title: Novel missense mutations in the human lysosomal sialidase gene in sialidosis patients and prediction of structural alterations of mutant enzymes.
abstract: Three novel missense mutations in the human lysosomal sialidase gene causing amino acid substitutions (P80L, W240R. and P316S) in the coding region were identified in two Japanese sialidosis patients. One patient with a severe, congenital form of type 2 sialidosis was a compound heterozygote for 239C-to-T (P80L) and 718T-to-C (W240R). The other patient with a mild juvenile-onset phenotype (type 1) was a homozygote for the base substitution of 946C-to-T (P316S). None of these mutant cDNA products showed enzymatic activity toward an artificial substrate when coexpressed in galactosialidosis fibroblastic cells together with protective protein/cathepsin A (PPCA). All mutants showed a reticular immunofluorescence distribution when coexpressed with the PPCA gene in COS-1 cells, suggesting that the gene products were retained in the endoplasmic reticulum/Golgi area or rapidly degraded in the lysosomes. Homology modeling of the structural changes introduced by the mutations predicted that the P80L and P316S transversions cause large conformational changes including the active site residues responsible for binding the sialic acid carboxylate group. The W240R substitution was deduced to influence the molecular surface structure of a limited region of the constructed models, which was also influenced by previously identified V217M and G243R transversions.
authors.affiliation: Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, Japan. kitoh@ph2.tokushima-u.ac.jp
authors.forename: Kohji
authors.initials: K
authors.lastname: Itoh
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Naganawa
authors.forename: Fumiko
authors.initials: F
authors.lastname: Matsuzawa
authors.forename: Seiichi
authors.initials: S
authors.lastname: Aikawa
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Doi
authors.forename: Naokazu
authors.initials: N
authors.lastname: Sasagasako
authors.forename: Takeshi
authors.initials: T
authors.lastname: Yamada
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Kira
authors.forename: Takuro
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Pshezhetsky
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Sakuraba
mesh_terms: D000328:Adult; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D007223:Infant; D008247:Lysosomes / Q000201:enzymology*; D008297:Male; D008958:Models, Molecular; D009081:Mucolipidoses / Q000201:enzymology / Q000235:genetics*; D009154:Mutation; D020125:Mutation, Missense / Q000235:genetics*; D009439:Neuraminidase / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D011247:Pregnancy; D011296:Prenatal Diagnosis; D011487:Protein Conformation; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D004247:DNA; D009439:Neuraminidase