Cloning, expression and chromosomal mapping of human lysosomal sialidase and characterization of mutations in sialidosis

Abstract

Sialidase (neuraminidase, EC 3.2.1.18) catalyses the hydrolysis of terminal sialic acid residues of glyconjugates. Sialidase has been well studied in viruses and bacteria where it destroys the sialic acid-containing receptors at the surface of host cells, and mobilizes bacterial nutrients. In mammals, three types of sialidases, lysosomal, plasma membrane and cytosolic, have been described. For lysosomal sialidase in humans, the primary genetic deficiency results in an autosomal recessive disease, sialidosis, associated with tissue accumulation and urinary excretion of sialylated oligosaccharides and glycolipids. Sialidosis includes two main clinical variants: late-onset, sialidosis type I, characterized by bilateral macular cherry-red spots and myoclonus, and infantile-onset, sialidosis type II, characterized by skeletal dysplasia, mental retardation and hepatosplenomegaly. We report the identification of human lysosomal sialidase cDNA, its cloning, sequencing and expression. Examination of six sialidosis patients revealed three mutations, one frameshift insertion and two missense. We mapped the lysosomal sialidase gene to human chromosome 6 (6p21.3), which is consistent with the previous chromosomal assignment of this gene in proximity to the HLA locus.

Molecular pathology of NEU1 gene in sialidosis

“…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].…”

“…One deletion (c.625delG) and one duplication (c.4 _ 7dupACTG) [Pshezhetsky et al, 1997] cause frameshifts and the appearance of premature truncation codons, probably leading to nonsensemediated decay of the mRNA and a complete loss of sialidase activity in the affected cells. 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.…”

“…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.…”

Molecular and structural studies of Japanese patients with sialidosis type 1

“…6). Neither mutation affected the residues involved in the predicted active site (R78, R97, D103, D135, R280, and R347) (Pshezhetsky et al 1997;Crennell et al 1993). …”

“…Recently, human lysosomal neuraminidase cDNA was isolated (Bonten et al 1996;Pshezhetsky et al 1997;Milner et al 1997), and studies for clarifying the pathogenesis of sialidosis were started. So far, six different mutations in the neuraminidase gene have been reported (Bonten et al 1996;Pshezhetsky et al 1997). However, little has been elucidated regarding the enzyme proteins caused by the mutant genes.…”