GD3- and O-acetylated GD3-gangliosides in the GM2 synthase-deficient mouse brain and their immunohistochemical localization

Abstract

Gangliosides in the brain of the knockout mouse deficient in the activity of β1,4 N-acetylgalactosaminyl transferase (β1,4 GalNAc-T)(GM2 synthase) consisted of nearly exclusively of GM3- and GD3-gangliosides as expected from the known substrate specificity of the enzyme and in confirmation of the initial reports from two laboratories that generated the mutant mouse experimentally. The total molar amount of gangliosides was approximately 30% higher in the mutant mouse brain than that in the wild-type brain. However, contrary to the initial reports, one-fourth of total GD3-ganglioside was O-acetylated. It reacted positively with an anti-O-acetylated GD3 monoclonal antibody and disappeared with a corresponding increase in GD3-ganglioside after mild alkaline treatment. The absence of O-acetylated GD3 in the initial reports can be explained by the saponification step included in their analytical procedures. Although quantitatively much less and identification tentative, we also detected GT3 and O-acetylated GT3. Anti-GD3 and anti-O-acetylated GD3 monoclonal antibodies gave positive reactions in the brain of mutant mouse as expected from the analytical results. Either antibody barely stained wild-type brain except for immunoreactivity of GD3 in the cerebellar Purkinje cells. The distributions of GD3 and O-acetylated GD3 in the brain of mutant mouse were similar but differential localization was noted in the cerebellar Purkinje cells and cerebral cortex.

Keywords: GM2-synthase; O-acetylated ganglioside; Sphingolipid; ganglioside; immunohistochemistry; knockout mouse.

Fig. 1.

Thin-layer chromatogram of brain gangliosides. Technical details are described in the text. A: Stained with the resorcinol spray; B: Stained with specific mouse monoclonal antibodies. Lane I: GalNAcT−/− mouse preparation after saponification; Lane II: GalNAcT−/− mouse preparation before saponification; Lane III: Preparation from Niemann-Pick type C brain as a control (This is only to show thin-layer chromatographic mobility of multiple gangliosides of known structures. The normal control brain used for quantitative analyses of gangliosides (Table I) was a wild-type mouse brain, which is not on this chromatogram.) Lane IV: Same as Lane II but stained with specific monoclonal antibody against O-acetylated GD3; Lane V: Same as Lane II but stained with specific monoclonal antibody against GD3. GalNAcT−/− brain contains essentially GD3, O-acetyl-GD3 and GM3 gangliosides. Upon saponification O-acetyl-GD3 disappears and the GD3 band becomes heavier (see Table I). We tentatively identify the band that runs slightly ahead of GD1b in Lane I as GT3, and the band running slightly ahead of GD1a in Lane II as O-acetylated GT3. Then, the mutant brain also contains detactable amounts of GT3 and O-acetylated GT3, and the latter is hydrolyzed to GT3 by saponificaiton. This interpretation can also be understood from the metabolic block in the GalNAcT−/− mouse.

Fig. 2.

Immunohistochemical localization of GD3 and O-acetyl-GD3 gangliosides in the brain of GalNAcT−/− mouse (a, b, e, f) and wild-type mouse (c, d). Sections were treated with monoclonal antibodies specific for GD3 (a, c, e) and O-acetyl-GD3 (b, d, f) and visualized with FITC-conjugated donkey anti-mouse IgG (H+L) antibody. Technical details are described in the text. Cerebellar cortex (a–d); M: molecular layer, P: Purkinje cell layer, G: granular layer, W: white matter. Cerebral cortex (e, f); I: molecular layer, II: external granular layer, III: external pyramidal layer, IV: internal granular layer, V: internal pyramidal layer.