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. 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 (1, 2). Tyrosine-sulfated proteins and/or tyrosylprotein sulfotransferase (TPST) 5 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 (3-5). 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 (3). 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 (7-9). 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 ϳ17-residue transmembrane domain, a membrane-proximal ϳ40-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 ϳ96% identical, and human and mouse TPST-2 have a similar degree of identity. TPST-1 is ϳ65-67% 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 (3). 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 ϳ18.5 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, pept...

Prostasin is a glycosylphosphatidylinositol-anchored serine protease, with epithelial sodium channel activation and tumor invasion suppression activities. We identified the bladder as an expression site of prostasin. In the mouse, prostasin mRNA expression was detected by reverse transcription and real-time polymerase chain reaction in the bladder, and the prostasin protein was localized by immunohistochemistry in the urothelial cells. In mice injected intraperitoneally with bacterial lipopolysaccharide (LPS), bladder prostasin mRNA expression was downregulated, whereas the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interferon-gamma (IFN-gamma), TNF-alpha, IL-1beta, and IL-6 was upregulated. Viral promoter-driven expression of the human prostasin homolog in the bladder of transgenic mice attenuated the LPS induction of iNOS but did not abolish the induction. LPS induction of COX-2, TNF-alpha, IL-1beta, and IL-6 expression, however, was not reduced by prostasin transgene expression. Liposome-mediated delivery of prostasin-expressing plasmid into mouse bladder produced similar attenuation effects on LPS-induced iNOS expression, while not affecting COX-2 or cytokine induction. Mice receiving plasmid expressing a catalytic mutant prostasin did not manifest the iNOS induction attenuation phenotype. We propose a proteolytic mechanism for prostasin to intercept cytokine signaling during LPS-induced bladder inflammation.

Tyrosine O-sulfation is a post-translational modification catalyzed by two tyrosylprotein sulfotransferases (TPST-1 and TPST-2) in the trans-Golgi network. Tpst2-deficient mice have male infertility, sperm motility defects, and possible abnormalities in sperm-egg membrane interactions. Studies here show that compared with wild-type sperm, fewer Tpst2-null sperm bind to the egg membrane, but more of these bound sperm progress to membrane fusion. Similar outcomes were observed with wild-type sperm treated with the anti-sulfotyrosine antibody PSG2. The increased extent of sperm-egg fusion is not due to a failure of Tpst2-null sperm to trigger establishment of the egg membrane block to polyspermy. Anti-sulfotyrosine staining of sperm showed localization similar to that of IZUMO1, a sperm protein that is essential for gamete fusion, but we detected little to no tyrosine sulfation of IZUMO1 and found that IZUMO1 expression and localization were normal in Tpst2-null sperm. Turning to a discovery-driven approach, we used mass spectrometry to characterize sperm proteins that associated with PSG2. This identified ADAM6, a member of the A disintegrin and A metalloprotease (ADAM) family; members of this protein family are associated with multiple sperm functions. Subsequent studies revealed that Tpst2-null sperm lack ADAM6 and ADAM3. Loss of ADAM3 is strongly associated with male infertility and is observed in knockouts of male germ line-specific endoplasmic reticulum-resident chaperones, raising the possibility that TPST-2 may function in quality control in the secretory pathway. These data suggest that TPST-2-mediated tyrosine O-sulfation participates in regulating the sperm surface proteome or membrane order, ultimately affecting male fertility.Tyrosine O-sulfation is a post-translational modification catalyzed by tyrosylprotein sulfotransferases (TPSTs) 3 (1).Although tyrosine O-sulfation was first described more than 50 years ago (2), the TPSTs were identified just a decade ago (3-5). Tyrosine-sulfated proteins and/or TPST activity have been observed in animals and plants but not in prokaryotes or fungi (1, 6). Most animals' genomes appear to have two genes encoding TPSTs, although only one Tpst gene has been identified in Drosophila (1, 6); a TPST also has recently been identified in Arabidopsis (7). The mammalian enzymes are known as TPST-1 and TPST-2; these two enzymes are broadly expressed in human and murine tissues and are co-expressed in the majority of cell types (1). Tyrosine O-sulfation occurs in the trans-Golgi network, with the luminally oriented catalytic domains of TPSTs mediating the transfer of sulfate from the universal sulfate donor 3Ј-phosphoadenosine 5Ј-phosphosulfate to tyrosine residues in polypeptides (3-5, 8 -10). TPST substrates include a variety of secreted and membrane-anchored proteins, including adhesion molecules, G-protein-coupled receptors, and extracellular matrix proteins. Tyrosine O-sulfation is implicated in protein-protein interactions and in optimization of protein function (1, 11)...

Fertilization-the fusion of gametes to produce a new organism-is the culmination of a multitude of intricately regulated cellular processes. In Caenorhabditis elegans, fertilization is highly efficient. Sperm become fertilization competent after undergoing a maturation process during which they become motile, and the plasma membrane protein composition is reorganized in preparation for interaction with the oocyte. The highly specialized gametes begin their interactions by signaling to one another to ensure that fertilization occurs when they meet. The oocyte releases prostaglandin signals to help guide the sperm to the site of fertilization, and sperm secrete a protein called major sperm protein (MSP) to trigger oocyte maturation and ovulation. Upon meeting one another in the spermatheca, the sperm and oocyte fuse in a specific and tightly regulated process. Recent studies are providing new insights into the molecular basis of this fusion process. After fertilization, the oocyte must quickly transition from the relative quiescence of oogenesis to a phase of rapid development during the cleavage divisions of early embryogenesis. In addition, the fertilized oocyte must prevent other sperm from fusing with it as well as produce an eggshell for protection during external development. This chapter will review the nature and regulation of the various cellular processes of fertilization, including the development of fertilization competence, gamete signaling, sperm-oocyte fusion, the oocyte to embryo transition, and production of an eggshell to protect the developing embryo.