Biomedical subjects

B D Shur

Publications and source records attributed to B D Shur.

At least 37 records · Page 2Linked to original sources

PubMed1994

Evidence that cell surface beta 1,4-galactosyltransferase spontaneously galactosylates an underlying laminin substrate during fibroblast migration.

beta 1,4-Galactosyltransferase is unusual among the glycosyltransferases in that a subpopulation exists on the cell surface in addition to its traditional biosynthetic location within the Golgi complex. On the cell surface, galactosyltransferase is expressed in spatially restricted, cell type-specific domains, where it functions as a receptor for extracellular oligosaccharide ligands during selected cellular interactions. For example, galactosyltransferase is found on the leading and trailing edges of migrating cells, where it facilitates lamellipodia formation and cell spreading by binding to specific N-linked oligosaccharides within laminin. Although the ability of galactosyltransferase to serve as a laminin receptor is well documented, it is unclear whether it functions solely in a lectin-like capacity to bind laminin glycoside ligands or uses its intrinsic catalytic activity to release itself from and modify its oligosaccharide substrate. In this study, we determined whether cell surface galactosyltransferase spontaneously galactosylates laminin matrices during cell migration using endogenous galactose donors. Cells were prelabeled with [3H]galactose, washed, and transferred in small clusters onto laminin matrices. The prelabeled cells migrated out from the cell cluster, during which time they deposited covalently bound [3H]galactose residues onto the laminin matrix. The degree of galactosylation was both laminin- and time-dependent and required actively migrating, intact cells. The radioactivity released from the 3H-galactosylated laminin by acid hydrolysis comigrated with authentic galactose standards on paper chromatography. In parallel assays, there was no radioactivity deposited on laminin matrices when cells were prelabeled with [3H]fucose or [3H]leucine. Furthermore, [3H]galactosylation was dependent upon galactosyltransferase-mediated cell migration, since prelabeled cells did not deposit [3H]galactose when migrating on fibronectin, upon which migration is integrin-dependent and galactosyltransferase-independent. These results raise the possibility that galactosyltransferase functions catalytically during cell migration, either to dissociate from its oligosaccharide ligand and/or to modify the extracellular matrix.

3T3 Cells↗

PubMed1994

Alteration of oligosaccharide biosynthesis by genetic manipulation of glycosyltransferases.

The alteration of oligosaccharide structures through genetic manipulation of glycosyltransferase activities is now a reality. It is apparent that this technique has greater consequences on oligosaccharide structure when an exogenous enzyme is introduced into cells, and in particular when this enzyme is responsible for a terminal glycosylation step. By contrast, only one study has examined the effects of overexpressing an endogenous glycosyltransferase, in which there was no detectable effect on glycosylation. However, there are still other key regulatory biosynthetic enzymes, such as GlcNAc transferase V and beta 1,3 GlcNAc transferase, whose overexpression may alter glycosylation. Both of these enzymes are required for the biosynthesis of polylactosaminoglycans (polymers of N-acetyllactosamine disaccharides), and their elevation in tumor cells correlates with increased expression of polylactosaminoglycans. Recently, the gene encoding GlcNAc transferase V has been isolated, but its transfection into cells and characterization of the resulting oligosaccharides awaits further study. Alternate strategies for modifying oligosaccharide structures could involve the introduction of more than one glycosyltransferase into cells to ensure the availability of biosynthetic intermediates. Alternatively, the disruption of specific glycosyltransferase genes by homologous recombination could be used to eliminate competing glycosyltransferases that act on a common substrate. Although oligosaccharide biosynthesis is directly dependent upon the presence or absence of specific glycosyltransferases, other factors also contribute to glycosylation. For example, the transport rate of a glycoprotein through the endoplasmic reticulum and Golgi complex, the levels of processing glycosidases, the availability of substrates, the host cell, and ultimately, the peptide backbone of the particular glycoprotein of interest are important contributors to the final outcome of oligosaccharide structure. Despite these complications, further study into the manipulation of glycosyltransferase genes may ultimately allow the controlled and predictable biosynthesis of glycoprotein sugar chains.

Animals↗

PubMed1994

Expressing murine beta 1,4-galactosyltransferase in HeLa cells produces a cell surface galactosyltransferase-dependent phenotype.

Beta 1,4-Galactosyltransferase is traditionally viewed as a biosynthetic component of the Golgi complex, but a portion of galactosyltransferase is also expressed on the cell surface, where it has been suggested to function as a receptor for extracellular oligosaccharide ligands. Although results from a variety of studies are consistent with a cell adhesion function for galactosyltransferase, the most rigorous test of surface galactosyltransferase function is to produce a surface galactosyltransferase-dependent phenotype in cells that normally express negligible levels of surface galactosyltransferase. In agreement with previous reports, human HeLa cells were found to express low levels of galactosyltransferase on their surface and, therefore, were stably transfected with cDNAs encoding murine galactosyltransferase. Murine galactosyltransferase was expressed both within the presumed Golgi complex and on the cell surface, as assayed by enzyme activity and with antiserum raised against the bacterially expressed murine enzyme. HeLa cell transfectants adhered more strongly to their extracellular substrates than did control transfectants, as evidenced by a flatter morphology in culture and a more rapid spreading upon plating. In contrast, cell spreading was low and similar among all cell types when plated on extracellular substrates that did not contain binding sites for galactosyltransferase. Antibodies and Fab fragments against recombinant murine galactosyltransferase inhibited the increased cell spreading characteristic of galactosyltransferase transfectants, as did soluble recombinant galactosyltransferase and a variety of galactosyltransferase perturbants. Thus, expression of heterologous galactosyltransferase produces a surface galactosyltransferase-dependent phenotype, confirming its function as a cell adhesion molecule.

Animals↗

PubMed1994

Localization of the long form of beta-1,4-galactosyltransferase to the plasma membrane and Golgi complex of 3T3 and F9 cells by immunofluorescence confocal microscopy.

beta-1,4-Galactosyltransferase (GalTase) is localized to two subcellular compartments, the Golgi complex, where it participates in cellular glycosylation, and the plasma membrane, where it functions as a receptor for oligosaccharide ligands on opposing cells or in the extracellular matrix. The gene for GalTase encodes two nearly identical proteins that differ only in their N-terminal cytoplasmic domains: both short and long GalTases share an 11-aa cytoplasmic tail, but long GalTase has an additional 13-aa sequence on its cytoplasmic domain. In this study, we investigated the subcellular distribution of endogenous long GalTase in untransfected F9 and 3T3 cells by using confocal microscopy and antibodies specific for the 13-aa sequence unique to long GalTase. Long GalTase was found in the Golgi complex as expected; long GalTase was also found on the plasma membrane in cell-type-specific distributions. In 3T3 cells, long GalTase was evident on the basal surface of cells possessing a migratory phenotype, being concentrated at the leading and trailing edges; nonmigratory cells had little detectable surface immunoreactivity. In F9 cells, long GalTase was localized on the plasma membrane, being concentrated at the apical aspect of intercellular junctions. These results demonstrate that in 3T3 and F9 cells, long GalTase is present on the cell surface in addition to the Golgi complex. The pattern of surface expression shows cell-type specificity that is consistent with GalTase function in cellular interactions.

3T3 Cells↗

PubMed1994

Glycobiology. The beginning of a sweet tale.

The diversity of complex carbohydrates has fascinated and frustrated glycobiologists for years. Now, manipulating oligosaccharide composition in the embryo promises new insights into their developmental functions.

Animals↗

PubMed1994

Molecular analysis of cell surface beta-1,4-galactosyltransferase function during cell migration.

Despite the identification and characterization of cell surface receptors for the extracellular matrix, it is unknown how their relative expression and cytoskeletal association regulate cell migration. Previous studies have identified beta-1,4-galactosyltransferase (GalTase; EC 2.4.1.38) on the surface of migrating cells, where it mediates cell migration on basal lamina matrices by associating with the cytoskeleton and binding to N-linked oligosaccharides in the E8 domain of laminin. In this study, the function of GalTase during cell migration was examined directly by analyzing the migration rate of stably transfected cell lines in which the relative level of surface GalTase and its ability to associate with the cytoskeleton were altered. We show here that the cytoskeleton contains a limiting, saturable, number of binding sites for surface GalTase. Furthermore, the rate of cell migration was inversely related to the ability of surface GalTase to associate with the cytoskeleton. Elevating surface GalTase in excess of the number of cytoskeleton-binding sites reduced the rate of cell migration, whereas decreasing the amount of surface GalTase available to bind the cytoskeleton increased migration rates. These results show that the rate of cell migration on basal lamina is directly dependent upon the expression of surface GalTase and the ability of this protein to associate with a limiting number of cytoskeleton-binding sites.

3T3 Cells↗

PubMed1994

Overexpressing sperm surface beta 1,4-galactosyltransferase in transgenic mice affects multiple aspects of sperm-egg interactions.

Sperm surface beta 1,4-galactosyltransferase (GalTase) mediates fertilization in mice by binding to specific O-linked oligosaccharide ligands on the egg coat glycoprotein ZP3. Before binding the egg, sperm GalTase is masked by epididymally derived glycosides that are shed from the sperm surface during capacitation. After binding the egg, sperm-bound oligosaccharides on ZP3 induce the acrosome reaction by receptor aggregation, presumably involving GalTase. In this study, we asked how increasing the levels of sperm surface GalTase would affect sperm-egg interactions using transgenic mice that overexpress GalTase under the control of a heterologous promoter. GalTase expression was elevated in many tissues in adult transgenic animals, including testis. Sperm from transgenic males had approximately six times the wild-type level of surface GalTase protein, which was localized appropriately on the sperm head as revealed by indirect immunofluorescence. As expected, sperm from transgenic mice bound more radiolabeled ZP3 than did wild-type sperm. However, sperm from transgenic animals were relatively unable to bind eggs, as compared to sperm from wild-type animals. The mechanistic basis for the reduced egg-binding ability of transgenic sperm was attributed to alterations in two GalTase-dependent events. First, transgenic sperm that overexpress surface GalTase bound more epididymal glycoside substrates than did sperm from wild-type mice, thus masking GalTase and preventing it from interacting with its zona pellucida ligand. Second, those sperm from transgenic mice that were able to bind the zona pellucida were hypersensitive to ZP3, such that they underwent precocious acrosome reactions and bound to eggs more tenuously than did wild-type sperm. These results demonstrate that sperm-egg binding requires an optimal, rather than maximal, level of surface GalTase expression, since increasing this level decreases sperm reproductive efficiency both before and after egg binding. Although sperm GalTase is required for fertilization by serving as a receptor for the egg zona pellucida, excess surface GalTase is counterproductive to successful sperm-egg binding.

Acrosome↗

PubMed1994

Perturbing cell surface beta-(1,4)-galactosyltransferase on F9 embryonal carcinoma cells arrests cell growth and induces laminin synthesis.

Cell growth and differentiation are influenced by intercellular contact, suggesting that cell adhesion molecules may be instrumental in triggering these events. F9 embryonal carcinoma cells are an ideal system in which to examine the function of cell adhesion molecules in growth and differentiation, since the relevant cell adhesion molecules and differentiation markers are well defined. Intercellular adhesion in F9 cells is mediated by uvomorulin, or E-cadherin, and cell surface beta-(1,4)-galactosyltransferase. Since previous studies suggested that neither F9 cell growth nor differentiation is directly dependent on uvomorulin function, in this study we examined whether cell surface galactosyltransferase plays any role in F9 cell growth or differentiation. A variety of galactosyltransferase perturbants, including anti-galactosyltransferase antibodies, UDPgalactose, and the substrate modifier protein alpha-lactalbumin, inhibited the growth of F9 cells, whereas control reagents did not. To examine this in more detail, we analyzed the effects of perturbing surface galactosyltransferase on progression through the F9 cell cycle. Anti-galactosyltransferase IgG treatment inhibited ornithine decarboxylase activity and lengthened the F9 cell cycle during G1 and G2, the latter mimicking the effects of retinoic acid, a reagent known to prolong the F9 cell cycle and induce differentiation. In contrast, anti-uvomorulin antibodies had no effect on F9 cell growth, ornithine decarboxylase activity, or progression through the cell cycle. Furthermore, perturbation of surface galactosyltransferase adhesions in F9 cell aggregates induced precocious F9 cell differentiation, as assayed by increased laminin synthesis, whereas control reagents had no effect. Thus, perturbing surface galactosyltransferase adhesions in F9 cells both decreases growth and stimulates synthesis of laminin. These results imply that interactions between surface galactosyltransferase and its oligosaccharide ligand during cell adhesion may affect the normal growth-regulatory and differentiation-inducing signals, as is seen, in part, during treatment with retinoic acid.

Animals↗

PubMed1994

Control of stable lamellipodia formation by expression of cell surface beta 1,4-galactosyltransferase cytoplasmic domains.

Mesenchymal cell migration on basal lamina is mediated, in part, by the binding of cell surface beta 1,4-galactosyltransferase (GalTase) to specific N-linked oligosaccharides in the E8 domain of laminin. On migrating cells, surface GalTase is anchored to the cytoskeleton; when GalTase is prevented from associating with the cytoskeleton, lamellipodia formation and subsequent migration are inhibited. To define better the involvement of GalTase-cytoskeleton interactions in cell motility, we examined the lamellipodia formation, polarity and migratory behavior of stably transfected 3T3 fibroblasts expressing increased or decreased levels of GalTase capable of interacting with the cytoskeleton. Initially, the motile behavior of individual cells was quantified in the absence of exogenous stimuli. Cells that overexpress GalTase binding sites for the cytoskeleton changed their polarity more frequently and translocated more erratically than did control cells when assayed on laminin substrata. These differences were not observed, however, when cells were plated on fibronectin, which does not contain binding sites for surface GalTase. GalTase-transfected cells were also assayed for their ability to polarize in response to a specific stimulus. In this case, the ability of a cell to reorient towards a gradient of platelet-derived growth factor was found to be directly proportional to the amount of GalTase associated with the cytoskeleton. Differences in response to platelet-derived growth factor were not due to differences in growth factor binding. Indirect immunofluorescence showed that altering the level of GalTase did not affect the ventrally distributed pool of GalTase stably associated with the cytoskeleton; however, stress fiber formation was inhibited. Thus, increasing surface GalTase binding sites for the cytoskeleton leads to erratic, multipolar behavior in the absence of any vectorial stimulus, but the ability to form a functional lamellipodium in response to a stimulus is dependent upon the amount of surface GalTase associated with the cytoskeleton. Apparently, cells are able to regulate cytoskeletal assembly and lamellipodial stability by altering the expression and/or affinity of appropriate matrix receptors, such as GalTase, and their corresponding binding sites in the cytoskeleton.

3T3 Cells↗

PubMed1993

Beta-1,4-galactosyltransferase expression during spermatogenesis: stage-specific regulation by t alleles and uniform distribution in + -spermatids and t-spermatids.

In the mouse, gamete recognition is mediated in part by the binding of sperm surface beta-1,4-galactosyltransferase (GalTase) to specific oligosaccharide residues on the zona pellucida glycoprotein ZP3 (D. J. Miller, M. B. Macek, and B. D. Shur. Nature 357, 589-593, 1992). The expression of GalTase on the sperm surface is regulated by alleles within the distal segment of the T/t complex and results in a haploid-specific increase in GalTase expression on spermatids and sperm from t-bearing males, suggesting that differences in sperm GalTase activity may contribute to t-sperm transmission ratio distortion (B.D. Shur and N. F. Scully. Genet. Res. Camb. 55, 177-181, 1990). In this study, we characterized the expression of GalTase RNA during wild-type and T/t-mutant spermatogenesis and analyzed the potential role of GalTase in transmission ratio distortion. Using northern blot analysis, S1 nuclease protection assays, and in situ hybridization, it was found that spermatogenic cells predominantly express the long form of the GalTase RNA, which encodes the GalTase protein that is preferentially targeted to the cell surface in somatic cells. In wild-type testes, GalTase RNA accumulates during the maturation of primary spermatocytes, reaches peak levels prior to meiosis, and decreases at meiosis. GalTase RNA accumulates to similar levels during the maturation of +/t and t/t primary spermatocytes, but unlike wild-type, the level of GalTase RNA in t-bearing spermatocytes remains elevated during meiotic division. Consequently, spermatids in t-mutant testes inherit higher levels of GalTase RNA than do wild-type spermatids, which likely accounts for the haploid-specific increase in surface GalTase activity characteristic of spermatids from t-bearing mice. The functional significance of the increased GalTase activity during t-sperm transmission ratio distortion was determined by examining the distribution of GalTase RNA and surface GalTase protein in haploid spermatids from heterozygous +/t males. Result show that+and t spermatids have similar levels of GalTase RNA assayed by quantitative in situ hybridization and similar levels of surface GalTase protein assayed by PCR genotyping of spermatids separated by fluorescence-activated cell sorting. These results indicate that although the expression of GalTase is regulated by alleles within the distal segment of the T/t complex, transmission ratio distortion in +/t mice is not likely due to haploid-specific differences in sperm surface GalTase activity.

Alleles↗

PubMed1993

Uvomorulin, LAMP-1, and laminin are substrates for cell surface beta-1,4-galactosyltransferase on F9 embryonal carcinoma cells: comparisons between wild-type and mutant 5.51 att- cells.

Intercellular adhesions in F9 embryonal carcinoma cells are primarily dependent upon two types of cell adhesion molecules: uvomorulin (Um or E-cadherin), which facilitates intercellular adhesion by homophilic binding, and cell surface beta-1,4-galactosyltransferase (GalTase), which binds terminal N-acetylglucosamine residues on consociate glycoprotein substrates on adjacent cell surfaces. The variant F9 cell line, 5.51 att-, undergoes initial cell aggregation, but fails to form the mature intercellular adhesions characteristic of wild-type F9 cells. The 5.51 att- cells show reduced Um expression; however, previous studies have shown that reduced levels of Um are not responsible for the att- mutant phenotype. Therefore, in this study we determined whether the att- mutant phenotype was the result of altered expression of surface GalTase or of its consociate glycoprotein substrates. Surface GalTase was found to be expressed normally on 5.51 att- cells. Furthermore, the initial intercellular adhesions characteristic of reaggregating 5.51 att- cells were dependent upon surface GalTase, since anti-GalTase antibodies inhibited 5.51 intercellular adhesion. Preliminary studies showed that the interaction of surface GalTase with its consociate glycoprotein substrates was abnormal on 5.51 att- cells. To define the biochemical basis for this observation, specific glycoprotein substrates for cell surface GalTase were identified and their expression and utilization were compared between wild-type and 5.51 att- cells. In wild-type cells, uvomorulin (Um), lysosome-associated membrane protein-1 (LAMP-1), and laminin (Lm) were shown to be substrates for cell surface GalTase, suggesting that they may participate in GalTase-specific adhesions. Moreover, the interaction between GalTase and these glycoproteins exhibited characteristic changes during retinoic acid-induced F9 cell differentiation. In contrast to that seen on wild-type cells, surface GalTase interaction with Um, LAMP-1, and Lm was atypical on 5.51 att- cells, resulting, in part, from increased synthesis of oligosaccharide substrates for surface GalTase, as determined by size exclusion and lectin affinity chromatography. N-Acetylglucosaminyltransferase activity was elevated in 5.51 att- cells and likely responsible for the increased expression of GalTase oligosaccharide substrates. These results suggest that mutant 5.51 att- cells are able to maintain residual intercellular adhesions because of increased expression of oligosaccharide substrates for cell surface GalTase. The impact of aberrant GalTase-specific adhesions on the ability of 5.51 att- cells to form mature intercellular adhesions and to differentiate in response to retinoic acid is discussed.

Animals↗

PubMed1993

Glycosyltransferases as cell adhesion molecules.

Specific glycosyltransferases are believed to be present on the cell surface, where they may function as cell adhesion molecules by binding oligosaccharide substrates on adjacent cell surfaces or in the extracellular matrix. The best studied of these is beta 1,4-galactosyltransferase, which mediates sperm binding to the egg coat and selected cell interactions with the basal lamina. Molecular models have recently been presented to account for the expression of beta 1,4-galactosyltransferase on the cell surface, thus enabling one to alter its surface expression and to examine the consequences on fertilization and development.

Animals↗

PubMed1993

Dominant negative mutation in cell surface beta 1,4-galactosyltransferase inhibits cell-cell and cell-matrix interactions.

In addition to its traditional location within the Golgi complex, beta 1,4-galactosyltransferase (GalTase) is also present on the cell surface, where it is thought to function as a cell adhesion molecule by binding to extracellular oligosaccharide ligands. Recent studies suggest that cells contain two forms of GalTase with distinct cytoplasmic domains. The longer form of GalTase contains a 13-amino acid cytoplasmic extension and is preferentially targeted to the plasma membrane, relative to the shorter GalTase protein that is confined primarily to the Golgi compartment. In this study, we created a dominant negative mutation that interferes with the function of cell surface GalTase by transfecting into cells cDNAs encoding truncated versions of the long form of GalTase containing the complete cytoplasmic and transmembrane domains, but devoid of the catalytic domain. In both F9 embryonal carcinoma cells and Swiss 3T3 fibroblasts, overexpressing the truncated long GalTase (TLGT) protein displaced the endogenous cell surface GalTase from its association with the cytoskeleton, resulting in a loss of intercellular adhesion and cell spreading specifically on matrices that use GalTase as a cell surface receptor. In contrast, overexpressing the analogous truncated short GalTase (TSGT) protein did not affect cell morphology or GalTase activity. In control assays, inducing the TLGT protein had no effect on cell interactions with fibronectin (which is independent of GalTase), or on the cytoskeleton attachment of another matrix receptor (beta 1 integrin), or on overall glycoprotein synthesis, thus eliminating nonspecific effects of the TLGT protein on cellular adhesion and metabolism. These results represent the first molecular manipulation of cell surface GalTase expression and confirm its function as a cell adhesion molecule. These studies further suggest that the cytoskeleton contains a defined, saturable number of binding sites for GalTase, which enables it to function as an adhesion molecule.

3T3 Cells↗

PubMed1993

Egg cortical granule N-acetylglucosaminidase is required for the mouse zona block to polyspermy.

The mammalian egg must be fertilized by only one sperm to prevent polyploidy. In most mammals studied to date, the primary block to polyspermy occurs at the zona pellucida, the mammalian egg coat, after exocytosis of the contents of the cortical granules into the perivitelline space. The exudate acts on the zona, causing it to lose its ability to bind sperm and to be penetrated by sperm previously bound to the zona. However, the cortical granule components responsible for the zona block have not been identified. Studies described herein demonstrate that N-acetylglucosaminidase is localized in cortical granules and is responsible for the loss in sperm-binding activity leading to the zona block to polyspermy. Before fertilization, sperm initially bind to the zona by an interaction between sperm surface GalTase and terminal N-acetylglucosamine residues on specific oligosaccharides of the zona glycoprotein ZP3 (Miller, D. J., M. B. Macek, and B. D. Shur. 1992. Nature (Lond.). 357:589-593). These GalTase-binding sites are lost from ZP3 after fertilization, an effect that can be duplicated by N-acetylglucosaminidase treatment. Therefore, N-acetylglucosaminidase, or a related glycosidase, may be present in cortical granules and be responsible for ZP3's loss of sperm-binding activity at fertilization. Of eight glycosidases assayed in exudates of ionophore-activated eggs, N-acetylglucosaminidase was 10-fold higher than any other activity. The enzyme was localized to cortical granules using immunoelectron microscopy. Approximately 70 or 90% of the enzyme was released from cortical granules after ionophore activation or in vivo fertilization, respectively. The isoform of N-acetylglucosaminidase found in cortical granules was identified as beta-hexosaminidase B, the beta, beta homodimer. Inhibition of N-acetylglucosaminidase released from activated eggs, with either competitive inhibitors or with specific antibodies, resulted in polyspermic binding to the zona pellucida. Another glycosidase inhibitor or nonimmune antibodies had no effect on sperm binding to activated eggs. Therefore, egg cortical granule N-acetylglucosaminidase is released at fertilization, where it inactivates the sperm GalTase-binding site, accounting for the block in sperm binding to the zona pellucida.

Animals↗

PubMed1993

Effects of overexpression of beta 1,4-galactosyltransferase on glycoprotein biosynthesis in F9 embryonal carcinoma cells.

beta 1,4-Galactosyltransferase (GalTase) plays a central role in the biosynthesis of N-acetyllactosamine-containing oligosaccharides. However, despite this seemingly important function, little is known about how changes in the levels of GalTase affect oligosaccharide biosynthesis. We have examined the effects of overexpressing GalTase on the glycosylation of endogenous glycoproteins in F9 mouse embryonal carcinoma cells. Cells transfected with either the short form of the GalTase cDNA (encoding a protein of 386 amino acids) or the long form of the GalTase cDNA (encoding a protein of 399 amino acids) had a 3-fold increase in total GalTase activity, relative to control F9 cells. Analysis of pronase-digested glycopeptides obtained from control and transfected cells after metabolic labelling with [6-3H]galactose revealed no significant qualitative or quantitative differences, as assessed by Bio-Gel P-6 gel filtration chromatography and Tomato lectin affinity chromatography. Furthermore, SDS-PAGE analysis of immunoprecipitated [3H]galactose-labelled lysosomal-associated membrane protein-1 (LAMP-1) glycoprotein showed no difference in amounts or mobility. Pronase digestion and subsequent analysis of the gel-fractionated LAMP-1 glycoproteins also indicated no differences between the various cell lines. The inability of elevated GalTase activity to affect glycosylation was not due to limiting levels of GalTase substrates, since an excess of substrates was detectable in lysed cells using either endogenous or exogenous GalTase and UDP-[3H]galactose. Finally, the subcellular distribution of GalTase, as assessed by sucrose gradient fractionation, was similar between all cell types, thus suggesting that GalTase was appropriately compartmentalized in the transfected cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗

PubMed1993

Cell surface beta 1,4 galactosyltransferase on primary spermatocytes facilitates their initial adhesion to Sertoli cells in vitro.

The Sertoli cell maintains the cytoarchitecture of the seminiferous epithelium and provides support for the developing germ cells through specialized adhesive junctions. Despite the importance of this adhesive interaction, our understanding of germ cell-Sertoli cell interactions is limited. Previous studies have shown that beta 1,4 galactosyltransferase (GalTase) is present on the surface of mature sperm, where it mediates sperm binding to the egg zona pellucida. Since GalTase is present on the surface of early spermatogenic cells long before it is required for sperm-egg recognition, we determined in this study whether GalTase on developing germ cells functions during adhesion to Sertoli cells. Consistent with such a function, GalTase was localized by indirect immunofluorescence to areas of putative germ cell-Sertoli cell contact. More directly, anti-GalTase IgG and Fab fragments inhibited the initial adhesion of spermatocytes to Sertoli cell monolayers; however, anti-GalTase antibodies were less able to inhibit spermatocyte-Sertoli cell adhesions after prolonged co-culture, presumably due to stabilization of the intercellular adhesion. After meiosis, surface GalTase begins to acquire its final distribution overlying the intact acrosome; there it was no longer able to facilitate germ cell adhesion to Sertoli cells. Indirect immunofluorescence and direct enzyme assays showed that Sertoli cells also expressed surface GalTase; however, most GalTase was confined to the basal cell surface where it was inaccessible to germ cells, but where it may function in adhesion to the underlying basal lamina. Preblocking studies confirmed that surface GalTase on the spermatocyte surface, rather than on the exposed apical Sertoli surface, mediated germ cell-Sertoli cell adhesion, presumably by binding to glycoside ligands on the Sertoli cell.

Animals↗

PubMed1993

Sperm require beta-N-acetylglucosaminidase to penetrate through the egg zona pellucida.

Fertilization in the mouse is initiated by sperm beta 1,4-galactosyltransferase (GalTase) binding to terminal N-acetylglucosamine residues on the zona pellucida glycoprotein ZP3. Binding of ZP3 induces exocytosis of the sperm acrosome, whose contents are believed to digest a penetration slit in the zona matrix through which sperm reach the egg. As a consequence of acrosomal exocytosis, GalTase is redistributed to the lateral aspect of the sperm head, where its function remains unknown. In this location, GalTase could conceivably impede zona penetration by binding to N-acetylglucosamine residues exposed on zona pellucida glycoproteins. Therefore, in this study we investigated the presence and function of acrosomal glycosidases capable of removing the GalTase-binding site from zona pellucida glycoproteins. beta-N-acetylglucosaminidase was found at very high levels in sperm, being more than 20-fold higher than other glycosidases assayed. The specific isozymic variant was identified as beta-hexosaminidase B. beta-N-acetylglucosaminidase was localized to sperm acrosomes by biochemical and indirect immunofluorescence studies and was released during the acrosome reaction, as expected for an enzyme involved in zona penetration. To determine if, in fact, acrosomal beta-N-acetylglucosaminidase facilitated penetration through the zona, an assay was developed using eggs that were rendered incapable of triggering the block to polyspermy. A specific competitive inhibitor of beta-N-acetylglucosaminidase activity, PUGNAC, inhibited sperm penetration of the zona in a dose-dependent manner, whereas a closely related beta-glucosidase inhibitor, PUGLU, had no effect on zona penetration or on beta-N-acetylglucosaminidase activity. Neither glycosidase inhibitor affected sperm motility or induction of the acrosome reaction. These results demonstrate that beta-N-acetylglucosaminidase is found in sperm acrosomes and is released during the acrosome reaction, at which time it facilitates sperm penetration through the zona. These results also imply that sperm have developed mechanisms to prevent the formation of stable interactions between surface receptors and their zona pellucida ligands during penetration.

Acetylglucosaminidase↗

PubMed1992

Complementarity between sperm surface beta-1,4-galactosyltransferase and egg-coat ZP3 mediates sperm-egg binding.

Despite its importance, the molecular basis of mammalian gamete recognition has remained unclear. The enzyme beta-1,4-galactosyltransferase (Gal-transferase) has been viewed traditionally as a biosynthetic component of the Golgi complex, but is also found on the surface of many cells where it can bind its specific glycoside substrate on adjacent cell surfaces or in the extracellular matrix. In mouse it has been suggested that Gal-transferase on the sperm head mediates fertilization by binding oligosaccharide residues in the egg coat, or zona pellucida, and that the ability of the zona pellucida to bind sperm is conferred by oligosaccharides of the ZP3 glycoprotein. However, it has not been confirmed that Gal-transferase and ZP3 are in fact complementary gamete receptors whose interaction mediates sperm-egg binding. Here we show that mouse sperm Gal-transferase specifically recognizes those oligosaccharides on ZP3 that have sperm-binding activity, but does not interact with other zona pellucida glycoproteins. In contrast, all zona pellucida glycoproteins are recognized by non-sperm Gal-transferase, demonstrating a more stringent substrate specificity for the sperm enzyme. This interaction is required for sperm-egg binding because blocking or removing the binding site for Gal-transferase on ZP3 inhibits its ability to bind sperm. After the release of the sperm acrosome, the transferase relocalizes to a new membrane domain where it can no longer bind to ZP3, which is consistent with the inability of acrosome-reacted sperm to bind ZP3 or to initiate binding to the zona pellucida. Following fertilization, ZP3 is modified by egg cortical granule secretions so that it loses sperm receptor activity, which can be accounted for by a selective loss of its binding site for sperm Gal-transferase. These results show that sperm surface beta-1,4-galactosyltransferase and the egg-coat glycoprotein ZP3 are complementary adhesion molecules that mediate primary gamete binding in the mouse.

Acrosome↗