The impact of three-dimensional structure on the expression of PlA alloantigens on human integrin beta 3.
We have compared the binding of affinity-purified anti-PlA1 IgG from seven nonrelated donors with chimeric integrin subunit beta 3 molecules expressed in the baculovirus-Spodoptera frugiperda insect cell system. Beta 3 chimeras were engineered to include segments of antigenic human beta 3 sequences spliced to intervening segments of nonantigenic Xenopus beta 3 sequence. Our results clearly show that antibodies from all seven donors will bind to nondenatured molecules containing the antigenic human beta 3 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 beta 3(288-490), 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 beta 3 is necessary for the expression of PlA epitopes, they also indicate that this polymorphic sequence alone is not sufficient. The requirement for additional human beta 3 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 beta 3(28-35) contained within the Cys26-Cys38 loop, binds to all chimeras containing this loop, even if the same chimeras are not recognized by anti-PlA1. Our results indicate that additional noncontiguous residues encompassed by the sequence 288-490 either directly contribute to the composition of the PlA1 epitope or, more likely, maintain the Cys26-Cys38 loop in a proper orientation with respect to the remainder of the beta 3 molecule and thereby maintain proper antigenic presentation of the sequences in that loop.
Honda
S
S
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92017, USA.
Honda
Y
Y
Bauer
B
B
Ruan
C
C
Kunicki
T J
TJ
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000276:immunology; D016824:Antigens, Human Platelet / Q000737:chemistry* / Q000276:immunology; D001483:Base Sequence; D016384:Consensus Sequence; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006801:Humans; D039661:Integrin beta3; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera
D015703:Antigens, CD; D016824:Antigens, Human Platelet; D000939:Epitopes; C061248:ITGB3 protein, human; D039661:Integrin beta3; D011993:Recombinant Fusion Proteins