The major objectives of this proposal are to gain fundamental understanding about the structure and steps in the biosynthesis and targeting of resident proteins in the Golgi apparatus. The work will focus on a specific recombinant glycosyltransferase, namely the murine UDPGal:beta-D-Gal alpha1,3 Galactosyltransferase (alpha1,3-GT). There is currently little information available about the biosynthesis of glycosyltransferases and the factors regulating their intracellular expression. Work in the previous funding period resulted in the cloning and sequencing of the cDNA for this enzyme from the mouse teratocarcinoma cell line F9. Both transient and permanent expression of the gene was accomplished in heterologous cell types, including COS-1 cells and Chinese hamster ovary (CHO) cells. In the proposed work the structure and biosynthesis of the enzyme in three different cell lines - F9 cells; F9 cells induced to permanently differentiate with retinoic acid (RA/F9 cells), a treatment causing elevated expression of both alpha1,3-GT transcripts and enzyme activity; and COS-1 cells transfected with cDNA for alpha1,3-GT - will be investigated.
The specific aims of this renewal application are to: 1. Determine the sites on the alpha1,3-GT for addition of Asn- and/or Ser/Thr-linked oligosaccharides, structurally characterize the oligosaccharides, and identify other potential posttranslational modifications; 2. Define the biosynthesis of the alpha1,3-GT by following it from its formation in the endoplasmic reticulum through the Golgi apparatus and to the cell exterior, where it appears to be eventually secreted; 3. Explore the possibility that there are structural elements within the alpha1,3-GT which target it to specific compartments of the Golgi apparatus using specific mutagenesis procedures and expression in COS-1 cells; and 4. Define the factors responsible for proteolysis and release of the enzyme from cells using the three cell lines mentioned above. Successful completion of the studies proposed will provide fundamental knowledge about the structure, biosynthesis, turnover, and intracellular expression of a developmentally-regulated glycosyltransferase in animal cells. This work will also provide new information about the biosynthesis of the Golgi apparatus and its components.
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