Domain structures and immunogenic regions of the 90-kDa heat-shock protein (HSP90). Probing with a library of anti-HSP90 monoclonal antibodies and limited proteolysis.

Domain structures of the 90-kDa heat-shock protein (HSP90) have been investigated with a library of anti-HSP90 monoclonal antibodies (mAbs) and by limited proteolysis with trypsin and chymotrypsin. Thirty-three mAbs were obtained by immunization with bacterially expressed human HSP90alpha and HSP90beta isoforms. Among them, ten and three mAbs reacted specifically with HSP90alpha and HSP90beta, respectively. Immunoblotting and enzyme-linked immunosorbent analyses revealed that major immunogenic domains were located at two restricted regions of HSP90alpha, i.e. amino acids 227-310 (designated Region I) and 702-716 (Region II), corresponding to a highly charged region and a region near the C terminus, respectively. Taken together with the characteristics of the amino acid sequences, these two immunogenic regions appeared to be exposed at the outer surface of HSP90. We further investigated the domain structures of HSP90 by limited proteolysis in combination with N-terminal sequencing and immunoblotting analyses. Tryptic cleavages of HSP90alpha at low concentrations revealed the existence of major susceptible sites at Arg400-Glu401, Lys615-Ala616, and Arg620-Asp621. Proteolysis at higher trypsin concentrations caused successive cleavages only toward the N-terminal direction from these sites, and Region I was included in the region selectively deleted during this process, thereby further suggesting its surface location. From these results, we propose three domain structures of HSP90 consisting of amino acids 1-400, 401-615, and 621-732. Differences in the protease sensitivity and immunogenicity further suggest that every domain is composed of two subdomains. This is the first study describing the domain structures and the immunogenic regions of HSP90.