Effect of Deletion of Glycoprotein lib Exon 28 on the Expression of the Platelet Glycoprotein IIb/IIIa Complex

We have isolated from an HEL cell cDNA library an alternatively spliced transcript for the platelet membrane glycoprotein Mb (GPIIb) that resulted from the deletion of the 34 amino acids of exon 28 of the GPIIb gene. Confirming an earlier report, we also detected this transcript in platelet mRNA. To determine the consequences of exon 28 deletion on the expression of the GPIIb/IIIa heterodimer, we expressed cDNA for GPIIb-28in COS-1 cells, either individually or simultaneously with a cDNA for GPIIIa. When recombinant GPIIb-28was expressed alone, it did not acquire resistance to the enzyme endo-β-N-acetylglucosaminidase H, was not cleaved into heavy and light chains, and was not transported to the cell surface. However, when recombinant GPIIb-28was coexpressed with recombinant GPIIIa, GPIIb/IIIa het-erodimers were assembled. Nevertheless, these heterodimers failed to complete posttranslational processing and were degraded intracellularly. Exon 28 contains one site for Asn-linked glycosylation. To determine if loss of this glycosylation site was responsible for the effects of exon 28 deletion, we removed the site from the exon 28 of intact GPIIb by oligonucleotide-mediated mutagenesis. However, absence of the carbohydrate appended to exon 28 did not prevent normal GPIIb/IIIa heterodimer expression. Our studies indicate that absence of the amino acids encoded by GPIIb exon 28 sufficiently perturbs the quaternary configuration of the GPIIb/IIIa heterodimer to impair its subsequent intracellular transport and processing. They also indicate that this alternatively spliced form of GPIIb mRNA, although present in megakaryocytes, is unlikely to make a significant contribution to the GPIIb/IIIa complexes expressed on platelets.© 1991 by The American Society of Hematology. 0006–4971 /91 / 7809-0033$3.00/0

我们从人红白血病（HEL）细胞的cDNA文库中分离出一种血小板膜糖蛋白IIb（GPIIb）的可变剪接转录本，它是由GPIIb基因的第28外显子的34个氨基酸缺失导致的。证实了早期的一份报告，我们也在血小板mRNA中检测到了这种转录本。为了确定第28外显子缺失对GPIIb/IIIa异二聚体表达的影响，我们在COS - 1细胞中表达GPIIb - 28的cDNA，要么单独表达，要么与GPIIIa的cDNA同时表达。当重组的GPIIb - 28单独表达时，它没有获得对内切 - β - N - 乙酰氨基葡萄糖苷酶H的抗性，没有被切割成重链和轻链，也没有被转运到细胞表面。然而，当重组的GPIIb - 28与重组的GPIIIa共表达时，GPIIb/IIIa异二聚体得以组装。尽管如此，这些异二聚体未能完成翻译后加工，并在细胞内降解。第28外显子包含一个天冬酰胺连接糖基化位点。为了确定这个糖基化位点的缺失是否是第28外显子缺失产生影响的原因，我们通过寡核苷酸介导的诱变从完整的GPIIb的第28外显子中去除了这个位点。然而，第28外显子上糖基的缺失并没有阻止正常的GPIIb/IIIa异二聚体表达。我们的研究表明，GPIIb第28外显子编码的氨基酸缺失充分扰乱了GPIIb/IIIa异二聚体的四级结构，从而损害其随后的细胞内转运和加工。它们还表明，这种GPIIb mRNA的可变剪接形式虽然存在于巨核细胞中，但不太可能对血小板上表达的GPIIb/IIIa复合物有显著贡献。©1991美国血液学会。0006–4971/91/7809 - 0033$3.00/0