Partial Characterization and Enrichment of a Membrane‐Bound Sialidase Specific for Gangliosides from Human Brain Tissue

Abstract

Gangliosides, constituents of surfaces of vertebrate cells, modulate important cellular functions. Ganglioside-specific sialidases that possibly control these processes have been observed in a number of tissues, but their characterization has proved difficult due to their low abundance and lability. Here we describe the partial isolation and characterization of a ganglioside sialidase from human brain grey matter. After membrane extraction with octylglucoside, the enzyme was purified about 1300-fold by ion-exchange, affinity and gel-permeation chromatographies. Although PAGE still showed several protein bands, specific photoaffinity labelling with iodinated S-N-acetyl-9-(4-azidosalicoylamido)-2,9-dideoxy-2,3-didehydroneuraminic acid identified a single polypeptide of 60 kDa likely to contain the active site of the sialidase. In the presence of 0.4 % octylglucoside, the purified sialidase desialylated gangliosides G,,, G,,,,, G,,, and G,,,, but was inactive towards G,,, GM2, colominic acid, sialyl-(a2-3)-lactose, 2-(4-methylumbelliferyl)-neuraminate, or the glycoprotein fetuin. The ganglioside sialidase activity was strongly inhibited by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, heparin and heparan sulfate. Because of its substrate and inhibitor profiles, the purified enzyme resembles the activity characterized previously in the plasma membrane of human neuroblastoma cells, but is distinct from a lysosomal activity. The purified brain sialidase thus appears to function in the selective desialylation of gangliosides with terminal sialic acid residues.

The Plasma Membrane-associated Sialidase MmNEU3 Modifies the Ganglioside Pattern of Adjacent Cells Supporting Its Involvement in Cell-to-Cell Interactions

“…Evidence of a possible involvement of this enzyme in the regulation of the sialic acid levels of the cell surface has been produced. For example, in neuroblastoma cells the enzyme triggers selective ganglioside desialylation, and such surface glycolipid modulations are involved in cell growth control and differentiation (23)(24)(25). The plasma membrane sialidase NEU3 3 is characterized by high substrate specificity for ganglioside substrates in the acidic range of pH (26).…”

Differential functional relevance of a plasma membrane ganglioside sialidase in cholinergic and adrenergic neuroblastoma cell lines

“…As shown previously in SK-N-MC neuroblastoma cells and human brain tissue, this enzyme is unique in that it accepts as substrates only gangliosides with terminal sialic acid residues, thereby creating as end products lactosylceramide or gangliosides GM1 or GM2 with`internal' sialic acids; sialylated glycoproteins or oligosaccharides, on the other hand, are not degraded [10,21]. The level of ganglioside sialidase expression, as measured by specific enzyme activity, showed different behavior in the cell lines in that it was low in sparse cultures of SK-N-MC cells but increased substantially when cells became confluent, reaching levels that were observed in the other neuroblastoma cell lines already at subconfluency.…”

“…The lysosomal activity, in contrast to that of the plasma membrane, also desialylated glycoproteins and the synthetic substrate 4-methylumbelliferylneuraminate [10,19,20]. Evidence for a cell surface orientation of the plasma membrane ganglioside sialidase was obtained from experiments that showed complete inhibition of the activity upon incubating intact cells at low temperature in the presence of millimolar concentrations of Cu 21 , whereas the lysosomal activity remained unaffected under these conditions [11,19]. The plasma membrane sialidase specifically cleaved sialic acid residues from a terminal position in the oligosaccharide portion of the gangliosides but did not attack gangliosides GM1 or GM2 with internal sialic acids.…”

“…Metabolic labeling studies in SK-N-MC neuroblastoma cells in the absence and presence of the extracellular sialidase inhibitor NeuAc2en confirmed such selective ganglioside desialylation by the cell surface enzyme [10]. A membrane-bound sialidase of identical specificity was recently characterized in human and bovine brain tissue [21,22], and the enzyme from both sources has just been cloned [23,24].…”

From the Ganglioside GQ1b? to Glycomimetic Antagonists of the Myelin-Associated Glycoprotein (MAG)

“…In general, the substrate specificity of mammalian sialidases is determined by the linkage type of the terminal sialic acid residue (2-3, 2-6 or 2-8) and does not depend on the structure of the underlying oligosaccharide. [46] Therefore, it cannot be excluded that the presented mimics are metabolically cleaved by sialidases. Nevertheless, this new class of MAG blockers constitute an important step toward the development of potent oligosaccharide mimics.…”