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Title: Expression of bovine non-classical major histocompatibility complex class 1 proteins in mouse P815 and human K562 cells

Author
item PARASAR, P - University Of Utah
item WILHELM, A - University Of Utah
item RUTIGLIANO, H - University Of Utah
item THOMAS, A - University Of Utah
item TENG, L - University Of Utah
item SHI, B - University Of Utah
item DAVIS, W - Washington State University
item Suarez, Carlos
item NEW, D - University Of Utah
item WHITE, K - University Of Utah
item DAVIES, CHRISTOPHER - University Of Utah

Submitted to: Research in Veterinary Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/6/2016
Publication Date: 6/8/2016
Citation: Parasar, P., Wilhelm, A., Rutigliano, H.M., Thomas, A.J., Teng, L., Shi, B., Davis, W.C., Suarez, C.E., New, D.D., White, K.L., Davies, C.J. 2016. Expression of bovine non-classical major histocompatibility complex class 1 proteins in mouse P815 and human K562 cells. Research in Veterinary Science. doi:10.1016/j.rvsc.2016.06.004.

Interpretive Summary: Major histocompatibility complex class I (MHC-I) proteins are components of vertebrate nucleated cells able to display peptide fragments of non-self-proteins from within the cell to cytotoxic T cells; this will trigger an immediate response from the immune system against a particular non-self-antigen displayed with via help of MHC class I protein. MHC-I proteins can be expressed as cell surface or secreted proteins. In this study we investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, by expressing the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA43 NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane.

Technical Abstract: Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, we expressed the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA43 NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane.