Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells. (August 2016)

• Record Type: Journal Article
• Authors: Parasar, Parveen; Wilhelm, Amanda; Rutigliano, Heloisa M.; Thomas, Aaron J.; Teng, Lihong; Shi, Bi; Davis, William C.; Suarez, Carlos E.; New, Daniel D.; White, Kenneth L.; Davies, Christopher J.
• Is Part Of: Research in veterinary science. Volume 107(2016)
• Page Start: 161
• Page End: 170
• Publication Date: 2016-08
• Subjects: Bovine leukocyte antigens (BoLA) -- Human K562 cells -- Major histocompatibility complex -- Mouse P815 cells -- Non-classical MHC class I; Veterinary medicine -- Periodicals; Veterinary Medicine -- Periodicals; Médecine vétérinaire -- Périodiques; Médecine vétérinaire -- Recherche -- Périodiques; Diergeneeskunde; 636.089
• DOI: 10.1016/j.rvsc.2016.06.004
• Languages: English
• ISSNs: 0034-5288

Abstract

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 BoLA-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. Highlights: Bovine non-classical MHC-I proteins can be either membrane associated or secreted. BoLA-NC1*00501, -NC3*00101 and -NC4*00201 were membrane associated proteins. The BoLA-NC1*00401 splice variant was expressed as a secreted protein. BoLA-NC2*00102 remained intracellular and may require a specific peptide antigen.