Targeted Disruption of Tyrosylprotein Sulfotransferase-2, an Enzyme That Catalyzes Post-translational Protein Tyrosine O-Sulfation, Causes Male Infertility

Abstract

Tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 and -2) expressed in all mammalian cells. Tyrosine sulfation plays an important role in the function of some known TPST substrates by enhancing protein-protein interactions. To explore the role of these enzymes in vivo and gain insight into other potential TPST substrates, TPST-2-deficient mice were generated by targeted disruption of the Tpst2 gene. Tpst2+/- mice appear normal and, when interbred, yield litters of normal size with a Mendelian distribution of the targeted mutation. Tpst2-/- mice have moderately delayed growth but appear healthy and attain normal body weight by 10 weeks of age. In contrast to Tpst1-/- males that have normal fertility, Tpst2-/- males are infertile. Tpst2-/- sperm are normal in number, morphology, and motility in normal media and appear to capacitate and undergo acrosomal exocytosis normally. However, they are severely defective in their motility in viscous media and in their ability to fertilize zona pellucida-intact eggs. Adhesion of Tpst2-/- sperm to the egg plasma membrane is reduced compared with wild type sperm, but sperm-egg fusion is similar or even increased. These data strongly suggest that tyrosine sulfation of unidentified substrate(s) play a crucial role in these processes and document for the first time the critical importance of post-translational tyrosine sulfation in male fertility. Tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 and -2) expressed in all mammalian cells. Tyrosine sulfation plays an important role in the function of some known TPST substrates by enhancing protein-protein interactions. To explore the role of these enzymes in vivo and gain insight into other potential TPST substrates, TPST-2-deficient mice were generated by targeted disruption of the Tpst2 gene. Tpst2+/- mice appear normal and, when interbred, yield litters of normal size with a Mendelian distribution of the targeted mutation. Tpst2-/- mice have moderately delayed growth but appear healthy and attain normal body weight by 10 weeks of age. In contrast to Tpst1-/- males that have normal fertility, Tpst2-/- males are infertile. Tpst2-/- sperm are normal in number, morphology, and motility in normal media and appear to capacitate and undergo acrosomal exocytosis normally. However, they are severely defective in their motility in viscous media and in their ability to fertilize zona pellucida-intact eggs. Adhesion of Tpst2-/- sperm to the egg plasma membrane is reduced compared with wild type sperm, but sperm-egg fusion is similar or even increased. These data strongly suggest that tyrosine sulfation of unidentified substrate(s) play a crucial role in these processes and document for the first time the critical importance of post-translational tyrosine sulfation in male fertility. Tyrosine O-sulfation is a widespread post-translational modification that was first described about 50 years ago (1Bettelheim F.R. J. Am. Chem. Soc. 1954; 76: 2838-2839Crossref Scopus (121) Google Scholar, 2Huttner W.B. Nature. 1982; 299: 273-276Crossref PubMed Scopus (183) Google Scholar). Tyrosine-sulfated proteins and/or tyrosylprotein sulfotransferase (TPST) 5The abbreviations used are: TPST, tyrosylprotein sulfotransferase; ZP, zona pellucida; LH, luteinizing hormone; FSH, follicle-stimulating hormone; CTβ, cholera toxin β-subunit; IVF, in vitro fertilization; PBS, phosphate-buffered saline; BSA, bovine serum albumin. activity have been described in many species throughout the plant and animal kingdoms, including Volvox carteri, one of the earliest multicellular organisms. At this time, 37 tyrosine-sulfated proteins have been identified in humans, many of which play important roles in inflammation, hemostasis, immunity, and other processes (3Moore K.L. J. Biol. Chem. 2003; 278: 24243-24246Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar, 4Önnerfjord P. Heathfield T.F. Heinegård D. J. Biol. Chem. 2004; 279: 26-33Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 5Choe H. Moore M.J. Owens C.M. Wright P.L. Vasilieva N. Li W. Singh A.P. Shakri R. Chitnis C.E. Farzan M. Mol. Microbiol. 2005; 55: 1413-1422Crossref PubMed Scopus (122) Google Scholar). These include certain adhesion molecules, G-protein-coupled receptors, coagulation factors, serpins, extracellular matrix proteins, hormones, and others. It has been demonstrated that some of these proteins require tyrosine sulfation for optimal function (3Moore K.L. J. Biol. Chem. 2003; 278: 24243-24246Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Nevertheless, it is very likely that we are only beginning to appreciate the complexity of the TPST substrate repertoire. In mice and humans, tyrosine O-sulfation is mediated by one of two tyrosylprotein sulfotransferases, called TPST-1 and TPST-2, which are localized to the trans-Golgi network (7Ouyang Y.B. Lane W.S. Moore K.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2896-2901Crossref PubMed Scopus (147) Google Scholar, 8Ouyang Y.B. Moore K.L. J. Biol. Chem. 1998; 273: 24770-24774Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 9Beisswanger R. Corbeil D. Vannier C. Thiele C. Dohrmann U. Kellner R. Ashman K. Niehrs C. Huttner W.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11134-11139Crossref PubMed Scopus (121) Google Scholar). Mouse TPST-1 and -2 are 370- and 376-residue type II transmembrane proteins, respectively. Each has a short N-terminal cytoplasmic domain, followed by a single ∼17-residue transmembrane domain, a membrane-proximal ∼40-residue stem region, and a luminal catalytic domain containing four conserved cysteine residues and two N-glycosylation sites. The amino acid sequence of human and mouse TPST-1 are ∼96% identical, and human and mouse TPST-2 have a similar degree of identity. TPST-1 is ∼65-67% identical to TPST-2 in both humans and mice. Both isoenzymes are broadly expressed in human and murine tissues and cell lines and are co-expressed in most, if not all, cell types (3Moore K.L. J. Biol. Chem. 2003; 278: 24243-24246Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). However, the relative abundance of the two proteins in tissues and cells is uncertain. The human TPST1 and TPST2 genes are on 7q11.21 and 22q12.1, respectively. The mouse Tpst1 and Tpst2 genes are ∼18.5 megabase pairs apart on chromosome 5. In vitro studies using synthetic peptide acceptors indicate that the two TPST isoenzymes differ in substrate preference. Peptides modeled on the N terminus of P-selectin glycoprotein ligand-1 are sulfated by the two isoenzymes with equal efficiency. In contrast, peptides modeled on sulfation sites of human C4 α chain and heparin cofactor II are sulfated more efficiently by TPST-1 than by TPST-2 (8Ouyang Y.B. Moore K.L. J. Biol. Chem. 1998; 273: 24770-24774Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Seibert et al. (10Seibert C. Cadene M. Sanfiz A. Chalt B.T. Sakmar T.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11031-11036Crossref PubMed Scopus (92) Google Scholar) studied sulfation of a peptide modeled on the N-terminal extracellular domain of human CCR5 that has four potential tyrosine sulfation sites. They showed that TPST-1 preferred Tyr14 over Tyr15 the sulfation TPST-2 preferred Tyr15 over Nevertheless, it is not known if TPST-1 and -2 have substrate in To gain an of the of we generated mice by targeted disruption of the Tpst1 or Tpst2 gene. studies of the Tpst1-/- mice and including on body weight and Y.B. C.E. Moore K.L. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). mice appear normal and, when interbred, yield litters of normal size with a Mendelian distribution of the targeted mutation. of the Tpst1 not showed that disruption of the Tpst1 not of the Tpst2 gene. Tpst1-/- mice appear healthy but have body weight than wild type mice. of Tpst1-/- males and was However, Tpst1-/- have litters than wild type to and we the and of Tpst2 mice. In this we in contrast to Tpst1-/- Tpst2-/- males are infertile. that Tpst2-/- males are and have normal sperm Tpst2-/- males are normal in number, morphology, and motility and appear to capacitate in vitro and undergo exocytosis in to normally. However, they are severely defective in motility in viscous media and in their ability to fertilize eggs. 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C.E. Moore K.L. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) and Tpst2 mice have been that are that are to both of one the Tpst1 or the Tpst2 has on or In both when are interbred, the targeted is the Mendelian that is for normal is not in However, other of the two lines are The that are not Tpst1 and Tpst2 are growth and the of the male of Tpst2-/- mice and wild type are the weeks of age. growth of Tpst2-/- mice wild type The in body weight weeks of when the body weight of male and is that of wild type However, the attain normal body 10 weeks of age. The body of male and Tpst1-/- mice are wild type to the time of weeks of age. However, the growth of Tpst1-/- mice that of wild type mice. Tpst1-/- mice have body weight 10 weeks of Y.B. C.E. Moore K.L. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). is the for Tpst1 the for the growth in Tpst2 mice the tyrosine-sulfated proteins described to (3Moore K.L. J. Biol. 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However, Tpst2-/- males are infertile. identified and that the of Tpst2-/- is an ability of sperm to the is sperm motility in viscous but not in with normal is in the ability of Tpst2-/- sperm to fertilize in vitro a reduced ability of sperm to to the plasma membrane of eggs. sperm a of viscous that sperm with motility that only a of sperm the the sperm with the and are and in a the sperm sperm and, when they to to the The motility is to in this viscous of the and the egg PubMed Scopus Google Scholar). data that the ability of Tpst2-/- sperm to in viscous is severely and a of the we have in Tpst2-/- sperm was in an to if Tpst2-/- sperm defective However, the of we not the or of sperm that have been described for Mol. Biol. 2003; Google Scholar, 2003; PubMed Scopus Google Scholar). The for this are uncertain. the of motility has been in to it has not been in the that were in with Tpst2-/- compared with wild type the for this is these data suggest the that Tpst2-/- males have a in function and/or is a which of the cell the egg by the sperm, of the of the by sperm, sperm-egg fusion of the sperm and egg plasma and egg Biol. 2002; PubMed Google Scholar, P. 2002; PubMed Scopus Google Scholar). that of the egg by Tpst2-/- sperm is severely data indicate that this is not to the of the sperm to the or undergo acrosomal The sperm appear to have a reduced ability to to the egg plasma and data on motility in viscous indicate that defective of by Tpst2-/- sperm is to a reduced ability to the In humans, 37 tyrosine-sulfated proteins have been identified to some of which have been to require tyrosine sulfation for optimal including certain adhesion molecules, G-protein-coupled receptors, coagulation factors, serpins, and The and are two proteins of importance in The human LH, FSH, and have been to sulfated a membrane-proximal in their N-terminal extracellular that are conserved in many including mice S. 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PubMed Scopus Google Scholar). of sperm proteins this but not it Nevertheless, a of the the of tyrosine-sulfated proteins expressed in the male and a more of the relative abundance and the of TPST-1 and At this it is not to the two the and substrates are not However, data in the domain some on the of the TPST in the male for both TPST isoenzymes have been in all of the in mice P. Biol. 2005; PubMed Scopus Google Scholar). In the TPST-1 but not TPST-2 were in type and and mice and in cells mice Biol. 2004; PubMed Scopus Google Scholar). However, in TPST-2 were in both cells and cells in and In that TPST-1 and TPST-2 have roles in vivo and document for the first time the critical importance of O-sulfation in male fertility. the degree of mouse and human TPST-2 and the of in mammalian suggest that tyrosine sulfation to male in of for for with sperm and of for with and and for for and