title: Functional characterization of HLA-DRA1*0101/DRB1*0401 molecules expressed in Drosophila melanogaster cells.
abstract: We have expressed the human MHC class II molecule, HLA-DRA1*0101/-DRB1*0401 (DRB1*0401), in Drosophila melanogaster Schneider 2 cells under control of the Drosophila metallothionein promoter. Upon induction with CuSO4, flow cytometry revealed expression of DRB1*0401 on the surface of the Drosophila cells at high levels. The membrane-bound class II molecules could present peptides specifically to DRB1*0401-restricted T cells. Drosophila-expressed DRB1*0401 molecules revealed a decreased N-linked glycosylation as compared to DRB1*0401 molecules purified from a human B-cell line. The purified DRB1*0401 molecules from Drosophila cells were dissociated into subunits in SDS-PAGE but could be stabilized with a peptide known to bind DRB1*0401 with a high affinity, indicating that the recombinant class II molecules from Drosophila cells are either empty or occupied by low affinity endogenous peptides. This assumption was further substantiated by the observation that the class II molecules from Drosophila cells had a much higher peptide-binding capacity than DRB1*0401 molecules derived from the human B-cell line. Otherwise, the two species of DRB1*0401 had similar peptide-binding specificities and affinities. The purified recombinant DRB1*0401 molecules also showed biological activity because immobilized complexes of DRB1*0401 and synthetic peptides specifically stimulated DRB1*0401-restricted T cells.
authors.affiliation: Department of Clinical Immunology, Rigshospitalet, The National University Hospital, Copenhagen, Denmark.
authors.forename: B E
authors.initials: BE
authors.lastname: Hansen
authors.forename: E C
authors.initials: EC
authors.lastname: Andersson
authors.forename: L S
authors.initials: LS
authors.lastname: Madsen
authors.forename: J
authors.initials: J
authors.lastname: Engberg
authors.forename: L
authors.initials: L
authors.lastname: Søndergaard
authors.forename: A
authors.initials: A
authors.lastname: Svejgaard
authors.forename: L
authors.initials: L
authors.lastname: Fugger
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017951:Antigen Presentation; D001402:B-Lymphocytes / Q000276:immunology; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D004331:Drosophila melanogaster; D004355:Drug Stability; D015870:Gene Expression; D006031:Glycosylation; D006684:HLA-DR Antigens / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D059805:HLA-DR alpha-Chains; D059811:HLA-DRB1 Chains; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D006684:HLA-DR Antigens; D059805:HLA-DR alpha-Chains; D059811:HLA-DRB1 Chains; C553505:HLA-DRB1*04:01 antigen; D010455:Peptides; D011994:Recombinant Proteins