Complete nucleotide sequences of major plasma protein genes of Bombyx mori

doi:10.1016/0167-4781(91)90048-Q

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression

Volume 1090, Issue 1, 27 August 1991, Pages 129-132

https://doi.org/10.1016/0167-4781(91)90048-Q Get rights and content

In the silkworm, Bombyx mori, a family of hormone-sensitive genes encodes major plasma proteins, termed ‘30K proteins’. We reported the organization of the 30K protein gene family together with the structure and expression of a member of the gene family (Mori, S. et al. (1991) J. Mol. Biol. 218, 7–12). In this communication, we present the complete structures of two other 30K protein genes in the family. Sequence analyses demonstrated that all three genes are similar to each other; a short first exon and protein-coding second exon are interspersed by a single intron. Structures homologous to the putative regulatory elements for the 30K protein gene expression are also found around each gene.

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Cited by (27)

Enhancement of human erythropoietin production in Chinese hamster ovary cells through supplementation of 30Kc19-30Kc6 fusion protein
2015, Process Biochemistry
Citation Excerpt :
Silkworm hemolymph consists of a group of structurally related proteins with a molecular weight of approximately 30 kDa, including 30Kc6, 30Kc12, 30Kc19, 30Kc21, and 30Kc23. During the fifth instar larval to early pupal stages, these “30 K proteins” are synthesized in fat body cells and accumulated in the hemolymph [24,25]. During metamorphosis from larva to pupa, these proteins are transferred from the hemolymph to fat body cells and are deposited there until use [26,27].
The importance of anti-apoptosis in mammalian cell culture has been widely recognized. We reported previously that expression of Bombyx mori 30 K genes in Chinese hamster ovary (CHO) cells increases recombinant protein production by inhibiting apoptosis and enhancing specific productivity. However, previous studies have shown expression of the anti-apoptotic 30Kc6 protein as inclusion bodies in Escherichia coli. 30Kc19 protein, another silkworm hemolymph protein, has cell-penetrating and recombinant protein productivity-improving properties, and we found that it improves soluble expression of its partner. In this study, we fused 30Kc6 with 30Kc19 as an expression partner to express a soluble fused protein and to deliver the protein to cells. Supplementing the recombinant 30Kc19-30Kc6 fusion protein in cell culture medium increased viability, effectively penetrated the cells, and inhibited CHO cell apoptosis by 68%. Moreover, the mitochondrial membrane potential and ATP generation also increased by 50% and 33%, respectively. Erythropoietin (EPO) productivity increased by > 30% because of the anti-apoptotic effect and increased specific productivity. These results demonstrate the potential use of this fusion protein as a supplement in mammalian cell culture during production of biopharmaceutical proteins.
Identification and characterization of a novel cell-penetrating peptide of 30Kc19 protein derived from Bombyx mori
2014, Process Biochemistry
Citation Excerpt :
These proteins have molecular weights of around 30 kDa, and 30Kc19 protein is the most abundant among 30K proteins (30Kc6, 30Kc12, 30Kc19, 30Kc21 and 30Kc23) in the hemolymph [37]. During the 5th instar larva to early pupa stage, these 30K proteins are synthesized in fat body cells and accumulate in the hemolymph [38,39]. They are then transferred from the hemolymph to fat body cells during metamorphosis from larva to pupa, and are deposited there until later use [40,41].
Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) have attracted increasing attention due to their high potential to deliver various, otherwise impermeable, bioactive agents, such as drugs and proteins across cell membranes. A number of CPPs have been discovered since then. Recently, 30Kc19 protein has attracted attention because it was the first cell-penetrating protein that has been found in insect hemolymph. Here, we report a cell-penetrating peptide derived from 30Kc19 protein, VVNKLIRNNKMNC, which efficiently penetrates cells when supplemented to medium for mammalian cell culture. Moreover, like other CPPs, this “Pep-c19” also efficiently delivered cell-impermeable cargo proteins, such as green fluorescent protein (GFP) into cells. In addition to the in vitro system, Pep-c19 exhibited the cell-penetrating property in vivo. When Pep-c19 was intraperitoneally injected into mice, Pep-c19 successfully delivered cargo proteins into various organ tissues with higher efficiency than the 30Kc19 protein itself, and without toxicity. Our data demonstrates that Pep-c19 has a great potential as a cell-penetrating peptide that can be used as a therapeutic tool to efficiently deliver different cell-impermeable cargo molecules into the tissues of various organs.
Identification of a functional element in the promoter of the silkworm (bombyx mori) fat body-specific gene Bmlp3
2014, Gene
Citation Excerpt :
Interestingly, changes in the level of 30 K protein mRNA in the fat body reflect changes in the hemolymph concentration of 30 K proteins, indicating that transcription is the major mode of 30 K protein gene regulation. B. mori hemolymph proteins have proven to be good models for studying the developmental regulation of gene expression (Izumi et al., 1981; Mori et al., 1991a, 1991b). The availability of the complete genome sequence of B. mori provides a unique opportunity to identify genes encoding 30 K proteins and determining how they are developmentally regulated.
30 K proteins are a group of structurally related proteins that play important roles in the life cycle of the silkworm Bombyx mori and are largely synthesized and regulated in a time-dependent manner in the fat body. Little is known about the upstream regulatory elements associated with the genes encoding these proteins. In the present study, the promoter of Bmlp3, a fat body-specific gene encoding a 30 K protein family member, was characterized by joining sequences containing the Bmlp3 promoter with various amounts of 5′ upstream sequences to a luciferase reporter gene. The results indicated that the sequences from − 150 to − 250 bp and − 597 to − 675 bp upstream of the Bmlp3 transcription start site were necessary for high levels of luciferase activity. Further analysis showed that a 21-bp sequence located between − 230 and − 250 was specifically recognized by nuclear factors from silkworm fat bodies and BmE cells, and could enhance luciferase reporter-gene expression 2.8-fold in BmE cells. This study provides new insights into the Bmlp3 promoter and contributes to the further clarification of the function and developmental regulation of Bmlp3.
A protein delivery system using 30Kc19 cell-penetrating protein originating from silkworm
2012, Biomaterials
Citation Excerpt :
30Kc19 is a member of the 30K protein family (30Kc6, 30Kc12, 30Kc19, 30Kc21 and 30Kc23), which is a family of similar structured proteins found in silkworm hemolymph that have molecular weights around 30 kDa [19,20]. These proteins are synthesized in fat body cells and accumulate in the hemolymph during the final instar (5th instar) larvae and the early pupal stage [21,22]. During metamorphosis from larvae to pupae, the 30K proteins are transferred from the hemolymph to fat body cells where they are deposited [23,24].
Cell-penetrating protein and its protein transduction domain have been used to deliver drugs and proteins into the cells via receptor-independent endocytosis. A number of cell-penetrating proteins including TAT derived from HIV-1 virus, VP22 from herpes simplex virus and Antennapedia from drosophila have been discovered. Here, we report a cell-penetrating protein, 30Kc19, originating from the hemolymph of silkworm, Bombyx mori. The 30Kc19 is the first cell-penetrating protein that has been found in insect hemolymph. When the 30Kc19 protein produced from recombinant Escherichia coli was supplemented into the medium for mammalian cell culture, 30Kc19 efficiently penetrated into various types of cells and localized at subcellular compartments including mitochondria and cytoplasm. 30Kc19 also delivered cargo proteins such as green fluorescence protein into the cells even though cargo proteins are not able to penetrate into cells by themselves. In addition to the in vitro system, 30Kc19 exhibited the protein transduction property in vivo. When 30Kc19 was intraperitoneally injected into mice, 30Kc19 delivered cargo proteins into various organ tissues of model animals without producing toxicity. Therefore, 30Kc19 has a great potential as a cell-penetrating protein that can be used as a medicinal tool to deliver cargo molecules including proteins into the target organ tissues in the body.
The homeodomain protein PBX participates in JH-related suppressive regulation on the expression of major plasma protein genes in the silkworm, Bombyx mori
2005, Insect Biochemistry and Molecular Biology
In the silkworm, Bombyx mori, major plasma proteins referred to as 30K proteins are the most abundant proteins in the hemolymph of final (fifth) instar larvae. Surgical extirpation of corpora allata, the source of a juvenile hormone (JH), causes rapid accumulation of 30K proteins in the hemolymph of fourth instar larvae. The 30K protein 6G1 (30K6G1) gene was repressed in primary cultured fat body cells treated with a JH analog (JHA), methoprene. To identify the JH response element present in the promoter region of the 30K6G1 gene, we performed transfection analyses of the 5′-deletion mutants of the 30K6G1 gene using primary cultured fat body cells, gel retardation assays and in vivo footprinting analysis. The results from those analyses revealed that a JH response element exists in the sequence between positions −147 and −140. When the promoter construct mutated at positions −143, −142, and −141 was transfected to fat body primary cultured cells, the suppression effect on the reporter gene expression caused by JHA was reduced. Gel retardation assay using specific antibody revealed that a PBX protein binds to the JH response element. Northern blot analysis revealed that the gene expression of Bombyx PBX is enhanced in the fat body cells by JHA treatment. These results indicate that PBX proteins are involved in the JH signaling pathway and play an important role in suppressing 30K protein gene expression in the fat body of B. mori.
Insect cytokine growth-blocking peptide triggers a termination system of cellular immunity by inducing its binding protein
2003, Journal of Biological Chemistry
Citation Excerpt :
By a combination of primary PCR using these primers and following 5′- and 3′-RACE PCR, GBP-binding protein cDNA was isolated and sequenced (Fig. 3A). The deduced 430-amino acid sequence with a molecular mass of 49.5 kDa is a novel protein containing a C-terminal region displaying limited homology to several insect lipoproteins such as 30k-lipoprotein (28) and microvitellogenin (29) (Fig. 3B). Further, this protein appears not to have a signal peptide and transmembrane domain.
Growth-blocking peptide (GBP) is a 25-amino acid cytokine found in lepidopteran insects that possesses diverse biological activities such as stimulation of immune cells (plasmatocytes), cell proliferation, and larval growth regulation. We found another novel function of GBP that induces a hemolysis of another class of blood cells (oenocytoids). In the lysate of oenocytoids we identified a GBP-binding protein that shows a specific affinity for GBP. The characterization of purified GBP-binding protein and its cDNA demonstrated it as a 49.5-kDa novel protein with a C-terminal region displaying limited homology to several insect lipoproteins. Results of Northern and Western blotting indicated that the GBP-binding protein should be synthesized only in blood cells. Immunoelectron microscopic analyses confirmed that indirect immunoreactive signals were mostly localized in oenocytoids. Kinetic and biological analyses of interaction between GBP and the binding protein showed their strong binding was followed by clearance of GBP from hemolymph, thus indicating that this protein might function as an inhibitory factor against GBP. Based on these results, we propose that insect cytokine GBP shows multifunctions even in cellular immunity: it serves to stimulate immune cells and afterward silences its own action by inducing the binding protein through specific hemolysis.

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: The sequence data in this paper have been submitted to the EMBL/Genbank Data Libraries under the accession numbers X54734 (21G1) and X54736 (19G1).

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Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression

Complete nucleotide sequences of major plasma protein genes of Bombyx mori

Biochim. Biophys. Acta

Dev. Biol.

Enhancement of human erythropoietin production in Chinese hamster ovary cells through supplementation of 30Kc19-30Kc6 fusion protein

Identification and characterization of a novel cell-penetrating peptide of 30Kc19 protein derived from Bombyx mori

Identification of a functional element in the promoter of the silkworm (bombyx mori) fat body-specific gene Bmlp3

A protein delivery system using 30Kc19 cell-penetrating protein originating from silkworm

The homeodomain protein PBX participates in JH-related suppressive regulation on the expression of major plasma protein genes in the silkworm, Bombyx mori

Insect cytokine growth-blocking peptide triggers a termination system of cellular immunity by inducing its binding protein