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 GMP-140. 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 GMP-140. 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 [33S]methionine-labelled GMP-140 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 GMP-140 will be sequenced and used to prepare synthetic 16-mer oligonucleotide probes from segments of limited codon degeneracy. Size-fractionated mRNA enriched for GMP-140 will be used to prepare a cDNA library in the expression vector Lambdagt11 or in the Okayama-Berg modified plasmid pBR322. The library will be screened with polyclonal antibodies to GMP-140 or with the synthetic oligonucleotide probes. The identity of cloned cDNAs for GMP-140 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.
