432 ISJ 14: 432-442, 2017 ISSN 1824-307X REVIEW Characterization and roles of lysozyme in molluscs W Jielian1,2, H Baoqing1 , W Chungen1, Y Peipei1 1School of Life Science, Education Ministry key Laboratory of Poyang Lake Environment and Resource Utilization, Nanchang University, Nanchang 330031, China 2Jiangxi Science＆Technology Normal University, Nanchang 330013, China Accepted October 31, 2017 Abstract Lysozyme can hydrolyse the β-1.4-glycosidic linkage between the N-acetylmuramic (NAM) and N-acetylglucosamine (NAG). The three major categories of lysozymes in molluscs are Goose-type, Chicken-type and Invertebrate-type lysozymes. The function of lysozymes is served as an innate immune protection against exogenous microbial invasion. The tyoical c-, g- and i-type mollusc lysozymes are secreting type, have signal peptide and eight, six and fourteen cysteine residues, respectively. The c- and g-type lysozymes are highly expressed in hepatopancreas, hemocytes and gills, and weakly expressed in foot and goand tissues of muscle. The i-type lysozyme gene is high expression in different tissues. The three type lysozymes exhibit antibacterial and digestive activity, and i-type lyaozyme also has antifungi activity. Furthermore, this review includes current knowledge regarding to the genomic structure, tissue distribution of mollusc lysozyme, the antimicrobial function and mechanism. The evolution of three type lysozymes in molluscs is also discussed. These lysozymes research may help to understand the basic knowledge and to use it in the production of molluscs. Key Words: c-type; g-type; i-type; mollusc; antimicrobial Introduction Molluscs possess as much as approximately 200,000 species, which widely distribute in various ecosystem, including terrestrial, freshwater and marine environments (Ponder and Lindberg, 2008), and rely on innate immune systems to mediate cellular and humoral components for defense against pathogens (Loker et al., 2004). In recent years, mollusc aquaculture has been facing a set back due to challenges emanating from pathogenic infections. Haliotis discus hannai suffers from abnormal deaths, and results in the considerable reduction of abalone output throughout the world (Zhang et al., 2004; Sawabe et al., 2007). The effector of mollusc immune is crucial to better understand the immune defense mechanisms and provides the potentially feasible solutions for disease control. The innate immune system is of great importance to protect invertebrate against a wide ___________________________________________________________________________ Corresponding author: W en Chungen School of Life Science Education Ministry key Laboratory of Poyang Lake Environment and Resource Utilization Nanchang University, Nanchang 330031, China E-mail: cgwen@ncu.edu.cn range of microbial pathogens and encompasses a complex array of defense reactions, in which mainly focusing on immune recognition, signal transduction and effector synthesis involved in cellular and humoral immunity in the field of mollusc immunity. Lysozyme is identified a classic mollusc immune effector in innate immune (Wang et al., 2013), which is originally found to dissolves bacterial cell walls in human saliva and tears (Haug et al., 2004), and was subsequently described in other vertebrates and invertebrate (Zhao et al., 2007; Whang et al., 2011; He et al., 2012; Wang et al., 2012; Umaasuthan et al., 2013). The enzyme is a ubiquitous bacteriolytic enzyme, which is produced by diverse groups of organisms, ranging from bacteria and bacteriophages to fungi, plants and animals (Bathige et al., 2013), is characterized by their ability to bacterial peptidoglycan between two amino sugars, N-acetylmuramic acid and N-acetylglucosamine and cause bacterial cell lysis (Chipman and Sharon, 1969; Prager and Jollès, 1996), and has bactericidal and digestive ability (Dobson et al., 1984; Itoh and Takahashi, 2007). Besides antimicrobial activity, lysozymes have also proved to perform many other functions, such as growth 433 stimulation, digestion, antiviral, anti-inflammatory, and even association with tumors (Irwin, 2004; Wang and Zhang, 2010; Lee et al. 2015; Xin et al., 2015), which are regarded to play important roles in the innate immunity and physiological activities, is a first line defensive protein that acts as a barrier to resist bacterial pathogen invasion in innate immune systems of invertebrates, and is widespread in many tissues and secretions (Bachali et al., 2002; Liu et al., 2006). Extensive studies have been devoted to their structure, catalytic mechanism, relationship between structure and activity, phylogeny, immunology, and genetics (Jollès, 1996). The types of lysozymes are different in amino acid sequences, biochemical and tissue distribution. The present review attempts to mainly focus on classification, distribution and function of mollusc lysozyme. It will help to improve the current knowledge about lysozyme of molluscs. Classification and charecteristics of mollusc lysozyme Lysozyme (EC 3.2.1.17) catalyzes the hydrolysis of 1, 4-beta-linkages between N-acetyl-dglucosamine (NAG) and N-acetylmuramic acid (NAM) in peptidoglycan heteropolymers of prokaryotic cell walls, and leads to the breakdown of bacterial cells (Fleming, 1922; Jollès and Jollès, 1984a). The enzyme are generally classified into six types based on differences in structural, catalytic and immunological characteristics, including chicken-type (c-type), goose-type (g-type), plant, bacteria, T4 phage, and invertebrate-type (i-type) lysozymes (Inouye et al., 1970; Matthews et al., 1981; Joskova et al., 2009). These types of lysozymes have been described in organisms (Jollès and Jollès, 1984). Three types lysozymes, c, g and i-type, have been recorded in molluscs (Wang et al. 2013; Guo et al., 2014; Zhu et al., 2016). The distribution and properties of lysozyme in molluscs are shown in Table 1. Table 1 the tissue distribution and characteristics of three types mollusca lysozymes species type distribution Lysozyme gene Accession number(s) Number of amino acids Haliotis discus discus c pallium, muscle, gill, digestive gland HdLysC ADR70995 146 Haliotis discus discus c pallium, muscle, gill, digestive gland HdLysC ADR70996 146 Ruditapes philippinarum c pallium, gill, hepatopancreas VpCLYZ-1 AGO06638 156 Ruditapes philippinarum c pallium, gill, hepatopancreas VpCLYZ-2 AGO06639 153 Mytilus galloprovincialis g crystalline, digestive gland MgLYZ1 AFF18185 206 Mytilus galloprovincialis g crystalline, digestive gland MgLYZ2 AFF18186 206 Argopecten irradians g pallium, gill, hepatopancreas - AY788903 200 Physella acuta g hepatopancreas PALYsG ADV36303 198 Chlamys farreri g hepatopancreas, gill CFLysG ABB53641 200 Mizuhopecten yessoensis g gill, pallium, hemocytes MyLysoG AEY77130 201 Meretrix meretrix i hepatopancreas, gill Mmelys ADL27913 146 Cristaria plicata i pallium, gill, hemocytes CpLYZ2 AFN66526 161 Cristaria plicata i pallium, gill, hemocytes CpLYZ1 AFN66527 160 Crassostrea gigas i digestive gland, basophil CGL BAF48044 142 Crassostrea gigas i gill, hemocytes CgLys BAD19059 137 Haliotis discus discus i pallium, muscle, gill, digestive gland AbLysG AGQ50336 131 Crassostrea virginica i digestive gland, hemocytes, cv-lysozym e 3 BAE93114 135 Ruditapes philippinarum i pallium, gill, hepatopancreas Tj-lysozym e BAC15553 136 Ruditapes philippinarum i mantles, gill, hepatopancreas RpiLYZ-2 AMS37097 156 434 The c-type lysozyme is originally isolated from chicken-egg (Itoh et al., 2007b), and subsequently is reported in other vertebrates and invertebrates, including amphibians, reptiles, mammalia, insect, crustacean and mollusc ( Jollès et al., 1996; Ito et al., 1999; Miyauchi et al., 2000; Olsen et al., 2003; Liu et al., 2006). The c-type lysozymes have two 2 catalytic residues (Glu53 and Asp70) and 8 cysteine residues that can form 4 disulfide bonds to stabilize the protein structure (Hikima et al., 2001; Jimènez-Cantizano et al., 2008; Ye et al., 2008). Several c-type lysozymes have recently been determined in Mytilus galloprovincialis (Wang et al., 2013), abalone Haliotis discus hannai (Umasuthan et al., 2013), and manila clam Venerupis philippinarum (Yang et al., 2017). Comparison with c-type lysozyme of vertebrates, that of mollusc counterparts have not been well characterized. The c-type lysozyme of H. discus hannai is firstly described, the full-length cDNA of HdLysC is 586 bp, and contains an open reading frame of 441 bp encoding a 147-amino acid protein with a calculated molecular mass of 15.64 kDa, an isoelectric point being 4.87, and a polyadenylation signal (AATAA). The genomic length of HdLysC is 2865 bp, and has four exons interrupted by three introns, 2 catalytic residues (Glu53 and Asp70), as well as the 8 cysteine residues involved in disulfide bond formation (Ding et al., 2011). The homologous structure of c-type lysozymes also exists in the genome of V. philippinarum and M. galloprovincialis (Wang et al., 2013; Yang et al., 2017). The genome of c-type lysozyme possess 4 exons interspaced by relatively large introns in vertebrate (Hikima et al., 2000), which do 3 exons separated by relatively smaller introns in invertebrate (Liu et al., 2006), and even is lost in Drosophila (Kylsten et al., 1992). The typical genomic structures of c-type lysozymes, number and size of both exons and introns, which exist in chicken, amphioxus, mosquito and silk moth, is also found in abalone H.discus hannai, and seem difference due to the changes of the introns length (Ding et al., 2011). The results indicate that the c-type lysozyme gene must have undergone unknown evolutionary events, e.g., a recombination, insertion or deletion in different lineages during evolution (Larsen et al., 2009). The lysozymes could usually be divided into the calcium binding and the noncalcium binding lysozymes according to the presence/absence of conversed calcium binding residue Asp (Nitta et al., 1987), which of birds and mammals belong to calcium binding lysozyme (Lemos et al., 1993), which of fish has not yet found calcium bindin lysozyme (Saurabh et al., 2008). Due to lack of calcium binding Asp residue, manila clam V. philippinarum is also categorized into the non-calcium binding lysozymes family (Yang et al., 2017). The g-type lysozyme is initially identified from egg of the whites Embden goose (Canfield, 1967), many of which is recently described from birds and fishes (Nakano and Graf; 1991; Thammasirirak, 2001; Larsen et al., 2009). The enzyme of molluscs is originally detected in Argopecten irradians (Zou et al. 2005; Zhao et al., 2007), and is subsequently reported in other molluscs (He et al. 2012; Wang et al., 2012; Zhang et al., 2012; Guo et al., 2014). The g-type lysozymes in birds and mammals are secreting type, and have four conserved cysteine in signal peptide that can make secreted proteins to form a more stable three-dimensional structure (Jollès and Jollès, 1975). All of known g-type lysozymes from Argopecten irradians, Chlamy farreri, Mytilus edulis, Physa acuta and H. discus hannai contain signal peptides, have similar three active center with (Glu82, Asp97, Asp108), and share one conserved cysteine that also exists in birds and mammals. The six conserved cysteines are observed in mollusc g-type lysozymes, except the lysozyme of Oncomelania hupensis that contains eight conserved cysteine. Comparison with other g-type lysozymes of scallops, that of O. hupensis has two additional cysteines (Zhang et al., 2012), and shares some features with other g-type lysozymes, such as the substrate binding sites, a signal peptide, the catalytic residues critical for the fundamental structure and function of g-type lysozymes (Nakai et al., 2005, 2007). P. acuta can survive better in polluted water environment than other snails. A better understanding the immune mechanisms of P.acuta may be lead to important advances in the innate immune system of invertebrate. Comparison with g-type lysozyme of other molluscs, the lysozyme of P. acuta shares the same substrate binding sites, the catalytic residues and the same six cysteines (Hikima et al., 2001; Zhao et al., 2007; Itoh et al., 2009). The same six cysteines also appear in P. acuta，which possibly constitute disulphide bridge to result in a compact structure, and are specific in molluscs (Zhao et al. 2005, 2007). The i-type lysozyme is originally described from starfish Asterias rubens (Jollès and Jollès,1975), which is identified in phylogenetically diverse organisms of invertebrates, including porifers, molluscs, annelids, nematodes, echinodermates, hemichordates, and arthropoda (Ito et al., 1999; Van Herreweghe and Michiels, 2012). The first i-type enzyme is described in marine shellfish (Hikima et al., 2001), and recently identified in other molluscs, including Chlamys islandica, Mytilus edulis, M. galloprovincialis, Crassostrea gigas, Crassostrea virginica, Ruditapes philippinarum (Nilsen et al. 1999; Olsen et al., 2003; Yue et al., 2011; Zhu et al., 2016). The complete amino acid sequence of the enzyme is cloned from Tapes japonica (Nilsen and Myrnes, 2001). The enzymes of T. japonica and C. islandica contain the same as fourteen cysteine residues (Ito et al., 1999; Nilsen et al., 1999), that of Meretrix meretrix, R. philippinarum and C. gigas have a signal peptide and fourteen conservative cysteine residue, and all structure domains are destabilase (Naoki et al., 2007; Xin et al., 2011; Yang et al., 2017). The typical g-type lysozyme of molluscs has six cysteine residues (Zou et al. 2005), which of numbers vary ranging from zero to ten in different species (Irwin and Gong 2003; Nilsen, 2003). The typical c-type lysozyme of molluscs has eight cysteine residues, which also exist in digestive organ (Xin et al., 2011; Yang et al., 2017). The high content of cysteine residues of g-type and i-type lysozymes 435 in molluscs are proposed to maintain more stable proteins that can possess a compacter structure in high osmolarity seawater and in the digestive (Ito et al., 1999; Zhao et al., 2007). Meanwhile, the three type lysozymes of molluscs are secreting type, and have signal peptide (Ito et al., 1999). The genetic structure of i-type lysozyme has is similar to that of c-type lysozyme, and both have 4 exons and 3 introns (Nilsen et al., 1999, 2001; Paskewitz et al., 2008). However, the lysozyme with 5 exons and 2 exons was also found in Mytilidae. The i-type lysozyme genome of M. edulis comprises 5 exons instead of the classical 4 exons of the c-type lysozyme gene (Bachali et al., 2002; Paskewitz et al., 2008). The maximum number of exons in g-type lysozyme genome possess 7 exons from human (Irwin and Gong, 2003), and chicken and mice have 6 exons (Nakano and Graf, 1991). The g-type lysozyme of abalone H. discus discus has 7 exons and 6 introns (Ding et al., 2011), and that of M. galloprovincialis do 6 exons and 5 introns (Hui et al., 2008). Therefore, it is suggested that i and c-type lysozyme may originate from the same ancestor. The genetype of lysozyme is more than 2 in molluscs (Li et al., 2008; Wang et al., 2012; Wen et al., 2015; Yang et al., 2017). Three hypotype of the g-type lysozymes are firstly found in one species of molluscs (Zhu et al., 2016), and c-, i- and phage-type lysozymes are described in R. philippinarum (Zhao et al., 2010; Ding et al., 2014). However, relatively little is known about the hypotype of lysozymes in vertebrates. Phylogenetic analysis showed that the major lysozyme genes were clustered into two main clades (Fig. 1) that include g-, c- and i-type lysozyme sequences. It is indicated that c- and i-type lysozyme belong to the near-edge parallel macromolecules, and the c- and g-type lysozyme is a parallel evolution. Except for abalone H. discus discus, the c- and i-type lysozymes were clustered into two main clades in molluscs. Phylogenetic analysis of lysozyme gene also showed that the i-type lysozymes were clustered into main clades. Chlamys islandica and Calyptogena sp were clustered to the corresponding subgroup in the phylogenetic tree (Fig. 2). Antimicrobial protection and mechanism Lysozyme is antibacterial and digestion of bacteria in the major functions, and widely distribute in the tissues or secretions of vertebrates and invertebrates (Hultmark et al., 1996; Irwin et al., 1996). The transcript expression of c-type lysozyme is obvious in kidney, spleen, brain and ovary tissues from Paralichthys olivaceus (Hikima et al., 2001). The g-type lysozyme gene replication is common in vertebrates, except for cartilaginous fish (Irwin, 2014). The expression of the g-type lysozyme is a high level in the kidney of Oncorhynchus (Miyauchi et al., 2000), by liquidchromatography tandem mass spectrometry (LC-MS/MS), that of c-type lysozyme increase significantly in the blood cells and blood lymphocytes of Biomphalaria glabrata (Mollusca) after stimulated by the live Bacillus megaterium (Cheng et al., 1978). The activity of mollusc lysozyme is detected in the hepatopancreas, hemolymph, gills, mantles, and digestive organs, by transcripts were detected inall tissues tested (He et al., 2012; Wang et al., 2012; Wen et al., 2015). The distribution of the lysozymes in molluscs is shown in Table 1. The c-type lysozyme transcripts are highly expressed in hepatopancreas, hemocytes and gills from V. philippinarum and H. discus hannai (Yu et al., 1999; Yang et al., 2017). The g-type lysozyme from the Fig. 1 Phylogenetic tree constructed by the neighbor-joining method in MEGA software based on the c, g, i-type lysozyme sequences. Bootstrap support values for the NJ tree are shown at the nodes (out of 1000 replicates). Ruditapes philippinarum AGO06638.1 Ruditapes philippinarum AGO06639.1 Cyclina sinensis AEG19518.1 Mytilus galloprovincialis AJQ21538.1 Ruditapes philippinarum AKN35212.1 Haliotis discus discus ADR70995.1 Crassostrea gigas BAF48044.2 Haliotis discus discus AGQ50336.1 Mytilus galloprovincialis AJQ21515.1 Meretrix meretrix ADL27913.1 Ruditapes philippinarum ACU83237.1 Chlamys islandica CAC34834.1 Haliotis discus discus AGQ50335.1 Mytilus galloprovincialis AFF18185.1 Physella acuta ADV36303.1 Mizuhopecten yessoensis AEY77130.1 Azumapecten farreri ABB53641.1 Argopecten irradians AAX09979.1 56 75 58 56 100 69 30 53 97 98 60 93 96 55 56 0.2 i-type lysozyme g-type lysozyme c-type lysozyme 436 Fig. 2 Phylogenetic tree constructed by the neighbor-joining method in MEGA software based on the i-type lysozyme sequences. Bootstrap support values for the NJ tree are shown at the nodes (out of 1,000 replicates). Mizuhopecten yessoensis is the highest expression in the hepatopancreas, gills and mantle (He et al. 2012). The i-type lysozymes of Meretrix meretrix and Octopus ocellatus mainly present in hepatopancreas, blood cells and gills (Hultmark et al., 1996; Zhao et al., 2010), that of C. virginica mainly exists in digestive gland and hemolymph (Xue et al., 2007), that of R. philippinarum is the highest expression in mantle (Zhu et al., 2016). The expression of i-type lysozyme in mantle is higher than that in gills, digestive glands and hemocytes from Crassostrea virginica, and is abundant in the tissues of gills, hepatopancreas and haemocytes from V. philippinarum (Itoh et al., 2007). The c- and g-type lysozymes are highly expressed in hepatopancreas, hemocytes and gills, and are weakly expressed in the tissues of muscle, foot and goand (Zou et al., 2005; Zhao et al., 2007; Ding et al., 2011; Wang et al., 2013; Umasuthan et al., 2013; Guo et al., 2014; Yang et al., 2017). The expression pattern of i-type lysozyme gene in different tissues probably indicate that the different biological functions of the enzyme occur during their evolution, that of g- and c-type lysozymes in different organs/tissues also suggested that they may serve as some extent reflect their functional role. The major biological role of lysozymes can act as antibacterial and immune-modulating agents (Hikima et al., 2001). The mRNA of lysozymes from Mizuhopecten yessoensis, H. discus hannai and M. galloprovincialis predominately express and execute its antibacterial activity in hepatopancreas, gills and mantle (Nilsen et al., 1999; Li et al., 2008; Wang et al., 2011; He et al., 2012). The expression of c-type lysozyme from C. farreri is in the hepatopancreas, gill and gonad, and the higher expression level in gills may contribute to the clearance of bacteria (Zhao et al., 2007). The g-type lysozyme possess combined features of the immune and digestion, and also gain the lytic activities to inhibit gram-positive and gram-negative bacteria in vitro, the g-type lysozyme s of C. farreri, M. galloprovincialis and M. yessoensis can inhibite Micrococcus lysodikicus, that of Physa acuta is beyond restraint to S. aureus (Zhao et al., 2007; Wang et al., 2013). The g-type lysozyme gene of O. hupensis is mainly expressed in hepatopancreas, and antibacterial activity was stronger than the c-t ype lysozyme (Zhang et al., 2012). The i-type lysozymes are detected in hemocytes from Ruditapes decussatus and R. philippinarum (Yue et al., 2011). The activity of i-type lysozyme in hemocytes from Mytilus edulis is higher than that from R. decussatus and R. philippinarum (Pipe, 1990; Carballal et al., 1997; Lopez et al., 1997). The gills often face to the invasion of all kinds of pathogens, which construct of only a single layer of fragile cells and covered with a thin layer of protective mucus, were constantly flushed with water that contained pathogens (Callewaert and Michiel, 2010). The antimicrobial activities of two lysozymes from V. philippinarum (rVpCLYZ-1 and rVpCLYZ-2) are investigated against Staphyloccocus aureus, Micrococcus luteus, Vibrio anguillarum, Enterobacter cloacae. rVpCLYZ-1 displays broad spectrum antibiotic activities, and they possess strong microbicidal activities against M. luteus and V. anguillarum. rVpCLYZ-2 has strong inhibitory activity against all detected bacteria, but is less effective against P. pastoris KM71. The turbidimetric assay is also performed to measure thelysozyme activity of rVpCLYZs against M. luteus and V. anguillarum (Yang et al., 2017). The recombinant CpLYZ1 has bacteriolytic activity against E. coli DH5a, A. Chlamys islandica CAC34834.1 Calyptogena sp. SB2001_1;AAN16211.1 Calyptogena sp. SB2001_1;AAN16212.1 Ruditapes philippinarum ACU83237.1 Meretrix meretrix ADL27913.1 Meretrix lusoria P86383.1 Crassostrea virginica BAE47520.1 Mytilus galloprovincialis AJQ21515.1 Haliotis discus discus AGQ50336.1 Ruditapes philippinarum AMS37097.1 Crassostrea gigas BAF48044.2 Crassostrea virginica BAE93114.1 Cyclina sinensis AEG19518.1 100 96 83 75 61 48 50 62 40 55 0.2 437 hydrophila, Staphyloccocus aureus, Streptococcus sp. and Staphylococcus epidermidis, and the bacteriolytic activity of CpLYZ1 against B. subtilis is the strongest, while the relative activity is 50 %. Its relative activity against E. coli DH5 a, A. hydrophila, S. aureus and Streptococcus sp. is 19 % - 28 %, and against S. epidermidis is only 16 %. The bacteriolytic activity of standard lysozyme against A. hydrophila, S. aureus, B. subtilis, Streptococcus sp. and S. epidermidis are higher than the recombinant CpLYZ1, but its bacteriolytic activity against E. coli DH5 a is lower than the recombinant CpLYZ1(Wu et al., 2013). Therefore, the lysozyme in gills of V. philippinarum shows strong antibacterial activity against Gram positive and Gram negative bacteria. The high expression level of mollusc lysozyme in gills implies that it has a significant contribution in prevention of microbial exploitation (Matsumoto et al., 2006). However, some i-type lysozymes from Venerupis philippinarum and Ruditapes decussates also express in haemocytes, and exhibit antibacterial activity against gram-positive bacteria and gram-negative bacteria (Lopes C, 1997; Itoh et al., 2007). Besides killing bacteria, the c-type lysozyme of R. philippinarum shows high antimicrobial activities, and the i-type lysozyme of V. philippinarum also has antifungi activity (Goto et al., 2007). Most lysozymes exhibit muramidase activity, and also do chitinase activity- enzymatic hydrolysis of chitin to produce N- acetyl glucosamine (Yang et al., 2017; Bathige et al., 2013). The result is probably the similarity between peptidoglycan (heteropolymer of β-1,4 linked Nacetylmuramic acid and N-acetylglucosamine), the natural substrate of lysozymes, chitin (homopolymer of β-1,4 linked N-acetylglucosamine), and the natural substrate of chitinases. Besides warding off pathogenic bacteria infections, the lysozymes have also other clear function of the chitinase activity, which of V. philippinarum, Tapes Japonica and Crassostrea virginica are reported to possess chitinase activity, (McHenery and Birkbeck, 1982; Ito et al. 1999; Nilsen et al. 1999; Miyauchi et al. 2000; Xue et al. 2004). The quaternary structure in Vp-ilys crystal is revealed dimer formation by Venerupis philippinarum lysozyme (Vp-ilys) molecules, which is assumed to result from the dissociation of the Vp-ilys dimer at high ionic strength with a high salt concentration (≥ 133 mM NaCl), thereby increasing chitinase and muramidase activity (Goto et al., 2007). The activity of lysozyme originated from glycosidic hydrolases is powerful to hydrolyze PGN and chitin (Takeshita et al., 2003; Goto et al., 2007; Callewaert and Michiels, 2010). The degradation of PGN and chitin in bacterial cell wall may lead to rapid killing of bacteria and fungi (Elmogy et al., 2015). The lysozymes serve as the function of important digestive enzymes in some animals (Dobson et al., 1984; Stewart et al., 1987; Lemos et al., 1993; Kornegay et al., 1994; Hultmark et al., 1996; Prager, 1996). While the enzymes are present in a high concentration, they are a major digestive enzyme in the true stomach of ruminants (Dobson et al., 1984; Jollès and Jollès, 1984; Irwin, 1996). Three-type lysozymes of molluscs are detected in digestive systems, and are regarded as digestive lysozymes (Nilsen et al., 1999; Olsena et al., 2003; Zhao et al., 2007). Digestive gland has an important lymphoid site in molluscs, and the hepatopancreas may act as a major site for the production of lysozymes (Mchenery et al., 1979; Jollés et al., 1996; Tan et al., 2007). The i-type lysozyme of C. gigas plays complementary role in digestive organs, it has been reported that the basophil cells have an intense enzyme activity, demonstrating that lysozyme is synthesized in the digestive tubule basophil cells. The i-type lysozyme genes in the hepatopancreas of Hyriopsis cumingii are down-regulated, which can inhibite bacteria to attack the host immune organs, and also promote the acid digestion of bacteria in molluscs (Zhang et al., 2010). Therefore, bacteria may protect themselves from lysozyme-induced digestion by down-regulating i-type lysozyme genes. The nutriments of molluscs are harvested to produce by autotrophic bacteria, the c- and i-type lysozyme of C. farreri are detected to serve as digestive lysozymes in digestive tract (Nilsen et al., 1999; Olsena et al., 2003; Zhao et al., 2007). It is postulated that lysozymes of deep-sea bivalves are similar to that of ruminants in digestive function (Jollès et al., 1996). The lysozyme of M. edulis is also involved in digestion, since lysozymes from the digestive gland-associated crystalline style are believed to be purified from the digestive gland (Olsen et al., 2003). In two i-type lysozymes (Cv-iLys1, 2) of eastern oyster C. virginica, Cv-iLys2 is mainly found in the digestive gland, which is lower amounts in the crystalline style, and is expressed in basophil cells of digestive tubules. In contrast, Cv-iLys1 is mainly found in lips and mantle, and is lower amounts in gills, style sac, midgut, digestive gland and gonads (Zobel et al., 1938; McHenery et al., 1985; Langdon et al., 1990). The molluscs are also ability to utilize bacteria as food. The deepwater molluscs rely on symbiotic bacteria in gills for nutrition (Jollès et al., 1996). The biochemical and molecular information about mollusc lysozymes is obtained from digestive systems (McHenery et al., 1979; Jollès et al., 1996; Ito et al., 1999; Miyauchi et al., 2000; Olsen et al. 2003; Liu et al., 2006). The lysozymes of molluscs not only possess combined features of immunity and digestion, but also can inhibit gram-positive and gram-negative bacteria. Therefore, it is suggested that the digestive lysozymes apparently evolve from parallel in different species, and acquire the ability to function in highly acidic and protease-rich environments (Jollès et al., 1984; Stewart et al., 1987; Kornegay et al., 1994; Prager, 1996; Regel et al., 1998). The lysozyme can also induce regulation of the synthesis and secretion of other immune factors in vivo of animal software (Zobel et al., 1983), and involve in digestion, promoting reproduction, stimulating growth, and cancer related functions, besides the common function of lysis of bacterial and fungal cell wall (Irwin, 2004; Zhang et al., 2005; Kanda et al., 2007). The lysozyme of O. hupensis not only has the function of resisting the removal of foreign pathogenic microorganisms, but also does the function of hydrolyzing fibrin. Other potential activities include isopeptidase activity and perhaps chitinase activity that is detected in both c-type (Chipman and Sharon, 1969; Callewaert and 438 Michiels, 2010) and i-type lysozymes (Jollès and Jollès, 1984; Takeshita et al., 2003; Goto et al., 2007; Xue er al., 2007). However, molluscs constantly encounter various potential pathogenic microorganisms in their living environment, and the content of lysozyme is affected by a variety of environmental factors and pathogens (Irwin et al., 1996). The lysozyme of M. meretrix shows strongly antibacterial activity against gram-positive and gram-negative bacteria, and the gene expression of lysozyme increases following Vibrio parahaemolyticus challenge, the recombinant g-type lysozyme shows strong antibacterial activity against Micrococcus luteus (Xin et al., 2011). The expression levels of c-type lysozymes increase after bacterial (Vibrio anguillarum) stimulation from V. philippinarum, H. discus hannai and Cyclina sinensis, and the recombinant lysozyme also shows bacteriolytic activity against both gram-positive and gram-negative bacteria (Goto et al., 2007; Yang et al., 2017). The two lysozymes are identied from V. philippinarum, the recombinant proteins of lysozymes (rVpCLYZ-1 and rVpCLYZ-2) possess strong microbicidal activities against M. luteus and fungi. Comparison with rVpCLYZ-1 and rVpCLYZ-2, the lysozyme from chicken egg-white shows lower activity against M. luteus (Yang et al., 2017). The mRNA expression of i-type lysozymes from M. galloprovincialis can be induced by Vibrio anguillarum (Hui et al., 2008). The lysozymes of R. philippinarum are designed as RpiLYZ-1, RpiLYZ-2, the expression of RpiLYZ-1, 2 are induced after Vibrio anguillarum stimulation, VpLYZ mRNAs are down-regulated sharply from 6 to 12 h post-infection. Then, the expression level increase to the peak at 72 h, and recover to the original level at 96 h (Yang et al., 2017). Therefore, mollusc lysozymes have obvious antibacterial activity against V. anguillarum (Bassem et al., 2006; Pan et al., 2010; Yue et al., 2011). While O. hupensis is infected by schistosome, the g-lysozyme gene expression significantly increase (Zhu et al., 2016), P. acuta (PALysG) possess to inhibite capacity against M. lysodikicus, and C. farreri ( CFLysG) can not inhibite S. aureus (Zhao et al., 2007). These results reveal that the c-type lysozyme is involved in the non-specific immune of molluscs. The external environment parameters, such as pH, temperature, and ion strength, can influence on the lytic activity of lysozymes (Ye et al., 2010). Generally, the optimal pH of the lytic activity is below 7 from mollusc lysozymes, c-type of M. galloprovincialis and R. philippinarum, g-type of O. hupensis, i-type of Crassostrea virginica (Umasuthan et al., 2013; Wang et al., 2013). While pH is less than 7, the lytic activity of g-type mollusc lysozymes changes to follow pH (Huang, 2014). However, the optimal pH of the lytic activity is generally ranging from 7 to 10 from c-type lysozymes of mammal and chicken (Hui et al., 2017; Yang et al., 2017). Moreover, high lytic activities are detected at pH 9.5 - 10. Similar phenomenon is also observed in lysozyme from chicken egg white with high activity at both pH 6.2 and 9.2 (Davies et al., 1969). The existence of a wide range of optimal conditions for the activity of c-type lysozyme is suggested that these conditions are perhaps species-specific (Bathige et al., 2013). The antibacterial activity of lysozyme in O. hupensis is examined. While the temperature is less than 50 ℃, the activity of lysozyme changes to follow temperature. Therefore, the optimum temperature of lysozyme activity was 50 ℃, and the optimum pH was 7.0 (Saurabh et al., 2008; Ye et al., 2008). At temperature ranging from 15 ℃ to 50 ℃, while the temperature increased, the bacteriolytic activity of i-type lysozyme from Cristaria plicata gradually increased. The relative activity declined when the temperature was above 50 ℃. The effect of pH on the enzyme of Cristaria plicata between pH 4.5 - 8.5 shows that pH of the highest activity was 5.5. The optimal pH and temperature for the enzyme activity of C. plicata were 5.5 and 50 °C (Wu et al., 2013; Dai et al., 2015). Meanwhile, the activity of i-type lysozyme from V. philippinarum is high in low temperature, and the optimal temperature is 20 ℃. The lysozyme of V. philippinarum has activity at low temperature, which is in agreement with the characteristic of coldblooded aquatic animals (Yang et al., 2017). The expression profiles of mollusc lysozymes further indicate the coexistence of multiple types of lysozymes in molluscs. The most known function of lysozyme is antibacterial activity by catalyzing the hydrolysis of bacterial cell walls, and can kill bacteria using non-enzymatic bactericidal domains (Dobson et al., 1984; Stewart et al., 1987; Lemos et al., 1993). Meanwhile, the mechanisms of action are different for gram-positive bacteria and gram-negative bacteria, the cell walls of gram-positive bacteria are exposed so that lysozyme can act directly on the cell walls and cause lysis of cell walls, and the cell wall components of gram negative bacteria, such as lipopolysaccharide (LPS), PliI and MliC/PliC, affect the cell wall of bacteria (Callewaert et al., 2008; Vanderkelen et al., 2011). Therefore, lysozyme should be combined with other components of the immune system in order to lysis the cell wall structure of gram negative bacteria, resulting in bacterial lysis death (Cheetham et al., 1992). The lytic activity of lysozyme against bacteria and fungi is suggested to be associated with the muramidase and chitinase activities. The c-type lysozymes typically possess muramidase activity that cleaves the -1, 4-glycosidic bond of peptidoglycan (PGN) in microbial cell walls, and cause the lysis of bacteria (Vocadlo et al., 2001; Supungul et al., 2010). The lysozyme is also served as a model for studies on enzyme structure and function (Peters et al., 1989; Prager and Jollès, 1996). Typical i-type lysozymes exhibit muramidase activity and generate bactericidal activity by hydrolyzing the cell wall, which show bacteriolytic activity against both Gram-positive and Gram-negative bacteria (Zhao et al., 2010; Zhou et al., 2017). Conclusion and perspective Lysozymes are present in variety of organisms, ranging from viruses to plants and animals. Although all lysozymes perform the same enzymatic function, and exhibit overall similarity in three dimensional (3D) structures, the primary amino acid sequences of these lysozymes is rarely the same. It is speculated that the 3D structure and function of the enzymes 439 are analogous, and the genes of the enzymes are not homologous. The various types of lysozymes are generated by convergence during evolution, and can coexist in the same taxon. For example, the c- and g-type lysozymes are in vertebrates. The c- and i-type lysozymes are present in arthropod, the c-, i- and g-type lysozymes exist in molluscs. The question of evolutionary relationship is raised among different types of lysozymes. The phylogenetic tree analysis shows that i-type lysozyme is more closely related to c-type one than g-type one in molluscs. The partial sequence of i- and c-type lysozyme gene is homology. The central exon of lysozyme genome from M. galloprovincialis is homologous to the second exon of that from chicken, and both belong to the c-type lysozyme (Wang et al., 2013). It is suggested that c- and i-type lysozyme belong to the near - edge parallel macromolecules, and is believed that c- and i-type lysozyme gene evolved from a single complete gene. The i-type of C. gigas, g-type of H. discus discus and M. galloprovincialis, was also clustered to the corresponding subgroup in the phylogenetic tree. However, the other evolutionary relationship of three type lysozymes also is supposed (Jollès and Jollès, 1984; Bachali et al., 2002). Some studies assumed that i-type lysozymes were more closely related to g-type lysozymes, and suggested that c-type was basal (implication ancestral) to g- and i-type lysozymes (Hikima et al., 2003). The i- and g-type lysozymes diverge from an ancestor of c-type. Others believe the g-type lysozyme is considered as the common ancestor to c- and i-type ones (Thunnissen et al., 1995). The i-, g- and c-type lysozymes are detected in molluscs, and this may provide some clues to clarify the relationship of the three types of lysozyme (Xin et al., 2011). These results consist with the notion that the three type lysozymes diverge from a common precursor, and c-type lysozyme is closed to the ancestor. Further, the molluscs encounter a greater range of bacterial strains or species in the marine environment, and the varied composition and structure of the bacterial cell wall may promote a type of ‘substrate-induced evolution’ of lysozymes (Jollès and Jollès, 1984). Two conserved amino acid Glu54 and Asp70 are critical for the c-type lysozyme lytic activity to bacterial cell wall, and the motif that flanking Asp70 is also conserved in the c-type lysozyme (Vocadlo et al., 2001). These results indicate that the mature c-type lysozyme of molluscs may possess the antimicrobial activity as well as that of other species. Other potential activities, such as isopeptidase activity and perhaps chitinase activity, are detected in c-type lysozymes (Chipman and Sharon, 1969; Callewaert and Michiels, 2010). In conclusion, the c-type lysozymes are characterized from some molluscs, and their expression profiles and antimicrobial activities are also investigated. These results provide helpful evidence for further understanding the innate immunity of molluscs. More investigation should be directed to understand the interaction mechanisms of c-type lysozymes with membranes or cell walls of bacteria. O. ocellatus has three conservative enzyme activity center (Glu40, Glu49, Ser52) and 12 conserved cysteines that form 4 pairs of protein disulfide bonds and the stable conformation. The characteristics of i-type mollusc lysozyme structure possess two catalytic domains exhibiting muramidase and isopeptidase activities (Jollès and Jollès, 1984b; Ito et al., 1999; Takeshita et al., 2003; Xue er al., 2007; Goto et al., 2007). Although lysozyme research is described in 1960s, the data about lysozyme is increasingly abundant. So far, the lysozymes are studied to remain one of the hot spots in life science, that of some animals has been studied more thoroughly, and that of molluscs still needs further to do improvement. The origin and evolution of mollusc type lysozyme will especially require more experimental data and bioinformatic analyses. 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