Mutations inside but not outside the peptide binding cleft of the H-2 Ld molecule affect CTL recognition and binding of the nucleoprotein peptide from the lymphocytic choriomeningitis virus.
In order to investigate the role of residues inside and outside the peptide binding cleft of the Ld molecule in peptide presentation to cytotoxic T lymphocytes (CTL), we constructed a series of point mutations in the Ld gene. We determined the effects of the mutations in the Ld molecule on the binding and recognition of an Ld-restricted CTL epitope derived from the nucleoprotein (NP) of the lymphocytic choriomeningitis virus (LCMV). Each of the mutations within the Ld peptide binding cleft resulted in a complete loss of CTL recognition. Addition of the LCMV NP peptide to cells expressing these mutants did not increase surface Ld expression, suggesting that the mutations altered peptide binding. Mutations involving pockets D and E within the cleft affected LCMV peptide binding and recognition as drastically as those in pocket B, which was predicted to interact with a main anchor residue of the peptide. In striking contrast, the mutations located outside the cleft did not change either recognition or binding. These results demonstrate that the Ld residues in the peptide binding cleft are the main determinants dictating LCMV NP peptide binding, and that the residues in each of the pockets within the cleft play a role in this interaction. Surprisingly, one mutation outside the peptide binding cleft, T92S, abrogated CTL lysis of target cells treated with the LCMV NP peptide, but not virus-infected cells. These data show that this mutation selectively altered the presentation of the LCMV NP peptide introduced to the cell exogenously, but not endogenously. This implies that the pathway by which peptides associate with class I molecules within the cell differs from that of exogenous peptide binding.
Hioe
C E
CE
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill.
McKinney
D M
DM
Frelinger
J A
JA
McMillan
M
M
D000595:Amino Acid Sequence; D000818:Animals; D017951:Antigen Presentation; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D000939:Epitopes / Q000378:metabolism; D005260:Female; D006183:H-2 Antigens / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D007739:L Cells; D008217:Lymphocytic choriomeningitis virus / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009698:Nucleoproteins / Q000276:immunology*; D017354:Point Mutation; D011485:Protein Binding; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D000939:Epitopes; D006183:H-2 Antigens; D009698:Nucleoproteins