RESUMO
Mucopolysaccharidosis (MPS) is a metabolic disorder resulting from a deficiency of lysosomal enzymes. It is an autosomal recessive disorder with similar incidences in men and women. Mucopolysaccharidosis type IV A is caused by a deficiency of N-acetylgalactosamine-6-sulfatase, which deficiency is, in turn, caused by alterations in the GALNS gene. It is marked by a short stature, a pigeon chest, frontal bossing, kyphosis, and a flat nasal bridge. Intraorally, macroglossia, hypodontia, dentinogenesis imperfecta, a broad mouth, and an anterior open bite are some of the common features. The present paper reports on a case of MPS in a 5-year-old male patient, along with providing a review of the literature and insight into the oral manifestations related to MPS IV A, also called Morquio A syndrome, and its dental treatment. It aims to highlight the clinical recommendations for oral health care in such cases during different phases of MPS IV A treatment.
Assuntos
Condroitina Sulfatases , Mucopolissacaridose IV , Masculino , Humanos , Criança , Feminino , Pré-Escolar , Mucopolissacaridose IV/genética , Mucopolissacaridose IV/terapia , Condroitina Sulfatases/genética , Condroitina Sulfatases/metabolismo , Atenção à SaúdeRESUMO
Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder (LSD) caused by mutations in gene encoding for GALNS enzyme. Lack of GALNS activity leads to the accumulation of glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate. Although enzyme replacement therapy has been approved since 2014 for MPS IVA, still there is an unmet medical need to have improved therapies for this disorder. CRISPR/Cas9-based gene therapy has been tested for several LSDs with encouraging findings, but to date it has not been assayed on MPS IVA. In this work, we validated for the first time the use of CRISPR/Cas9, using a Cas9 nickase, for the knock-in of an expression cassette containing GALNS cDNA in an in vitro model of MPS IVA. The results showed the successful homologous recombination of the expression cassette into the AAVS1 locus, as well as a long-term increase in GALNS activity reaching up to 40% of wild-type levels. We also observed normalization of lysosomal mass, total GAGs, and oxidative stress, which are some of the major findings regarding the pathophysiological events in MPS IVA. These results represent a proof-of-concept of the use of CRISPR/Cas9 nickase strategy for the development of a novel therapeutic alternative for MPS IVA.
Assuntos
Condroitina Sulfatases , Mucopolissacaridose IV , Humanos , Mucopolissacaridose IV/genética , Mucopolissacaridose IV/terapia , Sistemas CRISPR-Cas , Edição de Genes , Condroitina Sulfatases/genética , Condroitina Sulfatases/metabolismo , Condroitina Sulfatases/uso terapêutico , Sulfato de Queratano/metabolismo , Sulfato de Queratano/uso terapêutico , Glicosaminoglicanos/genética , Glicosaminoglicanos/metabolismoRESUMO
Mucopolysaccharidosis IV A (MPS IV A, Morquio A disease) is a lysosomal storage disease (LSD) produced by mutations on N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Recently an enzyme replacement therapy (ERT) for this disease was approved using a recombinant enzyme produced in CHO cells. Previously, we reported the production of an active GALNS enzyme in Escherichia coli that showed similar stability properties to that of a recombinant mammalian enzyme though it was not taken-up by culture cells. In this study, we showed the production of the human recombinant GALNS in the methylotrophic yeast Pichia pastoris GS115 (prGALNS). We observed that removal of native signal peptide and co-expression with human formylglycine-generating enzyme (SUMF1) allowed an improvement of 4.5-fold in the specific GALNS activity. prGALNS enzyme showed a high stability at 4 °C, while the activity was markedly reduced at 37 and 45 °C. It was noteworthy that prGALNS was taken-up by HEK293 cells and human skin fibroblasts in a dose-dependent manner through a process potentially mediated by an endocytic pathway, without any additional protein or host modification. The results show the potential of P. pastoris in the production of a human recombinant GALNS for the development of an ERT for Morquio A.
Assuntos
Condroitina Sulfatases/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Células Cultivadas , Condroitina Sulfatases/química , Condroitina Sulfatases/genética , Condroitina Sulfatases/isolamento & purificação , Endocitose , Estabilidade Enzimática , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Pichia/genética , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Sulfatases/genética , Sulfatases/metabolismo , TemperaturaRESUMO
BACKGROUND: Lysosomal storage diseases (LSD) are a group of genetic conditions which could present a vast spectrum of abnormalities that may include skeletal abnormalities, organ dysfunction, neuronal involvement, and tissue accumulation of complex molecules, among other manifestations. Definitive diagnosis of LSD is generally obtained by specific enzyme assays performed in leukocytes, fibroblasts, or more recently, dried-blood filter paper (DBFP) samples. METHODS: We recently introduced dried-leukocytes filter paper (DLFP) as an alternative source of enzyme to assay heparan sulfamidase and galactocerebrosidase activities, which could not be measured in DBFP samples using fluorometric methods. We present a new fluorometric methods on DLFP samples, for evaluation of α-glucosidase (GAA), ß-glucosidase (GBA), and N-acetylgalactosamine-6-sulfatase (GALNS) activities, key enzyme assays for the identification of patients with Pompe disease (PD), Gaucher disease (GD), and Morquio A disease (MD), respectively. RESULTS: We show a clear discrimination between confirmed PD, GD, and MD patients and healthy controls. CONCLUSIONS: We conclude that the assays of GAA, GBA, and GALNS on DLFP are reliable and useful methods for the identification of PD, GD, and MD diseases, respectively. As sample preparation is feasible in standard biochemical laboratories and transportation is very simple, it could enable patients living in remote areas to be investigated, diagnosed and eventually treated with the specific therapies available for these diseases.
Assuntos
Ensaios Enzimáticos/métodos , Doença de Gaucher/diagnóstico , Doença de Depósito de Glicogênio Tipo II/diagnóstico , Leucócitos/enzimologia , Mucopolissacaridose IV/diagnóstico , Fitas Reagentes/análise , Estudos de Casos e Controles , Condroitina Sulfatases/metabolismo , Dessecação , Ensaios Enzimáticos/instrumentação , Doença de Gaucher/sangue , Doença de Depósito de Glicogênio Tipo II/sangue , Humanos , Leucócitos/patologia , Mucopolissacaridose IV/sangue , Papel , alfa-Glucosidases/metabolismo , beta-Glucosidase/metabolismoRESUMO
Mucopolysaccharidosis IV A (MPS IV A) is a lysosomal storage disease produced by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme. Although genotype-phenotype correlations have been reported, these approaches have not enabled to establish a complete genotype-phenotype correlation, and they have not considered a ligand-enzyme interaction. In this study, we expanded the in silico evaluation of GALNS mutations by using several bioinformatics tools. Tertiary GALNS structure was modeled and used for molecular docking against galactose-6-sulfate, N-acetylgalactosamine-6-sulfate, keratan sulfate, chondroitin-6-sulfate, and the artificial substrate 4-methylumbelliferyl-ß-D-galactopyranoside-6-sulfate. Furthermore, we considered the evolutionary residue conservation, change conservativeness, position within GALNS structure, and the impact of amino acid substitution on the structure and function of GALNS. Molecular docking showed that amino acids involved in ligand interaction correlated with those observed in other human sulfatases, and mutations within the active cavity reduced affinity of all evaluated ligands. Combination of several bioinformatics approaches allowed to explaine 90% of the missense mutations affecting GALNS, and the prediction of the phenotype for another 21 missense mutations. In summary, we have shown for the first time a docking evaluation of natural and artificial ligands for human GALNS, and proposed an update in genotype-phenotype correlation for Morquio A, based on the use of multiple parameters to predict the disease severity.
Assuntos
Condroitina Sulfatases/genética , Condroitina Sulfatases/metabolismo , Biologia Computacional/métodos , Modelos Moleculares , Mucopolissacaridose IV/enzimologia , Fenótipo , Filogenia , Condroitina Sulfatases/química , Análise por Conglomerados , Genótipo , Humanos , Simulação de Dinâmica Molecular , Estrutura Molecular , Mutação de Sentido Incorreto/genética , Ligação Proteica , Conformação ProteicaRESUMO
The production and characterization of an active recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in Escherichia coli BL21(DE3) has been previously reported. In this study, the effect of the signal peptide (SP), inducer concentration, process scale, and operational mode (batch and semi-continuous) on GALNS production were evaluated. When native SP was presented, higher enzyme activity levels were observed in both soluble and inclusion bodies fractions, and its removal had a significant impact on enzyme activation. At shake scale, the optimal IPTG concentrations were 0.5 and 1.5 mM for the strains with and without SP, respectively, whereas at bench scale, the highest enzyme activities were observed with 1.5 mM IPTG for both strains. Noteworthy, enzyme activity in the culture media was only detected when SP was presented and the culture was carried out under semi-continuous mode. We showed for the first time that the mechanism that in prokaryotes recognizes the SP to mediate sulfatase activation can also recognize a eukaryotic SP, favoring the activation of the enzyme, and could also favor the secretion of the recombinant protein. These results offer significant information for scaling-up the production of human sulfatases in E. coli.
Assuntos
Condroitina Sulfatases/metabolismo , Meios de Cultura/metabolismo , Escherichia coli/metabolismo , Sinais Direcionadores de Proteínas , Condroitina Sulfatases/química , Condroitina Sulfatases/genética , Escherichia coli/genética , Humanos , Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Mucopolysaccharidosis (MPS) IVA is an autosomal recessive disorder caused by deficiency of the lysosomal enzyme N-acetylgalatosamine-6-sulfate sulfatase (GALNS), which leads to the accumulation of keratan sulfate and chondroitin 6-sulfate, mainly in bone. To explore the possibility of gene therapy for Morquio A disease, we transduced the GALNS gene into HEK293 cells, human MPS IVA fibroblasts and murine MPS IVA chondrocytes by using adeno-associated virus (AAV)-based vectors, which carry human GALNS cDNA. The effects of the promoter and the cotransduction with the sulfatase-modifying factor 1 gene (SUMF1) on GALNS activity levels was evaluated. Downregulation of the cytomegalovirus (CMV) immediate early enhancer/promoter was not observed for 10 days post-transduction. The eukaryotic promoters induced equal or higher levels of GALNS activity than those induced by the CMV promoter in HEK293 cells. Transduction of human MPS IVA fibroblasts induced GALNS activity levels that were 15-54% of those of normal human fibroblasts, whereas in transduced murine MPS IVA chondrocytes, the enzyme activities increased up to 70% of normal levels. Cotransduction with SUMF1 vector yielded an additional four-fold increase in enzyme activity, although the level of elevation depended on the transduced cell type. These findings suggest the potential application of AAV vectors for the treatment of Morquio A disease, depending on the combined choice of transduced cell type, selection of promoter, and cotransduction of SUMF1.
Assuntos
Condroitina Sulfatases/genética , Condroitina Sulfatases/metabolismo , Dependovirus/genética , Mucopolissacaridose IV/genética , Regiões Promotoras Genéticas/genética , Sulfatases/genética , Sulfatases/metabolismo , Animais , Linhagem Celular , Vetores Genéticos/genética , Humanos , Camundongos , Mucopolissacaridose IV/enzimologia , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , TransfecçãoRESUMO
Morquio A is an autosomal recessive disease caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), leading to the lysosomal accumulation of keratan-sulfate and chondroitin-6-sulfate. We evaluated in HEK293 cells the effect of the cytomegalovirus immediate early enhancer/promoter (CMV) or the elongation factor 1alpha (EF1alpha) promoters, and the coexpression with the sulfatase modifying factor 1 (SUMF1) on GALNS activity. Four days postransfection GALNS activity in transfected cells with CMV-pIRES-GALNS reached a plateau, whereas in cells transfected with EF1alpha-pIRES-GALNS continued to increase until day 8. Co-transfection with pCXN-SUMF1 showed an increment up to 2.6-fold in GALNS activity. Finally, computational analysis of transcription factor binding-sites and CpG islands showed that EF1alpha promoter has long CpG islands and high-density binding-sites for Sp1 compared to CMV. These results show the advantage of the SUMF1 coexpression on GALNS activity and indicate a considerable effect on the expression stability using EF1alpha promoter compared to CMV.
Assuntos
Condroitina Sulfatases/metabolismo , Expressão Gênica , Fator 1 de Elongação de Peptídeos/genética , Regiões Promotoras Genéticas/genética , Sulfatases/metabolismo , Sítios de Ligação , Linhagem Celular , Condroitina Sulfatases/genética , Biologia Computacional , Ilhas de CpG/genética , Citomegalovirus/genética , Humanos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Plasmídeos/genética , Fator de Transcrição Sp1/metabolismo , TransfecçãoRESUMO
INTRODUCTION: Mucopolysaccharidosis IVA (Morquio A) is caused by a deficiency of N-acetylgalactosamine-6-sulphate-sulphatase, a lysosomal enzyme required for the stepwise degradation of keratan-sulfate and chondroitin-6-sulfate. A deficiency in this enzyme results in an accumulation of glycosaminoglycans in several tissues. Currently, no effective therapies exist and only supportive measures are used to treat some manifestations of the disease. An ideal therapy is one that can be administrated early in life, has low mortality, and leads to long-term expression of the enzyme. Gene therapy emerges as a potential alternative to correct the genetic defect in MPS IVA. OBJECTIVE: Adenoassociated virus-derived expression vectors (AAV) were constructed to correct in vitro the enzyme deficiency in mucopolysaccharidosis IVA. MATERIALS AND METHODS: Adenoasociated virus-derived vectors containing the human GALNS gene and driven by the citomegalivirus immedited-early promoter were constructed using a free-adenoviral protocol. HEK293 cells and human skin Morquio A fibroblasts were transfected with the recombinat vectors. Enzyme activity was measured in cells 24 and 48 hours post-transfection. RESULTS: Free-adenovirus recombinant AAV vectors were obtained with titres up to 2.08x1010 capsids/mL. HEK293 cells and Morquio A fibroblasts transfected with vectors showed GALNS activity up to 3.05 nmoles/mg/h 48 hours post-transfection. CONCLUSION: The AAV mediated the in vitro expression of GALNS enzyme in the transfected cells. These results are the first step towards a gene therapy alternative to Morquio A disease using adenoassociated virus-derived vectors.
Assuntos
Condroitina Sulfatases/genética , Dependovirus/genética , Terapia Genética , Vetores Genéticos/genética , Mucopolissacaridose IV , Células Cultivadas , Condroitina Sulfatases/metabolismo , Dependovirus/metabolismo , Técnicas de Transferência de Genes , Vetores Genéticos/metabolismo , Humanos , Mucopolissacaridose IV/genética , Mucopolissacaridose IV/terapia , TransfecçãoRESUMO
Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Mutation screening of the GALNS was performed by genomic PCR and direct sequence analyses in 20 MPS IVA patients from Latin America. In this study, 12 different gene mutations including nine unreported ones were identified in 16 severe and four attenuated patients and accounted for 90.0% of the unrelated mutant alleles. The gene alterations were missense mutations except one insertion. Six recurrent mutations, p.A75G, p.G116S, p.G139S, p.N164T, p.R380S, and p.R386C, accounted for 5.0, 10.0, 5.0, 7.5, 5.0, and 32.5% of the unrelated mutant alleles, respectively. The p.R386C mutation was identified in all Latin American populations studied. Eleven mutations correlated with a severe form, while one mutation, p.R380S, was associated with an attenuated form. MPS IVA patients had an elevation of urine and plasma keratan sulfate (KS) concentrations compared with those of the age-matched control. KS concentrations in severe patients were higher than those in attenuated patients. These data provide evidence for extensive allelic heterogeneity and presence of a common mutation in Latin American patients. Accumulation of mutations with clinical description and KS concentration will lead us to predict clinical severity of the patient more precisely.