Role of non-anchor residues of Db-restricted peptides in class I binding and TCR triggering.
To understand better, the role of non-anchor residues of class I restricted T cell epitopes in class I binding and TCR stimulation, a panel of peptides was synthesized in which each of the non-anchor positions of the Db-restricted influenza peptide, ASNENMETM, was changed to each of the 20 natural amino acids (AAs). The relative affinity of all the peptides for Db was determined and their ability to stimulate anti-ASNENMETM cytotoxic T cell hybridomas was also assessed. The results illustrated that for Db binding, the AAs with the most solvent exposure had the smallest effect on binding. Changes at other positions affected binding to different degrees. Results for the recognition by the T cell hybridomas indicated that a peptide-MHC complex represents a multitude of epitopes, as each hybridoma recognized a different subset of peptides. Most changes in the highly solvent-exposed residues negatively affected recognition by all hybridomas while changes in other positions affected each hybridoma differently, independent of the direction of the side chain of the AA at that position. Furthermore, the use of saturating concentrations of low and high binding peptides showed that, as long as the class I-peptide complex is formed, the T-cell receptor does not differentiate between high and low binding peptides. This indicates that, although the stability of the class I-peptide complex is highly dependent on peptide affinity, the class I MHC conformation induced by low affinity peptides does not necessarily differ significantly from that induced by high affinity peptides. The results of peptide-class I recognition by one ASNENMETM-specific hybridoma was used to construct a peptide that differed from ASNENMETM at four of the nine residues, yet stimulated the hybridoma to a level comparable to ASNENMETM. In addition, peptides bearing the canonical Db-binding motif but unable to bind to the class I molecule with high affinity could be made to bind Db, by changing unfavorable AAs to favourable ones at appropriate positions. The extended motif determined was used to identify more accurately the peptides derived from Coxsakie b3 virus that would bind Db. It was also shown that some of the canonical characteristics of the peptide motif could be obviated and still obtain high affinity binding, provided optimal AAs, were present at secondary anchor positions.
Sigal
L J
LJ
School of Biological Sciences, University of Nebraska 68588-0118, USA.
Wylie
D E
DE
D000595:Amino Acid Sequence; D000818:Animals; D006183:H-2 Antigens / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D064532:Histocompatibility Antigen H-2D; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D009980:Influenza A virus / Q000276:immunology; D051379:Mice; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000276:immunology* / Q000378:metabolism / Q000502:physiology; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology* / Q000502:physiology*; D011485:Protein Binding / Q000276:immunology; D011487:Protein Conformation; D016601:RNA-Binding Proteins; D011948:Receptors, Antigen, T-Cell / Q000276:immunology / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014758:Viral Core Proteins / Q000276:immunology* / Q000378:metabolism / Q000502:physiology
D006183:H-2 Antigens; D064532:Histocompatibility Antigen H-2D; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D010455:Peptides; D016601:RNA-Binding Proteins; D011948:Receptors, Antigen, T-Cell; D014758:Viral Core Proteins