BIOSYNTHESIS OF A PLATELET GRANULE MEMBRANE PROTEIN

血小板颗粒膜蛋白的生物合成

• 批准号: 3347198
• 负责人: RODGER PAUL MCEVER
• 金额: $ 10.92万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1988-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3347198
• 关键词: RNA directed DNA polymerase; affinity chromatography; bacteriophage M13; bacteriophage lambda; carbohydrate structure; complementary DNA; electrophoresis; erythroleukemia; genetic library; genetic translation; histochemistry /cytochemistry; human tissue; immunochemistry; megakaryocytes; methionine; microsomes; molecular cloning; monoclonal antibody; oligonucleotides; platelets; proteolysis; radiotracer; secretion; vascular endothelium

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.

膜糖蛋白的生物合成、结构和功能