Mouse Na⁺/K⁺-ATPase β1-subunit has a K⁺-dependent cell adhesion activity for β-GlcNAc-terminating glycans

Abstract

A 48-kDa ␤-N-acetylglucosamine (GlcNAc)-binding protein was isolated from mouse brain by GlcNAc-agarose column chromatography. The N-terminal amino acid residues showed the protein to be a mouse Na ؉ ͞K ؉ -ATPase ␤1-subunit. When the recombinant FLAG-␤1-subunit expressed in Sf-9 cells was applied to a GlcNAcagarose column, only the glycosylated 38-and 40-kDa proteins bound to the column. In the absence of KCl, little of the proteins bound to a GlcNAc-agarose column, but the 38-and 40-kDa proteins bound in the presence of KCl at concentrations above 1 mM. Immunohistochemical study showed that the ␤1-subunit and GlcNAc-terminating oligosaccharides are at the cell contact sites. Inclusion of anti-␤1-subunit antibody or chitobiose in cell aggregation assays using mouse neural cells resulted in inhibition of cell aggregation. These results indicate that the Na ؉ ͞K ؉ -ATPase ␤1-subunit is a potassium-dependent lectin that binds to GlcNActerminating oligosaccharides: it may be involved in neural cell interactions.GlcNAc-binding lectin ͉ cell surface ͉ mouse brain N -linked glycosylation is the most frequent protein modification in eukaryotes, and a variety of functions are associated with the oligosaccharide structures (1, 2). High mannosetype oligosaccharides, an initial N-glycosylation form, are involved in the quality control of nascent proteins (3). Some of the oligosaccharides are then processed to mature complex-type oligosaccharides by the trimming of mannose residues by mannosidases I and II followed by the addition of GlcNAc residues by ␤-N-acetylglucosaminyltransferases I, II, IV, and V. In most cases, the GlcNAc residues of the outer branches are galactosylated and then sialylated. However, glycoproteins from mammalian brains contain N-linked oligosaccharides terminating with GlcNAc residues in relatively higher proportions (4, 5) in contrast to those from other tissues. Although the mechanism to bear the GlcNAc-terminating oligosaccharides is unknown, this is partly due to the different expression of ␤-1,4-galactosyltransferases in the brain from other tissues (6, 7). The presence of GlcNAc residues at the nonreducing termini of N-linked oligosaccharides may have some biological functions in the brain. In an effort to elucidate the biological functions of such unique oligosaccharides, we have demonstrated that the growth and neurite extension of mouse primary cultured neural cells and mouse and human neuroblastoma cells are regulated through their GlcNAc-terminating oligosaccharides expressed at the cell surface by culturing them in dishes coated with Psathyrella velutina lectin (PVL), a ␤-GlcNAc-binding lectin (8). These results suggest that GlcNAc-binding proteins are present at the cell surface and͞or extracellular matrices and are involved in cellular interactions. In the present study, we have isolated and identified a major GlcNAc-binding protein from mouse brain and shown its involvement in cellular interactions. Materials and Methods Isolation and Identification of a GlcNAc-Binding Protein from Mou...

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