RESUMO
The arylsulfatase A (ARSA) gene is observed to be deficient in patients with metachromatic leukodystrophy (MLD), a type of lysosomal storage disease. MLD is a severe neurodegenerative disorder characterized by an autosomal recessive inheritance pattern. This study aimed to map the most deleterious mutations at the metal binding sites of ARSA and the amino acids in proximity to the mutated positions. We utilized an array of computational tools, including PredictSNP, MAPP, PhD-SNP, PolyPhen-1, PolyPhen-2, SIFT, SNAP, and ConSurf, to identify the most detrimental mutations potentially implicated in MLD collected from UniProt, ClinVar, and HGMD. Two mutations, D29N and D30H, as being extremely deleterious based on assessments of pathogenicity, conservation, biophysical characteristics, and stability analysis. The D29 and D30 are located at the metal-interacting regions of ARSA and found to undergo post-translational modification, specifically phosphorylation. Henceforth, the in-depth effect of metal binding upon mutation was examined using molecular dynamics simulations (MDS) before and after phosphorylation. The MDS results exhibited high deviation for the D29N and D30H mutations in comparison to the native, and the same was confirmed by significant residue fluctuation and reduced compactness. These structural alterations suggest that such mutations may influence protein functionality, offering potential avenues for personalized therapeutic and providing a basis for potential mutation-specific treatments for severe MLD patients.
Assuntos
Cerebrosídeo Sulfatase , Leucodistrofia Metacromática , Mutação , Humanos , Sítios de Ligação , Cerebrosídeo Sulfatase/genética , Cerebrosídeo Sulfatase/metabolismo , Cerebrosídeo Sulfatase/química , Leucodistrofia Metacromática/genética , Leucodistrofia Metacromática/metabolismo , Metais/metabolismo , Metais/química , Simulação de Dinâmica MolecularRESUMO
Metachromatic leukodystrophy (MLD) is a rare leukoencephalopathy caused by pathogenic mutations in the ARSA gene. It manifests as severe motor symptoms, mental problems, and sometimes, seizures. We aimed to investigate the phenotypic manifestations and genetic causes of MLD in an Iranian family. We present the case of a 3-year-old girl who presented with hypotonia, muscular atrophy, and seizures. Neurological and neuromuscular examinations were performed to evaluate clinical characteristics. Whole exome sequencing (WES) was used to detect disease-causing variants. In silico analysis was performed to predict the pathogenicity of this variant. GROMACS software was utilized for molecular dynamic simulation (MDS). Neurological studies revealed marked slowing of motor conduction velocities and an increased motor unit action potential duration. Brain MRI scan revealed white matter abnormalities. By applying WES, we identified a novel homozygous missense variant (NM_000487.6, c.938G > C, p.R313P) in ARSA. Direct sequencing identified this homozygous variant in her asymptomatic younger sister, whereas both parents carried a heterozygous variant. This mutation has not been reported in genetic databases or in literature. In silico analysis predicted that any variation in this DNA position would cause disease, as it is highly conserved. The c.938G > C variant was classified as a pathogenic variant according to ACMG/AMP guidelines. MDS analysis indicated that c.938G > C had a significant impact on both the structure and stabilization of ARSA, ultimately resulting in impaired protein function. The identification of this variant expands the spectrum of ARSA gene mutations associated with MLD and highlights the importance of genetic testing for the diagnosis of MLD.
Assuntos
Leucodistrofia Metacromática , Humanos , Feminino , Pré-Escolar , Leucodistrofia Metacromática/diagnóstico , Leucodistrofia Metacromática/genética , Leucodistrofia Metacromática/patologia , Cerebrosídeo Sulfatase/genética , Cerebrosídeo Sulfatase/química , Cerebrosídeo Sulfatase/metabolismo , Irã (Geográfico) , Mutação , ConvulsõesRESUMO
A novel enzyme cleavable linker for antibody-drug conjugates is reported. The 3-O-sulfo-ß-galactose linker is cleaved sequentially by two lysosomal enzymes - arylsulfatase A and ß-galactosidase - to release the payload in targeted cells. An α-HER2 antibody-drug conjugate synthesised using this highly hydrophilic dual-cleavable linker exhibited excellent cytotoxicity and selectivity.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Cerebrosídeo Sulfatase/química , Imunoconjugados/química , Trastuzumab/farmacologia , beta-Galactosidase/química , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cerebrosídeo Sulfatase/metabolismo , Relação Dose-Resposta a Droga , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imunoconjugados/metabolismo , Estrutura Molecular , Trastuzumab/química , Trastuzumab/metabolismo , beta-Galactosidase/metabolismoRESUMO
BACKGROUND: Metachromatic leukodystrophy (MLD) is a rare inherited lysosomal disorder caused by mutations in ARSA. The biological processes of MLD disease caused by candidate pathogenic mutations in the ARSA gene remain unclear. METHODS: We used whole-exome sequencing (WES) and Sanger sequencing to identify the pathogenic mutation in a Chinese family. Literature review and protein three-dimensional structure prediction were performed to analyze the potential pathogenesis of the identified mutations. Overexpression cell models of wild-type and mutated ARSA genes were constructed. The accumulated sulfatides and expression profiles in the cell models were detected, and a series of bioinformatics analyses were carried out to compare the biological changes caused by the candidate pathogenic mutations. RESULTS: We identified an ARSA c.925G>A homozygous mutation from a Chinese late-infantile MLD patient, the first report of this mutation in East Asia. The literature and protein structure analysis indicated that three types of mutations at c.925G (c.925G>A, c.925G>T, c.925G>C) were pathogenic. The overexpression of wild-type or mutated ARSA genes influenced the accumulation of sulfatides. The co-expression modules in the mutated cell models were constructed by genes related to calcium signaling and vesicle transport. CONCLUSION: Our results identified a pathogenic mutation, ARSA homozygosity c.925G>A, from a Chinese MLD family. The pathogenic mechanism of the ARSA mutation in MLD was identified, which may suggest new approaches to diagnosis and treatment.
Assuntos
Cerebrosídeo Sulfatase/genética , Leucodistrofia Metacromática/genética , Mutação de Sentido Incorreto , Cerebrosídeo Sulfatase/química , Cerebrosídeo Sulfatase/metabolismo , Pré-Escolar , Homozigoto , Humanos , Leucodistrofia Metacromática/patologia , Masculino , Domínios ProteicosRESUMO
Metachromatic Leukodystrophy (MLD) and Multiple Sulfatase Deficiency (MSD) are rare and ultra-rare lysosomal storage diseases. Due to enzyme defects, patients are unable to split the sulfategroup from the respective substrates. In MSD all sulfatases are affected due to a defect of the Sulfatase Modifying Factor 1 (SUMF1) gene coding for the formylglycine generating enzyme (FGE) necessary for the modification of the active site of sulfatases. In MLD mutations in the arylsulfatase A (ARSA) gene cause ARSA deficiency with subsequent accumulation of 3-sulfogalactocerebroside especially in oligodendrocytes. The clinical consequence is demyelination and a devastating neurological disease. Enzyme replacement therapy (ERT) with recombinant human arylsulfatase A (rhARSA), gene therapy, and stem cell transplantation are suggested as new therapeutic options. The aim of our study was to characterize rhARSA concerning its substrate specificity using analytical isotachophoresis (ITP). Substrate specificity could be demonstrated by sulfate splitting from the natural substrates 3-sulfogalactocerebroside and ascorbyl-2-sulfate and the artificial substrate p-nitrocatecholsulfate, whereas galactose-6-sulfate, a substrate of galactose-6sulfurylase, was totally resistant. In contrast, leukocyte extracts of healthy donors were able to split sulfate also from galactose-6-sulfate. The ITP method allows therefore a rapid and simple differentiation between samples of MLD and MSD patients and healthy donors. Therefore, the isotachophoretic diagnostic assay from leukocyte extracts described here provides a fast and efficient way for the diagnosis of MLD and MSD patients and an elegant system to differentiate between these diseases in one assay.
Assuntos
Cerebrosídeo Sulfatase/química , Ensaios Enzimáticos/métodos , Isotacoforese/métodos , Leucócitos/enzimologia , Leucodistrofia Metacromática/enzimologia , Doença da Deficiência de Múltiplas Sulfatases/enzimologia , Sulfatases/química , Cerebrosídeo Sulfatase/genética , Cerebrosídeo Sulfatase/metabolismo , Humanos , Cinética , Leucodistrofia Metacromática/diagnóstico , Leucodistrofia Metacromática/genética , Doença da Deficiência de Múltiplas Sulfatases/diagnóstico , Doença da Deficiência de Múltiplas Sulfatases/genética , Proteínas Recombinantes/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sulfatases/genética , Sulfatases/metabolismo , Sulfatos/química , Sulfatos/metabolismoRESUMO
Metachromatic leukodystrophy (MLD) is a neurodegenerative disorder characterized by progressive demyelination resulting from impaired degradation and thus the accumulation of cerebroside-3-sulfate (sulfatide). It is caused by the deficiency of arylsulfatase A (ARSA) enzyme which is encoded by the ARSA gene. The present study reports the clinical, molecular, and bioinformatic investigation of three patients belonging to a consanguineous family with late-infantile MLD disorder. The results revealed a novel homozygous missense mutation c.699C>A (p.His231Gln) in exon 4 of ARSA gene in the three patients inherited from their heterozygous parents. Interestingly, this novel mutation is the second mutation identified in the substrate-binding site of ARSA protein and it was classified as damaging and deleterious by several bioinformatics tools. The c.699C>A (p.His231Gln) leads to changes in the pre-mRNA secondary structure and in the ARSA protein 3D structure with a significant root mean square deviation value which could probably affect its stability and function.
Assuntos
Cerebrosídeo Sulfatase/genética , Leucodistrofia Metacromática/genética , Mutação de Sentido Incorreto , Sítios de Ligação , Cerebrosídeo Sulfatase/química , Cerebrosídeo Sulfatase/metabolismo , Pré-Escolar , Consanguinidade , Éxons , Feminino , Heterozigoto , Homozigoto , Humanos , Lactente , Leucodistrofia Metacromática/patologia , Masculino , Simulação de Dinâmica Molecular , Linhagem , Ligação ProteicaRESUMO
A deficiency of the enzyme arylsulfatase A (ARSA) causes a progressive neurodegenerative lysosomal storage disease known as metachromatic leukodystrophy (MLD). Diagnosis is based on the onset of neurological symptoms, presence of gait abnormalities, spasticity, decreased muscle stretch reflexes and neuro-radiological evidence of demyelination. The purpose of the present study was to identify any mutation in the candidate ARSA gene in a family of late infantile MLD patients. The diagnosis of suspected MLD patients was confirmed by a MRI report and low ARSA enzymatic activity in leukocytes. Sanger sequencing of full-length coding regions of ARSA gene was performed. Changes in the nucleotide sequence were determined by comparing the obtained data with the wild-type sequence. mRNA expression was analysed using real-time PCR. A novel base pair substitution at position c.338T>C (p.L113P) of ARSA gene was observed in the family and was confirmed in a normal population via ARMS-PCR and Sanger sequencing. The mRNA expression of ARSA gene showed a significant difference between normal and carrier individuals (p = 0.0008). In silico analysis by POLYPHEN, a pathogenicity prediction tool, predicted the possible damaging nature of this mutation. I-TASSER, a protein-modelling server, demonstrated the effects of this mutation on different domains of the ARSA protein, which plays a crucial role in the structural and functional integrity of enzyme. The novel p.L113P mutation in a Pakistani family with late infantile MLD has a pathogenic and destructive effect on the protein structure and function of ARSA. It is the first case reported in a Pakistani population using genetic analysis.
Assuntos
Cerebrosídeo Sulfatase/genética , Leucodistrofia Metacromática/genética , Mutação , Adulto , Células Cultivadas , Cerebrosídeo Sulfatase/química , Cerebrosídeo Sulfatase/metabolismo , Criança , Feminino , Humanos , Lactente , Leucócitos/metabolismo , Leucodistrofia Metacromática/patologia , Masculino , Paquistão , Linhagem , Domínios ProteicosRESUMO
The lysosomal storage disorder (LSD) metachromatic leukodystrophy (MLD) is caused by a deficiency of the soluble, lysosomal hydrolase arylsulfatase A (ASA). The disease is characterized by accumulation of 3-O-sulfogalactosylceramide (sulfatide), progressive demyelination of the nervous system and premature death. Enzyme replacement therapy (ERT), based on regular intravenous injections of recombinant functional enzyme, is in clinical use for several LSDs. For MLD and other LSDs with central nervous system (CNS) involvement, however, ERT is limited by the blood-brain barrier (BBB) restricting transport of therapeutic enzymes from the blood to the brain. In the present study, the potential of different types of surfactant-coated biodegradable nanoparticles to increase brain delivery of ASA was evaluated. Three different strategies to bind ASA to nanoparticle surfaces were compared: (1) adsorption, (2) high-affinity binding via the streptavidin-biotin system, and (3) covalent binding. Adsorption allowed binding of high amounts of active ASA. However, in presence of phosphate-buffered saline or serum rapid and complete desorption occurred, rendering this strategy ineffective for in vivo applications. In contrast, stable immobilization with negligible dissociation was achieved by high-affinity and covalent binding. Consequently, we analyzed the brain targeting of two stably nanoparticle-bound ASA formulations in ASA-/- mice, an animal model of MLD. Compared to free ASA, injected as a control, the biodistribution of nanoparticle-bound ASA was altered in peripheral organs, but no increase of brain levels was detectable. The failure to improve brain delivery suggests that the ASA glycoprotein interferes with processes required to target surfactant-coated nanoparticles to brain capillary endothelial cells.
Assuntos
Encéfalo/metabolismo , Cerebrosídeo Sulfatase/administração & dosagem , Nanopartículas/administração & dosagem , Tensoativos/administração & dosagem , Animais , Avidina/química , Biotinilação , Cerebrosídeo Sulfatase/química , Cerebrosídeo Sulfatase/genética , Cerebrosídeo Sulfatase/farmacocinética , Feminino , Ácido Láctico/química , Leucodistrofia Metacromática/tratamento farmacológico , Leucodistrofia Metacromática/metabolismo , Camundongos Knockout , Nanopartículas/química , Poloxâmero/administração & dosagem , Poloxâmero/química , Poloxâmero/farmacocinética , Poliésteres/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polissorbatos/administração & dosagem , Polissorbatos/química , Polissorbatos/farmacocinética , Albumina Sérica Humana/química , Tensoativos/química , Tensoativos/farmacocinéticaRESUMO
Arylsulfatase A (ASA) deficiency is the cause of metachromatic leucodystrophy (MLD), a lysosomal storage disease associated with severe neurological disorders. Poly(butyl cyanoacrylate) (PBCA) nanoparticles overcoated with polysorbate 80 enabled the delivery of several drugs across the blood-brain barrier to the brain suggesting that these nanoparticles also may transport ASA across this barrier. The objective of this research, therefore, was to evaluate the feasibility of loading ASA onto PBCA nanoparticles. A stable ASA-loaded PBCA nanoparticle formulation was developed that could be easily freeze-dried and stored over a period of more than 8 weeks. The maximum loading capacity for this enzyme was -59 microg per 1 mg of PBCA. In the presence of 3% sucrose as a lyoprotector the activity of freeze-dried ASA was found to be 100% recoverable.
Assuntos
Cerebrosídeo Sulfatase/uso terapêutico , Embucrilato/química , Terapia de Reposição de Enzimas/métodos , Catecóis , Cerebrosídeo Sulfatase/administração & dosagem , Cerebrosídeo Sulfatase/química , Cromatografia Gasosa , Cromatografia em Gel , Portadores de Fármacos , Composição de Medicamentos , Eletroquímica , Excipientes , Liofilização , Indicadores e Reagentes , Cinética , Nanopartículas , Ligação Proteica , Solubilidade , Propriedades de Superfície , Adesivos TeciduaisRESUMO
Arylsulfatase A (ARSA) is a lysosomal sulfatase that catalyzes the hydrolysis of cerebroside sulfate. Its deficiency results in Metachromatic Leukodystrophy, whereas a minor condition called ARSA pseudodeficiency occurs in healthy individuals, which has been associated with the substitution of the glycosylated Asn350 by a Ser and with the loss of the polyadenylation signal. In this work, we have investigated ARSA dynamics employing molecular dynamics simulations in response to (1) different pH's, as, beyond its natural lysossomal environment, it has been recently identified in cytoplasmatic medium and (2) glycan occupancies, including its normal glycosylation state, presenting three high mannose-type oligosaccharides. Accordingly, four systems were studied considering ARSA under different conditions: (1) nonglycosylated at pH â¼ 7 (ARSApH7); (2) non-glycosylated at pH â¼ 5 (ARSApH5); (3) triple glycosylated at pH â¼ 5 (ARSAglyc,pH5); and (4) ARSA-N350S mutant at pH â¼ 5 (ARSAN350S,pH5). Lowering pH and increasing glycosylation was found to reduce the flexibility of the enzyme. In addition, at acidic pH, the glycosylated enzyme presented a higher secondary conformational stability when compared to its nonglycosylated counterpart, supporting experimental findings on triple glycosylation as the essential state of ARSA. The N350S mutant exhibited a consistent degree of unfolding, which may be related to its in vitro reduced stability. Finally, the obtained data are discussed in the search for structural evidences able to contribute to the understanding of biological activity of ARSA and molecular etiology of ARSA pseudodeficiency, as determined by ARSA-N350S in the absence of polyadenylation defect.
Assuntos
Cerebrosídeo Sulfatase/química , Cerebrosídeo Sulfatase/metabolismo , Cerebrosídeo Sulfatase/genética , Glicosilação , Humanos , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Mutação , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Fluorescent dye conjugates of arylsulfatase A (ASA) from rabbit liver were prepared at pH 9.0 in 0.1 M sodium bicarbonate buffer. The modification of amino or sulphadryl groups by dichlorotriazinylamino-fluorescein or Lucifer yellow fluorescent dyes did not alter the characteristic features of the enzyme molecule such as enzyme activity, dimerization of the protein molecule at pH 4.5 and anomalous kinetics of the native enzyme. The fluorescence intensity of the Lucifer yellow enzyme conjugates were quenched when the pH of the protein solution was changed from pH 7.5 to 4.5. Therefore, the Lucifer yellow enzyme conjugate can be used to study the kinetics of pH-dependent association and dissociation of the ASA. Availability of such fluorescent dyes conjugates of ASA or other lysosomal enzyme may be used as a biological tracer to study the receptor dependent endocytosis of enzyme molecules.
Assuntos
Cerebrosídeo Sulfatase/química , Endocitose , Corantes Fluorescentes/química , Imagem Molecular/métodos , Animais , Proteínas de Transporte/química , Fibroblastos/química , Fluoresceínas/química , Humanos , Concentração de Íons de Hidrogênio , Isoquinolinas/química , Fígado/enzimologia , Lisossomos/enzimologia , Coelhos , Pele/citologiaRESUMO
Metachromatic leukodystrophy is an autosomal recessive neurodegenerative disorder of the myelin metabolism due to the impaired function of the lysosomal enzyme arylsulfatase A. Three major clinical variants of metachromatic leukodystrophy (MLD) have been described: late infantile, juvenile, and late onset. The infantile form, whose clinical onset is usually before the age of 2 years, is the most frequent. The juvenile form manifests itself between 3 and 16 years and the late-onset form manifests at any time after puberty. As of today, more than 150 mutations causing MLD have been identified in the ARSA gene that encodes arylsulfatase A. In this paper, we report our experience with the diagnosis of MLD in seven Italian patients from unrelated families. We found 11 different mutations, four of which have not been previously described: c.1215_1223del9 (p.406_408del), c.601 T>C (p.Tyr201His), c.655 T>A (p.Phe219Ile), and c.87C>A (p.Asp29Glu). Our data show once more that there are still several mutations to be discovered in the ARSA gene and there are rarely repeating ones found in the population. The predictive value of the enzyme activity tests in regard to clinical manifestations is extremely limited.
Assuntos
Cerebrosídeo Sulfatase/genética , Leucodistrofia Metacromática/genética , Mutação , Sequência de Aminoácidos , Cerebrosídeo Sulfatase/química , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Itália , Leucodistrofia Metacromática/diagnóstico , Masculino , Dados de Sequência Molecular , LinhagemRESUMO
Cystine knots or nested disulfides are structurally difficult to characterize, despite current technological advances in peptide mapping with high-resolution liquid chromatography coupled with mass spectrometry (LC-MS). In the case of recombinant human arylsulfatase A (rhASA), there is one cystine knot at the C-terminal, a pair of nested disulfides at the middle, and two out of three unpaired cysteines in the N-terminal region. The statuses of these cysteines are critical structure attributes for rhASA function and stability that requires precise examination. We used a unique approach to determine the status and linkage of each cysteine in rhASA, which was comprised of multi-enzyme digestion strategies (from Lys-C, trypsin, Asp-N, pepsin, and PNGase F) and multi-fragmentation methods in mass spectrometry using electron transfer dissociation (ETD), collision induced dissociation (CID), and CID with MS(3) (after ETD). In addition to generating desired lengths of enzymatic peptides for effective fragmentation, the digestion pH was optimized to minimize the disulfide scrambling. The disulfide linkages, including the cystine knot and a pair of nested cysteines, unpaired cysteines, and the post-translational modification of a cysteine to formylglycine, were all determined. In the assignment, the disulfide linkages were Cys138-Cys154, Cys143-Cys150, Cys282-Cys396, Cys470-Cys482, Cys471-Cys484, and Cys475-Cys481. For the unpaired cysteines, Cys20 and Cys276 were free cysteines, and Cys51 was largely converted to formylglycine (>70%). A successful methodology has been developed, which can be routinely used to determine these difficult-to-resolve disulfide linkages, ensuring drug function and stability.
Assuntos
Cerebrosídeo Sulfatase/química , Cisteína/química , Dissulfetos/química , Fragmentos de Peptídeos/química , Mapeamento de Peptídeos/métodos , Sequência de Aminoácidos , Cerebrosídeo Sulfatase/metabolismo , Cromatografia Líquida/métodos , Cisteína/metabolismo , Dissulfetos/metabolismo , Humanos , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Arylsulfatase A is an endogenous enzyme that is responsible for the catabolism and control of sulfatides in humans. Its deficiency results in the accumulation of sulfatides in the cells of the central and peripheral nervous system leading to the destruction of the myelin sheath and resulting in metachromatic leukodystrophy (MLD), a neurodegenerative lysosomal storage disease. A recombinant human form of this glycoprotein (rhASA) is currently under development as an enzyme replacement therapy. At neutral and alkaline pH, this protein exists as a homodimer but converts to an octameric state in the mildly acidic environment of the lysosome, and a failure to form an octamer results in suboptimal catalytic activity (most likely due to a diminished protection from lysosomal proteases). Despite the obvious importance of the rhASA oligomerization process, its mechanistic details remain poorly understood. In this work, we use size exclusion chromatography (SEC) and electrospray ionization mass spectrometry (ESI MS) to monitor the dimer-to-octamer transition as a function of both solution pH and protein concentration. While SEC clearly shows different profiles (i.e., retention time differences) for rhASA when the chromatography is performed at neutral and lysosomal pH, consistent with changing oligomerization states, no resolved peaks could be observed for either octamer or dimer when analyzed at intermediate pH (5.5-6.5). This could be interpreted either as the result of a rapid dimer-to-octamer interconversion on the chromatographic time scale or as a consequence of the presence of previously unidentified intermediate species (e.g., tetramer and/or hexamer). In contrast, ESI MS provides strong evidence of the dimer-to-octamer transition state that occurs when the analysis is performed within a narrow pH range (6.0-7.0). Octamer assembly was shown to be a highly cooperative process with no intermediate states that are populated to detectable levels. A tetrameric state of rhASA exists at equilibrium with a dimer at neutral pH but does not appear to be involved in the octamer assembly process.
Assuntos
Cerebrosídeo Sulfatase/química , Cromatografia em Gel/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Cerebrosídeo Sulfatase/análise , Humanos , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/análise , Proteínas Recombinantes/químicaRESUMO
Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder of the brain caused by mutations in the gene encoding the lysosomal sulfatase, arylsulfatase A (ASA). It is not possible to treat the brain in MLD with recombinant ASA, because the enzyme does not cross the blood-brain barrier (BBB). In the present investigation, a BBB-penetrating IgG-ASA fusion protein is engineered and expressed, where the ASA monomer is fused to the carboxyl terminus of each heavy chain of an engineered monoclonal antibody (MAb) against the human insulin receptor (HIR). The HIRMAb crosses the BBB via receptor-mediated transport on the endogenous BBB insulin receptor, and acts as a molecular Trojan horse to ferry the ASA into brain from blood. The HIRMAb-ASA is expressed in stably transfected Chinese hamster ovary cells grown in serum free medium, and purified by protein A affinity chromatography. The fusion protein retains high affinity binding to the HIR, EC50 = 0.34 ± 0.11 nM, and retains high ASA enzyme activity, 20 ± 1 units/mg. The HIRMAb-ASA fusion protein is endocytosed and triaged to the lysosomal compartment in MLD fibroblasts. The fusion protein was radio-labeled with the Bolton-Hunter reagent, and the [(125) I]-HIRMAb-ASA rapidly penetrates the brain in the Rhesus monkey following intravenous administration. Film and emulsion autoradiography of primate brain shows global distribution of the fusion protein throughout the monkey brain. These studies describe a new biological entity that is designed to treat the brain of humans with MLD following non-invasive, intravenous infusion of an IgG-ASA fusion protein.
Assuntos
Anticorpos Monoclonais/farmacocinética , Antígenos CD/metabolismo , Encéfalo/metabolismo , Cerebrosídeo Sulfatase/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Receptor de Insulina/metabolismo , Proteínas Recombinantes de Fusão/farmacocinética , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Barreira Hematoencefálica/metabolismo , Química Encefálica , Células CHO , Cerebrosídeo Sulfatase/química , Cerebrosídeo Sulfatase/genética , Cricetinae , Cricetulus , Humanos , Radioisótopos do Iodo/química , Radioisótopos do Iodo/farmacocinética , Leucodistrofia Metacromática , Macaca mulatta , Masculino , Ligação Proteica , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Distribuição TecidualRESUMO
Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder caused by mutations in arylsulfatase A (ARSA) gene. No work on molecular genetics of MLD has been reported from India and the mutational spectrum in Indian patients is not known. The present study was undertaken to identify mutations in arylsulfatase A gene in Indian MLD patients, to evaluate genotype-phenotype correlation, and to see the effect of the novel mutants on the protein. Twenty MLD patients (16 families) were screened by ARMS PCR for the most common mutation (c.459+1G>A). Pseudodeficiency alleles were tested by RFLP method whereas rare and novel mutations were scanned by Conformation Sensitive Gel Electrophoresis (CSGE), followed by sequencing. The genotype-phenotype correlation was also attempted. Protein homology modeling analysis was carried out for two novel missense mutations identified, to assess the effect of these mutations on the protein conformation. Nine pathogenic alleles were found in 13 patients (65%). Four previously reported mutations and five novel variants were identified. Five patients (35%) were found to have pseudodeficiency alleles, c.1049A>G (p.Asn350Ser) and c.1524+95A>G. Genotype-phenotype correlation was found to be difficult to establish. Protein modeling studies showed that the mutations cause loss of interactions leading to conformational change in ASA protein. The study identified the mutational spectrum of Indian MLD patients, which will be helpful in genetic counseling, carrier detection and establishing prenatal diagnosis. Homology modeling helped to study conformational change in protein and has implications in generating novel therapeutic molecules.
Assuntos
Cerebrosídeo Sulfatase/genética , Leucodistrofia Metacromática/genética , Idade de Início , Alelos , Encéfalo/patologia , Cerebrosídeo Sulfatase/química , Criança , Pré-Escolar , Análise Mutacional de DNA , Genótipo , Humanos , Índia/epidemiologia , Lactente , Leucodistrofia Metacromática/etnologia , Leucodistrofia Metacromática/patologia , Imageamento por Ressonância Magnética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Arylsulfatase A is an oligomeric lysosomal enzyme. In the present study, we use this enzyme as a model protein to examine how heteromerization of wild-type and misfolded endoplasmic reticulum-degraded arylsulfatase A polypeptides affects the quality control of wild-type arylsulfatase A subunits. Using a conformation sensitive monoclonal antibody, we show that, within heteromers of misfolded and wild-type arylsulfatase A, the wild-type subunits are not fully folded. The results obtained show that arylsulfatase A polypeptide complexes, rather than the monomers, are subject to endoplasmic reticulum quality control and that, within a heteromer, the misfolded subunit exerts a dominant negative effect on the wild-type subunit. Although it has been shown that mature lysosomal arylsulfatase A forms dimers at neutral pH, the results obtained in the present study demonstrate that, in the early biosynthetic pathway, arylsulfatase A forms oligomers with more than two subunits.
Assuntos
Cerebrosídeo Sulfatase/metabolismo , Retículo Endoplasmático/metabolismo , Dobramento de Proteína , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , Cerebrosídeo Sulfatase/química , Cerebrosídeo Sulfatase/genética , Cricetinae , Dimerização , Lisossomos/metabolismo , Modelos Biológicos , Mutação/genéticaRESUMO
We have previously isolated and purified a goat sperm protein of 70 kDa molecular weight designated as P70 and characterized it as an inhibitor of Na(+),K(+)-ATPase. Our study reveals that the first 10 amino acid residues from the N-terminal end of P70 has high degree of homology with arylsulphatase A from mice, pig and human. Indirect immunofluorescence study shows the presence of the protein on goat sperm surface. Furthermore, live goat sperm and the extract of peripheral sperm plasma membrane proteins exhibit arylsulphatase A's desulphation activity. The P70 remains on the head surface of in vitro capacitated cauda epididymal sperm as shown by positive immunofluorescence staining of cauda sperm. Immunoblot and flow cytometric studies corroborate the above findings. The presence of P70 on capacitated cauda sperm surface suggest a possible role of this protein in sperm zona pellucida binding. In the present report we demonstrate arylsulphatase A like activity in P70 and describe its localization and expression in goat sperm.
Assuntos
Cerebrosídeo Sulfatase/metabolismo , Inibidores Enzimáticos/metabolismo , Cabras , Proteínas de Membrana/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Espermatozoides/química , Reação Acrossômica , Animais , Cerebrosídeo Sulfatase/química , Cerebrosídeo Sulfatase/genética , Epididimo/citologia , Humanos , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Peso Molecular , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Capacitação Espermática , Espermatozoides/citologia , Espermatozoides/metabolismoRESUMO
The sulfatase family of enzymes catalyzes hydrolysis of sulfate ester bonds of a wide variety of substrates. Seventeen genes have been identified in this class of sulfatases, many of which are associated with genetic disorders leading to reduction or loss of function of the corresponding enzymes. Amino acid sequence homology suggests that the enzymes have similar overall folds, mechanisms of action, and bivalent metal ion-binding sites. A catalytic cysteine residue, strictly conserved in prokaryotic and eukaryotic sulfatases, is post-translationally modified into a formylglycine. Hydroxylation of the formylglycine residue by a water molecule forming the activated hydroxylformylglycine (a formylglycine hydrate or a gem-diol) is a necessary step for the enzyme's sulfatase activity. Crystal structures of three human sulfatases, arylsulfatases A and B(ARSA and ARSB), and estrone/dehydroepiandrosterone sulfatase or steroid sulfatase (STS), also known as arylsulfatase C, have been determined. While ARSA and ARSB are water-soluble enzymes, STS has a hydrophobic domain and is an integral membrane protein of the endoplasmic reticulum. In this article, we compare and contrast sulfatase structures and revisit the proposed catalytic mechanism in light of available structural and functional data. Examination of the STS active site reveals substrate-specific interactions previously identified as the estrogen-recognition motif. Because of the proximity of the catalytic cleft of STS to the membrane surface, the lipid bilayer has a critical role in the constitution of the active site, unlike other sulfatases.
Assuntos
Sulfatases/química , Sulfatases/metabolismo , Aminoácidos/química , Neoplasias da Mama/enzimologia , Catálise , Domínio Catalítico , Cerebrosídeo Sulfatase/química , Cerebrosídeo Sulfatase/metabolismo , Cristalografia por Raios X , Feminino , Humanos , Técnicas In Vitro , Masculino , Modelos Moleculares , N-Acetilgalactosamina-4-Sulfatase/química , N-Acetilgalactosamina-4-Sulfatase/metabolismo , Gravidez , Neoplasias da Próstata/enzimologia , Estrutura Terciária de Proteína , Eletricidade Estática , Esteril-Sulfatase/química , Esteril-Sulfatase/metabolismoRESUMO
Carbohydrate residues on membrane proteins from sperm are important in gamete interaction. In recent years, Arylsulfatase A (AS-A) has been acquiring an important role from the various putative gamete interaction responsibles in sperm. The aim of this study was to determine if the capacitated boar sperm Arylsulfatase-A (AS-A), contains D-mannose, N-acetylglucosamine and/or sialic acid residues by its purification using affinity chromatography with Concanavalia ensiformis Agglutinin(Con-A) or Wheat Germ Agglutinin (WGA) as ligands. Sperm samples were capacitated in TALP-HEPES medium. Protein extract was added to the affinity columns. Sequencing of retained proteins was done after SDS-PAGE. Total capacitated sperm proteins electrophoresis showed molecular masses between 14 kDa and 102 kDa. A major band of 68 kDa, and 2 minor bands of 52 kDa and 47 kDa were observed. They were AS-A, hyaluronidase and lactadherin, respectively. The Con-A-retained proteins (RP) pattern showed bands from 14 to 98 kDa. After sequencing and BLAST analysis, the 62 kDa band corresponded to Arylsulfatase-A. The WGA RP fraction showed bands from 14 to 100 kDa. The 65 kDa band corresponded to AS-A. This study showed that AS-A has mannose, N-acetylglucosamine and/or sialic acid residues as part of its glycosilation. In this study AS-A was isolated from boar capacitated sperm by affinity chromatography using separately Con-A and WGA, indicating that there are mannose, N-acetylglucosamine and/or sialic acid residues in its glycosilation. AS-A is a membrane protein of capacitated sperm. Further investigation is needed to fully characterize the glycosidic residues bore by AS-A and to determine its function.