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J Balsamo

Publications and source records attributed to J Balsamo.

At least 37 records · Page 2Linked to original sources
PubMed1991

Antibodies to the retina N-acetylgalactosaminylphosphotransferase inhibit neurite outgrowth.

The neural retina N-acetylgalactosaminylphosphotransferase (GalNAcPTase) is a cell surface molecule (Balsamo and Lilien, 1980, 1983; Balsamo et al., 1986a) that is tightly associated with, and glycosylates, the calcium-dependent, cell-cell adhesion molecule, N-cadherin (Balsamo and Lilien, 1990). N-cadherin has been implicated in neuronal attachment and neurite outgrowth when at the surface of cells (Bixby et al., 1987, 1988; Matsunaga et al., 1988; Neugebauer et al., 1988; Tomaselli et al., 1988). The intimate association of the GalNAcPTase and N-cadherin prompted us to test the possibility that the GalNAcPTase is also involved in the process of neurite outgrowth. We tested the effect of one polyclonal and two monoclonal anti-GalNAcPTase antibodies in cultures of chick neural retina cells extending neurites on substrates requiring N-cadherin, beta integrin receptors, or the chicken homologue of L1, G4. The length and number of neurites produced were dramatically reduced on all of these substrates by the polyclonal and one of the monoclonal anti-GalNAcPTase antibodies. The second monoclonal antibody bound to the cell surface but was not inhibitory, indicating that it reacts with a different epitope. The mechanism through which the retina cell surface GalNAcPTase may modulate neurite outgrowth on many substrates is discussed.

Animals↗
PubMed1991

Immunolocalization of N-acetylgalactosaminylphosphotransferase in the adult retina and subretinal space.

The cell surface N-acetylgalactosaminylphosphotransferase (GalNAcPTase) modulates N-cadherin-mediated adhesion among embryonic chick retinal cells (Balsamo et al., 1990). We are investigating the potential role of this transferase in modulating adhesive interactions in the adult retina. Using a previously characterized monoclonal anti-GalNAcPTase, we have used immunohistochemical and immunoblot techniques to localize and characterize the transferase in the retinas of the post metamorphic frog (Xenopus laevis), adult cow, and adult cynomolgus macaque. On frozen sections, anti-GalNAcPTase specifically labels the outer segments of photoreceptors in all species. Immunolabel appears at the surface of, or between rod outer segments in all species. The nerve fiber layer also shows high immunoreactivity in all species. Monkey cone outer segments are also highly immunoreactive. Photoreceptor inner segments are clearly immunoreactive in the cow retina. Immunoblots of purified cow rod outer segments show immunolabeled bands near 220 kDa, which is the molecular mass of the GalNAcPTase used as immunogen. Purified Xenopus rod outer segments are not immunoreactive on blots, while soluble interphotoreceptor matrix (IPM) shows immunoreactive bands principally at 113-130, and 166 kDa. Cow soluble IPM shows immunoreactivity at 180 kDa. Based on these findings, we propose that the GalNAcPTase, or a fragment thereof, is a component of the IPM, and perhaps of the photoreceptor outer segment as well.

Animals↗
PubMed1991

Antibodies to the retina N-acetylgalactosaminylphosphotransferase modulate N-cadherin-mediated adhesion and uncouple the N-cadherin transferase complex from the actin-containing cytoskeleton.

Embryonic chick neural retina cells have at their surface an N-Acetylgalactosaminylphosphotransferase (GalNAcPTase) which is associated with, and glycosylates, the calcium-dependent cell-cell adhesion molecule, N-cadherin (Balsamo, J., and J. Lilien. 1990. J. Biol. Chem. 265:2923-2928). In this manuscript, we demonstrate that antibodies directed against the GalNAcPTase, as well as anti-N-cadherin antibodies, are able to inhibit adhesion of chick neural retina cells to a cell monolayer, to immobilized N-cadherin, or to immobilized anti-N-cadherin antibody. These results indicate that anti-GalNAcPTase antibodies modulate the function of N-cadherin, interfering with the formation of N-cadherin-mediated adhesions. We also demonstrate that actin is associated with the N-cadherin/GalNAcPTase complex and that binding of anti-GalNAcPTase antibodies to intact cells results in dissociation of actin from the complex. We suggest that the GalNAcPTase modulates N-cadherin function by altering its interaction with the cytoskeleton.

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PubMed1990

N-cadherin is stably associated with and is an acceptor for a cell surface N-acetylgalactosaminylphosphotransferase.

Calcium-dependent cell-cell adhesion among embryonic chick neural retina cells is mediated by N-cadherin, a 130,000-Da integral membrane protein. We have reported that the ability of chick neural retina cells to form calcium-dependent cell-cell adhesion is also correlated with the presence of an N-acetylgalactosaminyltransferase at the cell surface (Balsamo, J., and Lilien, J. (1982) J. Biol. Chem. 257, 349-354). This enzyme transfers N-acetylgalactosaminephosphate to endogenous acceptors (Balsamo, J., Pratt, R. S., and Lilien, J. (1986) Biochemistry 25, 5402-5407) and is tightly associated with these acceptors forming a complex which can be isolated from neutral detergent extracts of plasma membranes. We now report that N-cadherin is present in the complex and is an acceptor for the N-acetylgalactosaminylphosphotransferase. Antibodies prepared against N-cadherin precipitate the transferase containing complexes from neutral detergent extracts; however, when the complexes are dissociated by treatment with sodium dodecyl sulfate prior to immunoprecipitation, only N-cadherin is precipitated. Similarly, anti-transferase antibodies immunoprecipitate N-cadherin containing complexes prior to disruption of the complex with sodium dodecyl sulfate. Catalysis of the transferase reaction in the complex results in the transfer of N-acetylgalactosamine phosphate to oligosaccharide chains on N-cadherin. Glycosylation of N-cadherin by the transferase also occurs in situ; labeling of retina cells with [32P] orthophosphate results in N-cadherin molecules containing terminal N-acetylgalactosamine linked to an oligosaccharide chain via a phosphodiester bond.

Animals↗
PubMed1990

Synaptic localization and neural regulation of an N-acetylgalactosaminyl transferase in skeletal muscle.

We have previously documented the properties of a approximately 220-kDa cell surface glycosyltransferase that transfers N-acetylgalactosamine to oligosaccharide chains (Balsamo et al., 1986b). Because N-acetylgalactosamine-terminated carbohydrates are concentrated at the neuromuscular junction (Scott et al., 1988), we assayed skeletal muscle for the presence of the N-acetylgalactosaminyl transferase. Using immunohistochemical methods, we found that the enzyme is localized at neuromuscular junctions on normal adult rat muscle fibers. Biochemical assays confirm that junctional areas are highly enriched in the approximately 220-kDa immunoreactive species as well as in enzyme activity associated with the approximately 220-kDa species. This restricted distribution is dependent on synaptic integrity, as the enzyme appears extrasynaptically on denervation. These results provide a plausible metabolic basis for the localization of a synapse-specific carbohydrate and demonstrate that the expression of a glycosyltransferase is regulated by synaptic interactions.

Animals↗
PubMed1987

Immunologically unique and common domains within a family of proteins related to the retina Ca2+-dependent cell adhesion molecule, NcalCAM.

Rabbit polyclonal antibodies raised to gp90, a fragment of the embryonic chick neural retina Ca2+-dependent adhesive molecule, gp130, recognize gp130 and inhibit Ca2+-dependent cell-cell adhesion. When tested against a panel of 10-day embryonic tissues, one of these antisera recognizes a component with a molecular weight identical to that of gp130 in embryonic chick cerebrum, optic lobe, hind brain, spinal cord and neural retina only; the second antiserum recognizes a similar component in all of the embryonic chick tissues tested. These data imply the existence of an extended family of closely related cell surface components with immunologically distinct subgroups each of which may mediate Ca2+-dependent cell-cell adhesion. As the term CAM, or cell adhesion molecule, has become common usage we propose to refer to these molecules as calCAMs, reflecting their calcium dependence. Analysis of fragments and endoglycosidase digests of NcalCAM have allowed a comparison of its structure with similar molecules from different tissues and species that have been implicated in Ca2+-dependent cell-cell adhesion.

Animals↗
PubMed1986

Chick neural retina N-acetylgalactosaminyltransferase/acceptor complex: catalysis involves transfer of N-acetylgalactosamine phosphate to endogenous acceptors.

Homogenates of embryonic chick neural retina prepared in 1% Triton X-100 have the ability to transfer N-acetyl[32P]galactosamine [( 32P]GalNAc) from beta-32P-labeled uridine diphosphate N-acetylgalactosamine [( beta-32P]UDP-GalNAc) to endogenous macromolecular acceptors. The phosphotransferase activity sediments as three distinct peaks upon centrifugation on sucrose gradients. These peaks are coincident with the transferase/acceptor complexes previously described [Balsamo, J., & Lilien, J. (1982) J. Biol. Chem. 257, 345-354]. The parameters of the 32P transfer reaction closely parallel those observed with UDP-[3H]GalNAc as substrate when the densest particles, H, are used as a source of transferase/acceptors. Treatment of 3H- and 32P-labeled products with alpha-N-acetylgalactosaminidase removes [3H]GalNAc residues and exposes 32P-labeled groups. These data suggest that the sugar-phosphate is transferred intact, resulting in a terminal phosphodiester linkage. The resistance of the macromolecular products to digestion by endoglycosidase F and its sensitivity to hydrolysis under mild alkaline conditions suggest that the alpha-linked sugar is transferred to an oligosaccharide chain attached to the protein core via an O-serine or threonine residue. Characterization of the 32P- and 3H-labeled H particle products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a series of coincident high molecular weight polypeptides.

Animals↗
PubMed1986

Identification of the chick neural retina cell surface N-acetylgalactosaminyltransferase using monoclonal antibodies.

Intact embryonic chick neural retina cells have at their surface an N-acetylgalactosaminyltransferase which catalyzes the incorporation of N-acetylgalactosamine from UDP-N-acetylgalactosamine into endogenous macromolecular acceptors. The enzyme along with its endogenous acceptors can be isolated as a particulate complex following treatment of membrane-enriched fractions with Triton X-100. In this paper we report on two separate fusions generating monoclonal antibodies: one using as immunogen the particulate complex and the second using as immunogen a soluble N-acetylgalactosaminyltransferase found in tissue-culture-conditioned medium which lacks endogenous acceptor activity. Antibodies from both fusions recognize an antigen which is tightly associated with the particulate transferase/acceptor complex and a soluble antigen having N-acetylgalactosaminyltransferase activity toward exogenously added acceptors. The antibodies recognize a component of ca Mr 220,000, which shows N-acetylgalactosaminyltransferase activity after SDS-gel electrophoresis and transfer to nitrocellulose. This component comigrates on two-dimensional gel electrophoresis with an iodinatable cell surface component whose presence at the cell surface correlates with endogenous transferase activity. We conclude that the antibodies recognize the transferase enzyme itself. Immunohistochemical analysis shows that the enzyme is initially localized throughout the embryonic neural retina in a pattern indicative of a cell surface disposition but becomes restricted to the outer plexiform layer and to outer segments in the adult.

Animals↗
PubMed1982

An N-acetylgalactosaminyltransferase and its acceptor in embryonic chick neural retina exist in interconvertible particulate forms depending on their cellular location.

The N-acetylgalactosaminyltransferase activity and its associated endogenous acceptor present in neural retina cells from embryonic chicks can be separated into three distinct forms which migrate to different densities upon centrifugation in sucrose gradients. Two of these forms, termed L and H, are also present in preparations of plasma membranes. Dissociation of tissues into single cells by trypsin results in populations lacking the cell-surface forms of transferase activity towards endogenous acceptor. This loss can be prevented by including 1 mM Ca2+ in the trypsin-dissociation medium. Reacquisition of cell-surface activity among trypsin dispersed cells occurs with time in culture. In the presence of cycloheximide, the less dense particulate form, L. Appears first, with a concomitant decrease in the cytosol form. Upon removal of the block to protein synthesis, L is transformed into the denser from, H. Our studies suggest that peak H of transferase/acceptor polypeptide. The possible involvement of the transferase/acceptor in adhesion among neural retina cells is discussed.

Animals↗
PubMed1975

The binding of tissue-specific adhesive molecules to the cell surface. A molecular basis for specificity.

Factors present in supernatants prepared from neural retina or cerebral lobe tissue cultures bind specifically to cells of the same type and promote cellular aggregation; the basis for the specificity of these factor-cell interactions has been investigated. Pronase digestion destroys binding of protein but not the carbohydrate portion of factors. Digestion with a mixture of protease-free glycosidases destroys both protein and carbohydrate binding. Purified beta-N-acetylhexosaminidase reduces binding of retina factor by 80%. The enzymatic activity which destroys binding of cerebral lobe factor to cerebral cells appears to be alpha-mannosidase activity. Further, paper chromatography of the enzymatic digestion products of the binding factors reveals that N-acetylgalactosamine residues are released from the retina factor while mannosamine residues are released from the cerebral lobe factor. Inhibition of binding of factors to cells by monosaccharides is consistent with the enzyme data. N-Acetylgalactosamine maximally inhibits binding of factor to retina cells while mannosamine inhibits factor binding to cerebral cells. These data suggest that the specificity of cellular adhesion is determined, at least in part, by the sequence of sugars in an oligosaccharide residue of a complex glycoprotein.

Animals↗
PubMed1974

Embryonic cell aggregation: kinetics and specificity of binding of enhancing factors.

A new assay for tissue-type-specific adhesive macromolecules is described. This assay measures the binding to whole cells of radioactive macromolecules in aggregation-enhancing supernatants. We show for two tissue types, embryonic chick neural retina and cerebrum, that binding is tissue-type specific. Specific binding can be distinguished from nonspecific binding by its sensitivity to displacement with horse serum and by the kinetics of binding. Specific binding appears to be a cooperative phenomenon.

Animals↗