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Mol. Cells 2000; 10(2): 186-192

Published online April 30, 2000

© The Korean Society for Molecular and Cellular Biology

Immunological Detection of Serpin in the Fall Webworm, Hyphantria cunea and Its Inhibitory Activity on the Prophenoloxidase System

Doo-Sang Park, Sang Woon Shin, Soon-Duck Hong, and Ho-Yong Park

Abstract

We previously identified a serine type protease inhibitor (serpin) cDNA, using peR-based differential display, in the fall webworm which was up-regulated following a bacterial challenge (Shin et al., 1998). The serpin cDNA was inserted into an expression vector and the serpin protein was expressed in Escherichia coli. In order to investigate the action of serpin in vivo, we examined the concentration of serpin protein in the larvae of Hyphantria cunea by Western blot analysis using a polyclonal antibody raised in a rabbit injected with recombinant serpin. H. cunea serpin was found mainly in the plasma with a molecular mass of 56.6 kDa on SDS-PAGE followed by Western blot analysis. The concentration of serpin in the plasma was slightly increased following bacterial challenge. A new 50.5 kDa (approx.) band was detected post E. coli and distilled water injection. Both E. coli and distilled water injection induced increased phenoloxidase (PO) activity in the plasma, although E. coli injection produced a larger increase in activity. Hyphantria serpin probably participates in negative regulation of the prophenoloxidase (proPO) cascade. Recombinant serpin inhibits PO activity in the hemocyte lysate fraction activated by LPS. There is a similarity between the P2-P2' region (NKFG) of the serpin reactive site loop and the S2-S2' region (NRFG) of the insect proPO maturation site. This indicates a form of competitive inhibition of serpin against a protease involved in the activation of proPO. A tyrosine residue in the P11 region of serpin, which is conserved in the S11 regions of all known proPOs maturation sites, provides further support for this hypothesis.

Article

Research Article

Mol. Cells 2000; 10(2): 186-192

Published online April 30, 2000

Copyright © The Korean Society for Molecular and Cellular Biology.

Immunological Detection of Serpin in the Fall Webworm, Hyphantria cunea and Its Inhibitory Activity on the Prophenoloxidase System

Doo-Sang Park, Sang Woon Shin, Soon-Duck Hong, and Ho-Yong Park

Abstract

We previously identified a serine type protease inhibitor (serpin) cDNA, using peR-based differential display, in the fall webworm which was up-regulated following a bacterial challenge (Shin et al., 1998). The serpin cDNA was inserted into an expression vector and the serpin protein was expressed in Escherichia coli. In order to investigate the action of serpin in vivo, we examined the concentration of serpin protein in the larvae of Hyphantria cunea by Western blot analysis using a polyclonal antibody raised in a rabbit injected with recombinant serpin. H. cunea serpin was found mainly in the plasma with a molecular mass of 56.6 kDa on SDS-PAGE followed by Western blot analysis. The concentration of serpin in the plasma was slightly increased following bacterial challenge. A new 50.5 kDa (approx.) band was detected post E. coli and distilled water injection. Both E. coli and distilled water injection induced increased phenoloxidase (PO) activity in the plasma, although E. coli injection produced a larger increase in activity. Hyphantria serpin probably participates in negative regulation of the prophenoloxidase (proPO) cascade. Recombinant serpin inhibits PO activity in the hemocyte lysate fraction activated by LPS. There is a similarity between the P2-P2' region (NKFG) of the serpin reactive site loop and the S2-S2' region (NRFG) of the insect proPO maturation site. This indicates a form of competitive inhibition of serpin against a protease involved in the activation of proPO. A tyrosine residue in the P11 region of serpin, which is conserved in the S11 regions of all known proPOs maturation sites, provides further support for this hypothesis.

Mol. Cells
Nov 30, 2021 Vol.44 No.11, pp. 781~860
COVER PICTURE
3D quantitative images of the vesicular structure and the nucleolus using label free optical diffraction tomography (Kim et al., pp. 851-860).

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