Identification of phosphatase that dephosphorylates xylose in the glycosaminoglycan-protein linkage region of proteoglycans

Recently, we demonstrated that FAM20B is a kinase that phosphorylates the xylose (Xyl) residue in the glycosaminoglycan-protein linkage region of proteoglycans. The phosphorylation of Xyl residues by FAM20B enhances the formation of the linkage region. Rapid dephosphorylation is probably induced just after synthesis of the linker and just before polymerization initiates. Indeed, in vitro chondroitin or heparan sulfate polymerization does not occur when the Xyl residue of the tetrasaccharide linkage region is phosphorylated. However, the Enzyme responsible for the dephosphorylation of Xyl remains unknown. Here, we identified a novel protein that dephosphorylates the Xyl residue and designated it 2-phosphoxylose Phosphatase. The Phosphatase efficiently removed the phosphate from the phosphorylated trisaccharide, Galβ1-3Galβ1-4Xyl(2-O-phosphate), but not from phosphorylated tetrasaccharide, GlcUAβ1-3Galβ1-3Galβ1-4Xyl(2-O-phosphate). Additionally, RNA interference-mediated inhibition of 2-phosphoxylose Phosphatase resulted in increased amounts of GlcNAcα1-4GlcUAβ1-3Galβ1-3Galβ1-4Xyl(2-O-phosphate), Galβ1-3Galβ1-4Xyl(2-O-phosphate), and Galβ1-4Xyl(2-O-phosphate) in the cells. Gel filtration analysis of the glycosaminoglycan chains synthesized in the knockdown cells revealed that these cells produced decreased amounts of glycosaminoglycan chains and that the chains had similar lengths to those in the mock-transfected cells. Transcripts encoding this Phosphatase were ubiquitously, but differentially, expressed in human tissues. Moreover, the Phosphatase localized to the Golgi and interacted with the glucuronyltransferase-I involved in the completion of the glycosaminoglycan-protein linkage region. Based on these findings, we conclude that transient phosphorylation of the Xyl residue in the glycosaminoglycan-protein linkage region controls the formation of glycosaminoglycan chains of proteoglycans.

Chondroitin Sulfate; Glycosaminoglycan; Glycosyltransferases; Heparan Sulfate; Phosphatase; Phosphorylation; Proteoglycan; Proteoglycan Synthesis.