Abstract
The T cell co-receptor, CD8, binds to the alpha 3 domain of HLA class I (Salter, R.D., R.J. Benjamin, P.K. Wesley, S.E. Buxton, T.P.J. Garrett, C. Clayberger, A.M. Krensky, A.M. Norman, D.R. Littman, and P. Parham. 1990. Nature [Lond.]. 345:41; Connolly, J.M., T.A. Potter, E.M. Wormstall, and T.H. Hansen. 1988. J. Exp. Med. 168:325; and Potter, T.A., T.V. Rajan, R.F. Dick II, and J.A. Bluestone. 1989. Nature [Lond.]. 337:73). To identify regions of CD8 that are important for binding to HLA class I, we performed a mutational analysis of the CD8 molecule in the immunoglobulin (Ig)-like variable domain. Our mutational analysis was based on our finding that using a cell-cell adhesion assay murine CD8 (Lyt-2) did not bind to human class I. Since the interaction of human CD8 with HLA class I is species specific, we substituted nonconservative amino acids from mouse CD8 and analyzed the ability of the mutated CD8 molecules expressed in COS 7 cells to bind HLA class I-bearing B lymphoblastoid cells, UC. Mutants with the greatest effect on binding were located in a portion of the molecule homologous to the first and second hypervariable regions of an antibody combining site. In addition, a panel of 12 anti-CD8 monoclonal antibodies were used to stain the 10 CD8 mutants, and amino acids that affected antibody binding were localized on the crystal structure of the Bence-Jones homodimer, REI. Support for an Ig-like structure of CD8 can be found in the pattern of substitutions affecting antibody binding. This work supports the similar tertiary structure of the CD8 alpha-terminal domain and an Ig variable domain.
Highlights
The human CD8 molecule is expressed either as an al a homodimer or as an a/(3 heterodimer
We identified two regions of the CD8 molecule that are important for the species-specific interaction of CD8a with HLA class I
Because the least homology between human and mouse CD8 exists in the Ig-like domain (42% vs. 64-80% for the cytoplasmic and transmembrane regions, respectively), we hypothesized that this difference could lead to differences in ability of these two molecules to bind to HLA class I
Summary
Human CD8a cDNA [23]. Was subcloned into the HindIII-BamHI site of Bluescript SK. Mouse CD8a was cut out of the PM7-CD8L vector [24] with. XbaI and Pstl and subcloned into the BamHI-Pstl sites of Bluescript. Human and mouse CD8 were excised from the Bluescript vector with BamHI-HindIII, and HindIII-Xba I, respectively. The inserts were blunted with the Klenow fragment of DNA polymerase I, ligated to EcoRI adaptors (New England Biolabs, Beverly, MA), and subcloned into the EcoRI site of the pCDILSRa296 vectors [25]. CD8 mutants Ml-M10 were generated by oligonucleotide-directed mutagenesis with single-stranded phagemid
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