CHARACTERIZATION OF HUMAN LYSOSOMAL NEURAMINIDASE DEFINES THE MOLECULAR-BASIS OF THE METABOLIC STORAGE DISORDER SIALIDOSIS

Neuraminidases (sialidases) have an essential role in the removal of t erminal sialic acid residues from sialoglyconjugates and are distribut ed widely in nature. The human lysosomal enzyme occurs in complex with p-galactosidase and protective protein/cathepsin A (PPCA), and is def icient in two genetic disorders: sialidosis, caused by a structural de fect in the neuraminidase gene, and galactosialidosis, in which the lo ss of neuraminidase activity is secondary to a deficiency of PPCA. We identified a full-length cDNA clone in the dbEST data base, of which t he predicted amino acid sequence has extensive homology to other mamma lian and bacterial neuraminidases, including the F(Y)RIP domain and '' Asp-boxes.'' In situ hybridization localized the human neuraminidase g ene to chromosome band 6p21, a region known to contain the HLA locus. Transient expression of the cDNA in deficient human fibroblasts showed that the enzyme is compartmentalized in lysosomes and restored neuram inidase activity in a PPCA-dependent manner. The authenticity of the c DNA was verified by the identification of three independent mutations in the open reading frame of the mRNA from clinically distinct sialido sis patients. Coexpression of the mutant cDNAs with PPCA failed to gen erate neuraminidase activity, confirming the inactivating effect of th e mutations. These results establish the molecular basis of sialidosis in these patients, and clearly identify the cDNA-encoded protein as l ysosomal neuraminidase.