Cloning, Expression, and Functional Analyses of MHC Class II A in Ayu, Plecoglossus altivelis

The MHC (major histocompatibility complex) class II family plays a major role in vertebrate immune systems and is normally found only on antigen-presenting cells. In this study, we identified the gene encoding the α (PaMHCIIα) chain of MHC class II molecules in ayu (Plecoglossus altivelis). Phylogenetic analysis showed that PaMHCIIα was tightly grouped with MHC class IIα in rainbow smelt (Osmerus mordax) and salmon. PaMHCIIα transcripts were detected in all tested tissues, with high levels in the spleen, head kidney, and gill; expression increased significantly upon Vibrio anguillarum infection. PaMHCIIα mRNA expression was also up-regulated in monocytes/macrophages (MO/MФ) after in vitro stimulation with V. anguillarum. We prepared antibodies for detecting N-glycosylation of PaMHCIIα or neutralizing PaMHCIIα on ayu MO/MФ. Western blot analysis showed that native PaMHCIIα was indeed N-glycosylated. Moreover, PaMHCIIα neutralization not only significantly inhibited ayu MO/MФ phagocytosis, but also reduced the ability of V. anguillarum to induce MO/MФ-cytokine expression. Overall, these findings reveal that the important role of PaMHCIIα against bacterial challenge, and it likely contributes to pathogen responsiveness in ayu MO/MФ.

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Ao, J.Q., Ling, E., & Yu, X.Q. (2008). A Toll receptor from Manduca sexta is in response to Escherichia coli infection. Molecular Immunology, 45(2), 543-552.https://doi.org/10.1016/j.fsi.2014.10.012

Chen, J., Chen, Q., Lu, X.J., & Li, C.H. (2014). LECT2 improves the outcomes in ayu with Vibrio anguillarum infection via monocytes/macrophages. Fish & Shellfish Immunology, 41(2), 586-592. https://doi.org/10.1016/j.fsi.2014.10.012

Cosson, P., & Bonifacino, J.S. (1992). Role of transmembrane domain interactions in the assembly of class II MHC molecules. Science, 258(5082), 659-662.http://dx.doi.org/10.1126/science.1329208

Cuesta, A., Esteban, M.A., & Meseguer, J. (2006). Cloning, distribution and up-regulation of the teleost fish MHC class II alpha suggests a role for granulocytes as antigen-presenting cells. Molecular Immunology, 43(8), 1275-1285. https://doi.org/10.1016/j.molimm.2005.07.004

Di Pucchio, T., Chatterjee, B., Smed-Sörensen, A., Clayton, S., Palazzo, A., Montes, M., ... & Connolly, J. E. (2008). Direct proteasome-independent cross-presentation of viral antigen by plasmacytoid dendritic cells on major histocompatibility complex class I. Nature Immunology, 9(5), 551-557.http://dx.doi.org/10.1038/ni.1602

Ishikawa, S., Kowal, C., Cole, B., Thomson, C., & Diamond, B. (1995). Replacement of N-glycosylation sites on the MHC class II E alpha chain. Effect on thymic selection and peripheral T cell activation. The Journal of Immunology, 154(10), 5023-5029.

Jiang, J., Li, C., Zhang, Q., & Wang, X. (2013). Cloning and characterization of major histocompatibility complex class II genes in the stone flounder Kareius bicoloratus (Pleuronectidae). Genetics and Molecular Research, 12(4), 5820-5832. http://dx.doi.org/10.4238/2013.November.22.9

Lam, F.W., Wu, S.Y., Lin, S.J., Lin, C.C., Chen, Y.M., Wang, H.C., ... & Lin, J.H.Y. (2011). The expression of two novel orange-spotted grouper (Epinephelus coioides) TNF genes in peripheral blood leukocytes, various organs, and fish larvae. Fish & Shellfish Immunology, 30(2), 618-629. https://doi.org/10.1016/j.fsi.2010.12.011

Letunic, I., Doerks, T., & Bork, P. (2014). SMART: recent updates, new developments and status in 2015. Nucleic acids research, 43(D1), D257-D260. https://doi.org/10.1093/nar/gku949

Li, C., Chen, J., Shi, Y., & Li, M. (2009). Characterization of Listonella anguillarum as the aetiological agent of vibriosis occurred in cultured ayu (Plecoglossus altivelis) in Ninghai country, China. Acta Microbiologica Sinica, 49(7), 931-937.https://dx.doi.org/10.3321/j.issn:0001-6209.2009.07.014

Li, C.H., Lu, X.J., Li, M.Y., & Chen, J. (2015). Cathelicidin modulates the function of monocytes/macrophages via the P2X7 receptor in a teleost, Plecoglossus altivelis. Fish & Shellfish Immunology, 47(2), 878-885. https://doi.org/10.1016/j.fsi.2015.10.031

Liu, X., Zhan, Z., Li, D., Xu, L., Ma, F., Zhang, P., ... & Cao, X. (2011). Intracellular MHC class II molecules promote TLR-triggered innate immune responses by maintaining activation of the kinase Btk. Nature Immunology, 12(5), 416-424. https://dx.doi.org/10.1038/ni.2015

Luo, W., Zhang, J., Wen, J.F., Liu, H., Wang, W.M., & Gao, Z.X. (2014). Molecular cloning and expression analysis of major histocompatibility complex class I, IIA and IIB genes of blunt snout bream (Megalobrama amblycephala). Developmental & Comparative Immunology, 42(2), 169-173. https://doi.org/10.1016/j.dci.2013.08.011

Nag, B., Wada, H.G., Arimilli, S., Fok, K., Passmore, D., Sharma, S.D., & McConnell, H.M. (1994). The role of N-linked oligosaccharides of MHC class II antigens in T cell stimulation. Journal of Immunological Methods, 172(1), 95-104.https://dx.doi.org/10.1016/0022-1759(94)90382-4

Nath, S., Kales, S., Fujiki, K., & Dixon, B. (2006). Major histocompatibility class II genes in rainbow trout (Oncorhynchus mykiss) exhibit temperature dependent downregulation. Immunogenetics, 58(5-6), 443-453. https://doi.org/10.1007/s00251-006-0094-5

Pang, J.C., Gao, F.Y., Lu, M.X., Ye, X., Zhu, H.P., & Ke, X.L. (2013). Major histocompatibility complex class IIA and IIB genes of Nile tilapia Oreochromis niloticus: genomic structure, molecular polymorphism and expression patterns. Fish & Shellfish Immunology, 34(2), 486-496. https://doi.org/10.1016/j.fsi.2012.11.048

Piani, A., Hossle, J.P., Birchler, T., Siegrist, C.A., Heumann, D., Davies, G., ... & Lauener, R.P. (2000). Expression of MHC class II molecules contributes to lipopolysaccharide responsiveness. European Journal of Immunology, 30(11), 3140-3146. http://dx.doi.org/10.1002/1521-4141(200011)30:11<3140::AID-IMMU3140>3.0.CO;2-O

Rong, Y.J., Lu, X.J., & Chen, J. (2016). Molecular characterization of E-type prostanoid receptor 4 (EP4) from ayu (Plecoglossus altivelis) and its functional analysis in the monocytes/macrophages. PLoS One,11(1), e0147884. https://doi.org/10.1371/journal.pone.0147884

Scharsack, J.P., Kalbe, M., & Schaschl, H. (2007). Characterization of antisera raised against stickleback (Gasterosteus aculeatus) MHC class I and class II molecules. Fish & Shellfish Immunology, 23(5), 991-1002. https://doi.org/10.1016/j.fsi.2007.03.011

Schmid, D., Pypaert, M., & Münz, C. (2007). Antigen-loading compartments for major histocompatibility complex class II molecules continuously receive input from autophagosomes. Immunity, 26(1), 79-92. https://doi.org/10.1016/j.immuni.2006.10.018

Sültmann, H., Mayer, W.E., Figueroa, F., O'hUigin, C., & Klein, J. (1993). Zebrafish Mhc class II α chain-encoding genes: polymorphism, expression, and function. Immunogenetics, 38(6), 408-420.http://dx.doi.org/10.1007/BF00184521

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M.,& Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28(10), 2731-2739. https://doi.org/10.1093/molbev/msr121

Thankappan, A., Fuller, J.R., Godwin, U.B., Kearse, K.P.,& McConnell, T.J. (2006). Characterization of glycans on major histocompatibility complex class II molecules in channel catfish, Ictalurus punctatus. Developmental & Comparative Immunology, 30(9), 772-782. https://doi.org/10.1016/j.dci.2005.10.006

Ting, J.P.Y., & Trowsdale, J. (2002). Genetic control of MHC class II expression. Cell, 109(2), S21-S33. https://doi.org/10.1016/S0092-8674(02)00696-7

van Lierop, M.J.C., Knight, J., Secombes, C.J., Hermsen, T.T., Groeneveld, A., & Stet, R.J. (1998). Production and characterisation of an antiserum raised against recombinant rainbow trout (Oncorhynchus mykiss) MHC class II beta-chain (MhcOnmy-DAB). Fish &Shellfish Immunology, 8(4), 231-243.https://doi.org/10.1006/fsim.1997.0129

Wang, T., Tan, S., & Cai, Z. (2015). Characterization and expression of MHC class II alpha and II beta genes in mangrove red snapper (Lutjanus argentimaculatus).Molecular Immunology, 68(2), 373-381. https://doi.org/10.1016/j.molimm.2015.09.018

Xu, T., Sun, Y., Shi, G., Cheng, Y., & Wang, R. (2011). Characterization of the major histocompatibility complex class II genes in miiuy croaker. PLoS One, 6(8), e23823. https://doi.org/10.1371/journal.pone.0023823 Yu, S., Ao, J., & Chen, X. (2010). Molecular characterization and expression analysis of MHC class İİ α and β genes in large yellow croaker (Pseudosciaena crocea). Molecular Biology Reports, 37(3), 1295-1307. https://doi.org/10.1007/s11033-009-9504-8

Zhang, X.H., Shi, Y.H., & Chen, J. (2015). Molecular characterization of a transmembrane C-type lectin receptor gene from ayu (Plecoglossus altivelis) and its effect on the recognition of different bacteria by monocytes/macrophages. Molecular Immunology, 66(2), 439-450. https://doi.org/10.1016/j.molimm.2015.05.009