The long-term goal of this research proposal is to understand whether there is regulation of the biosynthesis of different glycosaminoglycans through direct regulation of their biosynthetic enzymes (enzyme substrates and/or gene expression) and/or through membrane compartmentation of their specific substrates and enzymes. We hope that this will allow us an understanding of the regulation of the biosynthesis of proteoglycans under physiological and pathological conditions. Towards achieving this goal we will: (a) continue our studies on the N-heparan sulfate sulfotransferase, a key enzyme in the biosynthesis of heparan sulfate. Antibodies against the enzyme will be obtained and used to localize the enzyme within the Golgi apparatus by immunoelectromicroscopy. We will also clone its gene and express it in Chinese hamster ovary cell mutants deficient in this enzyme. This should allow one to understand the role of the enzyme and its product in many biological functions attributed to heparan sulfate proteoglycans. (b) Continue our studies on the purification of the Golgi adenosine 3' phosphate 5' phosphosulfate (PAPS) transporter. We will use our recently reconstituted PAPS transport assay into proteoliposomes to monitor the purification of the transporter. For this we will use and antibody against the carboxyatractyloside binding region of the transporter as well as 3' 5'-ADP-Sepharose affinity chromatography. Antibodies against the purified PAPS transporter will be used to study its sub-Golgi localization and its relationship with the N-heparan sulfate sulfotransferase within the Golgi membrane. (c) Continue with our studies on the ATP transporters of the Golgi apparatus and rough endoplasmic reticulum membrane. We will reconstitute these transporters into proteoliposomes and purify them by affinity chromatography. Antibodies against the transporters will be made to study their subcellular localization and the structural relationship among them as well as with the ATP/ADP transporter in mitochondria. (d) Purify the O-heparan sulfate sulfotransferase from rat liver Golgi membranes and prepare antibodies against the enzyme. These will be used to determine its subcellular localization, and whether or not it occurs within the membrane in a complex with the N-heparan sulfate sulfotransferase and the PAPS transporter. Concurrently, we will attempt to complement these studies with mutant Chinese hamster ovary cells deficient in this enzyme and the PAPS transporter. By expressing these genes in these mutants we should be able to understand the specific roles of proteoglycan sulfation in biological systems.
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