Tolliod-like gene in Crassostrea ariakensis: Molecular cloning, structural characterization and expression by RLO stimulation

The BMP1/TLD-like proteinases are pleiotropic, astacin-like metalloproteinases. They play central roles in regulating the formation of the extracellular matrix (ECM) and signaling through various TGFβ-like proteins in morphogenetic and homeostatic events. Here we describe the cloning, structural characterization and expression of Tolloid-like gene in the oyster, Crassostrea ariakensis (CaTLL). The full-length cDNA of CaTLL spans 3492 nucleotides including an open reading frame of 2811 nucleotides which encodes a hypothetical protein of 936 amino acids, with a molecular mass of approximately 103 kDa. The CaTLL molecule possessed structural features of several motifs including an N-terminal signal peptide sequence, a prodomain with an RTRR motif, an astacin-like domain that contains a conserved zinc-binding motif HELGHVIGFWHEH, five CUBs and two EGF domains with the arrangement CUB-CUB-EGF-CUB-EGF-CUB-CUB. The proteolytic domain of Ca-Tolloid shares more than 30% identity with other astacins of various animals from squail to mammals, indicating its conserved catalytic ability. RT-PCR and quantitative real-time PCR analyses revealed that CaTLL showed the lowest expression level in hemocytes of normal groups, but was affected significantly by the challenge of an obligate intracellular Gram-negative bacterium, Rickettsia-like organisms, suggesting that Ca-Tolloid might be involved in the molluscan immune response, and its function is more diverse than previously assumed.