J. Kopitz et al., PARTIAL CHARACTERIZATION AND ENRICHMENT OF A MEMBRANE-BOUND SIALIDASESPECIFIC FOR GANGLIOSIDES FROM HUMAN BRAIN-TISSUE, European journal of biochemistry, 248(2), 1997, pp. 527-534
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.