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, wid ely distributed in nature, which remove sialic acid residues from glycoprot eins and glycolipids, All of the sialidase so far characterized at the mole cular 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 pol ypeptide of 380 amino acids with two Asp block consensuses and the YRIP seq uence in the amino terminal part of the primary structure. Here me demonstr ate that NEU2 encodes a functional sialidase, NEU2 was expressed in COS7 ce lls, giving rise to a dramatic increase in the sialidase activity measured in cell extracts with the artificial substrate CR;IU-NANA. Using a rabbit p olyclonal antiserum, on Western blots a protein band with a molecular weigh t of about 42 kDa was detectable, and its cytosolic localization was demons trated with cell fractionation experiments, These results were confirmed us ing immunohistochemical techniques. NEU2 expression in E,coli cells allow e d purification of the recombinant protein. As already observed in the enzym e expressed in COS7 cells, NEU2 pH optimum corresponds to 5.6 and the polyp eptide showed a K-m for 4-MU-NANA of 0.07 mM. In addition, based on the det ectable similarities between the NEU2 amino acid sequence and bacterial sia lidases, a prediction of the three-dimensional structure of the enzyme was carried out using a protein homology modeling approach.