Mouse Na+/K+-ATPase beta1-subunit has a K+-dependent cell adhesion activity for beta-GlcNAc-terminating glycans

Abstract

A 48-kDa beta-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 beta1-subunit. When the recombinant FLAG-beta1-subunit expressed in Sf-9 cells was applied to a GlcNAc-agarose 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 beta1-subunit and GlcNAc-terminating oligosaccharides are at the cell contact sites. Inclusion of anti-beta1-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 beta1-subunit is a potassium-dependent lectin that binds to GlcNAc-terminating oligosaccharides: it may be involved in neural cell interactions.

Fig. 1.

Isolation of GlcNAc-binding proteins from mouse brain by GlcNAc-agarose column chromatography. The blots containing proteins in the pass-through (lane A) and bound (lane B) fractions were stained with Coomassie brilliant blue. The blots containing proteins in the bound fraction (lane C), purified mouse brain Na⁺/K⁺-ATPase that bound to a GlcNAc-agarose column (lane D), mouse brain plasma membrane proteins that bound to an AsAg-transferrin-Sepharose column (lane E), and mouse brain plasma membrane proteins (lane F) were incubated with anti-canine Na⁺/K⁺-ATPase antibody. The positions of the α- and β-subunits are indicated with arrows and molecular masses.

Fig. 2.

Immunoblot and lectin blot analyses of the FLAG-β1-subunit expressed in Sf-9 cells. The blot containing purified FLAG-β1-subunit proteins was incubated with anti-FLAG antibody (lane A), anti-rat β1-subunit antibody (lane B), or Con A (lane C). The purified FLAG-β1-subunit proteins were subjected to GlcNAc-agarose column chromatography, and blots containing proteins in the pass-through (lane D) and bound (lane E) fractions were incubated with anti-rat β1-subunit antibody.

Fig. 3.

Effect of K⁺ concentration in the buffer on the binding of the 38- and 40-kDa FLAG-β1-subunit proteins to GlcNAc-agarose. Purified FLAG-β1-subunit proteins were applied to a GlcNAc-agarose column in the presence of increasing concentrations of KCl. Relative bindings of the FLAG-β1-subunit to the column were determined by anti-rat β1-subunit antibody reactivity toward the 38- and 40-kDa proteins in the bound fractions. Three independent experiments were conducted, and the mean values are shown with standard errors.

Fig. 4.

Cell aggregation activity of the β1-subunit. The extent of cell aggregation is represented as the ratio of the total particle number at indicated incubation time (N_t) to the initial particle number (N₀). The N_t/N₀ values represent the means ± standard errors of three independent experiments. Effects of antibodies and carbohydrates on aggregate formation of mouse embryonic neural cells and neuro-2a cells are shown in A and B, respectively. Cells were incubated in the absence of additions (a) or in the presence of 5 mM EDTA (b), anti-N-CAM antibody (c), rabbit anti-rat β1-subunit antibody (d), normal rabbit IgG (e), chitobiose (f), lactose (g), or mannose (h). ▪, ▴, and • indicate concentrations of 0.4, 2.0, and 10.0 μg/ml antibody, or 2, 10, and 20 mM carbohydrate, respectively. (C) Aggregates of control neuro-2a cells as a representative (a), and neuro-2a cells in the presence of 10.0 μg/ml anti-rat β1-subunit antibody (d) and 20 mM chitobiose (f) after 2-hr incubation. (Bar, 50 μm.)

Fig. 5.

Localization of the β1-subunit and GlcNAc-terminating oligosaccharides in cell aggregates. Fixed aggregates of mouse embryonic neural cells (a–c) and neuro-2a cells (d–f) were incubated with anti-rat β1-subunit antibody and visualized (a, b, d, and e). Fixed aggregates of neuro-2a cells were incubated with PVL and visualized (g and h). Nuclei and β-tubulin were stained blue and green, respectively (b, c, e, f, h, and i).

Fig. 6.

Effect of chitobiose on Na⁺/K⁺-ATPase activity. Na⁺/K⁺-ATPase was purified from mouse brain, and its activity was determined in the presence of chitobiose. C and C′ indicate the activities obtained in the presence and absence of KCl, respectively.