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进行。 这些 糖蛋白可能控制颗粒的包装和分泌 内容. 我们用免疫亲和法分离了一种糖蛋白 仅在α-颗粒中发现的来自人血小板的层析 膜，如通过免疫细胞化学确定的。 蛋白质也是 存在于巨核细胞、内皮细胞和人体内 红白血病（HEL）细胞系。 我们建议将 这种分泌颗粒膜蛋白的生物合成和结构， 称为糖蛋白IIa（GP IIa）。 首先，我们将确定其氨基 酸和碳水化合物组成。 接下来，[35S]中的脉冲追踪研究 甲硫氨酸标记的HEL细胞和内皮细胞将被执行， 评估蛋白水解修饰和糖基化在 GP IIa的加工。 NH2-和NH3-的部分氨基酸测序 蛋白质的COOH末端将用于制备合成的 并在兔子体内产生抗肽抗体。 Poly（A）+mRNA 从HEL细胞中分离的蛋白质将用于指导体外翻译 [35S]甲硫氨酸标记的GP IIa在微粒体膜的存在下。 蛋白质在膜中的取向将通过 蛋白酶处理，然后用抗 完整的蛋白质和NH 2-和COOH-末端肽。 氰 GP IIa的溴化物裂解产物将被测序并用于制备 由有限密码子区段合成的16聚体寡核苷酸探针 简并 富含GP IIa的大小分级mRNA将用于 在表达载体λ gt11中或在 Okayama-Berg修饰的质粒pBR322。 图书馆将被筛选， GP IIa的多克隆抗体或合成的寡核苷酸 probes. GP IIa克隆cDNA的身份将通过以下方法确认： 杂交选择体外翻译。 如果序列是不完整的， 将使用核苷酸限制性片段构建长度cDNA， 用poly（A）+mRNA和逆转录酶延伸的引物。 将全长cDNA的限制性片段克隆到M13噬菌体中， 重叠DNA片段测序。 推导的完整氨基酸 蛋白质的序列将用于预测其 膜的结构和取向。 结构数据还将 与在其它膜结构域的糖蛋白中发现的相比， 特别注意可能用作分类的片段 信号. 最终，所获得的信息将阐明 的分泌颗粒膜糖蛋白，以及机制， 这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.