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Conformational change in lysosomal neuraminidase caused by V217M mutation. The amino acid residues influenced by the change from V217 (green) to M217 (red) are shown; those colored blue are the wild-type residues and those colored yellow are the mutant residues. The active site residues are colored magenta
Source publication
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 649G-to-A and 727G-to-A, which result in amino acid alterations V217M and G243R, respectively. Using homology modeling, the structure of human lysosom...
Context in source publication
Context 1
... to the sequence alignment (Milner et al. 1997), we built a model of the human lysosomal neuraminidase without the N-terminal 47 residues, corresponding to the signal peptide (Milner et al. 1997;Vinogradova et al. 1998). The model was, thus, composed of the 368 amino acids from residue 48 to the terminal residue 415, the amino acid se- quence homology with the aligned fragment of Salmonella typhimurium LT2 being 39.7%. The modeled lysosomal neuraminidase retains a basic structure composed of six four-stranded antiparallel -sheets, and resembles a six- bladed propeller with the predicted active site cleft in the middle (Fig. 4). Both V217M and G243R are located in the -sheet of the third -sheet-loop unit (Fig. 4). The V217M transversion was deduced to alter the arrangement of 11 amino acid residues around the mutation site (Fig. 5). As to the G243R transversion, it was predicted to cause a drastic structural change with a wide range of up to 38 residues (Fig. 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. ...
Citations
... The expressed neuraminidase containing the V217M variant showed some residual enzyme activity, while the G243R mutant lost all of its enzyme activity. 13 Therefore, we speculate that both the residual enzyme activity and the conformational change caused by the mutant protein determine the clinical phenotype of the patient. ...
Purpose:
Sialidosis type 2 has variants that are both catalytically inactive (severe), while sialidosis type 1 has at least one catalytically active (mild) variant. This study aimed to discuss the structural changes associated with these variants in a newly reported family carrying NEU1 variants and explore the clinical characteristics of different combinations of variants in sialidosis type 1.
Methods:
First, whole-exome sequencing and detailed clinical examination were performed on the family. Second, structural analysis, including energy, flexibility and polar contacts, was conducted for several NEU1 variants, and a sialidase activity assay was performed. Third, previous NEU1 variants were systematically reviewed, and the clinical characteristics of patients in the severe-mild and mild-mild groups with sialidosis type 1 were analyzed.
Results:
We report a novel family with sialidosis type 1 and the compound heterozygous variants S182G and V143E. The newly identified V143E variant was predicted to be a mild variant through structural analysis and was confirmed by sialidase activity assay. The cherry-red spot was more prevalent in the severe-mild group, and ataxia was more common in the mild-mild group. Impaired cognition was found only in the severe-mild group. Moreover, patients with cherry-red spots and abnormal EEGs and VEPs had a relatively early age of onset, whereas patients with myoclonus had a late onset.
Conclusion:
Changes in flexibility and local polar contacts may be indicators of the NEU1 pathogenicity. Sialidosis type 1 can be divided into two subgroups according to the variant combinations, and patients with these two subtypes have different clinical characteristics.
... Sialidosis (SiD) (OMIM #256550) is a NEU1 deficiency caused by autosomal recessive NEU1 gene (Locus, 6p21.33) mutations, and it is associated with excessive accumulation of sialylglycoconjugates, including sialylglycopeptides and sialyloligosaccharides, in neurosomatic tissues and urinary secretion of sialyloligosaccharides [29,[32][33][34][35][36][37][38][39][40][41][42]. SiD is classified into two clinical phenotypes according to the age of onset and severity caused by heterogeneous NEU1 mutations, including missense ones. ...
... SiD is classified into two clinical phenotypes according to the age of onset and severity caused by heterogeneous NEU1 mutations, including missense ones. Late-onset type 1 SiD is a relatively mild form characterized by macular cherry-red spots and myoclonus [29,37,38,42], and early infantileonset type 2 SiD is a severe condition distinguished by the appearance of dysmorphic manifestations in addition to neurological symptoms and cherry-red spots [34,35]. The type 2 SiD is further divided into three clinical subtypes: congenital, infantile, and juvenile [34]. ...
Neuraminidase 1 (NEU1) is a lysosomal sialidase that cleaves terminal α-linked sialic acid residues from sialylglycans. NEU1 is biosynthesized in the rough endoplasmic reticulum (RER) lumen as an N-glycosylated protein to associate with its protective protein/cathepsin A (CTSA) and then form a lysosomal multienzyme complex (LMC) also containing β-galactosidase 1 (GLB1). Unlike other mammalian sialidases, including NEU2 to NEU4, NEU1 transport to lysosomes requires association of NEU1 with CTSA, binding of the CTSA carrying terminal mannose 6-phosphate (M6P)-type N-glycan with M6P receptor (M6PR), and intralysosomal NEU1 activation at acidic pH. In contrast, overexpression of the single NEU1 gene in mammalian cells causes intracellular NEU1 protein crystallization in the RER due to self-aggregation when intracellular CTSA is reduced to a relatively low level. Sialidosis (SiD) and galactosialidosis (GS) are autosomal recessive lysosomal storage diseases caused by the gene mutations of NEU1 and CTSA, respectively. These incurable diseases associate with the NEU1 deficiency, excessive accumulation of sialylglycans in neurovisceral organs, and systemic manifestations. We established a novel GS model mouse carrying homozygotic Ctsa IVS6 + 1 g/a mutation causing partial exon 6 skipping with simultaneous deficiency of Ctsa and Neu1. Symptoms developed in the GS mice like those in juvenile/adult GS patients, such as myoclonic seizures, suppressed behavior, gargoyle-like face, edema, proctoptosis due to Neu1 deficiency, and sialylglycan accumulation associated with neurovisceral inflammation. We developed a modified NEU1 (modNEU1), which does not form protein crystals but is transported to lysosomes by co-expressed CTSA. In vivo gene therapy for GS and SiD utilizing a single adeno-associated virus (AAV) carrying modNEU1 and CTSA genes under dual promoter control will be created.
... Moreover, prolonged visual evoked potential latency and giant cortical potential SSEP are also helpful for early diagnosis [6,8,14] . In neuroimaging, diffuse brain atrophy (particularly the cerebellum) can be evident in the advanced stage of type 1 sialidosis [15] . In addition, accumulation of sialyloligosaccharides can be observed in the central nervous system of sialidosis patients by pathological examination [15] . ...
... In neuroimaging, diffuse brain atrophy (particularly the cerebellum) can be evident in the advanced stage of type 1 sialidosis [15] . In addition, accumulation of sialyloligosaccharides can be observed in the central nervous system of sialidosis patients by pathological examination [15] . ...
... To date, more than 30 NEU1 mutations have been identified as responsible for type 1 sialidosis (Table 1) [4,7,8,[11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] . Missense variants are the most common pathogenic mutations, and few exonic duplications or deletions have been reported [11] . ...
Background:
Type 1 sialidosis, also known as cherry-red spot-myoclonus syndrome, is a rare autosomal recessive lysosomal storage disorder presenting in the second decade of life. The most common symptoms are myoclonus, ataxia and seizure. It is rarely encountered in the Chinese mainland.
Case summary:
A 22-year-old male presented with complaints of progressive myoclonus, ataxia and slurred speech, without visual symptoms; the presenting symptoms began at the age of 15-year-old. Whole exome sequencing revealed two pathogenic heterozygous missense variants [c.239C>T (p.P80L) and c.544A>G (p.S182G) in the neuraminidase 1 (NEU1) gene], both of which have been identified previously in Asian patients with type 1 sialidosis. All three patients identified in Mainland China come from three unrelated families, but all three show the NEU1 mutations p.S182G and p.P80L pathogenic variants. Increasing sialidase activity through chaperones is a promising therapeutic target in sialidosis.
Conclusion:
Through retrospective analysis and summarizing the clinical and genetic characteristics of type 1 sialidosis, we hope to raise awareness of lysosomal storage disorders among clinicians and minimize the delay in diagnosis.
... Table 2 shows these neurometabolic disorders and the main diagnostic procedures. (11,(24)(25)(26)(27)(28)(29)(30)(31) Iran J Child Neurol. Summer 2020 Vol. 14 No. 3 ...
Neurometabolic disorders are hereditary conditions mainly affect the function of the brain and the nervous system. The prevalence of these disorders is 1 in 1,000 live births. Such disorders, at different ages, could manifest as sepsis, hypoglycemia, and other neurologic disorders. Having similar manifestations leads to delayed diagnosis of neurometabolic disorders. A number of neurometabolic disorders have known treatments; however, to prevent long-term complications the key factors are early diagnosis and treatment. Although a large number of neurometabolic diseases have no treatment or cure, the correct and on-time diagnosis before death is important for parents to have plans for prenatal diagnosis. Different diagnostic procedures could be offered to parents, enzymatic procedures, and determining metabolites in plasma, urine, and CSF, and molecular genetic diagnosis. Molecular genetic diagnostic procedures are expensive and could not be offered to all parents. Therefore, we aimed to design algorithms to diagnose neurometabolic disorders according to some frequent and characteristic signs and symptoms. By designing these algorithms and using them properly, we could offer diagnostic enzymatic panels. These enzymatic panels are inexpensive; thereby reducing the financial burden on the parents. Also, having an early diagnosis according to these panels could lead to offering more accurate and less expensive molecular genetic tests.
... Table 2 shows these neurometabolic disorders and the main diagnostic procedures. (11,(24)(25)(26)(27)(28)(29)(30)(31) Iran J Child Neurol. Summer 2020 Vol. 14 No. 3 ...
Neurometabolic disorders are hereditary conditions mainly affect the function of the brain and the nervous system. The prevalence of these disorders is 1 in 1,000 live births. Such disorders, at different ages, could manifest as sepsis, hypoglycemia, and other neurologic disorders. Having similar manifestations leads to delayed diagnosis of neurometabolic disorders. A number of neurometabolic disorders have known treatments; however, to prevent long-term complications the key factors are early diagnosis and treatment. Although a large number of neurometabolic diseases have no treatment or cure, the correct and on-time diagnosis before death is important for parents to have plans for prenatal diagnosis. Different diagnostic procedures could be offered to parents, enzymatic procedures, and determining metabolites in plasma, urine, and CSF, and molecular genetic diagnosis. Molecular genetic diagnostic procedures are expensive and could not be offered to all parents. Therefore, we aimed to design algorithms to diagnose neurometabolic disorders according to some frequent and characteristic signs and symptoms. By designing these algorithms and using them properly, we could offer diagnostic enzymatic panels. These enzymatic panels are inexpensive; thereby reducing the financial burden on the parents. Also, having an early diagnosis according to these panels could lead to offering more accurate and less expensive molecular genetic tests.
... A list of the NEU1 mutations identified in these patients and their position in the primary structure of the protein are given in Figure 1A,B. For six of the patients, the mutations have been reported earlier [8,[16][17][18][19]. The remaining six patients, not described in the literature, carry novel NEU1 mutations (p.Ala167Val, p.Tyr268Cys, p.Ser410Arg-fs), as well as a splice variant G > A at intron 3 + (g.1635G > A-fs), five of which in compound heterozygosity with mutations shared by other sialidosis patients ( Figure 1B). ...
Congenital deficiency of the lysosomal sialidase neuraminidase 1 (NEU1) causes the lysosomal storage disease, sialidosis, characterized by impaired processing/degradation of sialo-glycoproteins and sialo-oligosaccharides, and accumulation of sialylated metabolites in tissues and body fluids. Sialidosis is considered an ultra-rare clinical condition and falls into the category of the so-called orphan diseases, for which no therapy is currently available. In this study we aimed to identify potential therapeutic modalities, targeting primarily patients affected by type I sialidosis, the attenuated form of the disease. We tested the beneficial effects of a recombinant protective protein/cathepsin A (PPCA), the natural chaperone of NEU1, as well as pharmacological and dietary compounds on the residual activity of mutant NEU1 in a cohort of patients’ primary fibroblasts. We observed a small, but consistent increase in NEU1 activity, following administration of all therapeutic agents in most of the fibroblasts tested. Interestingly, dietary supplementation of betaine, a natural amino acid derivative, in mouse models with residual NEU1 activity mimicking type I sialidosis, increased the levels of mutant NEU1 and resolved the oligosacchariduria. Overall these findings suggest that carefully balanced, unconventional dietary compounds in combination with conventional therapeutic approaches may prove to be beneficial for the treatment of sialidosis type I.
... The c.650T.C mutant showed a 44% residual activity compared to the wild-type enzyme. Even if the residual activity is still quite high compared to non-transfected cells, the mutation results in the p.V217A amino acid change, involving the same residue of the p.V217M substitution already described in a patient carrying sialidosis type I [34] and present in the HGMD database. This mutation is supposed to alter the interaction between NEU1 and PPCA, thus preventing the correct transport and maturation of the enzyme [34]. ...
... Even if the residual activity is still quite high compared to non-transfected cells, the mutation results in the p.V217A amino acid change, involving the same residue of the p.V217M substitution already described in a patient carrying sialidosis type I [34] and present in the HGMD database. This mutation is supposed to alter the interaction between NEU1 and PPCA, thus preventing the correct transport and maturation of the enzyme [34]. This evidence strongly suggests that also the c.650T.C mutation may affects the NEU1-PPCA interaction and could represent a pathological allele responsible for the mild form of the disease. ...
... In particular the two residues V217 and D234, together with the residues G218, L231, W240, G243 and A298 define a region of the NEU1 protein that could represent an important site in the surface interaction with PPCA partner ( Figure 2C). As already described in another case of sialidosis [34], both mutants showed reduced NEU1 protein levels by Western-blot analysis ( Figure 3B) which correlate with the reduction in sialidase activity. Moreover, their subcellular localization resulted altered compared to the wild-type protein and the mutant NEU1 proteins showed almost no signal in vesicular structures compatible with the lysosomal compartment ( Figure 4). ...
The NEU1 gene is the first identified member of the human sialidases, glycohydrolitic enzymes that remove the terminal sialic acid from oligosaccharide chains. Mutations in NEU1 gene are causative of sialidosis (MIM 256550), a severe lysosomal storage disorder showing autosomal recessive mode of inheritance. Sialidosis has been classified into two subtypes: sialidosis type I, a normomorphic, late-onset form, and sialidosis type II, a more severe neonatal or early-onset form. A total of 50 causative mutations are reported in HGMD database, most of which are missense variants. To further characterize the NEU1 gene and identify new functionally relevant protein isoforms, we decided to study its genetic variability in the human population using the data generated by two large sequencing projects: the 1000 Genomes Project (1000G) and the NHLBI GO Exome Sequencing Project (ESP). Together these two datasets comprise a cohort of 7595 sequenced individuals, making it possible to identify rare variants and dissect population specific ones. By integrating this approach with biochemical and cellular studies, we were able to identify new rare missense and frameshift alleles in NEU1 gene. Among the 9 candidate variants tested, only two resulted in significantly lower levels of sialidase activity (p<0.05), namely c.650T>C and c.700G>A. These two mutations give rise to the amino acid substitutions p.V217A and p.D234N, respectively. NEU1 variants including either of these two amino acid changes have 44% and 25% residual sialidase activity when compared to the wild-type enzyme, reduced protein levels and altered subcellular localization. Thus they may represent new, putative pathological mutations resulting in sialidosis type I. The in silico approach used in this study has enabled the identification of previously unknown NEU1 functional alleles that are widespread in the population and could be tested in future functional studies.
... The age of onset and severity of the clinical manifestations are correlated with NEU1 mutations (10,11) and the level of residual neuraminidase activity (10,12,13), indicating the existence of considerable genotype-phenotype correlation in this disease. To date, more than 40 mutations within the NEU1 gene have been identified in patients with sialidosis type I or type II (2,3,(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). ...
The case of a Japanese sialidosis type I patient with a novel NEU1 gene mutation is described. The patient developed an unsteady gait at age 14 and was referred to our hospital at age 16. On admission, subnormal intelligence, dysarthria, myoclonus, intentional tremors, limb and gait ataxia, hyperreflexia and macular cherry-red spots were observed. An enzymological analysis revealed a primary deficiency of neuraminidase. An NEU1 gene analysis identified two heterozygous missense mutations: p.P80L and p.D135N. The p.D135N mutation is a novel mutation that is considered to be associated with the mild clinical phenotype of sialidosis. Serial brain MRI showed diffuse brain atrophy progressing rapidly over the 41-month observation period.
... Table 2 shows these neurometabolic disorders and the main diagnostic procedures. (11,(24)(25)(26)(27)(28)(29)(30)(31) Iran J Child Neurol. Summer 2020 Vol. 14 No. 3 ...
Lysosomal storage disorders (LSDs) are relatively rare inborn errors of metabolism, resulting from the accumulation of substrates within the lysosomes.They represent a group of more than 40 distinct genetic disorders.Most of these disorders are inherited in an autosomal recessive manner, except Fabry’s disease and mucopolysaccharidoses type II (MPS II) which are inherited in an X-linked recessive manner.Most disorders present clinically with multi-system involvement.Common clinical features involve bony dysplasia, hepatosplenomegaly, central nervous system dysfunction, haematological abnormalities, and coarse hair and facial features.There are many phenotypical similarities within the categories.Potential treatments for some of these disorders are available in the form of enzyme replacement therapy and bone marrow transplantation.PCR-sequencing methods were used for genetic investigation of 236 pediatric cases referred or diagnosed in our department over a period of 3 years from Nov 2009 to Nov 2012.Detailed clinical data, including sex, age of onset of disease, age at diagnosis, mode of presentation, family history, consanguinity rates, and high-risk screening results were collected.Biochemical analysis was done in different laboratories in abroad.
... The p.P200L mutation (Fig. 1B1,B2), on the other hand, affects an amino acid localized in the putative sialidase-cathepsin A binding site, on the surface region of the molecule. Previously described mutations in this region usually result in complete or almost complete loss of enzyme activity and cause severe sialidosis type II phenotype (17,31). Our results are also consistent with this pattern, already observed by other authors: patient S2, who is homozygous for this alteration, presents typical severe clinical symptoms with psychomotor development delay and a series of skeletal abnormalities (see Table 3). ...
Coutinho MF, Lacerda L, Macedo-Ribeiro S, Baptista E, Ribeiro H, Prata MJ, Alves S. Lysosomal multienzymatic complex-related diseases: a genetic study among Portuguese patients.
The functional activity of lysosomal enzymes sialidase, β-galactosidase and N-acetylaminogalacto-6-sulfate-sulfatase in the cell depends on their association in a multienzyme complex with cathepsin A. Mutations in any of the components of this complex result in functional deficiency thereby causing severe lysosomal storage disorders. Here, we report the molecular defects underlying sialidosis (mutations in sialidase; gene NEU1), galactosialidosis (mutations in cathepsin A; gene PPGB) and GM1 gangliosidosis (mutations in β-galactosidase; gene GLB1) in Portuguese patients. We performed molecular studies of the PPGB, NEU1 and GLB1 genes in biochemically diagnosed Portuguese patients. Gene expression was determined and the effect of each mutation predicted at protein levels. In the NEU1 gene, we found three novel missense mutations (p.P200L, p.D234N and p.Q282H) and one nonsense mutation (p.R341X). In the PPGB gene, we identified two missense mutations, one novel (p.G86V) and one already described (p.V104M), as well as two new deletions (c.230delC and c.991-992delT) that give rise to non-functional proteins. We also present the first molecular evidence of a causal missense mutation localized to the cathepsin A active site. Finally, in the GLB1 gene, we found six different mutations, all of them previously described (p.R59H, p.R201H, p.H281Y, p.W527X, c.1572-1577InsG and c.845-846delC). Seven novel mutations are reported here, contributing to our knowledge of the mutational spectrum of these diseases and to a better understanding of the genetics of the lysosomal multienzymatic complex. The results of this study will allow carrier detection in affected families and prenatal molecular diagnosis, leading to the improvement of genetic counseling.
