| Sulfation is the second most common post-translational modification in the cell, requiring the synthesis of the high-energy sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). In higher organisms, PAPS synthesis is catalyzed by a single bifunctional enzyme, PAPS synthetase (PAPSS). In vertebrates, PAPS synthetase is coded by two genes, PAPSS1 and PAPSS2. The existence of two PAPS synthetase genes with different spatial and temporal expression patterns and unique subcellular localization suggests that these two isoforms have evolved to have distinct contributions to the sulfation pathway during development. To elucidate the functional role of PAPSS2 in mammalian development, the brachymorphic (bm) mouse was used to determine the molecular mechanism by which loss of PAPSS2 results in a post-natal chondrodysplasia. This analysis revealed that chondroitin sulfate proteoglycans (CSPGs) can directly interact with Indian hedgehog (Ihh), and that undersulfation of CSPGs in the bm mouse growth plate was associated with abnormal Ihh signaling and reduced chondrocyte proliferation. Studies of PAPSS1 nuclear localization and 35S-labeling experiments resulted in the identification of the first tyrosine-sulfated nuclear protein, thereby suggesting the existence of a nuclear sulfation pathway. Loss of function studies using RNAi against PAPSS1 showed that PAPSS1 activity can be efficiently downregulated and that loss of PAPSS1 activity in mouse embryonic stem (ES) cells resulted in changes to ES cell morphology. Finally, studies of the glycosaminoglycan (GAG) content of mouse ES cells revealed that ES cells have a unique fingerprint of sulfated GAG with high amounts of chondroitin-4-sulfate and the double sulfated chondroitin-4,6-sulfate. In summary, these studies have elucidated the distinct roles of PAPSS1 and PAPSS2 during mouse development revealing novel contributions of each isoform to the sulfate activation pathway. |
