Lack of Tyrosylprotein Sulfotransferase-2 Activity Results in Altered Sperm-Egg Interactions and Loss of ADAM3 and ADAM6 in Epididymal Sperm

Abstract

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)...

Protein disulfide isomerase homolog PDILT is required for quality control of sperm membrane protein ADAM3 and male fertility

“…The GPIase activity of tACE (i.e., the release of glycosylphosphatidylinositol-anchored proteins from the membrane) is implicated in ADAM3 modification during epididymal maturation (32). In addition to tissue-specific factors, recent studies indicated that the ubiquitously expressed proteins, TPST2 (protein-tyrosine sulfotransferase 2) and INPP5B (inositol polyphosphate-5-phosphatase B), are involved in ADAM3 quality control (13,33). Although aberrant folding and modification in cells can be associated with pathological conditions, including amyloid diseases such as Parkinson and Alzheimer's diseases (34,35), the complex relationships between different quality control systems in vivo is not fully understood (36).…”

Role of Posttranslational Protein Modifications in Epididymal Sperm Maturation and Extracellular Quality Control

“…Trimeric complexes between ADAM2-ADAM3-ADAM4, ADAM2-ADAM3-ADAM5, and ADAM2-ADAM3-ADAM6 have also been described [ 56 ]. Interestingly, ADAM3 and ADAM6 are lost from spermatozoa in mice lacking tyrosylprotein sulfotransferase-2 activity, which catalyzes the post-translational modifi cation of tyrosine O-sulfation, suggesting additional PTM of ADAM proteins contribute to their functionality or quality control [ 57 ]. Tyrosine O-sulfation has been shown in other cell systems to play a role in protein complex formation [ 58 ].…”

Testicular and epididymal ADAMs: expression and function during fertilization

“…81 Levels of ADAM18, ADAM24, ADAM26, and ADAM32, on the other hand, remain unchanged in Adam2 -/-and Adam3 -/-testicular cells and mature sperm. 64,66 The significant reductions in ADAM protein levels that have been reported in knockout mouse sperm are unsurprising given the various ADAM complexes that exist in mature sperm and the fact that many ADAMs are degraded if they are unable to form complexes with other ADAMs (Figure 2).…”

“…98 In relation to ADAM3 expression in sperm, it should be noted that male mice with deletions in Ace (encodes angioten-converting enzyme [ ) genes are infertile, and ADAM3 is commonly absent or defective in mature sperm from these mutant mice. 81,85,[99][100][101] The spermatogenesis-specific chaperone CLGN is required for the formation of the ADAM1A-ADAM2 complex, which regulates the integrity of ADAM3. 100,102 The testis-specific calreticulin-like chaperone CALR3, testis-specific protein disulfide iso merase homolog PDILT, and tyrosine-O-sulfation-catalyzing enzyme TPST2 are critical for the stability of ADAM3.…”