title: A soluble, single-chain Kd molecule produced by yeast selects a peptide repertoire indistinguishable from that of cell-surface-associated Kd.
abstract: Peptide binding to a soluble, single-chain Kd protein produced by the yeast strain Kluyveromyces lactis, and to Kd molecules on Kd-expressing cells (P815) was studied using radiolabeled Kd-restricted peptides. The stability of the peptide-Kd complexes formed was monitored in the absence and presence of unlabeled competitor peptides. Radioiodination of the Tyr anchor residue in position 2 of the peptide interferes with binding. A Kd-biased peptide library and a modified antigenic peptide in which a second Tyr was added in positions 6 and 8, respectively, were therefore used to assay binding. Recombinant and cell-associated Kd molecules are very similar in the following respects: the ease with which the proteins can be loaded with labeled peptide; the spectrum of peptides selected from a peptide library; the stability of the labeled peptide-Kd complex formed; and the ability to partially dissociate the class I-peptide complex with exogenous, unlabeled peptides. These results imply that measurements of peptide binding to soluble Kd molecules are a reliable indicator of the peptide-binding properties of Kd proteins on living cells. The large quantities of soluble recombinant Kd protein currently available represent an invaluable tool not only for dissecting the molecular mechanisms of antigen presentation but also for vaccinations and the design of T cell-specific toxins.
authors.affiliation: Unité de Biologie Moléculaire du Gène, INSERM U.277, Institut Pasteur, Paris, France.
authors.forename: J P
authors.initials: JP
authors.lastname: Abastado
authors.forename: D M
authors.initials: DM
authors.lastname: Ojcius
authors.forename: A
authors.initials: A
authors.lastname: Casrouge
authors.forename: P
authors.initials: P
authors.lastname: Yeh
authors.forename: T N
authors.initials: TN
authors.lastname: Schumacher
authors.forename: H L
authors.initials: HL
authors.lastname: Ploegh
authors.forename: P
authors.initials: P
authors.lastname: Kourilsky
mesh_terms: D000595:Amino Acid Sequence; D000954:Antigens, Surface / Q000378:metabolism; D001667:Binding, Competitive; D006183:H-2 Antigens / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D007716:Kluyveromyces / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
chemical_list: D000954:Antigens, Surface; D006183:H-2 Antigens; C024755:H-2K(K) antigen; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014443:Tyrosine