Receptor Tyrosine Phosphatase β (RPTPβ) Activity and Signaling Are Attenuated by Glycosylation and Subsequent Cell Surface Galectin-1 Binding

Abstract

O-Mannosyl-linked glycosylation is abundant within the central nervous system, yet very few glycoproteins with this glycan modification have been identified. Congenital diseases with significant neurological defects arise from inactivating mutations found within the glycosyltransferases that act early in the O-mannosyl glycosylation pathway. The N-acetylglucosaminyltransferase known as GnT-Vb or -IX is highly expressed in brain and branches O-mannosyl-linked glycans. Our results using SH-SY5Y neuroblastoma cells indicate that GnT-Vb activity promotes the addition of the O-mannosyl-linked HNK-1 modification found on the developmentally regulated and neuronspecific receptor protein-tyrosine phosphatase ␤ (RPTP␤). These changes in glycosylation accompany decreased cell-cell adhesion and increased rates of migration on laminin. In addition, we show that expression of GnT-Vb promotes its dimerization and inhibits RPTP␤ intrinsic phosphatase activity, resulting in higher levels of phosphorylated ␤-catenin, suggesting a mechanism by which GnT-Vb glycosylation couples to changes in cell adhesion. GnT-Vb-mediated glycosylation of RPTP␤ promotes galectin-1 binding and RPTP␤ levels of retention on the cell surface. N-Acetyllactosamine, but not sucrose, treatment of cells results in decreased RPTP retention, showing that galectin-1 binding contributes to the increased retention after GnT-Vb expression. These results place GnT-Vb as a regulator of RPTP␤ signaling that influences cell-cell and cell-matrix interactions in the developing nervous system. Glycosylation is regulated spatially and temporally during the development of the nervous system (1). In particular, sulfated glycoconjugates, such as the human natural killer-1 epitope, are important for proper migration and adhesion during neural development (2, 3). The human natural killer-1 (HNK-1) 3 epitope was originally discovered using a monoclonal antibody raised against a specific T-lymphoblastoid cell type (4). Since its discovery in lymphocytes, the HNK-1 (also known as CD57) antibody has been shown to react with many neural cell types, including glial, neuroectoderm, and neuroendocrine cells (5-8). The HNK-1 epitope consists of a glucuronic acid, transferred by glucuronyltransferases (GlcATs), that is 3-sulfated by HNK-1 sulfotransferase (HNK1st), linked to a precursor N-acetyllactosamine structure (Fig. 1). Numerous glycoproteins bearing the HNK-1 epitope have been identified in the nervous system, such as neural cell adhesion molecule (9), L1 (10), neural-glia cell adhesion molecule (11), myelinassociated glycoprotein (12), tenascin R (13), and RPTP (receptor protein-tyrosine phosphatase) (14, 15). In certain cell types, such as neural crest cells, the expression of the HNK-1 antigen is highly regulated during development (2) and is often found on migratory cells (3). The HNK-1 antigen is linked to critical functions during the formation of the nervous system, such as cell-matrix interactions (16), cell-cell adhesion (17), memory, and synaptic plasticity (15,18,19...

Developmental Expression of the Neuron-specific N-Acetylglucosaminyltransferase Vb (GnT-Vb/IX) and Identification of Its in Vivo Glycan Products in Comparison with Those of Its Paralog, GnT-V

“…The conclusion from these experiments was that there was little overall change in the total amount of O-Man glycans when ␣-dystroglycan was not expressed, suggesting that the majority of this class of glycans is actually found expressed on glycoproteins other than ␣-dystroglycan. Our previous in vitro experiments identified RPTP as a potential target of the O-mannosylation pathway (48). Here, we provide the first in vivo evidence that RPTP is indeed a physiological substrate of the O-mannosylation pathway.…”

“…We show that Cat-315 reactivity detects the elaboration of O-mannosyl glycans on RPTP and that this reactivity is reduced in knock-out animals. The in vitro studies showed that overexpressing GnT-Vb in neuroblastoma cells resulted in altered association of RPTP on the cell surface, which inhibited its phosphatase activity, resulting in destabilized E-cadherin and inhibition of cell-cell adhesion, measured by either aggregation or migration assays (48). It will be interesting to determine whether other members of the RPTP family express O-Man glycans and if their functions are altered when specific O-Man glycans are deleted or overexpressed.…”

“…GnT-Vb is therefore responsible for the branching of at least some O-mannosyl linked glycans, and these branched structures are clearly found in significant amounts in rabbit brain. Our work has shown that in two cell types GnT-Vb overexpression decreased adhesion to and increased motility on laminin (48,51,53). Moreover, in a neuroblastoma cell line, these effects were mediated through the protein receptor tyrosine phosphatase (RPTP, also known as RPTP␤), a novel substrate glycoprotein for O-mannosyl glycosylation (48).…”

“…Our work has shown that in two cell types GnT-Vb overexpression decreased adhesion to and increased motility on laminin (48,51,53). Moreover, in a neuroblastoma cell line, these effects were mediated through the protein receptor tyrosine phosphatase (RPTP, also known as RPTP␤), a novel substrate glycoprotein for O-mannosyl glycosylation (48). In addition, we determined that the monoclonal antibody Cat-315 detects the O-mannosyl glycan on RPTP synthesized by GnT-Vb.…”

“…Knock-outs-Our previous work demonstrated that the monoclonal antibody Cat-315 detects a carbohydrate epitope on RPTP early in development (48). Cat-315-reactive punctae pre-patterned the surface of neurons prior to synaptogenesis and seemed to define nonsynaptic or perisynaptic sites on the surfaces of the cells.…”