Abstract
Processing of antigens for presentation to helper T cells by MHC class II involves HLA-DM (DM) and HLA-DO (DO) accessory molecules. A mechanistic understanding of DO in this process has been missing. The leading model on its function proposes that DO inhibits the effects of DM. To directly study DO functions, we designed a recombinant soluble DO and expressed it in insect cells. The kinetics of binding and dissociation of several peptides to HLA-DR1 (DR1) molecules in the presence of DM and DO were measured. We found that DO reduced binding of DR1 to some peptides, and enhanced the binding of some other peptides to DR1. Interestingly, these enhancing and reducing effects were observed in the presence, or absence, of DM. We found that peptides that were negatively affected by DO were DM-sensitive, whereas peptides that were enhanced by DO were DM-resistant. The positive and negative effects of DO could only be measured on binding kinetics as peptide dissociation kinetics were not affected by DO. Using Surface Plasmon Resonance, we demonstrate direct binding of DO to a peptide-receptive, but not a closed conformation of DR1. We propose that DO imposes another layer of control on epitope selection during antigen processing.
Highlights
Helper T cells recognize antigens as processed peptides bound to the groove of proteins of major histocompatibility complex class II (MHC II)
Soluble HLA-DO heterodimers were transiently expressed in Hi5 insect cells through a Bacculovirus vector and purified via its alpha chain His-tag by using the Ni-NTA affinity column through gravity flow
Our findings provide evidence to the contrary, demonstrating that DO interacts with DR molecules directly, and not through modulating the effect of DM
Summary
Helper T cells recognize antigens as processed peptides bound to the groove of proteins of major histocompatibility complex class II (MHC II). During transport through the endocytic pathway the majority of Ii is removed from MHC II molecules by low pH and acid proteases [3] leaving a proteolytic fragment of Ii called CLIP (class II-associated Ii peptide) bound to MHC class II [4]. Upon reaching the endosomal compartment the Ii chain is cleaved leaving behind only a small peptide fragment known as CLIP bound to the groove of the MHC molecule. DM transiently interacts with empty MHC class II to generate a peptide-receptive conformation, and plays an active role in the selection of specific peptide/MHC class II complexes during antigen processing [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30]
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