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Barry D Shur

Publications and source records attributed to Barry D Shur.

12 recordsLinked to original sources
PubMed2006

A beta1,4-galactosyltransferase is required for convergent extension movements in zebrafish.

Our understanding of how complex carbohydrates function during embryonic development is still very limited, primarily due to the large number of glycosyltransferases now known to be involved in their synthesis. To overcome these limitations, we have taken advantage of the zebrafish system to analyze the function of complex carbohydrates during development by down-regulating the expression of specific glycosyltransferases. Herein, we report the identification of the zebrafish ortholog of mammalian beta1,4-galactosyltransferase I, beta4GalT1, and its requirement for proper convergent extension movements during gastrulation. beta4GalT1 is expressed in the oocyte and throughout the embryo during the first 24 h of development. Knockdown of zebrafish beta4GalT1 by two independent morpholino oligonucleotides results in embryos with a truncated anterior-posterior axis, as well as elongated somites and moderate defects in the patterning of the head mesenchyme. Co-injection of zebrafish beta4GalT1 mRNA returns galactosyltransferase activity to control levels and rescues the defects produced by morpholino oligonucleotides. In situ hybridizations of various molecular markers reveal that the axial mesoderm of epiboly stage embryos is abnormally widened in beta4GalT1 morphants, indicative of abnormal convergent extension. Consistent with this, the rate of anterior-posterior axis elongation is reduced relative to control-injected embryos, similar to that seen in known convergent extension mutants. Among the many potential substrates for beta4GalT1 is laminin, a principle component of the extracellular matrix that supports cell movements such as those that occur during convergent extension. Previous in vitro studies have shown that the galactosylation status of laminin directly influences its ability to support cell spreading and migration. In this regard, laminin isolated from beta4GalT1 morphant embryos is poorly galactosylated, which may contribute to defective cell migration during convergent extension movements. This work demonstrates that zebrafish can be used to identify critical developmental roles for specific glycosyltransferases that would not be obvious otherwise, such as an absolute requirement for beta4GalT1 during convergent extension movements.

Amino Acid Sequence↗
PubMed2006

A beta1,4-galactosyltransferase is required for Bmp2-dependent patterning of the dorsoventral axis during zebrafish embryogenesis.

Complex carbohydrates are highly polymorphic macromolecules that are involved in diverse biological processes; however, a detailed understanding of their function remains obscure. To better define the roles of complex carbohydrates during vertebrate embryogenesis, we have initiated an analysis of glycosyltransferase function using the zebrafish system. In this study, we report the characterization of a zebrafish beta1,4-galactosyltransferase (GalT), which has substantial homology with mammalian beta4GalT5 and is expressed zygotically throughout the zebrafish embryo. Downregulating the expression of beta4GalT5 by injection of specific morpholino oligonucleotides results in dorsalized zebrafish embryos, suggesting a role of beta4GalT5 in Bmp2-mediated specification of the dorsoventral axis. Consistent with this, morpholino-injected embryos have ventrally expanded chordin expression and reduced activation of the Bmp-dependent transcription factors Smad1/5/8. Because other growth factors, such as Egf and Fgf, require binding to extracellular proteoglycans for delivery and/or binding to their cognate receptors, we examined whether proteoglycans isolated from control and morpholino-injected embryos show differential binding affinities for Bmp2. In this regard, proteoglycans isolated from beta4GalT5 morphant embryos are underglycosylated and are unable to bind recombinant Bmp2 as efficiently as proteoglycans from control-injected embryos, whereas the binding of Bmp7 is relatively unaffected. These results suggest that beta4GalT5 is a previously unidentified zebrafish galactosyltransferase that is essential for proper patterning of the dorsoventral axis by regulating Bmp2 signaling. Furthermore, this work demonstrates that a relatively simple carbohydrate modification to endogenous proteoglycans can modulate the specificity of cytokine signaling.

Amino Acid Sequence↗
PubMed2006

Identification of novel gamete receptors that mediate sperm adhesion to the egg coat.

Mammalian fertilization is initiated by the species-specific binding of sperm to the zona pellucida, or egg coat. Earlier studies suggested that sperm-egg adhesion in mouse is mediated by the binding of beta1,4-galactosyltransferase-I (GalT) on the sperm surface to specific glycoside ligands on the egg coat glycoprotein, ZP3. Binding of multiple ZP3 oligosaccharides induces GalT aggregation, triggering a pertussis toxin-sensitive G-protein cascade leading to induction of the acrosome reaction. Consistent with this, sperm bearing targeted deletions in GalT are unable to bind ZP3 nor undergo ZP3-dependent acrosomal exocytosis; however, GalT-null sperm are still able to bind to the egg coat. This indicates that sperm-egg binding requires at least two independent binding mechanisms: a GalT-ZP3-independent event that mediates initial adhesion, followed by a GalT-ZP3 interaction that facilitates acrosomal exocytosis. During the past few years, novel GalT-ZP3-independent gamete receptors have been identified that appear to participate in initial gamete adhesion. On such receptor is SED1, an EGF repeat and discoidin domain protein that coats sperm as they traverse through the epididymis, and which is required for sperm to bind the egg coat. Similarly, a novel egg coat ligand is present on ovulated oocytes, but not on ovarian eggs, and which also appears to function in initial sperm binding. The identification of novel gamete receptors that are required for sperm-egg binding opens up new avenues for the development of specific contraceptive strategies.

Animals↗
PubMed2004

SED1 function during mammalian sperm-egg adhesion.

A prerequisite for successful fertilization is the species-specific binding of sperm to the extracellular coat of the egg. Gamete binding triggers the release of sperm hydrolytic enzymes that digest a path through the egg coat, thus bringing sperm into proximity with the egg plasma membrane where gamete fusion occurs. Although some components of the sperm membrane and the egg coat that participate in sperm-egg interactions have been identified, results from targeted deletions and gene substitutions indicate that other, as yet unidentified, gamete receptors must contribute to sperm-egg binding. Recent studies implicate the bi-motif protein, SED1, as being required for successful sperm-egg adhesion in mouse. SED1 contains Notch-like EGF repeats as well as discoidin/F5/8 complement domains--motifs that mediate a variety of cell-cell and cell-matrix interactions. SED1's ability to promote gamete adhesion resides within its two discoidin/F5/8C domains, which are able to dock to substrates as diverse as phospholipid membranes and extracellular matrices. SED1 is also expressed in a wide range of tissues and epithelia, where it may function similarly as an adhesive protein facilitating cell-cell and/or cell-matrix interactions.

Amino Acid Sequence↗
PubMed2003

Sperm from beta1,4-galactosyltransferase I-null mice exhibit precocious capacitation.

Mammalian sperm must undergo a physiological maturation, termed capacitation, before they are able to fertilize eggs. Despite its importance, the molecular mechanisms underlying capacitation are poorly understood. In this paper, we describe the capacitation phenotype of sperm lacking the long isoform of beta1,4-galactosyltransferase I (GalT I), a sperm surface protein that functions as a receptor for the zona pellucida glycoprotein, ZP3, and as an inducer of the acrosome reaction following ZP3-dependent aggregation. As expected, wild-type sperm must undergo capacitation in order to bind the zona pellucida and undergo a Ca(2+) ionophore-induced acrosome reaction. By contrast, GalT I-null sperm behave as though they are precociously capacitated, in that they demonstrate maximal binding to the zona pellucida and greatly increased sensitivity to ionophore-induced acrosome reactions without undergoing capacitation in vitro. The loss of GalT I from sperm results in an inability to bind epididymal glycoconjugates that normally maintain sperm in an 'uncapacitated' state; removing these decapacitating factors from wild-type sperm phenocopies the capacitation behavior of GalT I-null sperm. Interestingly, capacitation of GalT I-null sperm is independent of the presence of albumin, Ca(2+) and HCO(3)(-); three co-factors normally required by wild-type sperm to achieve capacitation. This implies that intracellular targets of albumin, Ca(2+) and/or HCO(3)(-) may be constitutively active in GalT I-null sperm. Consistent with this, GalT I-null sperm have increased levels of cAMP that correlate closely with both the accelerated kinetics and co-factor-independence of GalT I-null sperm capacitation. By contrast, the kinetics of protein tyrosine phosphorylation and sperm motility are unaltered in mutant sperm relative to wild-type. These data suggest that GalT I may function as a negative regulator of capacitation in the sperm head by suppressing intracellular signaling pathways that promote this process.

Acrosome Reaction↗
PubMed2003

Characterization of a novel ZP3-independent sperm-binding ligand that facilitates sperm adhesion to the egg coat.

During mammalian fertilization, sperm adhere to the extracellular coat of the egg, or zona pellucida, in a species-specific manner. In mouse, evidence suggests that sperm recognize and bind to specific oligosaccharide ligands within the zona pellucida glycoprotein, ZP3, via beta1,4-galactosyltransferase I (GalT I), a lectin-like receptor on the sperm surface. Although in vitro experiments using isolated gametes lend support to this model, recent in vivo studies of genetically altered mice question whether ZP3 and/or GalT I are solely responsible for sperm-egg binding. In this regard, sperm from GalT I-null mice bind poorly to ZP3 and fail to undergo a zona-induced acrosome reaction; however, they still bind to the ovulated egg coat in vitro. In this report, we characterize a novel ZP3- and GalT I-independent mechanism for sperm adhesion to the egg coat. Results show that the ovulated zona pellucida contains at least two distinct ligands for sperm binding: a ZP3-independent ligand that is peripherally associated with the egg coat and facilitates gamete adhesion; and a ZP3-dependent ligand that is present in the insoluble zona matrix and is recognized by sperm GalT I to facilitate acrosomal exocytosis. The ZP3-independent ligand is not a result of contamination by egg cortical granules, nor is it the mouse homolog of oviduct-specific glycoprotein. It behaves as a 250 kDa, WGA-reactive glycoprotein with a basic isoelectric point, distinguishing it from the acidic glycoproteins that form the insoluble matrix of the egg coat. When eluted from isoelectric focusing gels, the acidic matrix glycoproteins possess sperm-binding activity for wild-type sperm, but not for GalT I-null sperm, whereas the basic glycoprotein retains sperm-binding activity for both wild-type and GalT I-null sperm. Thus, GalT I-null sperm are able to resolve gamete recognition into at least two distinct binding events, leading to the characterization of a novel, peripherally associated, sperm-binding ligand on the ovulated zona pellucida.

Acetylglucosaminidase↗
PubMed2003

Mutational analysis of the cytoplasmic domain of beta1,4-galactosyltransferase I: influence of phosphorylation on cell surface expression.

Beta1,4-galactosyltransferase I (GalT I) exists in two subcellular compartments where it performs two distinct functions. The majority of GalT I is localized in the Golgi complex where it participates in glycoprotein biosynthesis; however, a small portion of GalT I is expressed on the cell surface where it functions as a matrix receptor by binding terminal N-acetylglucosamine residues on extracellular glycoside ligands. The GalT I polypeptide occurs in two alternate forms that differ only in the length of their cytoplasmic domains. It is thought that the longer cytoplasmic domain is responsible for GalT I function as a cell surface receptor because of its ability to associate with the detergent-insoluble cytoskeleton. In this study, we demonstrate that the long GalT I cytoplasmic and transmembrane domains are capable of targeting a reporter protein to the plasma membrane, whereas the short cytoplasmic and transmembrane domains do not have this property. The surface-localized GalT I reporter protein partitions with the detergent-insoluble pool, a portion of which co-fractionates with caveolin-containing lipid rafts. Site-directed mutagenesis of the cytoplasmic domain identified a requirement for serine and threonine residues for cell surface expression and function. Replacing either the serine or threonine with aspartic acid reduces surface expression and function, whereas substitution with neutral alanine has no effect on surface expression or function. These results suggest that phosphorylation negatively regulates GalT I function as a surface receptor. Consistent with this, phosphorylation of the endogenous, full-length GalT I inhibits its stable expression on the cell surface. Thus, the 13 amino acid extension unique to the long GalT I isoform is required for GalT I expression on the cell surface, the function of which is regulated by phosphorylation.

3T3 Cells↗
PubMed2003

Identification of mouse sperm SED1, a bimotif EGF repeat and discoidin-domain protein involved in sperm-egg binding.

We report the identification of SED1, a protein required for mouse sperm binding to the egg zona pellucida. SED1 is homologous to a small group of secreted cell-matrix adhesive proteins that contain Notch-like EGF repeats and discoidin/F5/8 type C domains. SED1 is expressed in spermatogenic cells and is secreted by the initial segment of the caput epididymis, resulting in SED1 localization on the sperm plasma membrane overlying the acrosome. SED1 binds specifically to the zona pellucida of unfertilized oocytes, but not to the zona of fertilized eggs. Recombinant SED1 and anti-SED1 antibodies competitively inhibit sperm-egg binding, as do truncated SED1 proteins containing a discoidin/C domain. SED1 null males are subfertile and their sperm are unable to bind to the egg coat in vitro. These studies illustrate that Notch-like EGF and discoidin/C domains, protein motifs that facilitate a variety of cellular interactions, participate in gamete recognition as well.

Amino Acid Motifs↗
PubMed2003

Cell adhesion and fertilization: steps in oocyte transport, sperm-zona pellucida interactions, and sperm-egg fusion.

Fertilization in mammals requires the successful completion of many steps, starting with the transport of gametes in the reproductive tract and ending with sperm-egg membrane fusion. In this minireview, we focus on three adhesion steps in this multistep process. The first is oocyte "pick-up," in which the degree of adhesion between the extracellular matrix of the cumulus cells and oviductal epithelial cells controls the successful pick-up of the oocyte-cumulus complex and its subsequent transfer into the oviduct. The second part of this review is concerned with the interaction between the sperm and the zona pellucida of the egg. Evidence is discussed that a plasma membrane form of galactosyltransferase on the surface of mouse sperm binds to ZP3 in the zona pellucida and initiates an acrosome reaction. Additional evidence raises the possibility that initial sperm binding to the zona pellucida is independent of ZP3. Last, we address the relationship between sperm adhesion to the egg plasma membrane and membrane fusion, especially the role of ADAM family proteins on the sperm surface and egg integrins.

Animals↗
PubMed2002

Targeted mutations in beta1,4-galactosyltransferase I reveal its multiple cellular functions.

Beta1,4-galactosyltransferase I (GalT I) is one of the most extensively studied glycosyltransferases. It is localized in the trans-Golgi compartment of most eukaryotic cells, where it participates in the elongation of oligosaccharide chains on glycoproteins and glycolipids. GalT I has also been reported in non-Golgi locations, most notably the cell surface, where it has been suggested to function non-biosynthetically as a receptor for extracellular glycoside substrates. Cloning of the GalT I cDNAs revealed that the gene encodes two similar proteins that differ only in the length of their cytoplasmic domains. Whether these different GalT I proteins, or isoforms, have similar or different biological roles is a matter of active investigation. The functions of the GalT I proteins have been addressed by targeted mutations that eliminate either both GalT I isoforms or just the long GalT I isoform. Eliminating both GalT I proteins abolishes most, but not all, GalT activity, an observation that led to the realization that other GalT family members must exist. The loss of both GalT I isoforms leads to neonatal lethality due to a wide range of phenotypic abnormalities that are most likely the result of decreased galactosylation. When the long isoform of GalT I is eliminated, galactosylation proceeds grossly normal via the short GalT I isoform, but specific defects in cell interactions occur that are thought to depend upon a non-biosynthetic function of the long GalT I isoform.

Animals↗