Tyrosylprotein Sulfotransferase-2 Expression Is Required for Sulfation of RNase 9 and Mfge8 in Vivo

Abstract

Protein-tyrosine sulfation is mediated by two Golgi tyrosyl-protein sulfotransferases (TPST-1 and TPST-2) that are widely expressed in vivo. However, the full substrate repertoire of this enzyme system is unknown and thus, our understanding of the biological role(s) of tyrosine sulfation is limited. We reported that whereas Tpst1(-/-) male mice have normal fertility, Tpst2(-/-) males are infertile despite normal spermatogenesis. However, Tpst2(-/-) sperm are severely defective in their motility in viscous media and in their ability to fertilize eggs. These findings suggest that sulfation of unidentified substrate(s) is crucial for normal sperm function. We therefore sought to identify tyrosine-sulfated proteins in the male genital tract using affinity chromatography on PSG2, an anti-sulfotyrosine monoclonal antibody, followed by mass spectrometry. Among the several candidate tyrosine-sulfated proteins identified, RNase 9 and Mfge8 were examined in detail. RNase 9, a catalytically inactive RNase A family member of unknown function, is expressed only in the epididymis after onset of sexual maturity. Mfge8 is expressed on mouse sperm and Mfge8(-/-) male mice are subfertile. Metabolic labeling coupled with sulfoamino acid analysis confirmed that both proteins are tyrosine-sulfated and both proteins are expressed at comparable levels in wild type, Tpst1(-/-), and Tpst2(-/-) epididymides. However, we demonstrate that RNase 9 and Mfge8 are tyrosine-sulfated in wild type and Tpst1(-/-), but not in Tpst2(-/-) mice. These findings suggest that lack of sulfation of one or both of these proteins may contribute mechanistically to the infertility of Tpst2(-/-) males.