Protein purification, cDNA cloning and characterization of a protease inhibitor from the Indian tasar silkworm, Antheraea mylitta

Shrivastava, Binita; Ghosh, Ananta K.

doi:10.1016/s0965-1748(03)00117-6

Cited by 26 publications

(27 citation statements)

References 41 publications

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“…In total, 10 SPI domains were identified: TIL [46], Kunitz_BPTI [46], Kazal [46], Antistasin [46], Pacifastin [47], whey acidic protein (WAP) [48], [49], amfpi [50], Bowman-Birk [46], [51], serpin [45] and alpha-macroglobulin [52] (Table 1 and Table S1). These 10 SPI domains can be divided into three types: serpin, alpha-macroglobulin and canoniacal SPIs,.…”

Section: Resultsmentioning

confidence: 99%

Genome-Wide Identification and Immune Response Analysis of Serine Protease Inhibitor Genes in the Silkworm, Bombyx mori

et al. 2012

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In most insect species, a variety of serine protease inhibitors (SPIs) have been found in multiple tissues, including integument, gonad, salivary gland, and hemolymph, and are required for preventing unwanted proteolysis. These SPIs belong to different families and have distinct inhibitory mechanisms. Herein, we predicted and characterized potential SPI genes based on the genome sequences of silkworm, Bombyx mori. As a result, a total of eighty SPI genes were identified in B. mori. These SPI genes contain 10 kinds of SPI domains, including serpin, Kunitz_BPTI, Kazal, TIL, amfpi, Bowman-Birk, Antistasin, WAP, Pacifastin, and alpha-macroglobulin. Sixty-three SPIs contain single SPI domain while the others have at least two inhibitor units. Some SPIs also contain non-inhibitor domains for protein-protein interactions, including EGF, ADAM_spacer, spondin_N, reeler, TSP_1 and other modules. Microarray analysis showed that fourteen SPI genes from lineage-specific TIL family and Group F of serpin family had enriched expression in the silk gland. The roles of SPIs in resisting pathogens were investigated in silkworms when they were infected by four pathogens. Microarray and qRT-PCR experiments revealed obvious up-regulation of 8, 4, 3 and 3 SPI genes after infection with Escherichia coli, Bacillus bombysepticus, Beauveria bassiana or B. mori nuclear polyhedrosis virus (BmNPV), respectively. On the contrary, 4, 11, 7 and 9 SPI genes were down-regulated after infection with E. coli, B. bombysepticus, B. bassiana or BmNPV, respectively. These results suggested that these SPI genes may be involved in resistance to pathogenic microorganisms. These findings may provide valuable information for further clarifying the roles of SPIs in the development, immune defence, and efficient synthesis of silk gland protein.

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Section: Resultsmentioning

confidence: 99%

Genome-Wide Identification and Immune Response Analysis of Serine Protease Inhibitor Genes in the Silkworm, Bombyx mori

et al. 2012

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“…S2). Regarding their physiological functions, it has been reported that ISPI-1 and AmFPI-1 are involved in an insect defense mechanism as an inhibitor to regulate the proteolytic activity of a variety of proteases, including fungal protease (13,19), and Manduca CP8 with no inhibitor activity contributed to the activation of prophenoloxidase cascade in the hemolymph (12). However, it remains to be determined whether those three proteins also exhibit the same microbial binding and/or phagocytic activity as GmCP8.…”

Section: Discussionmentioning

confidence: 99%

“…Therefore, it was concluded that GmCP8 was a small compact protein that was not conjugated with any other molecule and that one of two GmCP8 isoforms harbored an amidated C-terminal serine as Gly 87 participated in the formation of a C-terminal amide. Additionally, it was determined that the amino acid sequence of GmCP8 exhibits a high degree of similarity with the inducible serine protease inhibitor-1 (ISPI-1; Swiss-Prot accession number P81905), the fungal protease inhibitor-1 (AmFPI-1; Swiss-Prot accession number B0JFB8), and CP8 (GenBank accession number EF467286) from three different lepidopteran insects, G. mellonella (13), Antheraea mylitta (19), and M. sexta (12), respectively (supplemental Fig. S2).…”

Section: Purification Of Gmcp8 and Its Aminomentioning

confidence: 99%

An Insect Multiligand Recognition Protein Functions as an Opsonin for the Phagocytosis of Microorganisms

Kim

Shin

Noh

et al. 2010

Journal of Biological Chemistry

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We characterize a novel pathogen recognition protein obtained from the lepidopteran Galleria mellonella. This protein recognizes Escherichia coli, Micrococcus luteus, and Candida albicans via specific binding to lipopolysaccharides, lipoteichoic acid, and ␤-1,3-glucan, respectively. As a multiligand receptor capable of coping with a broad variety of invading pathogens, it is constitutively produced in the fat body, midgut, and integument but not in the hemocytes and is secreted into the hemolymph. The protein was confirmed to be relevant to cellular immune response and to further function as an opsonin that promotes the uptake of invading microorganisms into hemocytes. Our data reveal that the mechanism by which a multiligand receptor recognizes microorganisms contributes substantially to their phagocytosis by hemocytes. A better understanding of an opsonin with the required repertoire for detecting diverse invaders might provide us with critical insights into the mechanisms underlying insect phagocytosis.

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“…[13] Therefore, a number of PPIs have been isolated and characterized from various plant sources. [14151617181920]…”

Section: Introductionmentioning

confidence: 99%

Structural and biophysical characterization of cajanus cajan protease inhibitor

et al. 2017

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Context:A large number of studies have proven that Protease inhibitors (PIs), specifically serine protease inhibitors, show immense divergence in regulation of proteolysis by targeting their specific proteases and hence, they play a key role in healthcare.Objective:We aimed to access in-vitro anticancer potential of PI from Cajanus cajan (CCPI). Also, crystallization of CCPI was targetted alongwith structure determination and its structure-function relationship.Materials and Methods:CCPI was purified from Cajanus cajan seeds by chromatographic techniques. The purity and molecular mass was determined by SDS-PAGE. Anticancer potential of CCPI was determined by MTT assay in normal HEK and cancerous A549 cells. The crystallization screening of CCPI was performed by commercially available screens. CCPI sequence was subject to BLASTp with homologous PIs. Progressive multiple alignment was performed using clustalw2 and was modelled using ab initio protocol of I-TASSER.Results:The results showed ~14kDa CCPI was purified in homogeneity. Also, CCPI showed low cytotoxic effects of in HEK i.e., 27% as compared with 51% cytotoxicity in A549 cells. CCPI crystallized at 16°C using 15% PEG 6000 in 0.1M potassium phosphate buffer (pH 6.0) in 2-3weeks as rod or needles visualized as clusters under the microscope. The molecular modelling revealed that it contains 3 beta sheets, 3 beta hairpins, 2 β-bulges, 6 strands, 3 helices, 1helix-helix interaction, 41 β-turns and 27 γ-turns.Discussion and Conclusion:The results indicate that CCPI may help to treat cancer in vivo aswell. Also, this is the first report on preliminary crystallization and structural studies of CCPI.

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Protein purification, cDNA cloning and characterization of a protease inhibitor from the Indian tasar silkworm, Antheraea mylitta

Cited by 26 publications

References 41 publications

Genome-Wide Identification and Immune Response Analysis of Serine Protease Inhibitor Genes in the Silkworm, Bombyx mori

Genome-Wide Identification and Immune Response Analysis of Serine Protease Inhibitor Genes in the Silkworm, Bombyx mori

An Insect Multiligand Recognition Protein Functions as an Opsonin for the Phagocytosis of Microorganisms

Structural and biophysical characterization of cajanus cajan protease inhibitor

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