It was generally believed that teleost fish possess only few immunoglobulin isotypes such as IgM and IgD. The newly discovered IgZ/IgZ-like molecules in zebrafish and several other fish species greatly enriched our knowledge of immunoglobulin family in lower vertebrates. In the present study, we report a second IgZ-like isotype in zebrafish, which was designated as IgZ-2, adding a novel member to the immunoglobulin family in teleost fish. The IgZ-2 heavy chain encoding gene exhibited 76.5% nucleotide sequence identity to the previously reported IgZ. The putative IgZ-2 protein consists of four constant regions in its extracellular region, a transmembrane domain, and a short cytoplasmic tail. A number of conserved domains or residues such as the four Ig domains, cysteines required for Ig fold, hydrophobic and hydrophilic residues consistent with the CART domain, and Thr, Ser, Tyr residues known to be essential for association with the B cell co-receptors CD79A/B, were identified. Phylogenetic analysis showed that IgZ-2 grouped with IgZ and other known teleost IgZ-like sequences. The IgZ-2 transcripts were widely expressed in immune-related tissues, and could be significantly up-regulated by in vivo stimulation with LPS in various tissues including head kidney, spleen, intestine, skin, and gill. However, hardly IgZ-2 transcripts were detected during embryonic development until 2 weeks after fertilization. Double immunofluorescence staining showed that IgZ-2 and IgM were co-localized on B cell surfaces. Flow cytometric analysis showed the percentage of IgZ-2-positive cells could be dramatically up-regulated by interleukin-4 (IL-4), a cytokine known to activate the proliferation and differentiation of B cells. These observations indicated that the newly cloned IgZ-2 could be a novel B cell receptor. Our results will add new insights into the immunoglobulin class diversity of teleost fish, and also help us understand better the evolutionary history of adaptive immunity from fish to mammals as a whole.
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