Characterization of chitinase‐like proteins (Cg‐Clp1 and Cg‐Clp2) involved in immune defence of the mollusc Crassostrea gigas

Abstract

Glycoside hydrolase family 18 (GH18) is a phylogenetically conserved group of proteins present in eukaryotes, prokaryotes and viruses. The GH18 family is characterized by a Glyco_18 domain adopting an (a ⁄ b) 8 triose phosphate isomerase-barrel structure that consists of a specific arrangement of eight parallel b-strands, forming the barrel core, surrounded by eight a-helices [1]. This family classification, based only on similarities in amino acid sequences, groups together chitinases and proteins devoid of catalytic activity due to the substitution of a critical amino acid in the catalytic centre. This latter singular class of proteins, called chitinase-like proteins (CLPs), has been identified only recently in plants [2], mammals [3], insects [4] and molluscs [5]. CLPs have been implicated in many biological processes, such as control of nodulation [2] and growth ⁄ differentiation balance during development in plants [6]. Insect CLPs such as imaginal disc growth factors represent the first proliferating polypeptides reported from invertebrates [7]. These mitogenic growth factors cooperate with insulin to stimulate proliferation, polarization and mobility of imaginal disc cells in vitro. Imaginal disc growth factors may also constitute morphogenetic factors controlling embryonic and larval development, and could stimulate the cell growth required for wound healing [8,9]. In mammals, CLPs such as YM1 ⁄ 2 and YKL-40 (40 kDa mammalian protein with the N-terminus YKL) [also known as human cartilage glycoprotein-39 (HC-gp39) in humans] are considered to be cytokines [10,11] Chitinase-like proteins have been identified in insects and mammals as nonenzymatic members of the glycoside hydrolase family 18. Recently, the first molluscan chitinase-like protein, named Crassostrea gigas (Cg)-Clp1, was shown to control the proliferation and synthesis of extracellular matrix components of mammalian chondrocytes. However, the precise physiological roles of Cg-Clp1 in oysters remain unknown. Here, we report the cloning and the characterization of a new chitinase-like protein (Cg-Clp2) from the oyster Crassostrea gigas. Gene expression profiles monitored by quantitative RT-PCR in adult tissues and through development support its involvement in tissue growth and remodelling. Both Cg-Clp1-and CgClp2-encoding genes were transcriptionally stimulated in haemocytes in response to bacterial lipopolysaccharide challenge, strongly suggesting that these two close paralogous genes play a role in oyster immunity.Abbreviations Cg-Clp1 ⁄ 2, Crassostrea gigas chitinase-like protein 1 ⁄ 2; CLP, chitinase-like protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GH, glycoside hydrolase; HC-gp39, human cartilage glycoprotein-39 (also called YKL-40); LPS, lipopolysaccharide; YKL-40, 40 kDa mammalian protein with the N-terminus YKL.

Age-dependent expression of stress and antimicrobial genes in the hemocytes and siphon tissue of the Antarctic bivalve, Laternula elliptica, exposed to injury and starvation

“…The upregulated expression of Le-chitinase in L. elliptica following injury is in line with findings in the oyster C. gigas (Badariotti et al 2006(Badariotti et al , 2007a. mRNA levels of chitinases increased in the hemocytes of C. gigas following bacterial challenge and involvement in development, tissue growth, and remodeling was reported.…”

Identification and expression analysis of cDNA encoding chitinase-like protein (CLP) gene in Japanese scallop Mizuhopecten yessoensis

“…The full-length cDNA contained a polyadenylation signal (AATAAA) in the 3'-end UTR, which is common among the mRNAs of inflammatory molecules (Shen et al, 2011). The low levels of sequence identity in the C-terminal regions, probably caused by the relatively low selection pressure during evolution, may contribute little to the structure and function of CLPs (Badariotti et al, 2007). The observations that My-Clp1 contained conserved domain structures and architectures, such as the Glyco_18 domains and active centers, provide further evidence for the high degree of sequence conservation of this gene throughout its evolutionary history because all CLPs from other selected species exhibited the same domains based on amino acid sequence homology.…”

“…This result is not in agreement with those reported for C. gigas, which indicate a relatively low expression level in the muscle tissue. Furthermore, it was reported that the Chit2 gene was highly expressed in the muscle tissue (Badariotti et al, 2006(Badariotti et al, , 2007(Badariotti et al, , 2011. This may be due to the lack of expression during specific stages of growth and indicates that the biological activities may be spatially partitioned among the family members.…”

“…The glycoside hydrolase family 18 (GH18), characterized by the ability to hydrolyze chitin enzymatically, has been identified in organisms that synthesize or metabolize chitin, such as insects, yeasts, and crustaceans, and those that do not synthesize or metabolize chitin, such as higher plants and mammals. The GH18 family consists of a specific arrangement of 8 centrally located parallel β-strands and 8 surrounding α-helices (Badariotti et al, 2007). The GH18 family members aid in the protection of organisms from harsh conditions and host anti-parasite/pathogen invasions because they are often constitutively expressed in macrophages and epithelial cells, which provide the first line of defense against exogenous agents, including chitin-containing pathogens (Homer et al, 2006;Mizoguchi, 2006).…”

“…YKL-40, which has been significantly correlated with the severity of asthma, plays an important role in the homeostasis of several organs in humans, where it regulates tissue remodeling as well as cell proliferation and survival in response to pathological conditions (Giannetti et al, 2004;Kelleher et al, 2005;Recklies et al, 2005;Johansen, 2006). In mollusks, 3 CLPs (Cg-Clp1, Cg-Clp2, and Cg-Clp3) have been identified from the oyster Crassostrea gigas (Badariotti et al, 2006(Badariotti et al, , 2007(Badariotti et al, , 2011. Cg-Clp1 was found to be involved in growth control and remodeling processes in a manner similar to its YKL-40 mammalian counterpart, and Cg-Clp3 plays a role in embryonic development, adult oyster growth, and tissue remodeling during metamorphosis and gonadal restructuring (Badariotti et al, 2006(Badariotti et al, , 2011.…”