Neu4, a Novel Human Lysosomal Lumen Sialidase, Confers Normal Phenotype to Sialidosis and Galactosialidosis Cells

Abstract

Three different mammalian sialidases have been described as follows: lysosomal (Neu1, gene NEU1), cytoplasmic (Neu2, gene NEU2), and plasma membrane (Neu3, gene NEU3). Because of mutations in the NEU1 gene, the inherited deficiency of Neu1 in humans causes the severe multisystemic neurodegenerative disorder sialidosis. Galactosialidosis, a clinically similar disorder, is caused by the secondary Neu1 deficiency because of genetic defects in cathepsin A that form a complex with Neu1 and activate it. In this study we describe a novel lysosomal lumen sialidase encoded by the NEU4 gene on human chromosome 2. We demonstrate that Neu4 is ubiquitously expressed in human tissues and has broad substrate specificity by being active against sialylated oligosaccharides, glycoproteins, and gangliosides. In contrast to Neu1, Neu4 is targeted to lysosomes by the mannose 6-phospate receptor and does not require association with other proteins for enzymatic activity. Expression of Neu4 in the cells of sialidosis and galactosialidosis patients results in clearance of storage materials from lysosomes suggesting that Neu4 may be useful for developing new therapies for these conditions.Sialidases or neuraminidases are glycohydrolytic enzymes that remove terminal sialic acid residues from sialylated glycoproteins, oligosaccharides, and glycolipids. Three different mammalian sialidases have been described as follows: lysosomal (Neu1, gene NEU1), cytoplasmic (Neu2, gene NEU2), and plasma membrane (Neu3, gene NEU3). Neu1 shows the highest activity against oligosaccharides and short glycopeptides (1) and is involved in lysosomal catabolism of sialylated glycoconjugates (reviewed in Ref. 2). Neu2 is active against ␣2-3-sialylated oligosaccharides, glycopeptides, and gangliosides (3-5).

Developmental Change of Sialidase Neu4 Expression in Murine Brain and Its Involvement in the Regulation of Neuronal Cell Differentiation

“…In the mouse, it is dominantly expressed in brain, but its sialidase activity is very weak compared with other mouse sialidases (4). In contrast, human NEU4 is expressed not only in brain, but also in liver, kidney, and colon (5)(6)(7). We have demonstrated that NEU4 has two isoforms, differing in the N-terminal 12-amino acid residues that act as a mitochondrial-targeting sequence (7).…”

Characterization of the human sialidase Neu4 gene promoter

“…It has been shown that lysosomal/mitochondrial sialidase 4 (Neu4) has activity against sialylated glycoproteins, oligosaccharides, and glycolipids in vitro. In Neu4 transfected Tay-Sachs neuroglia cells, clearance of accumulated G M2 ganglioside has been shown (Seyrantepe et al, 2004). Neu4 deficient mice also show an increased G D1a and a decrease in G M1 ganglioside levels in the brain, indicating the activity of Neu4 against gangliosides in vivo (Seyrantepe et al, 2008).…”

“…The data we obtained might be useful to discover small molecules, which control selective high expression of the human Neu4 gene, resulting in the normal morphological phenotype in the lysosomes of Tay-Sachs patients. cells was achieved by only 3-5% of Neu4-expressing cells, indicating the therapeutic potential of the sialidase Neu4 enzyme in sialidosis and galactosialidosis for enzyme replacement therapy (Seyrantepe et al, 2004). Sialidase Neu4-deficient (Neu4 -/-) mice showed vacuolization and lysosomal storage in lung and spleen cells.…”

“…It has been suggested that the long form localizes in mitochondria and the short form is associated with the endoplasmic reticulum (Bigi et al, 2010). Seyrantepe et al (2004) first biochemically characterized the Neu4 enzyme, which has an almost equal broad substrate specificity against glycoproteins (mucin), oligosaccharides (sialyllactose) and sialylated glycolipids (mixed bovine gangliosides), and synthetic substrates 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (4-MU-NANA or 4MU-NeuAc) at an acidic pH of 3.2 (Monti et al, 2004;Seyrantepe et al, 2004). Overexpression of the sialidase Neu4 enzyme in sialidase Neu1-deficient sialidosis fibroblasts has been associated with the clearance of accumulated substrates and normal morphological phenotype of the lysosomal compartment.…”

“…So far, 4 types of sialidases have been identified in humans and characterized according to their subcellular distribution, substrate specificity, and stability. These sialidases are lysosomal Neu1, cytosolic Neu2, plasma membrane Neu3, and lysosomal/mitochondrial membrane Neu4 (Seyrantepe et al, 2004).…”