Assessing high affinity binding to HLA-DQ2.5 by a novel peptide library based approach.

Here we report on a novel peptide library based method for HLA class II binding motif identification. The approach is based on water soluble HLA class II molecules and soluble dedicated peptide libraries. A high number of different synthetic peptides are competing to interact with a limited amount of HLA molecules, giving a selective force in the binding. The peptide libraries can be designed so that the sequence length, the alignment of binding registers, the numbers and composition of random positions are controlled, and also modified amino acids can be included. Selected library peptides bound to HLA are then isolated by size exclusion chromatography and sequenced by tandem mass spectrometry online coupled to liquid chromatography. The MS/MS data are subsequently searched against a library defined database using a search engine such as Mascot, followed by manual inspection of the results. We used two dodecamer and two decamer peptide libraries and HLA-DQ2.5 to test possibilities and limits of this method. The selected sequences which we identified in the fraction eluted from HLA-DQ2.5 showed a higher average of their predicted binding affinity values compared to the original peptide library. The eluted sequences fit very well with the previously described HLA-DQ2.5 peptide binding motif. This novel method, limited by library complexity and sensitivity of mass spectrometry, allows the analysis of several thousand synthetic sequences concomitantly in a simple water soluble format.

Design of new high-affinity peptide ligands for human leukocyte antigen-DQ2 using a positional scanning peptide library.

Human leukocyte antigen (HLA)-DQ2 (DQA1 x 0501/DQB1 x 0201) is associated with several immune disorders, including celiac disease, which is caused by an inappropriate T-cell response to gluten. Interference with peptide presentation by HLA-DQ2, for example, by the use of peptide blockers, is a possible treatment strategy for such HLA-associated disorders. A successful implementation of this strategy will depend on the identification of ligands that bind much better to HLA-DQ2 than the disease related epitopes. We have used a positional scanning nonapeptide library to determine the optimal amino acids for each position of the HLA-DQ2 binding frame. By combining the optimal residues in each position, we were able to design high affinity binders to HLA-DQ2. Interestingly, the decapeptide with highest affinity was composed of the most favorable residues in each position. This sequence bound 50-fold better than the immunodominant gluten epitope DQ2-alpha-I-gliadin, which makes it an interesting lead compound for the development of blockers. For some natural HLA-DQ2 ligands, the correlation between measured and predicted affinities was poorer, but notably these peptides did not have optimal amino acids at all positions. Our approach represents a straightforward strategy for developing high-affinity binders to HLA class II molecules.

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.