A haemocyte-expressed Methyltransf_FA domain containing protein (MFCP) exhibiting microbe binding activity in oyster Crassostrea gigas.

The Methyltransf&#95;FA domain is well-known as a key protein domain of enzyme synthesizing juvenile hormone, and Methyltransf&#95;FA domain containing proteins (MFCPs) are widely existed in vertebrates and invertebrates. In the present study, a CgMFCP with a single Methyltransf&#95;FA domain was screened from oyster Crassostrea gigas, and its open reading frame of CgMFCP was of 1128 bp, encoding a polypeptide of 376 amino acids with a signal peptide, a Methyltransf&#95;FA domain and a transmembrane region. CgMFCP was clustered with FAMeTs from insecta and crustacea of arthropod. The mRNA transcripts of CgMFCP were detected in different tissues, with the extremely high expression level in haemocytes, which was 131.36-fold (p < 0.05) of that in gills. The expression level of CgMFCP protein was verified to be highly expressed in haemocytes. The expression level of CgMFCP mRNA in primarily cultured haemocytes significantly up-regulated at 3 h, 24 h and 48 h post LPS stimulation, which was 3.25-fold (p < 0.01), 2.04-fold (p < 0.05) and 3.59-fold (p < 0.01) compared to that in blank group. After the oysters were stimulated with Vibrio splendidus in vivo, the expression level of CgMFCP mRNA in haemocytes was also significantly up-regulated at 3 h, 12 h, and 24 h, which was 4.22-fold (p < 0.05), 4.39-fold (p < 0.05) and 6.35-fold (p < 0.01) of that in control group, respectively. By flow cytometry analysis, anti-rCgMFCP can label 95% of oyster haemocytes. And by fluorescence microscope analysis, CgMFCP was mainly distributed in cytomembrane of haemocytes. The recombinant CgMFCP (rCgMFCP) exhibited higher affinity towards MAN and LPS in a dose-dependent manner, while relatively lower affinity to PGN and poly (I:C). rCgMFCP also displayed binding activities towards Gram-negative bacteria (Vibrio anguillarum and V. splendidus), Gram-positive bacteria (Staphylococcu aureu) and fungi (Pichia pastoris). These results collectively indicated that CgMFCP specifically expressed in haemocytes and functioned as a pattern recognition receptor by binding to various microbes in oyster C. gigas, which provided insight into the function of Methyltransf&#95;FA domain containing proteins.
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