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(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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: GM1 gangliosidosis is a rare lysosomal storage disorder caused by GLB1 mutations. Because of its extreme rarity and symptoms that overlap with other neurodegenerative diseases, its diagnosis is sometimes challenging, especially in the late infantile form with less severe phenotype. We aim to expand the clinical and genetic spectrum of late infantile GM1 gangliosidosis., Methods: We confirmed a diagnosis of GM1 gangliosidosis based on GLB1 mutations and/or the deficiency of β-galactosidase activity. We identified the first two cases by whole-exome sequencing, and then the other six cases by direct sequencing of GLB1 with enzyme analysis., Results: All eight patients presented with developmental delay or regression during late infancy and later developed epilepsy, mostly intractable generalized tonic seizures. No clinical signs of storage disorders were noted except for skeletal abnormalities. Interestingly, we found aspartate transaminase (AST) elevations alone with normal alanine transaminase (ALT) levels in all patients. The recurrent mutation, p.D448V in GLB1, accounted for 50.0% of total alleles in our cohort., Conclusions: With a high index of clinical suspicion, skeletal survey and AST level would be important for early diagnosis of GM1 gangliosidosis. In addition, we would highlight the clinical usefulness of whole-exome sequencing in the diagnosis of non-classical presentation of ultra-rare neurodegenerative disease in children., (Copyright © 2018 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

N-Functionalized amino(hydroxymethyl)cyclopentanetriols are potent inhibitors of β-d-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for G M1 -gangliosidosis-associated lysosomal acid β-galactosidase thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

From 1,2;3,4-di-O-isopropylidene-α-D-galactopyranose, a series of highly functionalized (hydroxymethyl)cyclopentanes was easily available. In line with reports by Reymond and Jäger on similar structures, these amine containing basic carbasugars are potent inhibitors of β-D-galactosidases and, for the first time, could be shown to act as pharmacological chaperones for G M1 -gangliosidosis-associated lysosomal acid β-galactosidase mutant R201C, thus representing a new structural type of pharmacological chaperones for this lysosomal storage disease., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This report is about the identification, synthesis and initial biological characterization of derivatives of 4-epi-isofagomine as pharmacological chaperones (PC) for human lysosomal β-galactosidase. The two epimers of 4-epi-isofagomine carrying a pentyl group at C-5a, namely (5aR)- and (5aS)-5a-C-pentyl-4-epi-isofagomine, were prepared by an innovative procedure involving in the key step the addition of nitrohexane to a keto-pentopyranoside. Both epimers were evaluated as inhibitors of the human β-galactosidase: the (5aR)-stereoisomer (compound 1) was found to be a very potent inhibitor of the enzyme (IC 50  = 8 nM, 30× more potent than 4-epi-isofagomine at pH 7.3) with a high selectivity for this glycosidase whereas the (5aS) epimer was a much weaker inhibitor. In addition, compound 1 showed a remarkable activity as a PC. It significantly enhanced the residual activity of mutant β-galactosidase in 15 patient cell lines out of 23, with enhancement factors greater than 3.5 in 10 cell lines and activity restoration up to 91% of normal. Altogether, these results indicated that (5aR)-5a-C-pentyl-4-epi-isofagomine constitutes a promising PC-based drug candidate for the treatment of GM1-gangliosidosis and Morquio disease type B., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

From an easily available partially protected formal derivative of 1-deoxymannojirimycin, by hydroxymethyl chain-branching and further elaboration, lipophilic analogs of the powerful β-d-galactosidase inhibitor 4-epi-isofagomine have become available. New compounds exhibit improved inhibitory activities comparable to benchmark compound NOEV (N-octyl-epi-valienamine) and may serve as leads towards improved and more selective pharmacological chaperones for GM1-gangliosidosis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Introduction: Galactosialidosis is a rare lysosomal storage disease caused by a combined deficiency of GM1 β-galactosidase (β-gal) and neuraminidase secondary to a defect of a lysosomal enzyme protective protein/cathepsin A (PPCA) and mutation in CTSA gene. Three subtypes are recognized: early infantile, late infantile, and juvenile/adult. There is no specific therapy for patients with galactosialidosis at this time., Objectives: The aim of this study was to determine the chaperone effect of N-octyl-4-epi-β-valienamine (NOEV) on β-gal proteins in skin fibroblasts of PPCA-deficit patients., Methods: β-Gal and neuraminidase activities were measured for the diagnosis of the patients with galactosialidosis. Western blotting for PPCA protein and direct sequencing for CTSA gene were performed. Cultured skin fibroblast were treated with NOEV., Results: We report four novel patients with galactosialidosis: one had the early infantile form and the other three had the juvenile/adult form. We found that NOEV stabilized β-gal activity in lysate from cultured skin fibroblasts from these patients. Treatment with NOEV significantly enhanced β-gal activity in cultured skin fibroblasts in the absence of PPCA., Conclusions: Our results indicate the possibility that NOEV chaperone therapy might have a beneficial effect, at least in part, for patients with galactosialidosis., (Copyright © 2015 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Electrophilic fluorination of an exocyclic methoxymethylene enol ether derived from N-tert-butyloxycarbonyl-1,5-dideoxy-1,5-imino-3,4-O-isopropylidene-D-erythro-pent-2-ulose (11) provided the 5-fluoro derivative of the powerful β-galactosidase inhibitor 4-epi-isofagomine (8). This structural alteration, in combination with N-alkylation, led to considerably improved α-galactosidase selectivity. New compounds may serve as leads en route to new pharmacological chaperones for Fabry's disease., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A seven-month-old male child was brought in for an eye test for poor vision and nystagmus noticed from four months of age. The child had delayed milestones of development and multiple (six times) episodes of unexplained lower respiratory tract infection (from two months of age) treated by pediatricians at different centers without complete cure. Fundus examination showed bilateral cherry-red spots at the macula. There were diffusely distributed hyper-pigmented patches (Mongolian spots) on the back and extensor aspect of the extremities. The case was sent back to the pediatricians for a re-evaluation to rule out storage disorder. Lysosomal enzyme assay in the leucocytes showed a significantly reduced β-galactosidase level (15.6 nmol/hr/mg protein in contrast to a normal range of 79.6 to 480.0). This confirmed the patient to be a case of lysosomal storage disease, the GM1 gangliosidosis (type I).

Lysosomes are cytoplasmic compartments that contain many acid hydrolases and play critical roles in the metabolism of a wide range of macromolecules. Deficiencies in lysosomal enzyme activities cause genetic diseases, called lysosomal storage disorders (LSDs). Many mutations have been identified in the genes responsible for LSDs, and the identification of mutations is required for the accurate molecular diagnoses. Here, we analyzed cell lines that were derived from two different LSDs, GM1 gangliosidosis and sialidosis. GM1 gangliosidosis is caused by mutations in the GLB1 gene that encodes β-galactosidase. A lack of β-galactosidase activity leads to the massive accumulation of GM1 ganglioside, which results in neurodegenerative pathology. Mutations in the NEU1 gene that encodes lysosomal sialidase cause sialidosis. Insufficient activity of lysosomal sialidase progressively increases the accumulation of sialylated molecules, and various clinical symptoms, including mental retardation, appear. We sequenced the entire coding regions of GLB1 and NEU1 in GM1 gangliosidosis and sialidosis patient cells, respectively. We found the novel mutations p.E186A in GLB1 and p.R347Q in NEU1, as well as many other mutations that have been previously reported. We also demonstrated that patient cells containing the novel mutations showed the molecular phenotypes of the corresponding disease. Further structural analysis suggested that these novel mutation sites are highly conserved and important for enzyme activity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The ubiquitous distribution of lysosomes and their heterogeneous protein composition reflects the versatility of these organelles in maintaining cell homeostasis and their importance in tissue differentiation and remodeling. In lysosomes, the degradation of complex, macromolecular substrates requires the synergistic action of multiple hydrolases that usually work in a stepwise fashion. This catalytic machinery explains the existence of lysosomal enzyme complexes that can be dynamically assembled and disassembled to efficiently and quickly adapt to the pool of substrates to be processed or degraded, adding extra tiers to the regulation of the individual protein components. An example of such a complex is the one composed of three hydrolases that are ubiquitously but differentially expressed: the serine carboxypeptidase, protective protein/cathepsin A (PPCA), the sialidase, neuraminidase-1 (NEU1), and the glycosidase β-galactosidase (β-GAL). Next to this 'core' complex, the existence of sub-complexes, which may contain additional components, and function at the cell surface or extracellularly, suggests as yet unexplored functions of these enzymes. Here we review how studies of basic biological processes in the mouse models of three lysosomal storage disorders, galactosialidosis, sialidosis, and GM1-gangliosidosis, revealed new and unexpected roles for the three respective affected enzymes, Ppca, Neu1, and β-Gal, that go beyond their canonical degradative activities. These findings have broadened our perspective on their functions and may pave the way for the development of new therapies for these lysosomal storage disorders.

GM1 gangliosidosis and Morquio B disease are autosomal recessive diseases caused by the defect in the lysosomal β-galactosidase (β-Gal), frequently related to misfolding and subsequent endoplasmic reticulum-associated degradation. Pharmacological chaperone (PC) therapy is a newly developed molecular therapeutic approach by using small molecule ligands of the mutant enzyme that are able to promote the correct folding and prevent endoplasmic reticulum-associated degradation and promote trafficking to the lysosome. In this report, we describe the enzymological properties of purified recombinant human β-Gal(WT) and two representative mutations in GM1 gangliosidosis Japanese patients, β-Gal(R201C) and β-Gal(I51T). We have also evaluated the PC effect of two competitive inhibitors of β-Gal. Moreover, we provide a detailed atomic view of the recognition mechanism of these compounds in comparison with two structurally related analogues. All compounds bind to the active site of β-Gal with the sugar-mimicking moiety making hydrogen bonds to active site residues. Moreover, the binding affinity, the enzyme selectivity, and the PC potential are strongly affected by the mono- or bicyclic structure of the core as well as the orientation, nature, and length of the exocyclic substituent. These results provide understanding on the mechanism of action of β-Gal selective chaperoning by newly developed PC compounds., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

We report herein a newly developed domino reaction that facilitates the synthesis of new 1,5-dideoxy-1,5-iminoribitol iminosugar C-glycosides 7a-e and 8. The key intermediate in this approach is a six-membered cyclic sugar nitrone that is generated in situ and trapped by an alkene dipolarophile via a [2 + 3] cycloaddition reaction to give the corresponding isooxazolidines 10a-e in a "one-pot" protocol. The iminoribitol C-glycosides 7a-e and 8 were found to be modest β-galactosidase (bGal) inhibitors. However, compounds 7c and 7e showed "pharmacological chaperone" activity for mutant lysosomal bGal activity and facilitated its recovery in GM1 gangliosidosis patient fibroblasts by 2-6-fold.

G(M1)-gangliosidosis is a rare progressive neurodegenerative disorder due to an autosomal recessively inherited deficiency of lysosomal β-galactosidase. We have identified seven American black bears (Ursus americanus) found in the Northeast United States suffering from G(M1)-gangliosidosis. This report describes the clinical features, brain MRI, and morphologic, biochemical and molecular genetic findings in the affected bears. Brain lipids were compared with those in the brain of a G(M1)-mouse. The bears presented at ages 10-14 months in poor clinical condition, lethargic, tremulous and ataxic. They continued to decline and were humanely euthanized. The T(2)-weighted MR images of the brain of one bear disclosed white matter hyperintensity. Morphological studies of the brain from five of the bears revealed enlarged neurons with foamy cytoplasm containing granules. Axonal spheroids were present in white matter. Electron microscopic examination revealed lamellated membrane structures within neurons. Cytoplasmic vacuoles were found in the liver, kidneys and chondrocytes and foamy macrophages within the lungs. Acid β-galactosidase activity in cultured skin fibroblasts was only 1-2% of control values. In the brain, ganglioside-bound sialic acid was increased more than 2-fold with G(M1)-ganglioside predominating. G(A1) content was also increased whereas cerebrosides and sulfatides were markedly decreased. The distribution of gangliosides was similar to that in the G(M1)-mouse brain, but the loss of myelin lipids was greater in the brain of the affected bear than in the brain of the G(M1) mouse. Isolated full-length cDNA of the black bear GLB1 gene revealed 86% homology to its human counterpart in nucleotide sequence and 82% in amino acid sequence. GLB1 cDNA from liver tissue of an affected bear contained a homozygous recessive T(1042) to C transition inducing a Tyr348 to His mutation (Y348H) within a highly conserved region of the GLB1 gene. The coincidence of several black bears with G(M1)-gangliosidosis in the same geographic area suggests increased frequency of a founder mutation in this animal population., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A growing body of evidence suggests that misfolding of a mutant protein followed by its aggregation or premature degradation in the endoplasmic reticulum is one of the main mechanisms that underlie inherited neurodegenerative diseases, including lysosomal storage diseases. Chemical or pharmacological chaperones are small molecules that bind to and stabilize mutant lysosomal enzyme proteins in the endoplasmic reticulum. A number of chaperone compounds for lysosomal hydrolases have been identified in the last decade. They have gained attention because they can be orally administrated, and also because they can penetrate the blood-brain barrier. In this article, we describe two chaperone candidates for the treatment of GM1-gangliosidosis. We also discuss the future direction of this strategy targeting other lysosomal storage diseases as well as protein misfolding diseases in general.

Chaperone therapy is a newly developed molecular therapeutic approach to lysosomal diseases, a group of human genetic diseases causing severe brain damage. Based on early molecular studies during the last decade of the 20th century and early years of the 21st century, mainly on Fabry disease and GM1-gangliosidosis, we found some mutant enzyme proteins were unstable in the cell, and unable to express catalytic activities. Subsequently galactose and other active-site binding substrate analogs were found stabilized and enhance the mutant enzyme activity in culture cells. We concluded that the mutant misfolding enzyme protein and substrate analog competitive inhibitor (chemical chaperone) form a stable complex to be transported to the lysosome, to restore the catalytic activity of mutant enzyme after spontaneous dissociation under the acidic condition. This gene mutation-specific molecular interaction is a paradoxical phenomenon that an enzyme inhibitor in vitro serves as an enzyme stabilizer in situ. First we developed a commercially available compound 1-deoxygalactonojirimycin (DGJ) for Fabry disease, and confirmed the above molecular phenomenon. Currently DGJ has become a new candidate of oral medicine for Fabry disease, generalized vasculopathy involving the kidneys, heart and central nervous system in the middle age. This drug development has reached the phase 3 of human clinical study. Then we found two valienamine derivatives, N-octyl-4-epi-β-valienamine (NOEV) and N-octyl-β-valienamine (NOV), as promising therapeutic agents for human β-galactosidase deficiency disorders (GM1-gangliosidosis and Morquio B disease) and β-glucosidase deficiency disorders (phenotypic variations of Gaucher disease), respectively. Originally NOEV and NOV had been discovered as competitive inhibitors, and then their paradoxical bioactivities as chaperones were confirmed in cultured fibroblasts from patients with these disorders. Subsequently GM1-gangliosidosis model mice have been used for confirmation of clinical effectiveness, adverse effects and pharmacokinetic studies. Orally administered NOEV entered the brain through the blood-brain barrier, enhanced β-galactosidase activity, reduced substrate storage, and improved neurological deterioration clinically. Computational analysis revealed pH-dependent enzyme-chaperone interactions. Our recent study indicated chaperone activity of a new DGJ derivative, MTD118, for β-galactosidase complementary to NOEV. NOV also showed the chaperone effect toward several β-glucosidase gene mutants in Gaucher disease. Furthermore a commercial expectorant drug ambroxol was found to be a chaperone for β-glucosidase. A few Gaucher patients responded to this drug with remarkable improvement of oculomotor dysfunction and myoclonus. We hope chaperone therapy will become available for some patients with Fabry disease, GM1-gangliosidosis, Gaucher disease, and other lysosomal storage diseases particularly with central nervous system involvement., (Copyright © 2013 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.)

Background: This paper aims to report GLB1 activities and mutation analysis of three patients from the mainland of China, one with Morquio B disease and two with GM1 gangliosidosis., Methods: GLB1 activity and GLB1 gene mutation were analyzed in the three patients who were clinically suspected of having Morquio B disease or GM1 gangliosidosis. Novel mutations were analyzed by aligning GLB1 homologs, 100 control chromosomes, and the PolyPhen-2 tool., Results: The enzymatic activity of GLB1 was found to be 5.03, 4.20, and 4.50 nmol/h/mg in the three patients, respectively. Patient 1 was a compound heterozygote for p.[Arg148Cys] and p.[Tyr485Cys] mutations in the GLB1 gene. Patient 2 was a compound heterozygote for p.[Tyr270Phe] and p.[Leu337Pro] mutations. Patient 3 was a homozygote for p.[Asp448Val] mutation. Three mutations (p.[Tyr485Cys], p.[Tyr270Phe] and p.[Leu337Pro]) were novel variants and were predicted to damage GLB1 function., Conclusions: The enzymatic activity and related gene analysis of β-galactosidase should be performed in clinically suspected individuals to confirm diagnosis. The three novel mutations, p.[Tyr485Cys], p.[Tyr270Phe], and p.[Leu337Pro], are thought to be disease-causing mutations.

Objective: To utilize high-throughput sequencing to determine the etiology of juvenile-onset neurodegeneration in a 19-year-old woman with progressive motor and cognitive decline., Methods: Exome sequencing identified an initial list of 133,555 variants in the proband's family, which were filtered using segregation analysis, presence in dbSNP, and an empirically derived gene exclusion list. The filtered list comprised 52 genes: 21 homozygous variants and 31 compound heterozygous variants. These variants were subsequently scrutinized with predicted pathogenicity programs and for association with appropriate clinical syndromes., Results: Exome sequencing data identified 2 GLB1 variants (c.602G>A, p.R201H; c.785G>T, p.G262V). β-Galactosidase enzyme analysis prior to our evaluation was reported as normal; however, subsequent testing was consistent with juvenile-onset GM1-gangliosidosis. Urine oligosaccharide analysis was positive for multiple oligosaccharides with terminal galactose residues., Conclusions: We describe a patient with juvenile-onset neurodegeneration that had eluded diagnosis for over a decade. GM1-gangliosidosis had previously been excluded from consideration, but was subsequently identified as the correct diagnosis using exome sequencing. Exome sequencing can evaluate genes not previously associated with neurodegeneration, as well as most known neurodegeneration-associated genes. Our results demonstrate the utility of "agnostic" exome sequencing to evaluate patients with undiagnosed disorders, without prejudice from prior testing results.

Competitive inhibitors of either α-galactosidase (α-Gal) or β-galactosidase (β-Gal) with high affinity and selectivity have been accessed by exploiting aglycone interactions with conformationally locked sp(2)-iminosugars. Selected compounds were profiled as potent pharmacological chaperones for mutant lysosomal α- and β-Gal associated with Fabry disease and GM(1) gangliosidosis.

Therapeutic chaperone effect of a valienamine derivative N-octyl 4-epi-β-valienamine (NOEV) was studied in G(M1)-gangliosidosis model mice. Phamacokinetic analysis revealed rapid intestinal absorption and renal excretion after oral administration. Intracellular accumulation was not observed after continuous treatment. NOEV was delivered to the central nervous system through the blood-brain barrier to induce high expression of the apparently deficient β-galactosidase activity. NOEV treatment starting at the early stage of disease resulted in remarkable arrest of neurological progression within a few months. Survival time was significantly prolonged. This result suggests that NOEV chaperone therapy will be clinically effective for prevention of neuronal damage if started early in life hopefully also in human patients with G(M1)-gangliosidosis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

G(M1) gangliosidosis and Morquio B are autosomal recessive lysosomal storage diseases associated with a neurodegenerative disorder or dwarfism and skeletal abnormalities, respectively. These diseases are caused by deficiencies in the lysosomal enzyme β-d-galactosidase (β-Gal), which lead to accumulations of the β-Gal substrates, G(M1) ganglioside, and keratan sulfate. β-Gal is an exoglycosidase that catalyzes the hydrolysis of terminal β-linked galactose residues. This study shows the crystal structures of human β-Gal in complex with its catalytic product galactose or with its inhibitor 1-deoxygalactonojirimycin. Human β-Gal is composed of a catalytic TIM barrel domain followed by β-domain 1 and β-domain 2. To gain structural insight into the molecular defects of β-Gal in the above diseases, the disease-causing mutations were mapped onto the three-dimensional structure. Finally, the possible causes of the diseases are discussed.

N-(Dansylamino)hexylaminocarbonylpentyl-1,5-dideoxy-1,5-imino-D-galactitol, a strong competitive inhibitor of β-galactosidase, enhances residual β-galactosidase activities in fibroblasts and serves as lead en route to diagnostic compounds for tracking the fate of mutant β-gal as well as aberrant GM1 gangliosides by live cell imaging., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Two simple and reliably accessible intermediates, N-carboxypentyl- and N-aminohexyl-1-deoxy-D-galactonojirimycin were employed for the synthesis of a set of terminally N-dansyl substituted derivatives. Reaction of the terminal carboxylic acid of N-carboxypentyl-1-deoxy-D-galactonojirimycin with N-dansyl-1,6-diaminohexane provided the chain-extended fluorescent derivative. Employing bis(6-dansylaminohexyl)amine, the corresponding branched di-N-dansyl compound was obtained. Partially protected N-aminohexyl-1-deoxy-D-galactonojirimycin served as intermediate for two additional chain-extended fluorescent 1-deoxy-D-galactonojirimycin (1-DGJ) derivatives featuring terminal dansyl groups in the N-alkyl substituent. These new compounds are strong inhibitors of d-galactosidases and may serve as leads en route to pharmacological chaperones for GM1-gangliosidosis., (Copyright © 2011. Published by Elsevier Ltd.)

β-Galactosidase deficiency is a group of lysosomal lipid storage disorders with an autosomal recessive trait. It causes two clinically different diseases, G(M1) -gangliosidosis and Morquio B disease. It is caused by heterogeneous mutations in the GLB1 gene coding for the lysosomal acid β-galactosidase. We have previously reported the chaperone effect of N-octyl-4-epi-β-valienamine (NOEV) on mutant β-galactosidase proteins. In this study, we performed genotype analyses of patients with β-galactosidase deficiency and identified 46 mutation alleles including 9 novel mutations. We then examined the NOEV effect on mutant β-galactosidase proteins by using six strains of patient-derived skin fibroblast. We also performed mutagenesis to identify β-galactosidase mutants that were responsive to NOEV and found that 22 out of 94 mutants were responsive. Computational structural analysis revealed the mode of interaction between human β-galactosidase and NOEV. Moreover, we confirmed that NOEV reduced G(M1) accumulation and ameliorated the impairments of lipid trafficking and protein degradation in β-galactosidase deficient cells. These results provided further evidence to NOEV as a promising chaperone compound for β-galactosidase deficiency., (© 2011 Wiley-Liss, Inc.)

Background: GM1 gangliosidosis (GM1) is an autosomal recessive lysosomal storage disease caused by deficiency of acid beta-galactosidase (GLB1; EC3.2.1.23). Here, we identify three novel mutations in the GLB1 gene from two Han Chinese patients with GM1 that appear correlated with clinical phenotype., Methods: One of the two Han Chinese patients with GM1 presented with the juvenile form, and the other with the infantile form with cardiac involvement. Sequencing of the entire GLB1 gene revealed three novel mutations (p.H102 D, p.G494V, c.495_497delTCT), which were absent in 94 normal controls. Transient expression of cDNA encoding these variants was performed in COS-1 cells to evaluate β-galactosidase activities., Results: The first case (patient 1) with the juvenile form contained two missense mutations, p.H102 D and p.A301V. Patient 2 diagnosed with the infantile form of the disease with cardiac involvement was compound heterozygous for p.G494V and c.495_497delTCT mutations. All mutant beta-galactosidases exhibited significantly reduced activity (12%, 0%, 0%, and 0% for p.H102 D, p.A301V, p.G494V, and c.495_497delTCT), compared with the wild-type beta-galactosidase cDNA clone. The mutations identified in patient 2 with cardiomyopathy were localized in the GLB1 gene region common to both lysosomal beta-galactosidase and elastin binding protein (EBP), and caused a deletion in the elastin-binding domain of EBP., Conclusions: All four mutations identified in Han Chinese patients induce significant suppression of β-galactosidase activity, correlating with severity of disease and presence of cardiomyopathy.

Objective: The Epstein-Barr virus (EBV) is utilized as a tool in the study of cellular biology because of its capacity to transform B-lymphocytes. For this reason, EBV is used in conservation of human B-lymphocytes for long periods for subsequent evaluation of lysosomal hydrolase activity. Lymphoblastoid cell lines have several advantages for use over other cell types, such as prompt availability and possibility to develop, characterize and standardize cell banks, to test effects of promising pharmaceutical reagents. The study below presents biochemical data that demonstrate validity of lymphoblastoid cell lines for diagnosis of GM1-gangliosidosis, Gaucher, Fabry and Pompe diseases and mucopolysaccharidosis type I., Materials and Methods: Cultures were prepared from peripheral blood, collected from 25 normal subjects and 13 affected individuals. Enzyme activities and immunohistochemistry (IHC) were measured. Activities of enzymes beta-galactosidase, beta-glucosidase, alpha-iduronidase, alpha-galactosidase and alpha-glucosidase were measured before and after cryopreservation for 180 days. Enzymatic activity was measured when transformation was confirmed by IHC., Results: We observed some significant alterations in enzymatic activity of non-cultured cells when compared to others that had been cultured for 12 days and kept frozen for 180 days., Conclusions: However, these alterations did not invalidate use of the technology of transformation of lymphoblastoid cell lines with EBV, to diagnose the diseases mentioned above, in view of the fact that the cultured cells, before and after freezing, demonstrated similar enzymatic activities.

Background: The glycosphingolipid storage disorder GM1-gangliosidosis is a severe neurodegenerative condition for which no therapy is currently available. Protein misfolding in lysosomal defects may have the potential to be corrected by chemical chaperones: in vitro and clinical approaches are being investigated., Aims: We investigated the in vitro effect of galactose on some lysosomal hydrolases, and its in vitro efficacy as a chemical chaperone in GM1-gangliosidosis., Methods: Galactose was added to the culture medium of fibroblasts from patients, controls and transfected COS-1 cells. Enzyme assays of lysosomal hydrolases, beta galactosidase in particular, were performed., Results: Our data show that galactose alters selectively alpha and beta galactosidases. A significant increase (2,5 fold) in beta galactosidase activity occurred when galactose was added to the cultured fibroblasts of an adult patient. Chemical chaperone therapy requires the presence of residual enzyme activity. The adult patient here reported is heterozygous for the p.T329A mutation that showed no beta galactosidase activity, and for the p.R442Q mutation with residual enzyme activity. The p.R442Q mutation was therefore selected as a potential target for the galactose chaperone; after the addition of galactose, COS-1 cells transfected with this mutation showed an increase in beta galactosidase activity from 6.9% to 12% of control values., Conclusions: These results suggest that galactose or its derivatives with potential chaperone properties could be used in the development of non-invasive therapies for GM1-gangliosidosis.

GM1-gangliosidosis is a lysosomal storage disease (LSD) caused by an autosomal recessive deficiency of lysosomal acid beta-galactosidase (betagal). This leads to accumulation of GM1-ganglioside and its asialo derivative GA1 in the central nervous system (CNS), and progressive neurodegeneration. Therapeutic AAV-mediated gene delivery to the brain for LSDs has proven very successful in several animal models. GM1-gangliosidosis is also a prime candidate for AAV-mediated gene therapy in the CNS. As global neuropathology characterizes the most severe forms of this disease, therapeutic interventions need to achieve distribution of betagal throughout the entire CNS. Therefore, careful consideration of routes of administration and target structures from where metabolically active enzyme can be produced, released and distributed throughout the CNS, is necessary. The goal of this study was to investigate the pattern and mechanism of distribution of betagal in the adult GM1-gangliosidosis mouse brain upon hippocampal injection of an AAV vector-encoding betagal. We found evidence that three different mechanisms contribute to its distribution in the brain: (1) diffusion; (2) axonal transport within neurons from the site of production; (3) CSF flow in the perivascular space of Virchow-Robin. In addition, we found evidence of axonal transport of vector-encoded mRNA.

G(M1)-gangliosidosis is a lysosomal storage disorder caused by a deficiency of ss-galactosidase activity. Human GM1-gangliosidosis has been classified into three forms according to the age of clinical onset and specific biochemical parameters. In the present study, a canine model for type II late infantile human GM1-gangliosidosis was investigated 'in vitro' in detail. For a better understanding of the molecular pathogenesis underlying G(M1)-gangliosidosis the study focused on the analysis of the molecular events and subsequent intracellular protein trafficking of beta-galactosidase. In the canine model the genetic defect results in exclusion or inclusion of exon 15 in the mRNA transcripts and to translation of two mutant precursor proteins. Intracellular localization, processing and enzymatic activity of these mutant proteins were investigated. The obtained results suggested that the beta-galactosidase C-terminus encoded by exons 15 and 16 is necessary for correct C-terminal proteolytic processing and enzyme activity but does not affect the correct routing to the lysosomes. Both mutant protein precursors are enzymatically inactive, but are transported to the lysosomes clearly indicating that the amino acid sequences encoded by exons 15 and 16 are necessary for correct folding and association with protective protein/cathepsin A, whereas the routing to the lysosomes is not influenced. Thus, the investigated canine model is an appropriate animal model for the human late infantile form and represents a versatile system to test gene therapeutic approaches for human and canine G(M1)-gangliosidosis.

Lysosomal beta-galactosidase is required for the degradation of GM1 ganglioside and other glycolipids and glycoproteins with a terminal galactose moiety. Deficiency of this enzyme leads to the lysosomal storage disorder, GM1 gangliosidosis, marked by severe neurodegeneration resulting in premature death. As a step towards preclinical studies for enzyme replacement therapy in an animal model of GM1 gangliosidosis, a feline beta-galactosidase cDNA was cloned into a mammalian expression vector and subsequently expressed in Chinese hamster ovary (CHO-K1) cells. The enzyme secreted into culture medium exhibited specific activity on two synthetic substrates as well as on the native beta-galactosidase substrate, GM1 ganglioside. The enzyme was purified from transfected CHO-K1 cell culture medium by chromatography on PATG-agarose. The affinity-purified enzyme preparation consisted mainly of the protein with approximate molecular weight of 94 kDa and displayed immunoreactivity with antibodies raised against a 16-mer synthetic peptide corresponding to C-terminal amino acid sequence deduced from the feline beta-galactosidase cDNA.

G(M1) gangliosidosis is an inherited, fatal neurodegenerative disease caused by deficiency of lysosomal beta-d-galactosidase (EC 3.2.1.23) and consequent storage of undegraded G(M1) ganglioside. To characterize the genetic mutation responsible for feline G(M1) gangliosidosis, the normal sequence of feline beta-galactosidase cDNA first was defined. The feline beta-galactosidase open reading frame is 2010 base pairs, producing a protein of 669 amino acids. The putative signal sequence consists of amino acids 1-24 of the beta-galactosidase precursor protein, which contains seven potential N-linked glycosylation sites, as in the human protein. Overall sequence homology between feline and human beta-galactosidase is 74% for the open reading frame and 82% for the amino acid sequence. After normal beta-galactosidase was sequenced, the mutation responsible for feline G(M1) gangliosidosis was defined as a G to C substitution at position 1448 of the open reading frame, resulting in an amino acid substitution at arginine 483, known to cause G(M1) gangliosidosis in humans. Feline beta-galactosidase messenger RNA levels were normal in cerebral cortex, as determined by quantitative RT-PCR assays. Although enzymatic activity is severely reduced by the mutation, a full-length feline beta-galactosidase cDNA restored activity in transfected G(M1) fibroblasts to 18-times normal. beta-Galactosidase protein levels in G(M1) tissues were normal on Western blots, but immunofluorescence analysis demonstrated that the majority of mutant beta-galactosidase protein did not reach the lysosome. Additionally, G(M1) cat fibroblasts demonstrated increased expression of glucose-related protein 78/BiP and protein disulfide isomerase, suggesting that the unfolded protein response plays a role in pathogenesis of feline G(M1) gangliosidosis.

GM1 gangliosidosis is an autosomal recessive disorder caused by the deficiency of lysosomal acid hydrolase beta-galactosidase (beta-Gal). It is one of the most frequent lysosomal storage disorders in Brazil, with an estimated frequency of 1:17,000. The enzyme is secreted and can be captured by deficient cells and targeted to the lysosomes. There is no effective treatment for GM1 gangliosidosis. To determine the efficiency of an expression vector for correcting the genetic defect of GM1 gangliosidosis, we tested transfer of the beta-Gal gene (Glb1) to fibroblasts in culture using liposomes. Beta-Gal cDNA was cloned into the expression vectors pSCTOP and pREP9. Transfection was performed using 4 microL lipofectamine 2000 and 1.5-2.0 microg DNA. Cells (2 x 10(5)/well) were harvested 24 h, 48 h, and 7 days after transfection. Enzyme specific activity was measured in cell lysate and supernatant by fluorometric assay. Twenty-four hours after transfection, treated cells showed a higher enzyme specific activity (pREP9-beta-Gal: 621.5 +/- 323.0, pSCTOP-beta-Gal: 714.5 +/- 349.5, pREP9-beta-Gal + pSCTOP-beta-Gal: 1859.0 +/- 182.4, and pREP9-ss-Gal + pTRACER: 979.5 +/- 254.9 nmol x h-1 x mg-1 protein) compared to untreated cells (18.0 +/- 3.1 for cell and 32.2 +/- 22.2 nmol x h-1 x mg-1 protein for supernatant). However, cells maintained in culture for 7 days showed values similar to those of untreated patients. In the present study, we were able to transfect primary patients' skin fibroblasts in culture using a non-viral vector which overexpresses the beta-Gal gene for 24 h. This is the first attempt to correct fibroblasts from patients with GM1 gangliosidosis by gene therapy using a non-viral vector.

Diagnosis of GM1 gangliosidosis (OMIM 230500) is usually based on the presence of physical signs of storage such as coarse facial features, corneal clouding, cherry red macula, hepatosplenomegaly and skeletal dysostosis. More rarely it can present as nonimmune hydrops. We describe a male patient with GM1 gangliosidosis born to healthy first-cousin parents of Indian Asian descent. The disease was recognized on the basis of diffuse vacuolization of cyto- and syncytiotrophoblasts, stromal cells and amniocytes on histological analysis of the placenta. The placental examination was prompted by the prenatal detection of intrauterine growth retardation (IUGR) and oligohydramnios at 32 weeks of gestation. The diagnosis of GM1 gangliosidosis was supported by both biochemical and molecular data. The beta-galactosidase enzymatic activity on leukocytes was severely reduced, while the neuraminidase activity on fibroblasts was normal, thereby excluding galactosialidosis. The molecular analysis of the beta-galactosidase gene (GLB1) revealed a previously unreported splicing mutation (IVS1+2 insT) in homozygous state. Our case further illustrates the value of histological examination of the placenta in the diagnosis of lysosomal storage disorders and shows that either hydrops or IUGR can be presenting features of GM1 gangliosidosis in the neonatal period.

GM1-gangliosidosis is a glycosphingolipid (GSL) lysosomal storage disease caused by autosomal recessive deficiency of lysosomal acid beta-galactosidase (betagal), and characterized by accumulation of GM1-ganglioside and GA1 in the brain. Here we examined the effect of neonatal intracerebroventricular (i.c.v.) injection of an adeno-associated virus (AAV) vector encoding mouse betagal on enzyme activity and brain GSL content in GM1-gangliosidosis (betagal(-/-)) mice. Histological analysis of betagal distribution in 3-month-old AAV-treated betagal(-/-) mice showed that enzyme was present at high levels throughout the brain. Biochemical quantification showed that betagal activity in AAV-treated brains was 7- to 65-fold higher than in wild-type controls and that brain GSL levels were normalized. Cerebrosides and sulfatides, which were reduced in untreated betagal(-/-) mice, were restored to normal levels by AAV treatment. In untreated betagal(-/-) brains, cholesterol was present at normal levels but showed abnormal cellular distribution consistent with endosomal/lysosomal localization. This feature was also corrected in AAV-treated mice. The biochemical and histological parameters analyzed in this study showed that normal brain neurochemistry was achieved in AAV-treated betagal(-/-) mice. Therefore we show for the first time that neonatal AAV-mediated gene delivery of lysosomal betagal to the brain may be an effective approach for treatment of GM1-gangliosidosis.

Epstein-Barr virus (EBV) infection in vitro causes transformation of B cells and generates B lymphoblastoid cell lines (LCLs). These LCLs have been widely used for the diagnostic of several genetic metabolic disorders. However, up to now, efficiency of LCL generation has been based on misleading subjective analysis. In this study, quantitative analyses have been performed to indicate efficiency of B-cell transformation to measuring human lysosomal acid hydrolases associated with: GM1-gangliosidosis type I, Gaucher disease and mucopolysaccharidosis type I. Peripheral blood mononuclear cells were isolated from 13 subjects, and LCLs were produced by culturing them with EBV for 12 days. Activities of the enzymes beta-galactosidase, beta-glucosidase and alpha-iduronidase were measured before and after cryopreservation in liquid nitrogen for 30 days. Efficiency of the B-cell transformation was screened every 4 days by the enumeration of cell proliferation, cell counts and changes in granularity estimated by flow cytometry. We observed the generation of 13 LCLs. Cell transformation was confirmed by the gradual increase of cellular clusters, cell size and granularity. In addition, we determined that the activity of the enzymes mentioned above did not change following cryopreservation. These data suggest that our enumerative approach for screening of EBV-LCLs is efficient for the enzymatic determination of human lysosomal acid hydrolases and may thus replace misleading subjective analyses.

This report describes a rapid and simple method for mutation screening of G(M1) gangliosidosis in Shiba dogs by direct amplification of DNA from canine whole-blood specimens using a novel polymerase chain reaction (PCR) reagent cocktail, which can eliminate the DNA extraction process and amplify the genomic DNA directly from human or murine whole blood. The strategy of this mutation screening is based on the identification of a nucleotide deletion by restriction enzyme analysis, coupled with the direct PCR amplification. The target sequence of the canine beta-galactosidase gene could be amplified directly from various forms of canine whole-blood specimens, including anticoagulated blood, blood stored frozen for 1 year, dried blood held in filter paper for 1 year at room temperature, and dry powder of blood stripped from Giemsa-stained blood films, which had been prepared 10 years earlier, resulting in the determination of genotypes in all the specimens. This method simplified the molecular diagnosis and carrier screening of G(M1) gangliosidosis in Shiba dogs, making it simple to examine specimens from the large, widely distributed population of these dogs.

Ten low molecular compounds analogous to galactose were screened for inhibition of human beta-galactosidase activity. Among them, 1-deoxy-galactonojirimycin and N-(n-butyl)-deoxy-galactonojirimycin showed an inhibitory effect at high concentrations. However, they restored mutant enzyme activities expressed in enzyme-deficient knockout mouse fibroblasts and human beta-galactosidosis fibroblasts at lower intracellular concentrations. This effect was more remarkable on G(M1)-gangliosidosis mutations (R201C, I51T, R201H, R457Q) than Morquio B disease mutations (W273L, Y83H). These low molecular compounds pass though the blood-brain barrier in mice. We hope that this new therapeutic approach will become clinically applicable in the near future.

Lysosomal enzymes sialidase (alpha-neuraminidase), beta-galactosidase, and N-acetylaminogalacto-6-sulfate sulfatase are involved in the catabolism of glycolipids, glycoproteins, and oligosaccharides. Their functional activity in the cell depends on their association in a multienzyme complex with lysosomal carboxypeptidase, cathepsin A. We review the data suggesting that the integrity of the complex plays a crucial role at different stages of biogenesis of lysosomal enzymes, including intracellular sorting and proteolytic processing of their precursors. The complex plays a protective role for all components, extending their half-life in the lysosome from several hours to several days; and for sialidase, the association with cathepsin A is also necessary for the expression of enzymatic activity. The disintegration of the complex due to genetic mutations in its components results in their functional deficiency and causes severe metabolic disorders: sialidosis (mutations in sialidase), GM1-gangliosidosis and Morquio disease type B (mutations in beta-galactosidase), galactosialidosis (mutations in cathepsin A), and Morquio disease type A (mutations in N-acetylaminogalacto-6-sulfate sulfatase). The genetic, biochemical, and direct structural studies described here clarify the molecular pathogenic mechanisms of these disorders and suggest new diagnostic tools.

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.

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.

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.

Mutations in the lysosomal acid beta-galactosidase (EC 3.2.1.23) underlie two different disorders: GM1 gangliosidosis, which involves the nervous system and visceral organs to varying extents, and Morquio's syndrome type B (Morquio B disease), which is a skeletal-connective tissue disease without any CNS symptoms. This article shows that transduction of human GM1 gangliosidosis fibroblasts with retrovirus vectors encoding the human acid beta-galactosidase cDNA leads to complete correction of the enzymatic deficiency. The newly synthesized enzyme is correctly processed and targeted to the lysosomes in transduced cells. Cross-correction experiments using retrovirus-modified cells as enzyme donors showed, however, that the human enzyme is transferred at low efficiencies. Experiments using a different retrovirus vector carrying the human cDNA confirmed this observation. Transduction of human GM1 fibroblasts and mouse NIH 3T3 cells with a retrovirus vector encoding the mouse beta-galactosidase cDNA resulted in high levels of enzymatic activity. Furthermore, the mouse enzyme was found to be transferred to human cells at high efficiency. Enzyme activity measurements in medium conditioned by genetically modified cells suggest that the human beta-galactosidase enzyme is less efficiently released to the extracellular space than its mouse counterpart. This study suggests that lysosomal enzymes, contrary to the generalized perception in the field of gene therapy, may differ significantly in their properties and provides insights for design of future gene therapy interventions in acid beta-galactosidase deficiency.

Objectives: Characterization of beta-galactosidase in leukocytes and fibroblasts of heterozygotes for GM1 type I., Design and Methods: Leukocyte and fibroblast beta-galactosidase activity was determined fluorimetrically using 4-methylumbelliferyl-beta-D-galactoside as an artificial substrate. Optimum pH, Km, Vmax and thermostability of the enzyme at 42 degrees C were determined., Results: The leukocyte and fibroblast enzyme of heterozygotes have an optimum pH of 4.0 and 4.2, respectively. In normal subjects, the optimum pH was 4.2 in both cells, according to previous studies. The Km of the enzyme of heterozygotes was determined to be 0.65 mM in leukocytes and 0.59 mM in fibroblasts. The Vmax was determined in 167.21 nmol/h/mg of protein in heterozygotes leukocytes and 541.2 nmol/h/mg of protein in heterozygotes fibroblasts compared to 291.7 and 1768.1 nmol/h/mg of protein in controls leukocytes and fibroblasts, respectively. When leukocyte and fibroblast heterozygote beta-galactosidase was preincubated at 42 degrees C, after 80 min the residual activity was determined to be 25 to 30% of the initial activity. These results are similar to the control group., Conclusions: We have found significant differences between the two groups in some investigated parameters. Both fibroblasts and leukocytes showed a virtually similar level of reliability as source of enzyme for the detection of heterozygotes.

GM1-gangliosidosis is a lysosomal storage disorder caused by deficiency of acid beta-galactosidase (GLB1). We report five new beta-galactosidase gene mutations in nine Italian patients and one fetus, segregating in seven unrelated families. Six of the eight patients with the infantile, severe form of the disease presented cardiac involvement, a feature rarely associated with GM1-gangliosidosis. Molecular analysis of the patients' RNA and DNA identified two new RNA splicing defects, three new and three previously described amino acid substitutions. Interestingly, all patients with cardiac involvement were homozygous for one of these mutations: R59H, Y591C, Y591N, or IVS14-2A>G. In contrast, all other patients were compound heterozygous for one of the following mutations: R201H, R482H, G579D, IVS8+2T>C. Although we could not directly correlate the presence of cardiac abnormalities with specific genetic lesions, the mutations identified in patients with cardiomyopathy fell in the GLB1 cDNA region common to the lysosomal enzyme and the Hbeta-Gal-related protein, also known as the elastin binding protein (EBP). Consequently, both molecules are affected by the mutations, and they may contribute differently to the occurrence of specific clinical manifestations., (Copyright 2000 Wiley-Liss, Inc.)

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.

Objectives: Some biochemical characteristics of the human leukocyte and fibroblast beta-galactosidase were studied., Design and Methods: Leukocyte and fibroblast enzyme activity was determined fluorometricaly using 4-methylumbelliferyl-beta-D-galactoside as artificial substrate. Optimum pH, Km, Vmax and thermostability of the enzyme at 42 degrees C were determined., Results: The leukocyte and fibroblast enzyme has an optimum pH at 4.2, which is in agreement with the lysosomal origin of the enzyme. The Km of the enzyme was 0.62 in leukocytes and 0.67 in fibroblasts, and Vmax was 289.9 nmol/h/mg of protein and 1779.2 nmol/h/mg of protein in the two tissues, respectively. When fibroblast or leukocyte beta-galactosidase was pre-incubated at 42 degrees C, it did not retain its activity because the residual activity after 80 minutes of pre-incubation at this temperature was lower than 30% of the initial activity both in leukocytes and fibroblasts., Conclusions: This was the first study of Km, Vmax and thermostability of beta-galactosidase performed on leukocytes and provided data for a better characterization of the enzyme beta-galactosidase, allowing the improvement of the analytical conditions.

Two benign mutations, C739T(R247W) and C745T(R249W), in the alpha-subunit of beta-hexosaminidase A (Hex A) have been found in all but one of the currently identified Hex A-pseudodeficient subjects. To confirm the relationship of the benign mutations and Hex A pseudodeficiency and to determine how the benign mutations reduce Hex A activity, we transiently expressed each of the benign mutations, and other mutations associated with infantile, juvenile, and adult onset forms of GM2 gangliosidosis, as Hex S (alphaalpha) and Hex A (alphabeta) in COS-7 cells. The benign mutations decreased the expressed Hex A and Hex S activity toward the synthetic substrate 4-methylumbelliferyl-6-sulfo-beta-N-acetylglucosaminide (4-MUGS) by 60-80%, indicating that they are the primary cause of Hex A pseudodeficiency. Western blot analysis showed that the benign mutations decreased the enzymatic activity by reducing the alpha-subunit protein level. No change in heat sensitivity, catalytic activity, or the substrate specificity to the synthetic substrates, 4-methylumbelliferyl-beta-N-acetylglucosaminide or 4-methylumbelliferyl-6-sulfo-beta-N-acetylglucosaminide, was detected. The effects of the benign mutations on Hex A were further analyzed in fibroblasts, and during transient expression, using pulse-chase metabolic labeling. These studies showed that the benign mutations reduced the alpha-subunit protein by affecting its stability in vivo, not by affecting the processing of the alpha-subunit, i.e. phosphorylation, targeting, or secretion. Our studies also demonstrated that these benign mutations could be readily differentiated from disease-causing mutations using a transient expression system.