Effect of single amino acid substitutions on the formation of the PlA and Bak alloantigenic epitopes.
The subunits that comprise the platelet-specific integrin alpha IIb beta 3 are polymorphic in nature, with several allelic forms present in the human gene pool. Minor changes in the secondary and tertiary structures of platelet membrane glycoproteins (GP) IIb and IIIa encoded by these alleles can result in an alloimmune reaction after transfusion or during pregnancy. To better understand the molecular structure of the PlA alloantigen system, located on GPIIIa, and the Bak alloantigen on GPIIb, we used a heterologous mammalian expression system to express these integrin subunits in their known polymorphic forms. An expression vector containing the PlA1 form of a GPIIIa cDNA, which encodes a leucine at amino acid 33 (Leu33), was modified to express the PlA2-associated form encoding a proline at amino acid 33 (Pro33). Similarly, a Baka GPIIb cDNA expressing an isoleucine at amino acid 843 (IIe843) was modified to express the Bakb form containing a serine at the same position (Ser843). Transfection of these vectors into COS cells resulted in the synthesis of GPIIb and GPIIIa molecules that were identical in size to those present in platelet lysates. Immunoprecipitation of the GPIIIa-transfected COS lysates with PlA)-specific alloantisera indicated that the Leu33 form was recognized only by anti-PIA1 sera while the Pro33 form was bound only by anti-PlA2 sera, showing that single amino acid polymorphisms are necessary and sufficient to direct the formation of the PlA1 and PlA2 alloepitopes. Similar experiments with Bak allele-specific expression vectors indicated that while the amino acid polymorphism (IIe843 in equilibrium Ser843) was necessary, posttranslational processing of pro-IIb was required for efficient exposure of both the Baka and Bakb alloepitopes.
Goldberger
A
A
Blood Center of Southeastern Wisconsin, Milwaukee 53233.
Kolodziej
M
M
Poncz
M
M
Bennett
J S
JS
Newman
P J
PJ
D000483:Alleles; D000595:Amino Acid Sequence; D016824:Antigens, Human Platelet / Q000235:genetics / Q000276:immunology*; D001792:Blood Platelets / Q000502:physiology*; D000939:Epitopes / Q000032:analysis; D005822:Genetic Vectors; D006801:Humans; D039661:Integrin beta3; D016023:Integrins / Q000097:blood* / Q000235:genetics / Q000276:immunology; D007519:Isoantigens / Q000235:genetics / Q000276:immunology*; D007930:Leucine; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics
C044561:3a alloantigen, human; D016824:Antigens, Human Platelet; D000939:Epitopes; C061248:ITGB3 protein, human; D039661:Integrin beta3; D016023:Integrins; D007519:Isoantigens; D046911:Macromolecular Substances; D010980:Platelet Membrane Glycoproteins; D012333:RNA, Messenger; D007930:Leucine