The long-term goals of this proposal are to understand whether the biosynthesis of glycosaminoglycans and their protein cores occurs through (I) pre- and/or post-transcriptional regulation of biosynthetic enzymes and (2) different cellular compartmentalization of these enzymes and their substrates. Towards achieving these goals we will: (a) Continue our studies on the cloning and expression of the Golgi membrane adenosine 3' phosphate 5' phosphosulfate (PAPS) transporter. Peptide sequence of the purified PAPS transporter will be used to obtain, via PCR, oligonucleotide probes to screen cDNA libraries to yield a full-length clone of this transporter. This will be used to transfect and to study the resulting phenotype of wild type and mutant Chinese hamster ovary cells and to prepare antibodies. This should help to understand the role of the PAPS transporter in the many biological functions attributed to sulfation of macromolecules and to study its sub- Golgi localization. (b) Continue with our studies on the heparin- and heparan sulfate-N-deacetylase/N-sulfotransferase, key enzyme activities in the biosynthesis of these macromolecules which were recently cloned and expressed in our laboratory and found to be single polypeptides with two enzymatic activities. The different domains for the two enzymatic activities will be determined by proteolysis and site directed mutagenesis. Antibodies against the enzymes will be used to study their sub-Golgi localization, and the relationship with other Golgi enzymes and the PAPS transporter. (c) Continue with our studies on the recently characterized and reconstituted rough endoplasmic reticulum ATP transporter. A liposome reconstitution assay will be used to purify the transporter; the purified protein will allow cloning and expression of it and a study of its role in lumenal RER protein folding. (d) Continue with our studies on the purified Golgi membrane UDP-Galactose transporter towards our goal of understanding regulation of galactosylation in vivo and the sub-Golgi localization of the transporter. For either, we will clone the transporter cDNA and express it in wild type and mutant CHO cells defective in the transporter as well as prepare antibodies as described for the Golgi PAPS transporter. (e) Continue with our efforts to clone and express O-sulfotransferases by probable sequence homology with the N-sulfotransferases, using trachea and aorta muscle cells, which have high levels of O- and low of N-sulfated proteoglycans. Together, the above approaches should continue to enable us to better understand the role of proteoglycans in their many biological roles.
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