Soluble HLA-DQ2 expressed in S2 cells copurifies with a high affinity insect cell derived protein.
We here describe that soluble HLA-DQ2 (sDQ2) molecules, when expressed in Drosophila melanogaster S2 insect cells without a covalently tethered peptide, associate tightly with the D. melanogaster calcium binding protein DCB-45. The interaction between the proteins is stable in S2 cell culture and during affinity purification, which is done at high salt concentrations and pH 11.5. After affinity purification, the sDQ2/DCB-45 complex exists in substantial quantities next to a small amount of free heterodimeric sDQ2 and large amounts of aggregated sDQ2 free of DCB-45. Motivated by the stable complex formation and our interest in the development of reagents which inhibit HLA-DQ2 peptide binding, we have further characterized the sDQ2/DCB-45 interaction. Several lines of evidence indicate that an N-terminal fragment of DCB-45 is involved in the interaction with the peptide binding groove of sDQ2. Further mapping of this fragment of 54 residues identified a pentadecapeptide with high affinity for sDQ2 which may serve as a lead compound for the design of HLA-DQ2 blockers.
Jüse
Ulrike
U
Centre for Immune Regulation, Institute of Immunology, University of Oslo, Oslo, Norway. ulrike.juse@medisin.uio.no
Fleckenstein
Burkhard
B
Bergseng
Elin
E
Sollid
Ludvig M
LM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002460:Cell Line; D002846:Chromatography, Affinity; D019281:Dimerization; D029721:Drosophila Proteins / Q000737:chemistry*; D004331:Drosophila melanogaster; D005813:Genes, Synthetic; D005822:Genetic Vectors / Q000235:genetics; D006683:HLA-DQ Antigens / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D059848:HLA-DQ alpha-Chains; D059866:HLA-DQ beta-Chains; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry*; D017924:Nucleopolyhedroviruses / Q000235:genetics; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012995:Solubility; D014161:Transduction, Genetic
D002135:Calcium-Binding Proteins; C115373:DNA supercoiling factor, Drosophila; D029721:Drosophila Proteins; D006683:HLA-DQ Antigens; D059848:HLA-DQ alpha-Chains; D059866:HLA-DQ beta-Chains; C053186:HLA-DQ2 antigen; C069050:HLA-DQA1 antigen; C069051:HLA-DQB1 antigen; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins