The long-term goal of these studies is to understand the regulation of the pathway for sulfation of macromolecules in mammalian cells. The emphasis of the current proposal is on SO4= transport mediated by DTDST (Diastrophic Dysplasia Sulfate Transporter), mutations in which cause a variety of chondrodysplasias. The goal is to provide an integrated understanding of the relationships among several steps in the sulfation pathway: SO4= transport, steady state intracellular [SO4=], synthesis of the universal SO4= donor PAPS (3'-phosphoadenosine-5'-phosphosulfate), PAPS transport into the trans-Golgi, and rate of sulfation of glycosaminoglycans (GAG) and protein tyrosine residues. The cells to be studied are human and rat chondrocytic and osteoblastic cell lines, which express DTDST and secrete sulfated macromolecules at a high rate. There are 6 Specific Aims. Specific Aim 1 is to quantify the role of plasma membrane sulfate transport in regulating the intracellular SO4= pool. This will be done by defining the catalytic mechanism (e.g., SO4= + H+ exchange for CI) of transport by DTDST and possible regulation of DTDST expression or activity. Specific Aim 2 is to evaluate the role of cysteine and methionine catabolism in supplying SO4= under conditions of varying supply of exogenous SO4= and to determine whether cells can up regulate amino acid catabolism in response to SO4= deficiency. Specific Aim 3 is to measure and understand the factors that govern the rate of synthesis of PAPS in intact cells, including the effect of cytoplasmic SO4= concentration and possible regulation of expression of PAPS synthases. Specific Aim 4 is to determine whether there is substrate channeling of SO4= to PAPS synthase, either from the plasma membrane transporter DTDST or from catabolism of amino acids. Specific Aim 5 is to use cell-free preparations to determine whether PAPS transport into the Golgi is rate-limiting for sulfation of macromolecules. Specific Aim 6 is to measure the possible effect of sulfation rate on the Golgi transit time of the major sulfated protein aggrecan. The general approach in these studies is to perform 35S labeling and pulse-chase experiments as well as RT-PCR and immunodetection of expression levels of key proteins in the sulfation pathway. The difference between the approach proposed here and all the preceding work in this area is that we propose to measure the levels of key intermediates (cytoplasmic SO4=, PAPS, labeled protein in the secretory pathway) and the rates of turnover of these intermediates. These experiments will provide new insights into the regulation of a basic pathway (SO4= supply to sulfotransferases) that is of importance not only in the skeletal system but also in many branches of human pathophysiology.

R01GM026861

1979-07-01

2008-02-29