Identification, recombinant expression and immunological study of Lja-SHP2 in Lampetra japonica.

Abstract

The protein tyrosine phosphatase SHP2 of higher vertebrates, encoded by ptpn11 gene, catalyzes the dephosphorylation of tyrosine phosphorylation site, and plays regulatory roles in various signaling pathways by cooperating with other protein tyrosine kinase. Previous studies have shown that SHP2 plays an important role in the activation and signal transduction of T and B cells in higher vertebrates. To study the role of a SHP2 homologous molecule of lampreys (Lja-SHP2) in immune response, we cloned and expressed the open reading frame sequence of Lja-SHP2 gene in prokaryotic expression vector pET-32a. The recombinant protein was successfully expressed in E. coli and the rabbit-derived polyclonal antibody was prepared. Lampetra japonica were immunized with mixed bacteria, and the mRNA and protein of Lja-SHP2 in immune-related cells and tissues were detected by real-time quantitative PCR and Western blotting after immunization. The Lja-SHP2 mRNA and protein were not significantly affected in leukocytes and supraneural myeloid bodies, but up-regulated significantly in gill tissues (P<0.05) after challenged by mixed bacteria, which indicated that Lja-SHP2 mainly participates in the immune response of gill tissues after mixed bacteria stimulation. To further investigate whether Lja-SHP2 level was affected in three lymphocyte subsets, the B-cell mitogen lipopolysaccharide (LPS) and T-cell mitogen phytohaemagglutinin (PHA) were employed to boost the immune response in L. japonica. LPS immune stimulation increased Lja-SHP2 in leucocytes significantly compared with the control group, and but had a marginal effect on Lja-SHP2 expression in gills and supraneural myeloid bodies. PHA immune stimulation could up-regulate Lja-SHP2 level in leukocytes, gill tissues and supraneural myeloid bodies. The change of Lja-SHP2 was especially dramatical in leukocytes, which was about 2.5 times higher than that in the control group, suggesting that Lja-SHP2 is involved in the lamprey immune response mediated by PHA. Consistent with the previous finding that PHA could induce the activation of VLRA+ lymphocytes, our results showed that Lja-SHP2 might be included in the immune response of VLRA+ lymphocytes mediated by PHA in gills. This research will benefit exploring the functions of Lja-SHP2 in the immune response of lamprey and will provide clues for understanding the phylogenesis of SHP2 molecular family, and its roles in the early occurrence and evolution of adaptive immune system in higher vertebrates.