title: Peptide binding to soluble HLA-DR4 molecules produced by insect cells.
abstract: HLA-DR4Dw4 molecules were expressed in insect Sf9 cells. The transmembrane and cytoplasmic domains of the DR4 alpha- and beta-chains were replaced by the carboxy terminal sequence of decay accelerating factor, leading to a phosphatidyl inositol glycan membrane anchor. This structure contains a cleavage site for phosphatidyl inositol-specific phospholipase C, allowing efficient solubilization of the rDR4 molecules. We present evidence that infected insect cells express properly associated surface heterodimers and are able to present antigenic peptides to DR4Dw4-restricted T cell clones. Phosphatidyl inositol-specific phospholipase-cleaved recombinant molecules exhibited in vitro binding characteristics similar to DR4 molecules purified from lymphoblastoid cells. In terms of peptide specificity, pH optimum, kinetics, and affinity they were indistinguishable within the limits of our assay system. However, the peptide binding capacity of the recombinant molecules was higher than that of native DR4 molecules.
authors.affiliation: Pharmaceutical Research New Technologies, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
authors.forename: A
authors.initials: A
authors.lastname: Scheirle
authors.forename: B
authors.initials: B
authors.lastname: Takacs
authors.forename: L
authors.initials: L
authors.lastname: Kremer
authors.forename: F
authors.initials: F
authors.lastname: Marin
authors.forename: F
authors.initials: F
authors.lastname: Sinigaglia
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D015804:HLA-DR4 Antigen / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007313:Insecta; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010455:Peptides / Q000378:metabolism*; D010716:Phosphatidylinositols; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility
chemical_list: D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D015804:HLA-DR4 Antigen; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D010716:Phosphatidylinositols; D011993:Recombinant Fusion Proteins