Yvonne van de Wal

Peptide binding characteristics of the coeliac disease-associated DQ(α1*0501, β1*0201) molecule

Genetic susceptibility to coeliac disease (CD) is strongly associated with the expression of theHLA-DQ2 (α1*0501, β1*0201) allele. There is evidence that this DQ2 molecule plays a role in the pathogenesis of CD as a restriction element for gliadin-specific T cells in the gut. However, it remains largely unclear which fragments of gliadin can actually be presented by the disease-associated DQ dimer. With a view to identifying possible CD-inducing antigens, we studied the peptide binding properties of DQ2. For this purpose, peptides bound to HLA-DQ2 were isolated and characterized. Dominant peptides were found to be derived from two self-proteins: in addition to several sizevariants of the invariant chain (li)-derived CLIP peptide, a relatively large amount of an major histocompatibility complex (MHC) class I-derived peptide was found. Analogues of this naturally processed epitope (MHClα46–63) were tested in a cell-free peptide binding competition assay to investigate the requirements for binding to DQ2. First, a core sequence of 10 amino acids within the MHClα46–63 peptide was identified. By subsequent single amino acid substitution analysis of this core sequence, five putative anchor residues were identified at relative positions P1, P4, P6, P7, and P9. Replacement by the large, positively charged Lys at these positions resulted in a dramatic loss of binding. However, several other non-conservative substitutions had little or no discernable effect on the binding capacity of the peptides.Substitutions at P1 and P4 were most critical, suggesting a more prominent role as anchor residues. Structural features of the DQ2 molecule that may relate to the binding motif and to gluten sensitivity are discussed.

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.

Increased HLA-DQ2-affinity of a synthetic gliadin peptide by acid-induced deamidation of glutamine residues

Presentation of antigenic gliadin peptides by the HLA-DQ2 molecule is considered as a key event in celiac disease pathogenesis. Chemical deamidation of the side chains of glutamine residues might have a strong influence on gliadin peptide binding to the DQ2 molecule. Glutamine deamidation of A-gliadin peptide (45-56) under acidic conditions corresponding to the gastric environment was studied using RP-HPLC, Edman degradation, capillary electrophoresis and electrospray mass spectrometry. Deamidation resulted in peptides with increased DQ2-affinities as assessed in a cell-free binding assay.

Characterization of HLA-DQ-Specific Peptide-Binding Motifs.

Several immunological disorders display a striking association with particular HLA alleles. Although the basis for these HLA-disease associations is not completely understood, it is likely that peptides bound to the disease-associated molecules play a role in pathogenesis. The function of HLA molecules is to bind and present peptide antigens to T-cells. Because of polymorphisms in the peptide-binding site, different HLA molecules bind different sets of peptides since the polymorphic residues within the binding pockets of the HLA molecule determine which amino acid side chains can be bound. Such preferences for certain amino acids (anchor residues) at particular sites in the HLAbound peptide determine the so-called allele-dependent peptide-binding motif (for a review see ref. 1). Such a motif can be used to predict whether a particular peptide will bind to a given HLA-molecule. The characterization of the peptide-binding motifs of disease-associated class II molecules, therefore, can be a useful tool to identify potential disease-inducing peptides and antigens. Peptide-binding motifs can be defined using different approaches: 1. Characterization of naturally processed HLA-bound peptides (2-8) 2. Analysis of large peptide pools from M13 bacteriophage peptide display libraries (9) or synthetic peptide libraries (10-12) 3. Analysis of peptide-binding requirements in assays using peptide analogs with single amino acid substitutions (13-15).