Molecular and structural studies of Japanese patients with sialidosis type 1

Abstract

To gain insight into the pathogenesis of sialidosis type 1, we performed molecular investigations of two unrelated Japanese patients. Both of them are compound heterozygotes for base substitutions of 649 G-to-A and 727 G-to-A, which result in amino acid alterations V217M and G243R, respectively. Using homology modeling, the structure of human lysosomal neuraminidase was constructed and the structural changes caused by these missense mutations were deduced. The predicted change due to V217M was smaller than that caused by G243R, the latter resulting in a drastic, widespread alteration. The overexpressed gene products containing these mutations had the same molecular weight as that of the wild type, although the amounts of the products were moderately decreased. A biochemical study demonstrated that the expressed neuraminidase containing a V217M mutation was partly transported to lysosomes and showed residual enzyme activity, although a G243R mutant was retained in the endoplasmic reticulum/Golgi area and had completely lost the enzyme activity. Considering the data, we surmise that the V217M substitution may be closely associated with the phenotype of sialidosis type 1 with a late onset and moderate clinical course.

Molecular pathology of NEU1 gene in sialidosis

“…In vitro expression studies demonstrated that the mutant enzyme is targeted to the lysosome and retains about 10% of residual activity . Finally, the c.880C4T (p.R294S) mutation was found in two unrelated individuals of AfroAmerican origin affected with type I sialidosis [Bonten et al, 2000], and a combination of c.649G4A (p.V217M) and c.727G4A (p.G243R) mutations were found in two unrelated sialidosis type I patients from Japan [Naganawa et al, 2000]. All other missense mutations detected in the sialidase gene were individual (Table 1).…”

“…Using homology modeling, a potential effect of missense mutations on the sialidase tertiary structure was estimated and was correlated with the residual activity, folding, and intracellular localization of the enzyme, as well as with the clinical phenotype [Bonten et al, 2000;Lukong et al, 2000Lukong et al, , 2001Naganawa et al, 2000;Itoh et al, 2002]. For example, Lukong et al [2000] built the structural model of human sialidase using the atomic coordinates of homologous sialidases from Micromonospora viridifaciens, Salmonella typhimurium, and Vibrio cholerae.…”

Novel missense mutations in the human lysosomal sialidase gene in sialidosis patients and prediction of structural alterations of mutant enzymes

“…Conservation of the basic structural fold of sialidases, which consists of six four-stranded antiparallel -sheets arranged as the blades of a propeller around an axis of symmetry passing through the active site, among various species has also been shown (Crennell et al 1993(Crennell et al , 1994Gaskell et al 1995), which makes it possible to model the tertiary structure of human lysosomal sialidase (Lukong et al 2000;Naganawa et al 2000). In the present study, we identified three new missense mutations resulting in amino acid substitutions, and characterized the mutant products biochemically.…”

“…Lysosomal sialidase cDNA, including the entire coding region, was obtained by reverse transcription (RT)-polymerase chain reaction (PCR), and the nucleotide sequence was determined according to a previous report (Naganawa et al 2000). As described in the Results section, 239C-to-T (P80L), 718T-to-C (W240R), and 946C-to-T (P316S) substitutions were detected in exons 2, 4, and 5, respectively, of the lysosomal sialidase gene.…”

“…In addition, the sequence revealed the presence of three potential N-glycosylation sites and a carboxy-terminal Y-X-Xhydrophobic residue motif similar to that found in other lysosomal integral membrane proteins (Guarnieri et al 1993;Sosa et al 1993). A number of mutations have been identified in sialidosis patients (Bonten et al 1996;Pshezhetsky et al 1997;Lukong et al 2000;Naganawa et al 2000).…”