B.J Hum Jochimsen Genet et(2002) al.: Stetteria 47:29–37 hydrogenophila © Jpn Soc Hum Genet and Springer-Verlag4600/29 2002 ORIGINAL ARTICLE Kohji Itoh · Yasunori Naganawa · Fumiko Matsuzawa Seiichi Aikawa · Hirofumi Doi · Naokazu Sasagasako Takeshi Yamada · Jun-ichi Kira · Takuro Kobayashi Alexey V. Pshezhetsky · Hitoshi Sakuraba Novel missense mutations in the human lysosomal sialidase gene in sialidosis patients and prediction of structural alterations of mutant enzymes Received: Stptember 21, 2001 / Accepted: November 2, 2001 Abstract Three novel missense mutations in the human changes including the active site residues responsible for lysosomal sialidase gene causing amino acid substitutions binding the sialic acid carboxylate group. The W240R sub- (P80L, W240R, and P316S) in the coding region were stitution was deduced to influence the molecular surface identified in two Japanese sialidosis patients. One patient structure of a limited region of the constructed models, with a severe, congenital form of type 2 sialidosis was a which was also influenced by previously identified V217M compound heterozygote for 239C-to-T (P80L) and 718T-to- and G243R transversions. C (W240R). The other patient with a mild juvenile-onset phenotype (type 1) was a homozygote for the base substitu- Key words Lysosomal sialidase · Sialidosis · Molecular tion of 946C-to-T (P316S). None of these mutant cDNA modeling · Protective protein/cathepsin A · Galacto- products showed enzymatic activity toward an artificial sialidosis substrate when coexpressed in galactosialidosis fibroblastic cells together with protective protein/cathepsin A (PPCA). All mutants showed a reticular immunofluorescence distri- bution when coexpressed with the PPCA gene in COS-1 Introduction cells, suggesting that the gene products were retained in the endoplasmic reticulum/Golgi area or rapidly degraded Lysosomal sialidase (neuraminidase, EC 3.2.1.18) catalyzes in the lysosomes. Homology modeling of the structural the hydrolysis of the terminal sialic acid residues of changes introduced by the mutations predicted that the sialylated glycoconjugates. In mammals, the enzyme associ- P80L and P316S transversions cause large conformational ates with acid -galactosidase (EC 3.2.1.23) and protec- tive protein/cathepsin A (PPCA, EC 3.4.16.1) to form a K. Itoh1 (*) · Y. Naganawa · H. Sakuraba multienzyme complex in lysosomes (d’Azzo et al. 1982; Department of Clinical Genetics, The Tokyo Metropolitan Institute Verheijen et al. 1982, 1985; Hiraiwa et al. 1988; van der of Medical Science, Tokyo Metropolitan Organization for Medical Horst et al. 1989). In paticular, association with PPCA Research, Tokyo, Japan is essential for expression of the sialidase activity (d’Azzo F. Matsuzawa · S. Aikawa · H. Doi et al. 1982; van der Spoel et al. 1998). Sialidosis is an auto- Celestar Lexico Sciences, Chiba, Japan somal recessive metabolic inborn error caused by a genetic N. Sasagasako defect of lysosomal sialidase, which is accompanied by accu- Department of Neurology, Hamanomachi Hospital, Federation of mulation in tissues and urinary excretion of sialyloligo- Public Services and Affiliated Personal Aid Association, Fukuoka, Japan saccharides (reviewed by Lowden and O’Brien 1979; Thomas 2000). The clinical phenotypes of sialidosis are T. Yamada · J. Kira · T. Kobayashi† Department of Neurology, Neurological Institute, Graduate School classified in two major groups according to the age of onset of Medical Sciences, Kyushu University, Fukuoka, Japan (†deceased) and severity: late-onset type 1 sialidosis is a relatively mild A.V. Pshezhetsky form characterized by macular cherry-red spots and myo- Service de Génétique Médicale, Départment de Pédiatrie, Hôpital clonus, and early infantile-onset type 2 sialidosis is a severe Sainte-Justine, Université de Montréal, Montréal, Canada condition distinguished by the appearance of dysmorphic manifestations in addition to neurological symptoms and Present address: cherry-red spots. Sialidosis type 2 is further divided into 1 Division of Medicinal Biotechnology, Institute for Medicinal three clinical subtypes: congenital, infantile, and juvenile. Resources, Faculty of Pharmaceutical Sciences, University of The congenital form is the most severe subtype, and is Tokushima, 1-78 Sho-machi, Tokushima 770-8505, Japan Tel. ϩ81-88-633-7290; Fax: ϩ81-88-633-7290 associated with hydrops fetalis, neonatal ascites, edema, e-mail: [email protected] inguinal hernia, hepatosplenomegaly, and death at an early 30 N. Matsuda et al.: EGF receptor and osteoblastic differentiation age. Another human sialidase deficiency, galactosialidosis, the nonconsanguineous parents’ request, as previously de- is caused by a primary defect of PPCA with combined de- scribed (Sasagasako et al. 1993). The proband of the family creases in lysosomal sialidase, -galactosidase, and cathep- was the second female child of the parents, who was found sin A activities, and has clinical manifestations similar to to have hydrops fetalis in the 33rd gestational week. At those of sialidosis (reviewed by d’Azzo et al. 2000). birth, severe subcutaneous edema, pleural effusion, ascites, In recent years, the human lysosomal sialidase gene has and hepatosplenomegaly were observed. She was diag- been cloned (Bonten et al. 1996; Pshezhetsky et al. 1997; nosed as having a congenital form of type 2 sialidosis, and Milner et al. 1997) on the basis of the genomic and struc- she subsequently developed respiratory failure and died 87 tural information on the sialidases obtained from a variety days after birth. The male fetus was artificially aborted of species, including viruses and bacteria (Roggentin et al. in the 21st gestational week. A pathological study revealed 1989; Rothe et al. 1991; Crennell et al. 1993, 1994; Gaskell slight generalized subcutaneous edema, mild pleural effu- et al. 1995). The human sialidase cDNA contains a 1.2-kb sion, ascites, and mild hepatosplenomegaly. Light micro- open reading frame and a polyadenylation site within the 3Ј scopic analysis disclosed a large number of vacuoles in the untranslated region. The deduced amino acid sequence con- cytoplasm of the liver, spleen, skin, bone marrow, lymph tains four Asp boxes (S/T-X-D-S-G-X-X-W/F), which are nodes, adrenal cortex, anterior horn cells of the spinal cord, repeated at topologically equivalent positions, and a (F)RIP and neuronal cells in the cerebrum. sequence motif, which is part of the active site. In addition, the sequence revealed the presence of three potential Patient 2. The other patient is the 24-year-old male off- N-glycosylation sites and a carboxy-terminal Y-X-X- spring of consanguineous parents. At the age of 14 years, hydrophobic residue motif similar to that found in other he developed gait disturbance and dysgraphia. Generalized lysosomal integral membrane proteins (Guarnieri et al. convulsive seizure attacks, visual disturbance, dysarthria, 1993; Sosa et al. 1993). A number of mutations have been and action myoclonus were also observed. Physical exami- identified in sialidosis patients (Bonten et al. 1996; Bonten nation at 18 years of age revealed bilateral cherry-red spots and d’Azzo 2000; Pshezhetsky et al. 1997; Lukong et al. and cerebellar dysfunction. He was diagnosed as having 2000; Naganawa et al. 2000). type 1 sialidosis. The progression of the symptoms was Conservation of the basic structural fold of sialidases, rapid. Difficulty in swallowing appeared by 18 years of age, which consists of six four-stranded antiparallel -sheets ar- and he suffered from recurrent bronchopneumonia. His ranged as the blades of a propeller around an axis of sym- elder brother died of sialidosis at 18 years of age, but metry passing through the active site, among various species detailed clinical data are not available. has also been shown (Crennell et al. 1993, 1994; Gaskell et al. 1995), which makes it possible to model the tertiary Cell culture structure of human lysosomal sialidase (Lukong et al. 2000; Naganawa et al. 2000). In the present study, we identified Human skin fibroblasts derived from the two patients with three new missense mutations resulting in amino acid sub- sialidosis were maintained in Ham’s F-10 medium supple- stitutions, and characterized the mutant products biochemi- mented with 10% fetal calf serum (FCS) and antibiotics. cally. Using homology modeling, we deduced the structural For transient expression of the wild-type and mutant changes in the structure of the sialidase molecule caused by sialidase cDNAs, a fibroblastic cell line derived from a the substitutions. galactosialidosis patient (ASVGS-1) (Shimmoto et al. 1993) or COS-1 cells (Gluzman 1988) was cultured in the same medium. Subjects and methods Gene analysis Subjects Total RNA and genomic DNA were isolated from the cul- Sialidosis patients were diagnosed biochemically by assay- tured fibroblasts as described previously (Davis et al. 1986). ing lysosomal enzymes (Suzuki et al. 1981; Suzuki 1987) in Lysosomal sialidase cDNA, including the entire coding cultured skin fibroblasts or amniocytes. All fibroblastic cell region, was obtained by reverse transcription (RT)- strains showed the isolated sialidase deficiency. Other polymerase chain reaction (PCR), and the nucleotide se- enzyme activities, including those of -galactosidase and quence was determined according to a previous report cathepsin A, were normal. (Naganawa et al. 2000). As described in the Results section, The research was carried out in accordance with the 239C-to-T (P80L), 718T-to-C (W240R),