Biomedical subjects

M J Wassler

Publications and source records attributed to M J Wassler.

4 recordsLinked to original sources

PubMed2001

Subcellular localization of beta1,4-galactosyltransferase on bull sperm and its function during sperm-egg interactions.

The process of sperm-oocyte recognition is a complex interaction between the plasma membrane of sperm and the extracellular matrix of the oocyte. The best studied mammalian system is the mouse, in which sperm plasma membrane receptors recognize specific oligosaccharides on the egg coat glycoprotein ZP3. A well-defined ZP3 receptor on mouse sperm is beta1,4-galactosyltransferase (GalT). In this study, we investigated the possibility that GalT is present on bull sperm, and that it may participate during bovine sperm-oocyte binding. Using Western immunoblotting, bull sperm were found to have a protein of molecular weight similar to mouse GalT at approximately 60 kDa. Immunogold low voltage scanning electron microscopy reveals that GalT epitopes are confined to the anterior cap of fresh or capacitated bull sperm. To investigate the function of bovine sperm GalT, fresh bull sperm were pretreated with either preimmune or anti-GalT antibody and added to in vitro-matured bovine oocytes. Sperm exposed to preimmune serum fertilized 82.7% (153 of 185) of the oocytes, whereas sperm exposed to anti-GalT antiserum fertilized only 42.3% (202 of 478) of the oocytes. We determined whether the inhibition of fertilization resulted from a direct inhibition of sperm-oocyte binding. The number of sperm bound to eggs was determined by low voltage scanning electron microscopy following pretreatment with preimmune or anti-GalT antibody. An average of 25.3+/-2.2 (mean +/- SEM) sperm bound per half-oocyte when treated with preimmune serum. In contrast, exposure of sperm to anti-GalT antiserum significantly lowered (P<0.001) the frequency of sperm binding to 9.9+/-0.8 bound per half-oocyte. These results show that GalT is present on the anterior cap of the bovine sperm head, where it participates in fertilization by facilitating sperm-oocyte binding. The function of GalT in both the murine and bovine systems suggests that it may serve as a generalized gamete receptor in mammals.

Animals↗

PubMed2001

Galactosyltransferase function during mammalian fertilization.

Gamete recognition has been studied extensively in the mouse. In this system, it is generally believed that sperm bind to a class of O-linked oligosaccharides on the zona pellucida glycoprotein, ZP3. The best characterized sperm receptor for ZP3 is beta1, 4-galactosyltransferase (GalT), which functions in a lectin-like capacity by binding to N-terminal N-acetylglucosamine residues on ZP3 oligosaccharides. Multivalent oligosaccharides on ZP3, as well as synthetic polymers terminating in N-acetylglucosamine aggregate GalT, leading to activation of a heterotrimeric G protein cascade and culminating in the acrosome reaction. Following fertilization, cortical granules release N-acetylglucosaminidase, which removes the binding site for sperm GalT and facilitates the zona block to polyspermic binding. Genetic manipulation of GalT expression has confirmed its function as a ZP3 receptor. Overexpressing GalT on sperm leads to increased binding of ZP3, increased G protein activation, and precocious acrosome reactions. In contrast, sperm from mice made null for GalT by homologous recombination are refractory to ZP3, in that they are unable to bind soluble ZP3 and fail to undergo the acrosome reaction in response to zona glycoproteins. Surprisingly, GalT null sperm still bind to the zona and achieve low rates of fertilization in vitro. This then suggests that sperm-egg binding involves receptor-ligand interactions independent of GalT and ZP3. The current model suggests that GalT functions as the ZP3 receptor that is responsible for inducing the acrosome reaction, whereas initial sperm-zona binding is dictated by other sperm surface receptors. Consistent with this, at least three other zona pellucida monosaccharides have been implicated in sperm binding, and novel sperm surface glycoproteins have been suggested to function in gamete binding. A large scaffolding protein has been identified that associates with the GalT cytoplasmic domain and may be responsible for orchestrating its signal transduction capacities that lead to the acrosome reaction.

Acrosome Reaction↗

PubMed2001

Functional interaction between the SSeCKS scaffolding protein and the cytoplasmic domain of beta1,4-galactosyltransferase.

The beta1,4-galactosyltransferase family contains at least seven unique gene products, of which beta1,4-galactosyltransferase I (GalT) is the most exhaustively studied. GalT exists in the Golgi complex, similar to many other glycosyltransferases, as well as on the cell surface, where it functions as a signaling receptor for extracellular glycoside ligands. When expressed on the surface, GalT associates with the cytoskeleton and, upon ligand-induced aggregation, induces cell-type specific intracellular signal cascades. In an effort to define the mechanisms by which surface GalT exerts these intracellular effects, we used the yeast two-hybrid system to identify proteins that specifically interact with the GalT cytoplasmic domain. The yeast two-hybrid screen identified two distinct clones (1.12 and 2.52) that showed identity to portions of SSeCKS (Src Suppressed C Kinase Substrate). SSeCKS is a previously defined kinase and cytoskeleton scaffolding protein whose subcellular distribution and functions are remarkably similar to those attributed to GalT. Both SSeCKS and GalT have been localized to the perinuclear/Golgi region as well as to filopodia/lamellipodia. SSeCKS and GalT have been implicated in regulating cell growth, actin filament dynamics, and cell spreading. Interestingly, 1.12 and 2.52-GFP constructs were localized to subcellular domains that correlated with the two purported subcellular distributions for GalT; 2.52 being confined to the Golgi, whereas 1.12 localized primarily to filopodia. Coimmunoprecipitation assays demonstrate stable binding between the GalT cytoplasmic domain and the 1.12 and 2.52 domains of SSeCKS in appropriately transfected cells. Similar assays demonstrate binding between the endogenous GalT and SSeCKS proteins also. Coimmunoprecipitation assays were performed in both directions and produced similar results (i.e. using either anti-GalT domain or anti-SSeCKS domain antibodies as the precipitating reagent). A functional interaction between the GalT cytoplasmic domain and SSeCKS was illustrated by the ability of either the 1.12 or 2.52 SSeCKS domain to restore a normal adhesive phenotype in cells overexpressing the TL-GFP dominant negative construct. TL-GFP is composed of the GalT cytoplasmic and transmembrane domains fused to GFP, and leads to a loss of cell adhesion on laminin by displacing the endogenous GalT from its cytoskeleton binding sites. This is the first reported interaction between a glycosyltransferase and a scaffolding protein, and suggests that SSeCKS serve to integrate the various functions ascribed to the GalT cytoplasmic domain.

A Kinase Anchor Proteins↗

PubMed2000

Clustering of cell surface (beta)1,4-galactosyltransferase I induces transient tyrosine phosphorylation of focal adhesion kinase and loss of stress fibers.

It is well appreciated that clustering of receptors for the extracellular matrix, most notably the integrins, elicits intracellular signal cascades. One of the first indications that integrin-dependent signaling has occurred is by the activation of focal adhesion kinase (FAK). Another, although less well understood, receptor for the extracellular matrix is (beta)1, 4-galactosyltransferase I (GalT). GalT participates during lamellipodia formation and cell migration by recognizing terminal N-acetylglucosamine residues on basal lamina glycosides. In this study, we investigated whether GalT is also capable of eliciting intracellular signal cascades, specifically FAK activation, in response to ligand binding and/or aggregation. 3T3 fibroblasts were treated with two different reagents capable of aggregating GalT, either antibodies raised against recombinant GalT or multivalent polymers of N-acetylglucosamine, and the effects on tyrosine phosphorylation were analyzed. Both reagents induced an initial tyrosine phosphorylation (1-2 minutes) and subsequent dephosphorylation (5-10 minutes) of proteins with molecular mass 67 and 125 kDa. These proteins were identified as paxillin and FAK, respectively, by immunoprecipitation with anti-paxillin and anti-FAK antibodies. Preimmune IgG, anti-GalT Fab fragments, irrelevant polymers and monomeric N-acetylglucosamine had no effect. The ability of GalT aggregation to induce transient tyrosine phosphorylation was dependent upon cell density. In addition, FAK dephosphorylation was found to be sensitive to the phosphatase inhibitor, sodium pervanadate. Similar to the integrins, GalT requires association with the cytoskeleton in order to function as a matrix receptor. To determine if the transient tyrosine phosphorylation of FAK was dependent upon GalT binding to the cytoskeleton, stably transfected fibroblasts expressing different amounts of GalT were treated with polymeric N-acetylglucosamine. Cells expressing increased levels of GalT associated with the cytoskeleton showed increased levels of FAK tyrosine phosphorylation and prolonged dephosphorylation, relative to control cells. In contrast, cells in which a dominant negative form of GalT prevents association with the cytoskeleton showed no or weak response to polymeric N-acetylglucosamine. Concomitant with the GalT-stimulated dephosphorylation of FAK, cells treated with anti-GalT antibodies or polymeric N-acetylglucosamine showed a loss of actin stress fibers and focal adhesions. Pervanadate treatment inhibited the GalT-dependent loss of actin stress fibers. To confirm the requirement of GalT in transient FAK phosphorylation and stress fiber reorganization in this system, we created cells homozygous null for the GalT isoform that functions as a matrix receptor. These cells were incapable of phosphorylating FAK in response to GalT agonists and, interestingly, showed a lack of lamellar stress fibers when cultured on basal lamina matrices. These data suggest that GalT function as a basal lamina receptor involves transient activation of FAK and an associated reorganization of stress fibers.

3T3 Cells↗