title: 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.
authors.affiliation: Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Hoffhines
authors.affiliation: Departments of Molecular and Cellular Biology, University of California, Davis, California 94616.
authors.forename: Constance H
authors.initials: CH
authors.lastname: Jen
authors.affiliation: Departments of Molecular and Cellular Biology, University of California, Davis, California 94616; Department of Chemistry, University of California, Davis, California 94616.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Leary
authors.affiliation: Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104; Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104; Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104; Oklahoma Center for Medical Glycobiology, Oklahoma City, Oklahoma 73104. Electronic address: kevin-moore@omrf.org.
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Moore
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004822:Epididymis / Q000378:metabolism; D047468:Immunoprecipitation; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D008894:Milk Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism*; D013431:Sulfates / Q000378:metabolism*; D015238:Sulfotransferases / Q000235:genetics* / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D000954:Antigens, Surface; D017931:DNA Primers; C490919:Mfge8 protein, mouse; D008894:Milk Proteins; D013431:Sulfates; D015238:Sulfotransferases; C529648:TPST2 protein, mouse; D012260:Ribonucleases