Molecular cloning and characterization of a plasma membrane-associated sialidase specific for gangliosides

Gangliosides are plasma membrane components thought to play important roles in cell surface interactions, cell differentiation, and transmembrane sign aling. A mammalian sialidase located in plasma membranes is unique in speci fically hydrolyzing gangliosides, suggesting crucial roles in regulation of cell surface functions. Here we describe the cloning and expression of a c DNA for the ganglioside sialidase, isolated from a bovine brain cDNA librar y based on the amino acid sequence of the purified enzyme from bovine brain . This cDNA encodes a 428-amino acid protein containing a putative transmem brane domain and the three Asp boxes characteristic of sialidases and shari ng 19-38% sequence identity with other sialidases, Northern blot and polyme rase chain reaction analyses revealed a general distribution of the gene in mammalian species, including man, and the mouse. In COS-7 cells transientl y expressing the sialidase, the activity was found to be 40-fold that of th e control level with ganglioside substrates in the presence of Triton X-100 , and the hydrolysis was almost specific to gangliosides other than GM1 and GM2, both alpha 2-->3 and alpha 2-->8 sialyl linkages being susceptible. T he major subcellular localization of the expressed sialidase was assessed t o be plasma membrane by Percoll density gradient centrifugation of cell hom ogenates and by immunofluorescence staining of the transfected COS-7 cells. Analysis of the membrane topology by protease protection assay suggested t hat this sialidase has a type I membrane orientation with its amino terminu s facing to the extracytoplasmic side and lacking a signal sequence.