Characterization of human lysosomal neuraminidase defines the molecular basis of the metabolic storage disorder sialidosis.

Abstract

Neuraminidases (sialidases) have an essential role in the removal of terminal sialic acid residues from sialoglycoconjugates and are distributed widely in nature. The human lysosomal enzyme occurs in complex with 13-galactosidase and protective protein/cathepsin A (PPCA), and is deficient in two genetic disorders: sialidosis, caused by a structural defect in the neuraminidase gene, and galactosialidosis, in which the loss of neuraminidase activity is secondary to a deficiency of PPCA. We identified a full-length cDNA clone in the dbEST data base, of which the predicted amino acid sequence has extensive homology to other mammalian and bacterial neuraminidases, including the F(Y)RIP domain and "Asp-boxes." In situ hybridization localized the human neuraminidase gene 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 neuraminidase activity in a PPCA-dependent manner. The authenticity of the cDNA was verified by the identification of three independent mutations in the open reading frame of the mRNA from clinically distinct sialidosis patients. Coexpression of the mutant cDNAs with PPCA failed to generate neuraminidase activity, confirming the inactivating effect of the mutations. These results establish the molecular basis of sialidosis in these patients, and clearly identify the cDNA-encoded protein as lysosomal neuraminidase.

Molecular pathology of NEU1 gene in sialidosis

“…Patients homozygous for c.625delG and c.4 _ 7dupACTG mutations belong to a severe infantile-onset form of sialidosis associated with dysostosis multiplex, dysmorphic phenotype, hepatosplenomegaly, and ascites [Pshezhetsky et al, 1997;Lukong et al, 2000]. Another deletion, c.1209delC [Bonten et al, 1996], caused a frameshift that extended the sialidase protein by 69 amino acids. Transient expression of the mutant protein showed that it does not have enzymatic activity and is not transported to the lysosome [Bonten et al, 1996].…”

“…Another deletion, c.1209delC [Bonten et al, 1996], caused a frameshift that extended the sialidase protein by 69 amino acids. Transient expression of the mutant protein showed that it does not have enzymatic activity and is not transported to the lysosome [Bonten et al, 1996]. Duplication c.1193 _ 1198dupACCACT [Bonten et al, 2000] caused an in-frame duplication of H399 and Y400 amino acid residues and produced a mutant protein with about 30% of residual activity.…”

“…All of them should result in a synthesis of truncated proteins without catalytic activity. In the case of the c.1129G>T (p.E377X) mutation, truncated protein expressed in human fibroblasts or COS cells lacked both lysosomal localization and catalytic activity [Bonten et al, 1996]. Siblings carrying this mutation in one allele showed a mild clinical phenotype because of the V54M missense mutation in the other allele that reduced enzymatic activity of the mutant sialidase in vitro to only~40% of the normal level [Bonten et al, 2000].…”

“…All reported mutations were experimentally veri¢ed at both the RNA and genomic DNA level. a The numbers for the nucleotide changes are reported in accordance with published DNA sequence [Pshezhetsky et al,1997] and GenBank entry for lysosomal sialidase (accession number NM _ 000434), the A of the initiator codon denoted as nucleotide +1.The following changes were made to mutations originally reported by Bonten et al [1996;. Reported as G378X (c.1127G4T) by Bonten et al [2000].…”

“…Cathepsin A was cloned and its mutations in galactosialidosis were first characterized 15 years ago [Galjart et al, 1988], but cloning of sialidase has been hampered for almost two decades by the low tissue content and instability of this enzyme. Three groups simultaneously identified the human sialidase cDNA and gene by a homologous search in the Expressed Sequence Tags Database (dbEST, National Center for Biotechnology Information) and direct sequencing of human chromosome 6 [Bonten et al, 1996;Milner et al, 1997;Pshezhetsky et al, 1997]. These studies paved the way for the characterization of the molecular basis of sialidosis.…”