Expression of a novel human sialidase encoded by the NEU2 gene.
Sialidases (E.C.3.2.1.18) belong to a group of glycohydrolytic enzymes, widely distributed in nature, which remove sialic acid residues from glycoproteins and glycolipids. All of the sialidase so far characterized at the molecular level share an Asp block, repeated three to five times in the primary structure, and an F/YRIP sequence motif which is part of the active site. Using a sequence homology-based approach, we previously identified a human gene, named NEU2, mapping to chromosome 2q37. NEU2 encoded protein is a polypeptide of 380 amino acids with two Asp block consensuses and the YRIP sequence in the amino terminal part of the primary structure. Here we demonstrate that NEU2 encodes a functional sialidase. NEU2 was expressed in COS7 cells, giving rise to a dramatic increase in the sialidase activity measured in cell extracts with the artificial substrate 4-MU-NANA. Using a rabbit polyclonal antiserum, on Western blots a protein band with a molecular weight of about 42 kDa was detectable, and its cytosolic localization was demonstrated with cell fractionation experiments. These results were confirmed using immunohistochemical techniques. NEU2 expression in E.coli cells allowed purification of the recombinant protein. As already observed in the enzyme expressed in COS7 cells, NEU2 pH optimum corresponds to 5.6 and the polypeptide showed a K(m)for 4-MU-NANA of 0.07 mM. In addition, based on the detectable similarities between the NEU2 amino acid sequence and bacterial sialidases, a prediction of the three-dimensional structure of the enzyme was carried out using a protein homology modeling approach.
Monti
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Telethon Institute of Genetics and Medicine (TIGEM), San Raffaele Biomedical Science Park, via Olgettina 58, 20132 Milan, Italy. monti@tigem.it
Preti
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Nesti
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Ballabio
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Borsani
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D003600:Cytosol / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012486:Salmonella typhimurium / Q000201:enzymology; D016415:Sequence Alignment; D014162:Transfection
C403285:NEU2 protein, human; D009439:Neuraminidase