The impact of three-dimensional structure on the expression of PIA alloantigens on human integrin β3

Abstract

We have compared the binding of affinity-purified anti-PIA1 IgG from seven nonrelated donors with chimeric integrin subunit β3 molecules expressed in the baculovirus-Sporfoptera frugiperda insect cell system. β3 chimeras were engineered to include segments of antigenic human β3 sequences spliced to intervening segments of nonantigenic Xenopus β3 sequence. Our results clearly show that antibodies from all seven donors will bind to nondenatured molecules containing the antigenic human β3 Cys26-Cys38 loop only when it is presented in a correct orientation that must be maintained by noncontiguous human sequences. Key downstream sequences are located within the region β3288-490, flanking either side of the putative long-range disulfide at Cys435. Although our results confirm unambiguously that the Leu/Pro polymorphism at position 33 in human β3 is necessary for the expression of PIA epitopes, they also indicate that this polymorphic sequence alone is not sufficient. The requirement for additional human β3 sequence transcends the need to maintain a correct orientation within the Cys26-Cys38 loop itself, because the murine monoclonal antibody SZ21, which recognizes the sequence β328-35 contained within the Cys26-Cys38 loop, binds to all chimeras containing this loop, even if the same chimeras are not recognized by anti-PIA1. Our results indicate that additional noncontiguous residues encompassed by the sequence 288-490 either directly contribute to the composition of the PIA1 epitope or, more likely, maintain the Cys26-Cys38 loop in a proper orientation with respect to the remainder of the β3 molecule and thereby maintain proper antigenic presentation of the sequences in that loop. © 1995 by The American Society of Hematology.