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Gangliosides are plasma membrane components thought to play important roles in cell surface interactions, cell differentiation, and transmembrane signaling. A mammalian sialidase located in plasma membranes is unique in specifically hydrolyzing gangliosides, suggesting crucial roles in regulation of cell surface functions. Here we describe the cloning and expression of a cDNA for the ganglioside sialidase, isolated from a bovine brain cDNA library based on the amino acid sequence of the purified enzyme from bovine brain. This cDNA encodes a 428-amino acid protein containing a putative transmembrane domain and the three Asp boxes characteristic of sialidases and sharing 19 -38% sequence identity with other sialidases. Northern blot and polymerase chain reaction analyses revealed a general distribution of the gene in mammalian species, including man, and the mouse. In COS-7 cells transiently expressing the sialidase, the activity was found to be 40-fold that of the 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 ␣233 and ␣238 sialyl linkages being susceptible. The major subcellular localization of the expressed sialidase was assessed to be plasma membrane by Percoll density gradient centrifugation of cell homogenates and by immunofluorescence staining of the transfected COS-7 cells. Analysis of the membrane topology by protease protection assay suggested that this sialidase has a type I membrane orientation with its amino terminus facing to the extracytoplasmic side and lacking a signal sequence.

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Plasma membrane-associated sialidase is a key enzyme for ganglioside hydrolysis, thereby playing crucial roles in regulation of cell surface functions. Here we demonstrate that mice overexpressing the human ortholog (NEU3) develop diabetic phenotype by 18 -22 weeks associated with hyperinsulinemia, islet hyperplasia, and increased ␤-cell mass. As compared with the wild type, insulin-stimulated phosphorylation of the insulin receptor (IR) and insulin receptor substrate I was significantly reduced, and activities of phosphatidylinositol 3-kinase and glycogen synthase were low in transgenic muscle. IR phosphorylation was already attenuated in the younger mice before manifestation of hyperglycemia. Transient transfection of NEU3 into 3T3-L1 adipocytes and L6 myocytes caused a significant decrease in IR signaling. In response to insulin, NEU3 was found to undergo tyrosine phosphorylation and subsequent association with the Grb2 protein, thus being activated and causing negative regulation of insulin signaling. In fact, accumulation of GM1 and GM2, the possible sialidase products in transgenic tissues, caused inhibition of IR phosphorylation in vitro, and blocking of association with Grb2 resulted in reversion of impaired insulin signaling in L6 cells. The data indicate that NEU3 indeed participates in the control of insulin signaling, probably via modulation of gangliosides and interaction with Grb2, and that the mice can serve as a valuable model for human insulin-resistant diabetes.Gangliosides are a family of sialic acid-containing glycosphingolipids present in the cell surface membranes. Several lines of evidence suggest their important functional roles in regulating a wide range of biological processes including cell growth, cell differentiation, and transmembrane signaling (1-3). Most of the observations on ganglioside function, however, have been performed so far by using bacterial sialidases and exogenous gangliosides to mimic in vivo expression. To obtain further insights into their physiological significance and regulation mechanism, it is necessary to focus on endogenous sialidases responsible for ganglioside hydrolysis inside the cells. Three types of mammalian sialidase have been cloned and established to have low identity with each other in their primary structure. They differ in enzymatic properties and subcellular localization, being mainly found in lysosomes (Neu1), cytosol (Neu2), and plasma membranes (Neu3). Other than the contribution of lysosomal sialidase to glycoconjugate catabolism in lysosomes, their cellular roles are not well understood. The fact that plasma membrane sialidase is unique in specifically hydrolyzing gangliosides (4 -10) and in its subcellular localization in plasma membranes, where levels of other glycosidases are very low, suggests participation in cell surface events through modulation of gangliosides. To obtain evidence of this, we previously cloned sialidase cDNAs of mammalian origin (8, 9, 11, 12), and we have recently investigated effects of overexpression in transgenic...

Sialidase expression levels are inversely correlated with the metastatic potential of mouse colon adenocarcinoma 26 sublines, as assessed by activity assays and RT-PCR, irrespective of total and cell surface sialic acid contents. Compared with low metastatic NL4 and NL44 cell lines, the highly metastatic NL17 and NL22 cells exhibit low expression of sialidases, accompanied with higher levels of sialylLe x and GM3. To investigate whether these properties of NL17 cells can be altered by sialidase overexpression, we transfected a cytosolic sialidase gene into NL17 cells. The result was markedly inhibited lung metastasis, invasion and cell motility with a concomitant decrease in sialylLe x and GM3 levels, in line with the case of spontaneously low metastatic sublines having relatively high endogenous sialidase levels, implying that sialidase level is a determining factor affecting metastatic ability. Treatment of the cells with antibodies against sialylLe x and GM3 affected cell adhesion and/or cell motility, providing evidence that desialylation of these molecules, as targets of sialidase, is involved in the suppression of metastasis. Aberrant sialylation in cancer cells is generally accepted as one characteristic feature associated with metastatic potential. 1-5 Cellular sialic acid contents are controlled mainly by expression levels of sialyltransferases and sialidases. To elucidate the molecular mechanisms and significance of aberrant sialylation, our studies have been focused on sialidase. In the 1970s, exogenous sialidases of microbial origin were often used for studies on treatment with cancer cells to enhance tumor growth regression or cellular immunity. However, observations on endogenous sialidase of malignant cells can facilitate further understanding of the pathologic desialylation. Mammalian sialidases have so far been classified into 3 or 4 types differing in subcellular localization and enzymatic properties. Three types, located mainly in cytosol, lysosomes and plasma membranes, have been cloned. 6 Although many functional aspects of each sialidase have yet to be clarified, they are likely to be involved not only in catabolism of glycoconjugates but also in functional modulation through desialylation of molecules, depending on their particular subcellular location and substrate specificity. The lysosomal form may have a major role in glycoprotein catabolism by collaborating with lysosomal proteases or endoglycosidase, as fragmentation into glycopeptides or oligosaccharide chains is required before cleavage by this sialidase. 7 Cytosolic sialidase may participate in regulatory desialylation because of its ability to act on native glycoproteins and gangliosides at neutral pH. 8 Plasma membrane sialidase hydrolyzes gangliosides specifically 9,10 and has been suggested to participate in cell surface events.To understand the role of sialidases in metastasis, we previously measured activity levels in transformed rat 3Y1 cell lines and found an inverse correlation with metastatic potential. 11 No significant...