title: Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells.
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.
authors.affiliation: Department of Animal, Dairy and Veterinary Sciences, 4815 Old Main Hill, Utah State University, Logan, UT, USA; Center for Integrated BioSystems, 4700 Old Main Hill, Utah State University, Logan, UT, USA.
authors.forename: Parveen
authors.initials: P
authors.lastname: Parasar
authors.affiliation: Department of Animal, Dairy and Veterinary Sciences, 4815 Old Main Hill, Utah State University, Logan, UT, USA; Center for Integrated BioSystems, 4700 Old Main Hill, Utah State University, Logan, UT, USA.
authors.forename: Amanda
authors.initials: A
authors.lastname: Wilhelm
authors.affiliation: Department of Animal, Dairy and Veterinary Sciences, 4815 Old Main Hill, Utah State University, Logan, UT, USA; School of Veterinary Medicine, 4815 Old Main Hill, Utah State University, Logan, UT, USA.
authors.forename: Heloisa M
authors.initials: HM
authors.lastname: Rutigliano
authors.affiliation: Department of Animal, Dairy and Veterinary Sciences, 4815 Old Main Hill, Utah State University, Logan, UT, USA; Center for Integrated BioSystems, 4700 Old Main Hill, Utah State University, Logan, UT, USA.
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Thomas
authors.affiliation: Department of Animal, Dairy and Veterinary Sciences, 4815 Old Main Hill, Utah State University, Logan, UT, USA; Center for Integrated BioSystems, 4700 Old Main Hill, Utah State University, Logan, UT, USA.
authors.forename: Lihong
authors.initials: L
authors.lastname: Teng
authors.affiliation: Department of Animal, Dairy and Veterinary Sciences, 4815 Old Main Hill, Utah State University, Logan, UT, USA; Center for Integrated BioSystems, 4700 Old Main Hill, Utah State University, Logan, UT, USA.
authors.forename: Bi
authors.initials: B
authors.lastname: Shi
authors.affiliation: Department of Veterinary Microbiology and Pathology, P.O. Box 647040, Washington State University, Pullman, WA, USA.
authors.forename: William C
authors.initials: WC
authors.lastname: Davis
authors.affiliation: USDA-ARS Animal Disease Research Unit, P.O. Box 646630, Washington State University, Pullman, WA, USA.
authors.forename: Carlos E
authors.initials: CE
authors.lastname: Suarez
authors.affiliation: Department of Veterinary Microbiology and Pathology, P.O. Box 647040, Washington State University, Pullman, WA, USA.
authors.forename: Daniel D
authors.initials: DD
authors.lastname: New
authors.affiliation: Department of Animal, Dairy and Veterinary Sciences, 4815 Old Main Hill, Utah State University, Logan, UT, USA; School of Veterinary Medicine, 4815 Old Main Hill, Utah State University, Logan, UT, USA; Center for Integrated BioSystems, 4700 Old Main Hill, Utah State University, Logan, UT, USA.
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: White
authors.affiliation: Department of Animal, Dairy and Veterinary Sciences, 4815 Old Main Hill, Utah State University, Logan, UT, USA; School of Veterinary Medicine, 4815 Old Main Hill, Utah State University, Logan, UT, USA; Center for Integrated BioSystems, 4700 Old Main Hill, Utah State University, Logan, UT, USA. Electronic address: chris.davies@usu.edu.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Davies
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002460:Cell Line; D005786:Gene Expression Regulation; D015395:Histocompatibility Antigens Class I / Q000145:classification / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins; D051379:Mice; D013045:Species Specificity; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins
keywords: Bovine leukocyte antigens (BoLA); Human K562 cells; Major histocompatibility complex; Mouse P815 cells; Non-classical MHC class I