Nerve Nerve cell function depends upon appropriate contacts between the neuron and other cells in its immediate environment (1). These include specialized glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system, which ensheathe the neuronal axon with myelin, an insulating structure of multilayered membranes (2). Myelin is required for efficient nerve impulse conduction but has other profound biological effects. The inability of nerves to regenerate after CNS injury in adults may be due largely to the axon's inability to grow when in contact with CNS myelin (3, 4). Identification of the cell-surface constituents on neuronal axons and myelin membranes that interact with each other to control cell behavior may facilitate efforts to enhance nerve regeneration as well as modulate myelination.Myelin-associated glycoprotein (MAG), a quantitatively minor protein constituent of CNS (1%) and peripheral nervous system (0.1%) myelin, is implicated in myelin-axonThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.interactions based on its in vivo location and in vitro binding properties. In the CNS, MAG is located exclusively on myelin membranes juxtaposed to axons, where it contributes to maintenance of the periaxonal cytoplasmic collar (5). Purified MAG incorporated into liposomes binds specifically to neuronal processes in cell culture (6)(7)(8). This binding is blocked by an anti-MAG monoclonal antibody (mAb 513) that also inhibits neuron-oligodendrocyte adhesion in vitro (7).One consequence of MAG-axon binding is inhibition of neurite outgrowth from CNS neurons. Cultured primary CNS neurons fail to extend neurites on a substratum of MAGexpressing CHO cells, whereas neurite extension proceeds readily on cells transfected with the reverse (control) construct (9). Recombinant MAG adsorbed to a culture surface inhibits neurite outgrowth from neuroblastoma cells in culture, as does a mixture of detergent-solubilized myelin proteins (10). Immunodepletion of MAG from the solubilized myelin proteins reduces their neurite outgrowth inhibitory activity by more than half. These data implicate MAG as a major neurite outgrowth inhibitory factor in myelin. If true, axonal ligands for MAG may be key elements controlling nerve regeneration in the CNS.

Pyrimidine dimer formation in response to UV radiation is governed by the thymine content of the potential dimer and the two flanking nucleotides. An enzymatic activity can be purified from Micrococcus luteus that cleaves the N-glycosyl bond between the 5' pyrimidine of a dimer and the corresponding sugar without rupture of a phosphodiester bond. We propose that strand scission at a dimer site by the M. luteus enzyme requires two activities, a pyrimidine dimer DNA-glycosylase and an apyrimidinic/apurinic endonuclease.

Past studies from this laboratory have suggested that mouse sperm binding to the egg zona pellucida is mediated by a sperm galactosyltransferase (GalTase), which recognizes and binds to terminal N-acetylglucosamine (GIcNAc) residues in the zona pellucida (ShuT, B. D., and N. G. Hall, 1982, J. Cell Biol. 95:567-573; 95:574-579). We now present evidence that directly supports this mechanism for gamete binding. GalTase was purified to homogeneity by sequential affinity-chromatography on GIcNAc-agarose and a-lactalbumin-agarose columns. The purified enzyme produced a dose-dependent inhibition of sperm binding to the zona pellucida, relative to controls. To inhibit sperm/zona binding, GalTase had to retain its native conformation, since neither heat-inactivated nor Mn+÷-deficient GalTase inhibited sperm binding. GalTase inhibition of sperm/zona binding was not due to steric blocking of an adjacent sperm receptor on the zona, since GalTase could be released from the zona pellucida by forced galactosylation with UDPGal, and the resulting galactosylated zona was still incapable of binding sperm. In control experiments, when UDPGal was replaced with the inappropriate sugar nucleotide, UDPglucose, sperm binding to the zona pellucida remained normal after the adsorbed GalTase was washed away.The addition of UDPGal produced a dose-dependent inhibition of sperm/zona binding, and also dissociated preformed sperm/zona adhesions by catalyzing the release of the sperm GalTase from its GIcNAc substrate in the zona pellucida. Under identical conditions, UDPglucose had no effect on sperm binding to the zona pellucida. The ability of UDPGal to dissociate sperm/zona adhesions was both time-and temperature-dependent. UDPGal produced nearly total inhibition of sperm/zona binding when the zonae pellucidae were first galactosylated to reduce the number of GalTase binding sites.Finally, monospecific anti-GalTase IgG and its Fab fragments produced a dose-dependent inhibition of sperm/zona binding and concomitantly blocked sperm GalTase catalytic activity. Preimmune IgG or anti-mouse brain IgG, which also binds to the sperm surface, had no effect. The sperm GalTase was localized by indirect immunofluorescence to a discrete plasma membrane domain on the dorsal surface of the anterior head overlying the intact acrosome. These results, along with earlier studies, show clearly that sperm GalTase serves as a principal gamete receptor during fertilization.

From a systematic search of the UniGene and dbEST databanks, using human beta 4-galactosyltransferase (beta 4GalT-I), which is recognized to function in lactose biosynthesis, as the query sequence, we have identified five additional gene family members denoted as beta 4GalT-II, -III, -IV, -V, and -VI. Complementary DNA clones containing the complete coding regions for each of the five human homologs were obtained or generated by a PCR-based strategy (RACE) and sequenced. Relative to beta 4GalT-I, the percent sequence identity at the amino acid level between the individual family members, ranges from 33% (beta 4GalT-VI) to 55% (beta 4GalT-II). The highest sequence identity between any of the homologs is between beta 4GalT-V and beta 4GalT-VI (68%). beta 4GalT-II is the ortholog of the chicken beta 4GalT-II gene, which has been demonstrated to encode an alpha-lactalbumin responsive beta 4-galactosyltransferase (Shaper et al., J. Biol. Chem., 272, 31389-31399, 1997). As established by Northern analysis, beta 4GalT-II and -IV show the most restricted pattern of tissue expression. High steady state levels of beta 4GalT-II mRNA are seen only in fetal brain and adult heart, muscle, and pancreas; relatively high levels of beta 4GalT-VI mRNA are seen only in adult brain. When the corresponding mouse EST clone for each of the beta 4GalT family members was used as the hybridization probe for Northern analysis of murine mammary tissue, transcription of only the beta 4GalT-I gene could be detected in the lactating mammary gland. These observations support the conclusion that among the six known beta 4GalT family members in the mammalian genome, that have been generated through multiple gene duplication events of an ancestral gene(s), only the beta 4GalT-I ancestral lineage was recruited for lactose biosynthesis during the evolution of mammals.

A 1.3-kilobase cDNA clone (7A) coding for bovine galactosyltransferase (glycoprotein 4-3-galactosyltransferase, EC 2.4.1.38) was isolated from a Xgtll expression library by immunological screening with monospecific polyclonal antisera to the affinity-purified bovine enzyme. The nucleotide sequence of this clone predicts an open reading frame that starts at the 5' end of the insert and codes for a polypeptide of 334 amino acids with Mr 37,645. Based on a Mr of 57,000 for the membrane-bound enzyme this clone accounts for approximately 61% of the coding sequence. Portions of the predicted amino acid sequence matched the six tryptic peptides isolated from affinity-purified bovine galactosyltransferase. Clone 7A hybridizes to a 4.8-kilobase bovine mRNA and identifies multiple EcoRI restriction fragments in bovine, murine, and human DNA.UDPgalactose:N-acetyl-D-glucosaminyl-glycopeptide 4-p-D-galactosyltransferase (glycoprotein 4-/-galactosyltransferase; EC 2.4.1.38) is a Golgi membrane-bound enzyme that participates in the biosynthesis of the carbohydrate moieties of glycoproteins and glycolipids (1, 2). Galactosyltransferase catalyzes the following reaction of UDPgalactose (UDP-Gal) and N-acetylglucosamine (GlcNAc): MnI2 UDP-Gal + GlcNAc Mn Gal(/31-+4)GlcNAc + UDP where the acceptor sugar, N-acetylglucosamine, may be either the free monosaccharide or the nonreducing terminal monosaccharide of a carbohydrate side chain of a glycoprotein or glycolipid (3). Galactosyltransferase can also interact with the regulatory protein a-lactalbumin to form the heterodimer, lactose synthetase (EC 2.4.1.22). This complex catalyzes the transfer of galactose from UDPgalactose to glucose, forming lactose (4). The net result of the specific interaction of galactosyltransferase with a-lactalbumin is to lower the Km for glucose so that lactose synthesis can take place at physiological concentrations of glucose.Galactosyltransferase has also been localized to the plasma membrane of a diverse variety of cells and tissues by immunohistochemical (5-9) and biochemical procedures [comprehensively reviewed in Pierce et al. (10) and Shur (11)]. This cell surface distribution has led to the postulate that, in addition to its biosynthetic role, galactosyltransferase has a functional role in intercellular recognition and/or adhesion (12). A detailed analysis of the cell surface galactosyltransferase localized to the plasma membrane overlying the intact acrosome of mouse sperm supports a functional role in sperm-egg recognition (7, 13). There is also evidence to suggest that galactosyltransferase might be functionally involved in the T/t complex of the mouse, a region in which mutations lead to abnormal embryonic development and sperm production (14).Because of the multiple functions of this membrane-bound enzyme and our long range goal of determining the relationship between the cell surface and intracellular form of galactosyltransferase, we have taken advantage of molecular cloning techniques to obtain structural information on this enzy...

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Myelin-associated glycoprotein (MAG, Siglec-4) is a quantitatively minor membrane component expressed preferentially on the innermost myelin wrap, adjacent to the axon. It stabilizes myelin-axon interactions by binding to complementary ligands on the axolemma. MAG, a member of the Siglec family of sialic acid-binding lectins, binds specifically to gangliosides GD1a and GT1b, which are the major sialoglycoconjugates on mammalian axons. Mice with a disrupted Galgt1 gene lack UDP-GalNAc:GM3/GD3 N-acetylgalactosaminyltransferase (GM2/GD2 synthase) and fail to express complex brain gangliosides, including GD1a and GT1b, instead expressing a comparable amount of the simpler gangliosides GM3, GD3, and O-acetyl-GD3. Galgt1-null mice produce similar amounts of total myelin compared to wild-type mice, but as the mice age, they exhibit axon degeneration and dysmyelination with accompanying motor behavioral deficits. Here we report that Galgt1-null mice display progressive and selective loss of MAG from the brain. At 1.5 months of age, MAG expression was similar in Galgt1-null and wild-type mice. However, by 6 months of age MAG was decreased approximately 60% and at 12 months of age approximately 70% in Galgt1-null mice compared to wild-type littermates. Expression of the major myelin proteins (myelin basic protein and proteolipid protein) was not reduced in Galgt1-null mice compared to wild type. MAG mRNA expression was the same in 12-month-old Galgt1-null compared to wild-type mice, an age at which MAG protein expression was markedly reduced. We conclude that the maintenance of MAG protein levels depends on the presence of complex gangliosides, perhaps due to enhanced stability when MAG on myelin binds to its complementary ligands, GD1a and GT1b, on the apposing axon surface.

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