Desialylation of extracellular GD1a-neoganglioprotein suggests cell surface orientation of the plasma membrane-bound ganglioside sialidase activity in human neuroblastoma cells.
The orientation of the catalytic site of a ganglioside-specific sialidase in the plasma membrane of SK-N-MC neuroblastoma cells was probed using water-soluble GD1a-neoganglioprotein substrate on intact cells and GM1-product detection by cholera toxin B. Desialylation of substrate was readily observed, whereas specific sialidase inhibitors prevented the reaction, and conditioned medium was inactive. Inhibitors of endocytosis and acidification had no effect on substrate degradation, and lowering temperature to 18 degrees C reduced activity but did not abolish it. We conclude that the ganglioside sialidase activity is cell surface-orientated and displays an in situ specificity that mirrors enzyme preparations in vitro.
Kopitz
J
J
University of Heidelberg, Department of Pathochemistry and Neurochemistry, Im Neuenheimer Feld 220, D-69120, Heidelberg, Germany. juergen_kopitz@med.uni-heidelberg.de
Oehler
C
C
Cantz
M
M
D020134:Catalytic Domain / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism*; D002772:Cholera Toxin / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D005679:G(M3) Ganglioside / Q000378:metabolism; D005732:Gangliosides / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D009439:Neuraminidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009447:Neuroblastoma / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D014407:Tumor Cells, Cultured
D017077:Culture Media, Conditioned; D004791:Enzyme Inhibitors; D005679:G(M3) Ganglioside; D005732:Gangliosides; D006023:Glycoproteins; C021298:ganglioside, GD1a; D002772:Cholera Toxin; D009439:Neuraminidase