BIOSYNTHESIS OF A PLATELET GRANULE MEMBRANE PROTEIN

膜糖蛋白的生物合成、结构和功能 仅限于分泌颗粒的研究尚未见报道。这些 糖蛋白可能控制颗粒的包装和分泌 内容。我们用免疫亲和的方法分离出一种糖蛋白。 仅存在于α颗粒中的人血小板层析 免疫细胞化学测定的细胞膜。这种蛋白质也是 存在于巨核细胞、内皮细胞和人类 红白血病(HEL)细胞株。我们建议将其描述为 这种分泌颗粒膜蛋白的生物合成和结构， 指定糖蛋白IIa(GP IIa)。首先，我们将测定它的氨基 酸和碳水化合物的组成。下一步，[35s]中的脉冲追逐研究 蛋氨酸标记的HEL细胞和内皮细胞将被用于 评估蛋白水解性修饰和糖基化在 GP IIa的加工。NH2-和NH2-的部分氨基酸序列分析 COOH-末端的蛋白质将用于制备合成的 多肽和提高兔抗肽抗体。Poly(A)mRNA 从HEL细胞中分离出来的将被用于指导体外翻译 [35S]在微粒体膜存在下蛋氨酸标记GP IIa。 膜中蛋白质的取向将通过以下方法进行检测 蛋白水解酶处理后与抗体的免疫沉淀 完整的蛋白质和NH2-和COOH-末端的多肽。氰化物 将对GP IIa的溴裂解产物进行测序并用于制备 从有限密码子片段合成16聚体寡核苷酸探针 堕落。富含GP IIa的分级大小的mRNA将用于 在表达载体lamda gt11中或在 Okayama-Berg修饰的pBR322。这座图书馆将用 抗GP IIa多克隆抗体或与合成的寡核苷酸结合 探测器。将通过以下方式确认GP IIa克隆的cDNA的身份 杂交选择的体外翻译。如果序列不完整，则为Full 长度的cdna将使用核苷酸限制片段构建为 一种含聚(A)mRNA和逆转录酶的延伸引物。 全长cDNA的限制性片段将被克隆到M13噬菌体和 对重叠的DNA片段进行测序。推导出的完整氨基酸 蛋白质的序列将被用来预测其 膜的结构和取向。结构数据还将 与在其他膜结构域的糖蛋白中发现的相比， 并特别注意可能用作排序的片段 信号。最终，所获得的信息将澄清其功能 分泌颗粒膜糖蛋白及其作用机制的研究 哪些蛋白质被导向分泌颗粒。

The biosynthesis, structure, and function of membrane glycoproteins restricted to secretory granules have not been studied. These glycoproteins may control the packaging and secretion of granule contents. We have isolated a glycoprotein by immunoaffinity chromatography from human platelets which is found only in Alpha-granule membranes as determined by immunocytochemistry. The protein is also present in megakaryocytes, endothelial cells, and in the human erythroleukemia (HEL) cell line. We propose to characterize the biosynthesis and structure of this secretory granule membrane protein, designated glycoprotein IIa (GP IIa). First, we will determine its amino acid and carbohydrate composition. Next, pulse-chase studies in [35S] methionine-labelled HEL cells and endothelial cells will be performed to assess the role of proteolytic modifications and glycosylation in the processing of GP IIa. Partial amino acid sequencing of the NH2- and COOH-terminal ends of the protein will be used to prepare synthetic peptides and raise anti-peptide antibodies in rabbits. Poly(A)+ mRNA isolated from HEL cells will be used to direct in vitro translation of [35S]methionine-labelled GP IIa in the presence of microsomal membranes. The orientation of the protein in the membrane will be examined by protease treatment followed by immunoprecipitation with antibodies to the intact protein and to the NH2- and COOH-terminal peptides. Cyanogen bromide cleavage products of GP IIa will be sequenced and used to prepare synthetic 16-mer oligonucleotide probes from segments of limited codon degeneracy. Size-fractionated mRNA enriched for GP IIa will be used to prepare a cDNA library in the expression vector Lamda gt11 or in the Okayama-Berg modified plasmid pBR322. The library will be screened with polyclonal antibodies to GP IIa or with the synthetic oligonucleotide probes. The identity of cloned cDNAs for GP IIa will be confirmed by hybrid-selected in vitro translation. If the sequence is incomplete, full length cDNA will be constructed using a nucleotide restriction fragment as a primer for extension with poly(A)+ mRNA and reverse transcriptase. Restriction fragments of full length cDNA will be cloned in M13 phage and overlapping DNA segments sequenced. The deduced complete amino acid sequence of the protein will be used to make predictions about its structure and orientation in the membrane. The structural data will also be compared with that found in glycoproteins of other membrane domains, with particular attention to segments which might serve as sorting signals. Ultimately, the information obtained will clarify the function of secretory granule membrane glycoproteins as well as the mechanisms by which proteins are directed to secretory granules.