PARTIAL CHARACTERIZATION AND ENRICHMENT OF A MEMBRANE-BOUND SIALIDASESPECIFIC FOR GANGLIOSIDES FROM HUMAN BRAIN-TISSUE

Gangliosides, constituents of surfaces of vertebrate cells, modulate i mportant cellular functions. Ganglioside-specific sialidases that poss ibly control these processes have been observed in a number of tissues , but their characterization has proved difficult due to their low abu ndance and lability, Hen we describe the partial isolation and charact erization of a ganglioside sialidase from human brain grey matter, Aft er membrane extraction with octylglucoside, the enzyme was purified ab out 1300-fold by ion-exchange, affinity and gel-permeation chromatogra phies. Although PAGE still showed several protein bands, specific phot oaffinity labelling with iodinated salicoylamido)-2,9-dideoxy-2,3-dide hydroneuraminic acid identified a single polypeptide of 60 kDa likely to contain the active site of the sialidase. In the presence of 0.4% o ctylglucoside, the purified sialidase desialylated gangliosides G(M3), G(D1a), G(D1b) and G(T1b), but was inactive towards G(M1), G(M2), col ominic acid, sialyl-(alpha 2-3)-lactose, 2-(4-methylumberlliferyl)-neu raminate, or the glycoprotein fetuin. The ganglioside sialidase activi ty was strongly inhibited by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, heparin and heparan sulfate. Because of its substrate and inhibi tor profiles, the purified enzyme resembles the activity characterized previously in the plasma membrane of human neuroblastoma cells, but i s distinct from a lysosomal activity. The purified brain sialidase thu s appears to function in the selective desialylation of gangliosides w ith terminal sialic acid residues.