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.
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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. (+info)
Processing of lysosomal beta-galactosidase. The C-terminal precursor fragment is an essential domain of the mature enzyme.
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Lysosomal beta-D-galactosidase (beta-gal), the enzyme deficient in the autosomal recessive disorders G(M1) gangliosidosis and Morquio B, is synthesized as an 85-kDa precursor that is C-terminally processed into a 64-66-kDa mature form. The released approximately 20-kDa proteolytic fragment was thought to be degraded. We now present evidence that it remains associated to the 64-kDa chain after partial proteolysis of the precursor. This polypeptide was found to copurify with beta-gal and protective protein/cathepsin A from mouse liver and Madin-Darby bovine kidney cells and was immunoprecipitated from human fibroblasts but not from fibroblasts of a G(M1) gangliosidosis and a galactosialidosis patient. Uptake of wild-type protective protein/cathepsin A by galactosialidosis fibroblasts resulted in a significant increase of mature and active beta-gal and its C-terminal fragment. Expression in COS-1 cells of mutant cDNAs encoding either the N-terminal or the C-terminal domain of beta-gal resulted in the synthesis of correctly sized polypeptides without catalytic activity. Only when co-expressed, the two subunits associate and become catalytically active. Our results suggest that the C terminus of beta-gal is an essential domain of the catalytically active enzyme and provide evidence that lysosomal beta-galactosidase is a two-subunit molecule. These data may give new significance to mutations in G(M1) gangliosidosis patients found in the C-terminal part of the molecule. (+info)
Characterization of beta-galactosidase mutations Asp332-->Asn and Arg148-->Ser, and a polymorphism, Ser532-->Gly, in a case of GM1 gangliosidosis.
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We have identified and characterized three missense mutations in a patient with type 1 G(M1) gangliosidosis, namely a substitution of G for A at nucleotide position 1044 (G1044-->A; in exon 10) on one allele, which converts Asp(332) into asparagine, and both a mutation (C492-->A in exon 4, leading to the amino acid change of Arg(148)-->Ser) and a polymorphism (A1644-->G in exon 15, leading to a change of Ser(532)-->Gly) on the other allele. This patient had less than 1% residual beta-galactosidase activity and minimally detectable levels of immunoreactive beta-galactosidase protein in fibroblasts. To account for the above findings, a series of expression and immunolocalization studies were undertaken to assess the impact of each mutation. Transient overexpression in COS-1 cells of cDNAs encoding Asp(332)Asn, Arg(148)Ser and Ser(532)Gly mutant beta-galactosidases produced abundant amounts of precursor beta-galactosidase, with activities of 0, 84 and 81% compared with the cDNA clone for wild-type beta-galactosidase (GP8). Since the level of vector-driven expression is much less in Chinese hamster ovary (CHO) cells than in COS-1 cells, and we knew that exogenous beta-galactosidase undergoes lysosomal processing when expressed in these cells, transient expression studies were performed of Arg(148)Ser and Ser(532)Gly, which yielded active forms of the enzyme. In this case, the Arg(148)Ser and Ser(532)Gly products gave rise to 11% and 86% of the control activity respectively. These results were not unexpected, since the Arg(148)Ser mutation introduced a major conformational change into the protein, and we anticipated that it would be degraded in the endoplasmic reticulum (ER), whereas the polymorphism was expected to produce near-normal activity. To examine the effect of the Asp(332)Asn mutation on the catalytic activity, we isolated CHO clones permanently transfected with the Asp(332)Asn and Asp(332)Glu constructs, purified the enzymes by substrate-analogue-affinity chromatography, and determined their kinetic parameters. The V(max) values of both mutant recombinant enzymes were markedly reduced (less than 0.9% of the control), and the K(m) values were unchanged compared with the corresponding wild-type enzyme isolated at the same time. Both the Arg(148)Ser beta-galactosidase in CHO cells and Asp(332)Asn beta-galactosidases (in COS-1 and CHO cells) produced abundant immunoreaction in the perinuclear area, consistent with localization in the ER. A low amount was detected in lysosomes. Incubation of patient fibroblasts in the presence of leupeptin, which reduces the rate of degradation of lysosomal beta-galactosidase by thiol proteases, had no effect on residual enzyme activity, and immunostaining was again detected largely in the perinuclear area (localized to the ER) with much lower amounts in the lysosomes. In summary, the Arg(148)Ser mutation has no effect on catalytic activity, whereas the Asp(332)Asn mutation seriously reduces catalytic activity, suggesting that Asp(332) might play a role in the active site. Immunofluorescence studies indicate the expressed mutant proteins with Arg(148)Ser and Asp(332)Asn mutations are held up in the ER, where they are probably degraded, resulting in only minimum amounts of the enzyme becoming localized in the lysosomes. These results are completely consistent with findings in the cultured fibroblasts. Our results imply that most of the missense mutations described in G(M1) gangliosidosis to date have little effect on catalytic activity, but do affect protein conformation such that the resulting protein cannot be transported out of the ER and fails to arrive in the lysosome. This accounts for the minimal amounts of enzyme protein and activity seen in most G(M1) gangliosidosis patient fibroblasts. (+info)
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.
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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. (+info)
GM1-gangliosidosis in Alaskan huskies: clinical and pathologic findings.
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Three Alaskan Huskies, two females and one male, were diagnosed with GM1-gangliosidosis. Clinically, diseased animals exhibited proportional dwarfism and developed progressive neurologic impairment with signs of cerebellar dysfunction at the age of 5-7 months. Skeletal lesions characterized by retarded enchondral ossification of vertebral epiphyses were revealed by radiographs of the male dog at 5.5 months of age. Histologic examination of the central nervous system (CNS) revealed that most neurons were enlarged with a foamy to granular cytoplasm due to tightly packed vacuoles that displaced the Nissl substance. Vacuoles in paraffin-embedded sections stained positively with Luxol fast blue and Grocott's method, and in frozen sections vacuoles were periodic acid-Schiff positive. Foamy vacuolation also occurred within neurons of the autonomic ganglia. Extracerebral cells such as macrophages and peripheral lymphocytes also displayed foamy cytoplasm and vacuolation. In the CNS of diseased animals, a mild demyelination and axonal degeneration was accompanied by a significant astrogliosis (P < 0.05) in the gray matter as compared with age- and sex-matched control dogs. There was also a significant loss (P < 0.05) of oligodendrocytes in the gray and white matter of affected animals as compared with controls. Ultrastructurally, the neuronal storage material consisted of numerous circular to concentric whorls of lamellated membranes or stacks of membranes in parallel arrays. GM1-gangliosidosis in Alaskan Huskies resembles beta-galactosidase deficiency in other canine breeds, and these CNS disorders may be a consequence of neuronal storage and disturbed myelin processing. (+info)
Signaling pathways transduced through the elastin receptor facilitate proliferation of arterial smooth muscle cells.
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In this report we demonstrate that soluble peptides, elastin degradation products stimulate proliferation of arterial smooth muscle cells. We show that these effects are due to generation of intracellular signals transduced through the cell surface elastin receptor, which consists of peripheral 67-kDa elastin-binding protein (EBP) (spliced variant of beta-galactosidase), immobilized to the transmembrane sialidase and the protective protein. We found that elastin receptor-transduced signaling triggers activation of G proteins, opening of l-type calcium channels, and a sequential activation of tyrosine kinases: FAK, c-Src, platelet-derived growth factor-receptor kinase and then Ras-Raf-MEK1/2-ERK1/2 phosphorylation cascade. This, in turn, causes an increase in expression of cyclins and cyclin-dependent kinases, and a consequent increase in cellular proliferation. The EBP-transduced signals also induce tyrosine kinase-dependent phosphorylation of beta-tubulin, LC3, microtubule-associated protein 1, and alpha-actin and troponin-T, which could be linked to reorganization of cytoskeleton. We have also disclosed that induction of these signals can be abolished by anti-EBP antibody or by galactosugars, which cause shedding of EBP from the cell surface. Moreover, elastin-derived peptides did not induce proliferation of EBP-deficient cells derived from patients bearing a nonsense mutation of the beta-galactosidase gene or sialidase-deficient cells from patients with congenital sialidosis. (+info)
Central nervous system inflammation is a hallmark of pathogenesis in mouse models of GM1 and GM2 gangliosidosis.
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Mouse models of the GM2 gangliosidoses [Tay-Sachs, late onset Tay-Sachs (LOTS), Sandhoff] and GM1 gangliosidosis have been studied to determine whether there is a common neuro-inflammatory component to these disorders. During the disease course, we have: (i) examined the expression of a number of inflammatory markers in the CNS, including MHC class II, CD68, CD11b (CR3), 7/4, F4/80, nitrotyrosine, CD4 and CD8; (ii) profiled cytokine production [tumour necrosis factor alpha (TNF alpha), transforming growth factor (TGF beta 1) and interleukin 1 beta (IL1 beta)]; and (iii) studied blood-brain barrier (BBB) integrity. The kinetics of apoptosis and the expression of Fas and TNF-R1 were also assessed. In all symptomatic mouse models, a progressive increase in local microglial activation/expansion and infiltration of inflammatory cells was noted. Altered BBB permeability was evident in Sandhoff and GM1 mice, but absent in LOTS mice. Progressive CNS inflammation coincided with the onset of clinical signs in these mouse models. Substrate reduction therapy in the Sandhoff mouse model slowed the rate of accumulation of glycosphingolipids in the CNS, thus delaying the onset of the inflammatory process and disease pathogenesis. These data suggest that inflammation may play an important role in the pathogenesis of the gangliosidoses. (+info)
Brain ceramide hexosides in Tay-Sachs disease and generalized gangliosidosis (GM1-gangliosidosis).
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The carbohydrate composition was determined for ceramide hexosides isolated from brains of patients with Tay-Sachs disease and generalized gangliosidosis (hereby named GM1-gangliosidosis). Gray matter of patients with each disease showed a characteristic abnormal ceramide hexoside pattern. In Tay-Sachs gray matter, ceramide trihexoside is the major component, whereas ceramide tetrahexoside is barely detectable. In GM1-gangliosidosis, ceramide tetrahexoside is the major ceramide hexoside, while ceramide trihexoside is present only in small amount. These two major components have been characterized as the asialo derivatives of, respectively, the "Tay-Sachs ganglioside" (GM2-ganglioside) and the normal major monosialoganglioside (GM1-ganglioside). In both diseases, more than half the ceramide monohexoside of gray matter was glucocerebroside. Gray matter ceramide dihexoside, present in both diseases at higher than normal levels, was mostly ceramide lactoside, with possibly a small amount of ceramide digalactoside. Sulfatide contained only galactose. The abnormal ceramide hexoside pattern is limited to gray matter: white matter showed normal ceramide hexosides, i.e. a preponderance of monohexosides and sulfatide, with no detectable glucocerebroside. (+info)