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.
Highlights
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 [7,8,9]
Peptides modeled on sulfation sites of human C4 ␣ chain and heparin cofactor II are sulfated more efficiently by TPST-1 than by TPST-2 [8]
Seibert et al [10] 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 as the initial sulfation site, whereas TPST-2 preferred Tyr15 over Tyr14
Summary
Gene Targeting—Mouse genomic clones were identified and plaquepurified from a 129S6/SvEv spleen Lambda FIX II genomic library (Stratagene) by high stringency hybridization with a full-length mouse TPST-2 cDNA probe. Conditioned media and the SDS extracts were clarified by centrifugation (16,000 ϫ g, 10 min), proteins were precipitated with cold acetone, and the precipitates were washed three times with cold acetone and air-dried These samples were hydrolyzed (0.2 M Ba(OH) in degassed H2O) for 18 h at 110 °C and neutralized by the slow addition of H2SO4 using phenol red as an indicator, as previously described [14]. For observation of sperm in normal viscosity medium, a 40-l drop of the swim-up sample in M199-BSA was placed in glass bottom culture dishes (MatTek Corp.) on a heatable mounting frame held at 37 °C by a Tempcontrol 37-2 digital temperature controller (Carl Zeiss). Cumulus cells were removed by brief incubation (Ͻ5 min) in Whitten’s-HEPES medium containing 3% BSA (Albumax I; Invitrogen) and 0.02% Type IV-S hyaluronidase from bovine testis (Sigma). This analysis assesses the differences between individual mice as well as differences between the two genotypes
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