or JA signaling.

Purification and Characterization of Recombinant Lipid Storage Protein-2 from Drosophila melanogaster

Lipid storage protein 2 (Lsd 2) is a conserved insect protein that belongs to the small PAT family of proteins. PAT proteins are found associated to the lipid droplets of adipocytes and play significant roles in the regulation of triacylglycerides metabolism. Here we describe the expression and purification of Lsd2, its reconstitution in lipoprotein particles, the location of putative lipid binding sites and its secondary structure. This study provides the starting point for future studies on the mechanism of function of Lsd2. The similarities and differences between Lsd1 and Lsd2, the only PAT proteins found in insects, are discussed.

Inhibition of HeLa Cell Apoptosis by Storage‐Protein 2

Apoptosis is a barrier to maintaining high viable cell densities in animal cell culture. Silkworm hemolymph and its 30K protein have been reported to exhibit anti-apoptotic activity in various mammalian and insect cell systems. The 30K protein is thermally unstable at temperatures higher than 60 degrees C; however, the silkworm hemolymph heat-treated at 70-80 degrees C still exhibited anti-apoptotic activity. This indicates that silkworm hemolymph contains another anti-apoptotic compound other than 30K protein. In this article, the anti-apoptotic molecule other than 30K protein was found from the silkworm hemolymph and identified. This molecule was storage-protein 2 (SP2), which has no homology with any known anti-apoptotic protein. This molecule was heat-stable up to 80 degrees C, while 30K protein lost its activity at temperatures higher than 60 degrees C. When apoptosis was induced by staurosporine in HeLa cells, SP2 protein suppressed nuclear fragmentation and apoptotic body formation. Moreover, the generation of reactive oxygen species after apoptosis induction was inhibited, which means the inhibition occurred in an early step of the apoptotic process. Inhibition of apoptosis by the SP2 protein would lead to the minimization of cell death during commercial mammalian cell culture.

Molecular cloning, expression, and characterization of a cDNA encoding the arylphorin-like hexameric storage protein from the mulberry longicorn beetle, Apriona germari.

We describe here the cloning, expression, and characterization of a cDNA encoding the arylphorin-like hexameric storage protein from the mulberry longicorn beetle, Apriona germari (Coleoptera, Cerambycidae). The complete cDNA sequence of A. germari hexamerin (AgeHex) is comprised of 2,160 bp with 720 amino acid residues. The deduced protein sequence of AgeHex is most similar to Tenebrio molitor hexamerin2 (65.3%). Phylogenetic analysis further confirmed the AgeHex is more closely related to T. molitor hexmerin2 and T. molitor early-staged encapsulation inducing protein than to the other insect storage proteins. Southern blot analysis suggested the presence of A. germari hexamerin gene as a single copy and Northern blot analysis confirmed fat body-specific expression at the transcriptional level. The cDNA encoding AgeHex was expressed as a 80-kDa protein in the baculovirus-infected insect cells. Western blot analysis using the polyclonal antiserum against recombinant AgeHex indicated that the AgeHex corresponds to storage protein 2 (SP2) present in the A. germari larval hemolymph.

CDNA sequence and gene expression of storage protein-2 — a juvenile hormone-suppressible hexamerin from the fall webworm, Hyphantria cunea Drury

We isolated and sequenced a cDNA clone corresponding to storage protein-2 (SP-2) from the fall webworm, Hyphantria cunea. The cDNA for SP-2 (2572 bp) codes for a 747-residue protein with a predicted molecular mass of 88.5 kDa. The calculated isoelectric point is 7.6. Multiple alignment analysis of amino acid sequence revealed that SP-2 is most similar to BJHSP2 (74.3% identity). According to both the phylogenetic analyses and criteria for amino acid composition, SP-2 belongs to the subfamily of moderately methionine-rich storage proteins (3.2% methionine, 11.8% aromatic amino acid). Topical application of the JH analog, methoprene, after head ligation of larvae, suppressed transcription of the SP-2 gene, indicating hormonal effects at the transcriptional level. The SP-2 transcript was detected by Northern blot analysis in Malpighian tubules, in addition to the fat body where it was most abundant. The local expression of SP-2 in Malpighian tubules suggests that it may have some function in that organ.

Storage proteins of the fall webworm, Hyphantria cunea drury

AbstractTwo storage proteins, storage protein‐1 (SP1) and storage protein‐2 (SP2), were found in hemolymph and fat body during the development of Hyphantria cunea, the fall webworm. Both storage proteins show similiar quantitative changes during development in males and females; however, SP1 is more abundant. The hemolymph of last instar larvae contains high concentrations of the storage proteins. However, following pupation, the storage proteins accumulate in fat bodies. SP1 peaks in the hemolymph of males and females late in last instar larvae (8‐day‐old 7th instar larvae).SP1 has a native molecular weight of 460,000 and consists of six identical subunits (Mr = 76,700), while SP2 has a molecular weight of 450,000 and is composed of two different subunits (Mr = 74,100 and 72,400). Both SP1 and SP2 are hexamers and are phosphorylated glycolipoproteins. The pl values of SP1 and SP2 were determined to be 5.70 and 5.50, respectively.Antibodies raised against SP1 react positively with vitellogenin and ovary extract, as well as with proteins in the hemolymph from last instar larvae and proteins in pupal fat bodies. Storage protein synthesis starts in fat bodies of a 4‐day‐old 7th instar larvae and in female peaks at 6–8 days of the 7th instar.

Linkage studies on the Storage protein-2 gene (Pst) in Bombyx mori

Linkage studies on the Storage protein-2 gene (Pst) in Bombyx mori