Characterization and functional analysis of the serpin-10 gene from oak silkworm, Antheraea pernyi (Lepidoptera: Saturniidae)

Saima KAUSAR,Guo-Qing WEI,Bao-Jian ZHU,Muhammad Nadeem ABBAS,Chao-Liang LIU,Cen QIAN,Yu SUN,Ya LIU,Lei WANG

doi:10.14411/eje.2017.055

Abstract

Serpin is a broadly distributed superfamily of proteins that have a crucial role in regulating various immune reactions. Herein we identified a serpin-10 gene from Antheraea pernyi that encodes a 1557 amino acid residue protein with a predicted molecular weight of 58.76 kDa. Recombinant Apserpin-10 protein was expressed in a prokaryotic expression system (Escherichia coli) and the purified protein was used to prepare rabbit anti-Apserpin-10 polyclonal antibodies. Quantitative real-time polymerase chain reaction and western blot analysis indicate that Apserpin-10 was transcribed in all the tissues examined, including haemolymph, malpighian tubules, fat body, silk gland, integument and mid gut; the greatest expression level of Apserpin-10 was recorded in the fat body and haemocytes. The comparison of different developmental stages showed that Apserpin-10 transcript level was highest in 5th instar larvae, while the lowest expression was recorded at the egg stage. We also investigated the expression patterns of Apserpin-10 in fat body and haemocyte samples, following administration of heat-inactivated gram-positive bacteria (Micrococcus luteus), gram negative bacteria (Escherichia coli), a fungus (Beauveria bassiana) and virus (nuclear polyhedrosis virus, NPV). A substantial up-regulation of Apserpin-10 expression was recorded following pathogen challenge in both the tissues tested. Further the knock down of Apserpin-10 led to down regulation of antimicrobial peptide genes. Altogether, our results indicate that Apserpin-10 is involved in the innate immunity of A. pernyi.

Highlights

Serpin is a widely distributed superfamily of proteins
The pattern in the spatial pattern of Apserpin-10 in tissues examined at different developmental stages could provide valuable clues about its biological role in A. pernyi
Apserpin-10 was expressed in all the tissues tested, the expression levels varied between tissues

Summary

Introduction

A variety of serpin and serpin-like genes are documented in both prokaryotes and eukaryotes, and according to estimates this family contains more than 1,500 members (Irving et al, 2000; Rawling et al, 2004). They mainly regulate serine and cysteine proteases in cells. They constitute a single chain, which forms a specific structure with a reactive center loop (RCL) close to the C-terminus, which provides a surface to bind with a target protease (Rawlings et al, 2004). The proteolytic inhibition starts by the formation of a complex between serpin and its target protein (Potempa et al, 1994). Protease inhibitor 10 plays a key role in the regulation of protease activity during haematopoiesis and apoptosis induced by TNF (Schleef & Chuang, 2000)

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