The goal of this proposal is to localize by a synthetic approach the 'continuous' antigenic sites of the Beta subunit of human major histocompatibility antigen HLA-DR2. The work is a collaboration between Z. Atassi (Baylor) and P. Cresswell (Duke University). The DR2 Beta subunits will be purified from a DR2 homozygous B-Lymphoblastoid cell line and will be used to raise rabbit and mouse antisera and to prepare DR2 Beta-specific rat and mouse monoclonal antibodies. These antisera, together with a panel of human alloantisera that are either specific to, or cross-reactive with, HLA-DR2 will be used to localize the antigenic sites on the DR2 Beta subunit. We shall employ the synthetic strategy we recently introduced for the localization of continuous antigenic sites in proteins. The approach consists of the synthesis of a series of consecutive overlapping peptides that together represent the entire primary structure of the protein. Thirty overlapping (5-residue overlaps) peptides representing the entire extracellular part of DR2 Beta subunit, the intracellular piece and the various sequences reported in the region 60-69 will be synthesized. The peptides will be studied for their ability to bind the aforementioned polyclonal and monoclonal antibodies. The peptides will also be used to determine the interacting surfaces on the DR2 Beta-subunit with the Alpha- and I-subunits. The approach will narrow the antigenic sites to within 9-13 residues. Further synthesis will be carried out around these indicated regions to achieve narrower delineation of the antigenic sites. Polyclonal and monoclonal antibodies will be prepared against immunochemically active, as well as other, peptides and will be studied for their ability to bind HLA-DR2 and DR2 Beta subunit. These studies will enable better understanding, in molecular terms of the role HLA-DR antigens in immune responses.
