A haemocyte-expressed Methyltransf_FA domain containing protein (MFCP) exhibiting microbe binding activity in oyster Crassostrea gigas. (September 2021) - Li, Jiaxin
Abstract: The Methyltransf_FA domain is well-known as a key protein domain of enzyme synthesizing juvenile hormone, and Methyltransf_FA domain containing proteins (MFCPs) are widely existed in vertebrates and invertebrates. In the present study, a Cg MFCP with a single Methyltransf_FA domain was screened from oyster Crassostrea gigas, and its open reading frame of Cg MFCP was of 1128 bp, encoding a polypeptide of 376 amino acids with a signal peptide, a Methyltransf_FA domain and a transmembrane region. Cg MFCP was clustered with FAMeTs from insecta and crustacea of arthropod. The mRNA transcripts of Cg MFCP 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 Cg MFCP protein was verified to be highly expressed in haemocytes. The expression level of Cg MFCP 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 Cg MFCP 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-r Cg MFCP can label 95% of oyster haemocytes. And by fluorescence microscope analysis, Cg MFCP was mainly distributed in cytomembrane of haemocytes. The recombinant Cg MFCP (r Cg MFCP) exhibited higher aﬃnity towards MAN and LPS in a dose-dependent manner, while relatively lower aﬃnity to PGN and poly (I:C). r Cg MFCP 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 Cg MFCP 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_FA domain containing proteins. Highlights: Cg MFCP with a single Methyltransf_FA domain was identified from C. gigas. The anti-r Cg MFCP can label 95% of oyster haemocytes by flow cytometry analysis. Cg MFCP mRNA expression in haemocytes was induced by LPS and V. splendidus stimulation. r Cg MFCP exhibited recognition and binding activity towards various PAMPs and microbes.