Impaired elastic-fiber assembly by fibroblasts from patients with either Morquio B disease or infantile GM1-gangliosidosis is linked to deficiency in the 67-kD spliced variant of beta-galactosidase

We have previously shown that intracellular trafficking and extracellular assembly of tropoelastin into elastic fibers is facilitated by the 67-kD elastin-binding protein identical to an enzymatically inactive, alternatively spliced variant of beta-galactosidase (S-Gal). In the present study, we investigated elastic-fiber assembly in cultures of dermal fibroblasts from patients with either Morquio B disease or GM1-gangliosidosis who bore different mutations of the beta-galactosidase gene. We found that fibroblasts taken from patients with an adult form of GM1-gangliosidosis and from patients with an infantile form, carrying a missense mutations in the beta-galactosidase gene-mutations that caused deficiency in lysosomal beta-galactosidase but not in S-Gal-assembled normal elastic fibers. In contrast, fibroblasts from two cases of infantile GM1-gangliosidosis that bear nonsense mutations of the beta-galactosidase gene, as well as fibroblasts from four patients with Morquio B who had mutations causing deficiency in both forms of beta-galactosidase, did not assemble elastic fibers. We also demonstrated that S-Gal-deficient fibroblasts from patients with either GM1-gangliosidosis or Morquio B can acquire the S-Gal protein, produced by coculturing of Chinese hamster ovary cells permanently transected with S-Gal cDNA, resulting in improved deposition of elastic fibers. The present study provides a novel and natural model validating functional roles of S-Gal in elastogenesis and elucidates an association between impaired elastogenesis and the development of connective-tissue disorders in patients with Morquio B disease and in patients with an infantile form of GM1-gangliosidosis.     

Processing of human beta-galactosidase in GM1-gangliosidosis and Morquio B syndrome

The nature of the molecular defect resulting in the beta-galactosidase deficiency in different forms of GM1-gangliosidosis and mucopolysaccharidosis IV B (Morquio B syndrome) was investigated. Normal and mutant cultured skin fibroblasts were labeled in vivo with [3H]leucine and immunoprecipitation studies with human anti-beta-galactosidase antiserum were performed, followed by polyacrylamide gel electrophoresis and fluorography. In Morquio B syndrome, the mutation does not interfere with the normal processing and intralysosomal aggregation of beta-galactosidase. In cells from infantile and adult GM1-gangliosidosis, 85-kDa precursor beta-galactosidase was found to be synthesized normally but more than 90% of the enzyme was subsequently degraded at one of the early steps in posttranslational processing. The residual 5-10% beta-galactosidase activity in adult GM1-gangliosidosis is 64-kDa mature lysosomal enzyme with normal catalytic properties but with a reduced ability of the monomeric form to aggregate into high molecular weight multimers. Knowledge of the exact nature of the molecular defect underlying beta-galactosidase deficiency in man may lead to a better understanding of the clinical and pathological heterogeneity among patients with different types of GM1-gangliosidosis and Morquio B syndrome.     

GM1 gangliosidosis and Morquio B disease: An update on genetic alterations and clinical findings

GM1 gangliosidosis and Morquio B syndrome, both arising from beta-galactosidase (GLB1) deficiency, are very rare lysosomal storage diseases with an incidence of about 1:100,000-1:200,000 live births worldwide. Here we report the beta-galactosidase gene (GLB1) mutation analysis of 21 unrelated GM1 gangliosidosis patients, and of 4 Morquio B patients, of whom two are brothers. Clinical features of the patients were collected and compared with those in literature. In silico analyses were performed by standard alignments tools and by an improved version of GLB1 three-dimensional models. The analysed cohort includes remarkable cases. One patient with GM1 gangliosidosis had a triple X syndrome. One patient with juvenile GM1 gangliosidosis was homozygous for a mutation previously identified in Morquio type B. A patient with infantile GM1 gangliosidosis carried a complex GLB1 allele harbouring two genetic variants leading to p.R68W and p.R109W amino acid changes, in trans with the known p.R148C mutation. Molecular analysis showed 27 mutations, 9 of which are new: 5 missense, 3 microdeletions and a nonsense mutation. We also identified four new genetic variants with a predicted polymorphic nature that was further investigated by in silico analyses. Three-dimensional structural analysis of GLB1 homology models including the new missense mutations and the p.R68W and p.R109W amino acid changes showed that all the amino acid replacements affected the resulting protein structures in different ways, from changes in polarity to folding alterations. Genetic and clinical associations led us to undertake a critical review of the classifications of late-onset GM1 gangliosidosis and Morquio B disease.     

Population analysis of the GLB1 gene in South Brazil

Infantile GM1 gangliosidosis is caused by the absence or reduction of lysosomal beta-galactosidase activity. Studies conducted in Brazil have indicated that it is one of the most frequent lysosomal storage disorders in the southern part of the country. To assess the incidence of this disorder, 390 blood donors were tested for the presence of two common mutations (1622-1627insG and R59H) in the GLB1 gene. Another group, consisting of 26 GM1 patients, and the blood donors were tested for the presence of two polymorphisms (R521C and S532G), in an attempt to elucidate whether there is a founder effect. The frequencies of the R59H and 1622-1627insG mutations among the GM1 patients studied were 19.2% and 38.5%, respectively. The frequency of polymorphism S532G was 16.7%, whereas R521C was not found in the patients. The overall frequency of either R59H or 1622-1627insG was 57.7% of the disease-causing alleles. This epidemiological study suggested a carrier frequency of 1:58. Seven different haplotypes were found. The 1622-1627insG mutation was not found to be linked to any polymorphism, whereas linkage disequilibrium was found for haplotype 2 (R59H, S532G) (p < 0.001). These data confirm the high incidence of GM1 gangliosidosis and the high frequency of two common mutations in southern Brazil.     

4-epi-Isofagomine derivatives as pharmacological chaperones for the treatment of lysosomal diseases linked to β-galactosidase mutations: Improved synthesis and biological investigations

(5aR)-5a-C-pentyl-4-epi-isofagomine 1 is a powerful inhibitor of lysosomal β-galactosidase and a remarkable chaperone for mutations associated with GM1-gangliosidosis and Morquio disease type B. We report herein an improved synthesis of this compound and analogs (5a-C-methyl, pentyl, nonyl and phenylethyl derivatives), and a crystal structure of a synthetic intermediate that confirms its configuration resulting from the addition of a Grignard reagent. These compounds were evaluated as glycosidase inhibitors and their potential as chaperones for mutant lysosomal galactosidases determined. Based on these results and on docking studies, the 5-C-pentyl derivative 1 was selected as the optimal structure for further investigations: this compound induces the maturation of mutated β-galactosidase in fibroblasts of a GM1-gangliosidosis patient and promote the decrease of keratan sulfate and oligosaccharide load in patient cells. Compound 1 is clearly capable of restoring β-galactosidase activity and of promoting maturation of the protein, which should result in significant clinical benefit. These properties strongly support the development of compound 1 for the treatment of GM1-gangliosidosis and Morquio disease type B patients harboring β-galactosidase mutations sensitive to pharmacological chaperoning.     

Molecular basis of GM1 gangliosidosis and Morquio disease, type B. Structure-function studies of lysosomal beta-galactosidase and the non-lysosomal beta-galactosidase-like protein

GM1 gangliosidosis and Morquio B disease are distinct disorders both clinically and biochemically yet they arise from the same beta-galactosidase enzyme deficiency. On the other hand, galactosialidosis and sialidosis share common clinical and biochemical features, yet they arise from two separate enzyme deficiencies, namely, protective protein/cathepsin A and neuraminidase, respectively. However distinct, in practice these disorders overlap both clinically and biochemically so that easy discrimination between them is sometimes difficult. The principle reason for this may be found in the fact that these three enzymes form a unique complex in lysosomes that is required for their stability and posttranslational processing. In this review, I focus mainly on the primary and secondary beta-galactosidase deficiency states and offer some hypotheses to account for differences between GM1 gangliosidosis and Morquio B disease.     

Turnover of beta-galactosidase in fibroblasts from patients with genetically different types of beta-galactosidase deficiency.

The turnover of lysosomal beta-galactosidase was studied in fibroblast cultures from patients with Gm1-gangliosidosis and combined beta-galactosidase and neuraminidase deficiency, which had 5-10% residual beta-galactosidase activity. beta-Galactosidase was specifically inactivated with the suicide substrate beta-D-galactopyranosylmethyl-p-nitro-phenyltriazene (beta-Gal-MNT) and from the subsequent restoration of enzyme activity in cell cultures turnover times were calculated. By using [3H]beta-Gal-MNT, the hydrolytic activity per molecule of beta-galactosidase was determined. 3H-labelled beta-D-galactopyranosylmethylamine, the precursor of [3H]beta-gal-MNT, was obtained by Raney-nickel-catalysed exchange with 3H2O. The rate of synthesis of beta-galactosidase in normal and all mutant cells tested was found to be 0.4-0.5 pmol/day per mg of cellular protein. The GM1-gangliosidosis cells tested contain the normal amount of 0.5 pmol of beta-galactosidase/mg of protein with a normal turnover time of about 10 days, but only 10% of beta-galactosidase activity per enzyme molecule. Cells with combined beta-galactosidase and neuraminidase deficiency contain only 0.3 pmol of beta-galactosidase/mg of protein with a decreased turnover time of 1 day and normal hydrolytic properties (200 nmol of 4-methylumbelliferyl galactoside/h pmol of beta-galactosidase).     

Molecular heterogeneity in human beta-galactosidase and neuraminidase deficiency

Human lysosomal beta-galactosidase and neuraminidase exist in a complex together with a 32-kilodalton (kd) glycoprotein. The latter protein was found to have a dual function: it is required for the aggregation of monomeric 64-kd beta-galactosidase into high molecular weight (600-700 kd) multimers and it is an essential subunit of neuraminidase together with a 76-kd polypeptide. The severe neurological disorder galactosialidosis, characterized by a coexistent deficiency of beta-galactosidase and neuraminidase, was found to be due to a genetic defect of the 32-kd protective protein. The molecular background of the clinical heterogeneity within this syndrome is described and will undoubtedly be further elucidated since we have recently isolated the gene coding for the protective protein. The sequence of normal and mutant (enzyme) proteins will also provide better insight into the characteristics of the beta-galactosidase-neuraminidase-protective protein complex. Another interesting model for the study of posttranslational processing is the defective phosphorylation of beta-galactosidase in cells from patients with GM1-gangliosidosis.     

Lysosomal neuraminidase. Catalytic activation in insect cells is controlled by the protective protein/cathepsin A

Lysosomal N-Acetyl-alpha-neuraminidase is active in complex with the protective protein/cathepsin A (PPCA) and beta-galactosidase. The interaction with PPCA is essential for the correct intracellular routing and lysosomal localization of neuraminidase, but the mechanism of its catalytic activation is unclear. To investigate this process, we have used the baculovirus expression system to co-express neuraminidase and PPCA precursors in insect cells, which resulted in high enzymatic activity of neuraminidase. Both the 34- and 20-kDa PPCA subunits were required for the activation. We further demonstrated that when expressed alone, the neuraminidase precursor remained dimeric (114 kDa) and had low enzymatic activity, but when co-expressed with PPCA and beta-galactosidase, it multimerized in a complex of approximately 1350 kDa, together with the other two proteins. The fully active neuraminidase co-precipitated with full-length PPCA and beta-galactosidase precursors. However, when co-expressed with the individual PPCA subunits, neuraminidase co-precipitated only with the small 20-kDa polypeptide, which therefore must contain a neuraminidase-binding site. Our finding suggests a model of activation of neuraminidase dependent on its oligomerization at acidic pH that is mediated by interaction with PPCA.     

Transport of human lysosomal neuraminidase to mature lysosomes requires protective protein/cathepsin A.

Human lysosomal N-acetyl-alpha-neuraminidase is deficient in two lysosomal storage disorders, sialidosis, caused by structural mutations in the neuraminidase gene, and galactosialidosis, in which a primary defect of protective protein/cathepsin A (PPCA) leads to a combined deficiency of neuraminidase and beta-D-galactosidase. These three glycoproteins can be isolated in a high molecular weight multi-enzyme complex, and the enzymatic activity of neuraminidase is contingent on its interaction with PPCA. To explain the unusual need of neuraminidase for an auxiliary protein, we examined, in transfected COS-1 cells, the effect of PPCA expression on post-translational modification, turnover and intracellular localization of neuraminidase. In pulse-chase studies, we show that the enzyme is synthesized as a 46 kDa glycoprotein, which is poorly phosphorylated, does not undergo major proteolytic processing and is secreted. Importantly, its half-life is not altered by the presence of PPCA. However, neuraminidase associates with the PPCA precursor shortly after synthesis, since the latter protein co-precipitates with neuraminidase using anti-neuraminidase antibodies. We further demonstrate by subcellular fractionation of transfected cells that neuraminidase segregates to mature lysosomes only when accompanied by wild-type PPCA, but not by transport-impaired PPCA mutants. These data suggest a novel role for PPCA in the activation of lysosomal neuraminidase, that of an intracellular transport protein.     

Mutation analyses in 17 patients with deficiency in acid beta-galactosidase: three novel point mutations and high correlation of mutation W273L with Morquio disease type B

An inherited deficiency in beta-galactosidase can result in GM1 gangliosidosis, with several phenotypes of generalized or chronic psychomotor deterioration, as well as in Morquio disease type B, a characteristic mucopolysaccharidosis free of neurological symptoms. We performed mutation analyses in 17 juvenile and adult patients from various European regions with a deficiency in beta-galactosidase and skeletal abnormalities. Fifteen of these had the Morquio B phenotype and have remained neurologically healthy until now while the two others exhibited psychomotor retardation of juvenile onset. A two-base substitution (851-852TG-->CT; W273L) was present in 14 of the 15 Morquio B cases. Even if one excludes alleles from patients with possible common descent, there was a much higher frequency (79%) among those with Morquio B phenotype for the W273L mutation than previously reported in the literature (37%). That the Morquio phenotype is also expressed in heterozygotes for W273L and alleles typically found in GM1 gangliosidosis makes it possible to predict the phenotype and reliably detect heterozygotes. A single French patient had a novel missense point mutation (Q408P) together with a known mutation (T500A) while the mentally retarded patients were both heterozygous for two mutations known in chronic GM1 gangliosidosis together with two novel missense point mutations (Y270D and H281Y) in the vicinity of W273L. Our results confirm the high impact of Trp 273 for the function of beta-galactosidase and the expression of the Morquio B phenotype. In addition, a second domain around the amino acids 400-500 may also be of significance.     

GM1-ganglioside-mediated activation of the unfolded protein response causes neuronal death in a neurodegenerative gangliosidosis

GM1-ganglioside (GM1) is a major sialoglycolipid of neuronal membranes that, among other functions, modulates calcium homeostasis. Excessive accumulation of GM1 due to deficiency of lysosomal beta-galactosidase (beta-gal) characterizes the neurodegenerative disease GM1-gangliosidosis, but whether the accumulation of GM1 is directly responsible for CNS pathogenesis was unknown. Here we demonstrate that activation of an unfolded protein response (UPR) associated with the upregulation of BiP and CHOP and the activation of JNK2 and caspase-12 leads to neuronal apoptosis in the mouse model of GM1-gangliosidosis. GM1 loading of wild-type neurospheres recapitulated the phenotype of beta-gal-/- cells and activated this pathway by depleting ER calcium stores, which ultimately culminated in apoptosis. Activation of UPR pathways did not occur in mice double deficient for beta-gal and ganglioside synthase, beta-gal-/-/GalNAcT-/-, which do not accumulate GM1. These findings suggest that the UPR can be induced by accumulation of the sialoglycolipid GM1 and this causes a novel mechanism of neuronal apoptosis.     

Novel mutations (Asn 484 Lys,Thr 500 Ala,Gly 438 Glu) in Morquio B disease

Primary deficiency of beta-galactosidase results in GM1 gangliosidosis and Morquio B disease. Of the more than 40 disease-causing mutations described in the Gal gene to date, about 75% are of the missense type and are scattered along the length of the gene. No single, major common mutation has been associated with GM1 gangliosidosis. However, a Trp 273 Leu mutation has been commonly found in the majority of patients with Morquio B disease defined genotypically to date.We now report three new mutations in three Morquio B patients where the Trp 273 Leu mutation is absent. Two of the mutations, C1502G (Asn 484 Lys) and A1548G (Thr 500 Ala), were found in twins (one male, one female) who display a mild form of Morquio B disease and keratan sulfate in the urine. In their fibroblasts, residual activity was 1.9% and 2.1% of controls. On Western blots, the 84-kDa precursor and the 64-kDa mature protein were barely detectable. The occurrence of a 45-kDa degradation product indicates that the mutated protein reached the lysosome but was abnormally processed. In the third case, we identified only a G1363A (Gly 438 Glu) mutation (a major deletion on the second allele has not been ruled out). This female patient too displays a very mild form of the disease with a residual activity of 5.7% of control values. In fibroblasts from this case, the 84-kDa precursor and the 45-kDa degradation product were present, while the mature 64-kDa form was barely detectable. The occurrence of these three mutations in the same area of the protein may define a domain involved in keratan sulfate degradation.     

Incorporation and degradation of GM1 ganglioside and asialoGM1 ganglioside in cultured fibroblasts from normal individuals and patients with beta-galactosidase deficiency

The uptake and degradation of GM1 ganglioside (GM1) and asialoGM1 ganglioside (GA1) were studied in cultured fibroblasts from normal individuals and patients with beta-galactosidase deficiency, using the lipid-loading test. The glycolipids were incorporated from the media into the fibroblasts and the terminal galactose was hydrolyzed in normal cells. The hydrolysis rates of GA1 were 80-86% of normal on the 3rd day after loading, while GM1 was hydrolyzed slowly; 35-54% on the 14th day. In infantile GM1 gangliosidosis and I-cell disease, little GM1 and GA1 was hydrolyzed on any day of culture, while fibroblasts from patients with adult GM1 gangliosidosis, Morquio disease type B and galactosialidosis hydrolyzed the lipids at nearly normal rates. The intracellular accumulation of the glycolipids, on the basis of protein content, was abnormally high in the case of infantile GM1 gangliosidosis and I-cell disease, but normal in the other disorders examined. These observations indicate that the in situ metabolism of GM1 and GA1 is probably normal in fibroblasts from patients with adult GM1 gangliosidosis, Morquio disease type B and galactosialidosis, although in vitro beta-galactosidase activities in these disorders are very low. The results are compatible with findings that GM1 and GA1 do not accumulate in the somatic organs of patients with adult GM1 gangliosidosis and galactosialidosis. In I-cell disease, however, the results of the loading test did not agree with the finding that there is little accumulation of glycolipids in postmortem tissues.     

Immunoelectron microscopical localization of lysosomal beta-galactosidase and its precursor forms in normal and mutant human fibroblasts

Immunoelectron microscopy was performed to study the biosynthesis of lysosomal beta-galactosidase (beta-gal) in normal and mutant human fibroblasts. Using polyclonal and monoclonal antibodies we show in normal cells precursor forms of beta-gal in the rough endoplasmic reticulum (RER) and in the Golgi apparatus throughout the stack of cisternae. In the lysosomes virtually all beta-gal exists as a high molecular weight multimer of mature enzyme. In the autosomal recessive disease GM1-gangliosidosis caused by a beta-gal deficiency and in galactosialidosis, associated with a combined deficiency of lysosomal neuraminidase and beta-gal, precursor forms of the latter enzyme are found in RER, Golgi and some labeling is present at the cell surface. The lysosomes remain unlabeled, indicative for the absence of enzyme molecules in this organelle. In galactosialidosis fibroblasts also no mature beta-gal is found in the lysosomes but in these cells the presence of the monomeric form can be increased by leupeptin (inhibition of proteolysis) whereas addition of a partly purified 32 kDa "protective protein" results in the restoration of high molecular weight beta-gal multimers in the lysosomes.     

GM1-gangliosidosis (genetic beta-galactosidase deficiency): identification of four mutations in different clinical phenotypes among Japanese patients

GM1-gangliosidosis is a genetic neurological disorder caused by mutations in the lysosomal acid beta-galactosidase gene. While its phenotypic expression is complex, it is usually classified as being of infantile, juvenile, or adult form, on the basis of age at onset, the rate of symptomatic progression, and severity of central nervous system involvement. We have analyzed the acid beta-galactosidase gene in 12 Japanese patients from nine families. The aim was to identify mutations in individual patients and then to examine possible correlation between the mutations and the clinical phenotypes. Northern blotting studies with a full-length human beta-galactosidase cDNA showed that the mRNA ranged from undetectable to substantially decreased in the infantile patients but was normal in quantity and size in all juvenile and adult patients. Four distinct missense mutations have been identified, each limited to the respective clinical forms within our small-size samples. In the infantile patient with decreased but detectable mRNA, a point mutation was found resulting in Arg49----Cys. In the infantile patient with nearly undetectable mRNA, mutation Arg457----Ter was identified. The mutation Arg201----Cys was found in all four of the juvenile patients, while all six adult patients were homozygous for the point mutation Ile51----Thr. The mutations found in the juvenile and adult patients alter restriction sites in the normal gene and thus are amendable to quick screening. The prediction that these mutations are responsible for the clinical disease was confirmed by no expression of the catalytic activity of the mutant proteins in the COS-I cell expression system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elimination of abnormal sialylglycoproteins in fibroblasts with sialidosis and galactosialidosis by normal gene transfer and enzyme replacement

Sialidosis and galactosialidosis are lysosomal storage diseases caused by the genetic defects of lysosomal sialidase (neuraminidase-1; NEU1) and lysosomal protective protein/cathepsin A (PPCA), respectively, associated with a NEU1 deficiency, excessive accumulation of sialylglycoconjugates, and development of progressive neurosomatic manifestations; in addition, the latter disorder is accompanied by simultaneous deficiencies of beta-galactosidase and cathepsin A. We demonstrated that a few soluble N-glycosylated proteins carrying sialyloligosaccharides sensitive to glycopeptidase F (GPF) can be specifically detected in cultured fibroblasts from sialidosis and galactosialidosis cases by blotting with a Maackia amurensis (MAM) lectin. We also examined the therapeutic effects of normal gene transfer and enzyme replacement by evaluating the decreases in sialylglycoconjugates accumulated in fibroblasts with these NEU1 deficiencies. The specific N-glycosylated proteins detected on MAM lectin blotting as well as the granular lysosomal fluorescence due to an avidin-FITC/biotinylated MAM lectin conjugate in sialidosis and galactosialidosis fibroblasts disappeared in parallel with the restoration of the intracellular NEU1 activity after transfection of the recombinant NEU1 fused to HA tag sequence and the wild-type PPCA cDNA as well as administration of the recombinant PPCA precursor protein. The detection method for the abnormal sialylglycoproteins in cultured cells involving MAM lectin was demonstrated to be useful not only for biochemical and diagnostic analyses of NEU1 deficiencies but also for therapeutic evaluation of these conditions.     

Mutations in acid beta-galactosidase cause GM1-gangliosidosis in American patients.

We describe four new mutations in the beta-galactosidase gene. These are the first mutations causing infantile and juvenile GM1-gangliosidosis to be described in American patients. Cell lines from two patients with juvenile and from six patients with infantile GM1-gangliosidosis were analyzed. Northern blot analysis showed the acid beta-galactosidase message to be of normal size and quantity in two juvenile and four infantile cases and of normal size but reduced quantity in two infantile cases. The mutations are distinct from the Japanese mutations. All are point mutations leading to amino acid substitutions: Lys577-->Arg, Arg590-->His, and Glu632-->Gly. The fourth mutation, Arg208-->Cys, accounts for 10 of 16 possible alleles. Two infantile cases from Puerto Rico of Spanish ancestry are homozygous for this mutation, suggesting that this allele may have come to South America and North America via Puerto Rico. That these mutations cause clinical disease was confirmed by marked reduction in catalytic activity of the mutant proteins in the Cos-1 cell expression system.