title: Class I MHC alpha 3 domain can function as an independent structural unit to bind CD8 alpha.
abstract: Functional interactions between CD8-dependent cytotoxic T cells and their targets require physical contact between CD8 and a non-polymorphic determinant on the alpha 3 domain of the class I MHC molecule. We developed a cell-free assay to directly monitor this molecular interaction, specifically excluding the participation of other cellular proteins and lipids. This assay employed a soluble CD8 derivative and a plate-bound HLA-A2.1 derivative, alpha 3/MalE, in which the alpha 3 domain has been expressed independently of its neighboring polypeptide domains on the native class I MHC molecule and beta 2-microglobulin (beta 2-m). These proteins were produced using eukaryotic and prokaryotic expression systems, respectively. Our data demonstrated specific, saturable binding between soluble CD8 alpha (sCD8 alpha) and alpha 3/MalE, and the Kd of this interaction was determined to be 4.5 x 10(-7) M. Monoclonal antibodies (mAb) directed against either CD8 or the alpha 3 domain of class I MHC inhibited binding; mAb directed against other sites on class I MHC and beta 2-m did not. Our data suggest that the interaction between CD8 alpha and the alpha 3 domain of class I MHC does not require the participation of neighboring class I sequences or beta 2-m.
authors.affiliation: Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA.
authors.forename: J
authors.initials: J
authors.lastname: Fayen
authors.forename: J H
authors.initials: JH
authors.lastname: Huang
authors.forename: H
authors.initials: H
authors.lastname: Meyerson
authors.forename: D
authors.initials: D
authors.lastname: Zhang
authors.forename: R
authors.initials: R
authors.lastname: Getty
authors.forename: N
authors.initials: N
authors.lastname: Greenspan
authors.forename: M
authors.initials: M
authors.lastname: Tykocinski
mesh_terms: D001483:Base Sequence; D001667:Binding, Competitive; D016827:CD8 Antigens / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D015789:HLA-A2 Antigen / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility
chemical_list: D016827:CD8 Antigens; D017931:DNA Primers; D015789:HLA-A2 Antigen; D011993:Recombinant Fusion Proteins