Soluble HLA-DQ2 expressed in S2 cells copurifies with a high affinity insect cell derived protein

Abstract

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.

Structural and Functional Studies of trans-Encoded HLA-DQ2.3 (DQA1*03:01/DQB1*02:01) Protein Molecule

“…aggregated DQ2.3 molecules. MS analysis showed that the second peak contained the DQ2.3 molecule and a protein identified as Drosophila super coiling factor (FlyBase database; FlyBaseID: FBgn0025682) (22), likely competing with the linked gluten peptide for binding to the peptide groove. The third peak contained the DQ2.3 molecule and was further purified before crystallization.…”

Use of MHC II Structural Features in the Design of Vaccines for Organ-Specific Autoimmune Diseases

“…The analysis provided above would also be very useful if one were to consider designing blocking peptides in order to contain or even eliminate autoimmune disease. A general problem with this approach is its non-specificity, which would block the allele-specific immune response to any foreign antigen [84][85][86]. Nevertheless, it might be possible to deliver sufficient quantities of such blocking antigen to the site(s) of autoimmune antigen presentation (e.g.…”