Cloning and characterization of an invertebrate type lysozyme from Venerupis philippinarum

Abstract

Lysozymes are key proteins to invertebrates in the innate immune responses against bacterial infections and providing nutrition as digestion enzymes. In the present study, an invertebrate type lysozyme (denoted as VpLYZ) was identified from Venerupis philippinarum haemocytes by cDNA library and RACE approaches. The full-length cDNA of VpLYZ consisted of 805 nucleotides with a canonical polyadenylation signal sequence AATAAA and a polyA tail, and an open-reading frame of 558 bp encoding a polypeptide of 185 amino acids with a calculated molecular mass of 20.87 kD and theoretical pI of 8.44. The high similarity of VpLYZ with other i-type lysozymes from mollusk indicated that VpLYZ should be a new member of i-type lysozyme family. Similar to most i-type lysozymes, VpLYZ possessed all conserved features critical for the fundamental structure and function of i-type lysozymes, such as three catalytic residues (Glu19, Asn72 and Ser75) and i-type specific motif CL(E/L/R/H)C(I/M)C. By semi-quantitative RT-PCR analysis, mRNA transcript of VpLYZ was found to be most abundantly expressed in the tissues of gills, hepatopancreas and haemocytes, weakly expressed in the tissues of muscle, foot and mantle. After clams were challenged by Vibrio anguillarum, the mRNA level of VpLYZ in overall haemocyte population was recorded by quantitative real-time RT-PCR. VpLYZ mRNA was down-regulated sharply from 6 h to 12 h post-infection. Then, the expression level increased to the peak at 72 h and recovered to the original level at 96 h. All these results indicated that VpLYZ was involved in the immune response against microbe infection and contributed to the clearance of bacterial pathogens.

Introduction

Lysozyme (muramidase, EC3.2.1.17) is a ubiquitous enzyme existing in numerous phylogenetically diverse organisms such as bacteria, bacteriophages, fungi, plants and animals (Jollès and Jollès, 1984). Specifically, the enzyme catalyzes the cleavage of the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in bacterial peptidoglycan, and causes cell lysis (Prager & Jollès, 1996, Qasba & Kumar, 1997). It has been widely accepted that lysozyme functioned as important digestive enzymes in some animals, especially for filter-feeding organisms (Nilsen et al., 1999, Xue et al., 2007). In addition to the digestive capability, accumulating evidences have also demonstrated that lysozyme could exert its function in innate immunity as antibacterial or immune-modulating agents (Grunclová et al., 2003, Hultmark, 1996, Kollien et al., 2003, Regel et al., 1998, Simser et al., 2004, Sun et al., 1991).

Based on the differences in structural, catalytic and immunological characters, lysozymes found so far are generally classified into six distinct types: chicken-type (c-type) lysozyme, goose-type (g-type) lysozyme, invertebrate-type (i-type) lysozyme, phage lysozyme, bacterial lysozyme and plant lysozyme (Bachali et al., 2002, Bachali et al., 2004, Fastrez, 1996 Beintema & Terwisscha van Scheltinga, 1996, Hikima et al., 2003, Hultmark, 1996, Ito et al., 1999, Jollès & Jollès, 1984, Jollès et al., 1996, Liu et al., 2006, Nilsen & Myrnes, 2001, Olsen et al., 2003, Prager, 1996, Qasba & Kumar, 1997). Unlike g-type and c-type lysozyme, i-type lysozyme is a novel family found solely in invertebrate. The first i-type lysozyme was purified from the starfish Asterias rubens by Jollès and Jollès (1975). Currently, about 20 i-type lysozymes have been identified from nematoda, mollusca, arthropoda and echinodermata, respectively.

As filter-feeding organisms, mollusks expose to various potential pathogens in the aquatic environment. For defensive and nutritional purposes, mollusk lysozymes are therefore expected to possess bactericidal effect in addition to digestive capability (Nilsen et al., 1999, Nilsen et al., 2003). In the present study, the full-length cDNA of an i-type lysozyme (denoted as VpLYZ) was identified from V. philippinarum, and the tissue distribution and expression profile of VpLYZ after being infected by Vibrio pathogen were also investigated, hopefully providing new insight into disease control of mollusk aquaculture.