| Research Abstract |
We have investigated structural-features, biosynthesis, and processing of three membrane- bound proteins including 5'-nucleotidase (5'-NT), Alkaline phosphatase (ALP), and carcinoembryonic antigen (CEA), demonstrating that those proteins are anchored to the plasma membrane via glycosyl- phosphatidylinositol (GPI).
1. Common features in primary Structures deduced from their cDNAs we isolated and sequenced cDNAs for 5'-NT and ALP from both the rat liver and human placenta. The two proteins are found to have a common feature in their primary structures deduced from the cDNAs; they contain hydrophobic domains with about 20 amino acids at both the NH2 and COOH termini, which are also observed in the primary structure of CEA.
2. Chemical analyses of purified proteins 1) Identification of the NH_2-terminal sequence: 5'-NT and ALP were purified and subjected to determination of their NH_2-terminal sequences. A comparison of the NH_2-terminal sequences between the cDNA-predicted precursors and th
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e purified proteins revealed that the NH_2-terminal hydrophobic domain in each precursor is a signal peptide which is cotranslationally cleaved. 2) Purification and characterization of the COOH-terminal domain: The purified proteins were cleaved into peptide fragments by treatment with protease or BRCN. The hydrophobic COOH- terminal fragment of each protein was purified by sequential chromatographies. Chemical analyses demonstrated that each fragment contains oligopeptide and GPI components such as ethanolamine, mannose, glucosamine, inositol and fatty acids. Each peptide sequence was determined and identified at positions prior to the COOH-terminal hydrophobic domain present in their precursors. The results indicate that the predicted COOH-terminal hydrophobic domain is proteolytically removed and replaced by GPI, which in turn functions as the membrane anchor.
3. Labeling experiments in cultured cells The presence of the GPI anchor in these proteins was confirmed by metabolic labeling experiments. Established cell lines of JEG-3 (for ALP) and QGP-1 (CEA) and isolated hepatocytes (5'-NT) were incubated with ^3H-labeled compounds such as ethanolamine, inositol and fatty acids. It was found that all the labeled compounds are incorporated into these proteins. Less
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