RESUMO
Hyaluronan and chondroitin/dermatan sulfate are glycosaminoglycans that play major roles in the biomechanical properties of a wide variety of tissues, including cartilage. A chondroitin/dermatan sulfate chain can be divided into three regions: (1) a single linkage region oligosaccharide, through which the chain is attached to its proteoglycan core protein, (2) numerous internal repeat disaccharides, which comprise the bulk of the chain, and (3) a single nonreducing terminal saccharide structure. Each of these regions of a chondroitin/dermatan sulfate chain has its own level of microheterogeneity of structure, which varies with proteoglycan class, tissue source, species, and pathology. We have developed rapid, simple, and sensitive protocols for detection, characterization and quantitation of the saccharide structures from the internal disaccharide and nonreducing terminal regions of hyaluronan and chondroitin/dermatan sulfate chains. These protocols rely on the generation of saccharide structures with free reducing groups by specific enzymatic treatments (hyaluronidase/chondroitinase) which are then quantitatively tagged though their free reducing groups with the fluorescent reporter, 2-aminoacridone. These saccharide structures are further characterized by modification through additional enzymatic (sulfatase) or chemical (mercuric ion) treatments. After separation by fluorophore-assisted carbohydrate electrophoresis, the relative fluorescence in each band is quantitated with a cooled, charge-coupled device camera for analysis. Specifically, the digestion products identified are (1) unsaturated internal Deltadisaccharides including DeltaDiHA, DeltaDi0S, DeltaDi2S, DeltaDi4S, DeltaDi6S, DeltaDi2,4S, DeltaDi2,6S, DeltaDi4,6S, and DeltaDi2,4,6S; (2) saturated nonreducing terminal disaccharides including DiHA, Di0S, Di4S and Di6S; and (3) nonreducing terminal hexosamines including glcNAc, galNAc, 4S-galNAc, 6S-galNAc, and 4, 6S-galNAc.
Assuntos
Condroitina/metabolismo , Dermatan Sulfato/metabolismo , Eletroforese em Gel de Poliacrilamida , Ácido Hialurônico/metabolismo , Acetilgalactosamina/metabolismo , Animais , Condro-4-Sulfatase/metabolismo , Dissacarídeos/química , Humanos , Monossacarídeos/química , Ratos , Espectrometria de Fluorescência , Especificidade por Substrato , Sulfatases/metabolismoRESUMO
An assay was developed, using two similar formats, to simultaneously measure both the lysosomal targeting receptor binding and enzyme activity of the recombinant human enzyme N-acetylgalactosamine-4-sulfatase. This assay also has potential application for all phosphorylated lysosomal enzymes that contain mannose-6-phosphate residues. The receptor was either purified from fetal bovine sera then adsorbed, or produced in situ by growing and fixing diploid human fibroblast-like cells, to a solid phase. The enzyme substrate was 4-methylumbelliferyl sulfate which fluoresces after cleavage of the sulfate moiety. Both the precursor and mature forms of the recombinant enzyme were used to demonstrate the specificity and usefulness of the assay. The assay is rapid and sensitive and has a wide dynamic range. Association between the receptor and the mannose-6-phosphate residues was abrogated in the presence of a competitive inhibitor, mannose 6-phosphate. However, partial activity was still measured when the mature enzyme was incubated in the presence of mannose 6-phosphate when using the fixed fibroblast format. This would indicate that the recombinant enzymes contain at least one terminal sugar moiety other than mannose 6-phosphate which can recognize receptors on the surface of human fibroblast-like cells. Other possible applications of this assay are also discussed.
Assuntos
Condro-4-Sulfatase/metabolismo , Receptor IGF Tipo 2/metabolismo , Adsorção , Animais , Ligação Competitiva , Bovinos , Linhagem Celular , Condro-4-Sulfatase/análise , Fibroblastos , Humanos , Himecromona/análogos & derivados , Himecromona/metabolismo , Cinética , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Espectrometria de Fluorescência/métodosRESUMO
Sulfatases contain an active site formylglycine residue that is generated by post-translational modification. Crystal structures of two lysosomal sulfatases revealed significant similarity to the catalytic site of alkaline phosphatase containing a serine at the position of formylglycine. To elucidate the catalytic mechanism of sulfate ester hydrolysis, the formylglycine of arylsulfatases A and B was substituted by serine. These mutants upon incubation with substrate were covalently sulfated at the introduced serine. This sulfated enzyme intermediate was stable at pH 5. At alkaline pH it was slowly hydrolyzed. These characteristics are analogous to that of alkaline phosphatase which forms a phosphoserine intermediate that is stable at pH 5, but is hydrolyzed at alkaline pH. In wild-type sulfatases the hydroxyl needed for formation of the sulfated enzyme intermediate is provided by the aldehyde hydrate of the formylglycine. The second, non-esterified hydroxyl of the aldehyde hydrate is essential for rapid desulfation of the enzyme at acidic pH, which most likely occurs by elimination. The lack of this second hydroxyl in the serine mutants explains the trapping of the sulfated enzyme intermediate. Thus, in acting as a geminal diol the formylglycine residue allows for efficient ester hydrolysis in an acidic milieu.
Assuntos
Alanina/análogos & derivados , Cerebrosídeo Sulfatase/metabolismo , Condro-4-Sulfatase/metabolismo , Glicina/análogos & derivados , Ésteres do Ácido Sulfúrico/metabolismo , Alanina/metabolismo , Fosfatase Alcalina/metabolismo , Sítios de Ligação , Catecóis/metabolismo , Cerebrosídeo Sulfatase/genética , Condro-4-Sulfatase/genética , Galactose/análogos & derivados , Glicina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , SerinaRESUMO
Severe Maroteaux-Lamy syndrome is usually fatal in teenage or early adult life. Until recently, allogeneic bone marrow transplantation was the only form of enzyme replacement. We report the first successful transplant using CD34 selected, mobilised allogeneic blood cells for an inborn error of metabolism. A busulphan, cyclophosphamide, melphalan and antithymocyte globulin conditioning regimen was used as myeloablative therapy. Allogeneic CD34 selected granulocyte colony-stimulating factor (G-CSF)-mobilised blood cells from a HLA-identical sibling were used for the transplant. Haemopoietic reconstitution occurred on day 10 post-transplant with normal N-acetylgalactosamine-4-sulphatase levels. Infectious and graft-versus-host disease (GVHD) complications were minimal. We suggest that CD34 selected, mobilised allogeneic blood cells are a safe form of enzyme replacement therapy in Maroteaux-Lamy syndrome and should be considered in other metabolic diseases where the benefits of haemopoietic transplantation are proven.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Mucopolissacaridose VI/terapia , Adolescente , Adulto , Obstrução das Vias Respiratórias/etiologia , Obstrução das Vias Respiratórias/terapia , Antígenos CD34/metabolismo , Separação Celular , Criança , Condro-4-Sulfatase/metabolismo , Sobrevivência de Enxerto , Hematopoese , Humanos , Masculino , Mucopolissacaridose VI/complicações , Mucopolissacaridose VI/enzimologia , Condicionamento Pré-Transplante , Transplante HomólogoRESUMO
Sulfatases undergo an unusual protein modification leading to conversion of a specific cysteine residue into alpha-formylglycine. This conversion is essential for catalytic activity. In arylsulfatase A the alpha-formylglycine is generated inside the endoplasmic reticulum at a late stage of protein translocation. Using in vitro translation in the presence of transport-competent microsomes we found that arylsulfatase B is also modified in a similar way by the formylglycine-generating machinery. Modification depended on protein transport and on the correct position of the relevant cysteine. Arylsulfatase A and B did not compete for modification, as became apparent in co-expression experiments. This could argue for an association of the modification machinery with the protein translocation apparatus.
Assuntos
Alanina/análogos & derivados , Cerebrosídeo Sulfatase/metabolismo , Condro-4-Sulfatase/metabolismo , Cisteína/metabolismo , Glicina/análogos & derivados , Alanina/metabolismo , Animais , Linhagem Celular , Cerebrosídeo Sulfatase/biossíntese , Cerebrosídeo Sulfatase/genética , Condro-4-Sulfatase/biossíntese , Condro-4-Sulfatase/genética , Cricetinae , Cisteína/genética , Retículo Endoplasmático/metabolismo , Células Eucarióticas/metabolismo , Glicina/metabolismo , Humanos , Processamento de Proteína Pós-Traducional , Fatores de TempoRESUMO
Mucopolysaccharidosis type VI (MPS VI) is a lysosomal storage disease caused by a deficiency of N-acetylgalactosamine-4-sulfatase (4S). A feline MPS VI model used to demonstrate efficacy of enzyme replacement therapy is due to the homozygous presence of an L476P mutation in 4-sulfatase. An additional mutation, D520N, inherited independently from L476P and recently identified in the same family of cats, has resulted in three clinical phenotypes. L476P homozygotes exhibit dwarfism and facial dysmorphia due to epiphyseal dysplasia, abnormally low leukocyte 4S/betahexosaminidase ratios, dermatan sulfaturia, lysosomal inclusions in most tissues including chondrocytes, corneal clouding, degenerative joint disease, and abnormal leukocyte inclusions. Similarly, D520N/D520N and L476P/D520N cats have abnormally low leukocyte 4S/betahexosaminidase ratios, mild dermatan sulfaturia, lysosomal inclusions in some chondrocytes, and abnormal leukocyte inclusions. However, both have normal growth and appearance. In addition, L476P/D520N cats have a high incidence of degenerative joint disease. We conclude that L476P/D520N cats have a very mild MPS VI phenotype not previously described in MPS VI humans. The study of L476P/D520N and D520N/ D520N genotypes will improve understanding of genotype to phenotype correlations and the pathogenesis of skeletal dysplasia and joint disease in MPS VI, and will assist in development of therapies to prevent lysosomal storage in chondrocytes.
Assuntos
Mucopolissacaridose VI/genética , Mucopolissacaridose VI/patologia , Mutação , Animais , Artrografia , Gatos , Condro-4-Sulfatase/metabolismo , Dermatan Sulfato/metabolismo , Modelos Animais de Doenças , Feminino , Genótipo , Humanos , Articulações/patologia , Leucócitos/enzimologia , Leucócitos/patologia , Masculino , Mucopolissacaridose VI/diagnóstico por imagem , Mucopolissacaridose VI/metabolismo , Linhagem , Fenótipo , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
We present the imaging findings in a patient with mucopolysaccharidosis (MPS) type VI (Maroteaux-Lamy syndrome) who developed holocord syringomyelia. This represents the only reported case of syrinx formation in a child with MPS VI. Clinical, neurologic and spinal magnetic resonance imaging findings are presented. The patient has maintained a stable clinical and neurologic course over the period following allogeneic bone marrow transplant.
Assuntos
Transplante de Medula Óssea , Mucopolissacaridoses/complicações , Siringomielia/diagnóstico , Siringomielia/etiologia , Condro-4-Sulfatase/metabolismo , Glicosaminoglicanos/urina , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Mucopolissacaridoses/metabolismoRESUMO
BACKGROUND: Mucopolysaccharidosis type VI (MPS VI) is the lysosomal storage disorder caused by the deficient activity of arylsulfatase B (ASB). In this study, we evaluated bone marrow transplantation (BMT) for the treatment of MPS VI and the effects of irradiation on the survival and engraftment of bone marrow-transplanted neonatal rats. METHODS: One- to 2-day-old MPS VI rats were injected with normal bone marrow after irradiation with 200, 400, or 800 cGy. Ninety percent of the animals receiving a single dose of 200 cGy (n=30) survived the procedure, whereas irradiation with 400 cGy (n=23) or 800 cGy (n=12) resulted in significant mortality (78% and 100%, respectively). Engraftment was monitored by determining ASB activities in peripheral white blood cells and by Y chromosome in situ hybridization analysis. Fifty-two percent of the animals from the 200-cGy group engrafted for up to 8 months after BMT; among the five animals that survived the 400-cGy dose, all engrafted. In comparison, only 20% of nonirradiated animals engrafted at low levels. Of the 24 engrafted animals that were monitored for 8 months after BMT, clinical and/or radiographic improvements were noted in only one (BMT animal 3). Enzymatic analysis revealed that the ASB activities in the reticuloendothelial organs of this animal, as well as two other engrafted but clinically unimproved animals (BMT animals 1 and 2), were normal or near normal; correspondingly, the glycosaminoglycan levels in these organs were significantly reduced. Consistent with the clinical and biochemical observations, light and electron microscopic findings were more improved in BMT animal 3 as compared with BMT animals 1 and 2, although a reduction of storage was evident in each of these transplant recipients, particularly in the trachea and aorta, two tissues that are characteristic sites of pathology in human patients. CONCLUSIONS: These results indicate that BMT in newborn MPS VI patients may prevent many of the pathological and clinical findings in this disorder, but is likely to have very limited and unpredictable effects on the skeletal abnormalities.
Assuntos
Animais Recém-Nascidos/fisiologia , Transplante de Medula Óssea , Mucopolissacaridose VI/terapia , Animais , Aorta/patologia , Aorta/ultraestrutura , Transplante de Medula Óssea/diagnóstico por imagem , Transplante de Medula Óssea/imunologia , Transplante de Medula Óssea/patologia , Gatos , Condro-4-Sulfatase/sangue , Condro-4-Sulfatase/metabolismo , Feminino , Sobrevivência de Enxerto/efeitos da radiação , Humanos , Leucócitos/enzimologia , Masculino , Microscopia Eletrônica , Mucopolissacaridose VI/diagnóstico por imagem , Mucopolissacaridose VI/patologia , Radiografia , Ratos , Ratos Mutantes , Ratos Sprague-Dawley , Traqueia/patologia , Traqueia/ultraestrutura , Irradiação Corporal TotalRESUMO
Mucopolysacchariodosis type VI (MPS VI) is the lysosomal storage disorder caused by the deficient activity of arylsulfatase B (ASB; N-acetylgalactosamine 4-sulfatase) and the subsequent accumulation of the glycosaminoglycan (GAG), dermatan sulfate. In this study, a retroviral vector containing the full-length human ASB cDNA was constructed and used to transduce skin fibroblasts, chondrocytes, and bone marrow cells from human patients, cats, or rats with MPS VI. The ASB vector expressed high levels of enzymatic activity in each of the cell types tested and, in the case of cat and rat cells, enzymatic expression led to complete normalization of 35SO4 incorporation. In contrast, overexpression of ASB in human MPS VI skin fibroblasts did not lead to metabolic correction. High-level ASB expression was detected for up to eight weeks in transduced MPS VI cat and rat bone marrow cultures, and PCR analysis demonstrated retroviral-mediated gene transfer to approximately 30-50% of the CFU GM-derived colonies. Notably, overexpression of ASB in bone marrow cells led to release of the enzyme into the media and uptake by MPS VI cat and rat skin fibroblasts and/or chondrocytes via the mannose-6-phosphate receptor system, leading to metabolic correction. Thus, these studies provide important rationale for the development of gene therapy for this disorder and lay the frame-work for future in vivo studies in the animal model systems.
Assuntos
Condro-4-Sulfatase/metabolismo , Terapia Genética/métodos , Vetores Genéticos , Glicosaminoglicanos/biossíntese , Mucopolissacaridose IV/enzimologia , Retroviridae , Animais , Sequência de Bases , Medula Óssea/metabolismo , Cartilagem/metabolismo , Gatos , Linhagem Celular , Condro-4-Sulfatase/biossíntese , Primers do DNA , Dermatan Sulfato/biossíntese , Fibroblastos/metabolismo , Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Humanos , Dados de Sequência Molecular , Mucopolissacaridose IV/terapia , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Pele/metabolismo , Especificidade da Espécie , Sulfatos/metabolismo , Transfecção/métodosRESUMO
Maroteaux-Lamy syndrome, or mucopolysaccharidosis type VI (MPS-VI), is a lysosomal storage disorder characterized by the defective degradation of dermatan sulfate due to the deficiency of N-acetylgalactosamine-4-sulfatase (4S). The clinical severity of MPS-VI ranges in a continuum from mildly affected to severely affected patients. Mutations in MPS-VI patient samples were identified by chemical cleavage and direct DNA sequencing of PCR products derived from patient cDNA. Five amino acid substitutions were identified (T92M, R95Q, Y210C, H393P, and L498P), individually introduced into the wild-type 4S cDNA by site-directed in vitro mutagenesis, and transfected into Chinese hamster ovary cells. Three of the five mutations (R95Q, Y210C, and H393P) were observed in >1 of 25 unrelated MPS-VI patients; however, the mutations were not found in 20 control individuals. The residual 4S activity and protein (biochemical phenotype) were determined for each mutant in order to confirm their identity as mutations and to dissect the contribution of each mutant allele to the overall clinical phenotype of the patient. For each patient, the combined biochemical phenotypes of the two 4S mutant alleles demonstrated a good correspondence with the observed clinical phenotype (with the possible exception of a patient who was a compound heterozygote for T92M and L498P). This preliminary correspondence between genotype and the phenotype in MPS-VI may, with further refinement, contribute to the assessment of therapeutic approaches for MPS-VI patients.
Assuntos
Condro-4-Sulfatase/genética , Mucopolissacaridose VI/genética , Mutação Puntual , Adolescente , Animais , Sequência de Bases , Células CHO , Criança , Pré-Escolar , Condro-4-Sulfatase/biossíntese , Condro-4-Sulfatase/metabolismo , Cricetinae , Primers do DNA , DNA Complementar , Humanos , Lactente , Dados de Sequência Molecular , Mucopolissacaridose VI/enzimologia , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , TransfecçãoRESUMO
We examined the in vivo role of Kurloff cells (KC), guinea pig natural killer cells, during the development of L2C leukemia, by analysing changes in the arylsulfatases (Asases) in the lysosomal Kurloff body. The Kurloff body is rich in acid phosphatase, esterase and proteoglycans, as are large granular lymphocyte granules. Moreover, the Kurloff body contains lysosomal Asase B, with unusual anionic isoforms. Injection of L2C cells elicited a three-fold increase in KC Asase activity on day 6. The increase in KC Asase activity was correlated with the number of circulating L2C cells. The basic Asase form (pl 8) was lost, and a concomitant increase in anionic isoforms (pl 5-6) was observed on day 6. The role of the latter in cytolysis was investigated by examining their capacity to lyse L2C target cells. We conclude that Asase participates in cytolysis when lysis is mediated by the complete assembly of cytolytic proteins. Changing and increasing KC Asase activity during leukemia development may be a marker for activated KC in vivo. These findings suggest that the cytolytic activation of KC occurs during the preleukemic period.
Assuntos
Condro-4-Sulfatase/metabolismo , Isoenzimas/metabolismo , Células Matadoras Naturais/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Animais , Ânions , Proteínas Sanguíneas/metabolismo , Linhagem Celular , Citosol/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Cobaias , Ativação Linfocitária , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Pré-Leucemia/sangue , Pré-Leucemia/enzimologiaRESUMO
A deficiency of the enzyme arylsulfatase B results in the lysosomal storage disorder Maroteaux-Lamy syndrome or mucopolysaccharidosis type VI. Severe, intermediate and mild forms of this autosomal recessively inherited disease can be clinically differentiated. To determine the molecular defect in a patient with the intermediate form of the disorder, DNA fragments generated from the patient's mRNA by reverse transcription and subsequent amplification by the polymerase chain reaction were subcloned and sequenced. The mRNA transcribed from one allele contains a 244-base pair deletion causing a frameshift and a truncation of the open reading frame. The C-terminal third of the encoded mutant polypeptide has a nonsense sequence. This mutation is due to a deletion of exon 5 in this allele. A silent A to G transition at nucleotide 1191 was present in the same allele, and the second allele was characterized by a T to C transition at nucleotide 1600 causing a mutation of the translational stop codon to a glutamine codon (*534Q) and extending the encoded polypeptide by 50 amino acids. Stable expression of the *534Q allele in LTK- cells resulted in a mutant precursor 4 kDa larger than the wild-type precursor. The majority of the mutant precursor appears to be degraded before reaching the trans Golgi. This is consistent with an altered polypeptide structure, where a number of missing or masked epitopes were observed in an enzyme immunobinding assay using a panel of monoclonal antibodies. Immunoquantification analysis showed that epitopes were most likely masked, as missing epitopes could be reformed by binding the mutant protein to a polyclonal antibody of arylsulfatase B. It is suggested that the additional amino acids at the C terminus of the arylsulfatase B polypeptide induce a protein conformational change. *534Q mutant polypeptide escaping degradation is sorted to dense lysosomes. The mutant polypeptide has an approximately 9-fold higher catalytic efficiency than wild-type arylsulfatase B.
Assuntos
Condro-4-Sulfatase/genética , Condro-4-Sulfatase/metabolismo , Mutação da Fase de Leitura , Mucopolissacaridose VI/enzimologia , Mucopolissacaridose VI/genética , Deleção de Sequência , Alelos , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Criança , Clonagem Molecular , Códon/genética , DNA/biossíntese , DNA/isolamento & purificação , Primers do DNA , Glutamina , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
High-titre stocks of an amphotropic retrovirus, constructed so as to express a full-length cDNA encoding the human lysosomal enzyme N-acetylgalactosamine-4-sulphatase (4-sulphatase) from the cytomegalovirus immediate early promoter, were used to infect skin fibroblasts from a clinically severe mucopolysaccharidosis type VI (MPS VI) patient. The infected MPS VI cells showed correction of the enzymic defect with the enzyme being expressed at high levels and in the correct subcellular compartment. Surprisingly this did not result in correction of glycosaminoglycan turnover as measured by accumulation of 35S in metabolically labelled cells. We demonstrate that this is apparently caused by an induced reduction of the activities of other lysosomal sulphatases, presumably due to competition for a sulphatase-specific processing mechanism by the over-expressed 4-sulphatase. The level of steroid sulphatase, which is a microsomal sulphatase, was also reduced. Infection of skin fibroblasts from a second, clinically mildly affected, MPS VI patient with the same virus also resulted in no significant change in the level of glycosaminoglycan storage. However, in this case the cause of the observed phenomenon was less clear. These results are of obvious practical importance when considering gene therapy for a sulphatase deficiency such as MPS VI and also provide possible new avenues for exploration of the processes involved in sulphatase synthesis and genetically determined multiple sulphatase deficiency.
Assuntos
Condro-4-Sulfatase/metabolismo , Lisossomos/enzimologia , Mucopolissacaridose VI/enzimologia , Condro-4-Sulfatase/genética , Terapia Genética , Glicosaminoglicanos/metabolismo , Humanos , Proteínas Recombinantes/metabolismo , Pele/enzimologia , TransfecçãoRESUMO
The influence of hypothyroidism in the adult rat on brain biochemistry was investigated. Hypothyroidism was induced in 6-month-old male rats by partial thyroidectomy coupled with the administration of 6-n-propyl-2-thiouracil (0.005%, w/v) in the drinking water. Age-matched euthyroid males served as the controls. Hypothyroidism resulted in brain region-specific changes in certain catabolic enzyme activities. Acid phosphatase activity was reduced in the cerebellum (by 34%) and the medulla (by 38%), whereas alkaline phosphatase activity was decreased in the midbrain (by 37%) and the subcortex (by 49%). A differential response was also observed in the case of aryl sulphatase activity: aryl sulphatase A (myelin-degradative activity) was diminished in the cerebellum (by 56%), whereas aryl sulphatase B remained unchanged in all regions. Acetylcholine esterase activity was reduced in the cerebellum (by 45%), the medulla (by 34%) and the subcortex (by 45%), whereas monoamine oxidase activity was affected in only one region, the cerebellum, where it was increased by (61%). The compromise of myelin and neurotransmitter degradative enzyme activities may place severe restrictions on normal brain function. The vulnerability of the adult rat cerebellum to the effects of thyroidectomy is commensurate with the known clinical signs of cerebellar dysfunction in adult hypothyroid man. These findings raise the possibility of an important role for the thyroid hormones in the mature brain.
Assuntos
Encéfalo/enzimologia , Hipotireoidismo/enzimologia , Acetilcolinesterase/metabolismo , Fosfatase Ácida/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Cerebelo/enzimologia , Cerebrosídeo Sulfatase/metabolismo , Condro-4-Sulfatase/metabolismo , Modelos Animais de Doenças , Masculino , Bulbo/enzimologia , Ratos , Ratos Sprague-Dawley , TireoidectomiaRESUMO
The presence and composition of arylsulfatases in secretions of various glands of the boar genital tract were studied. Arylsulfatase A was present in seminal plasma but not in extracellular fluids of the testis and epididymis nor in blood serum of boars. On the other hand, arylsulfatase B was present in both seminal plasma and extracellular fluids of the testis but was completely resorbed in the epididymis. The acrosomal arylsulfatase A did not leak out of spermatozoa before ejaculation. We conclude that arylsulfatases A and B present in seminal plasma are secreted by the seminal vesicles, for three reasons: 1) secretions from seminal vesicles contained 2.3-fold higher arylsulfatase activities than did those from seminal plasma, but had an identical composition; 2) cauda epididymal fluids did not contain arylsulfatase; and 3) other accessory glands of the boar genital tract did not secrete arylsulfatase. When intact boar spermatozoa were incubated with arylsulfatase A, complete desulfation of seminolipid was observed. The most important arguments favoring our hypothesis that desulfation of seminolipid does not start before ejaculation are the following: 1) desulfoseminolipid is not detectable in epididymal or freshly ejaculated sperm samples; 2) the acrosomal arylsulfatase A cannot desulfate seminolipid present at the surface of the plasma membrane of intact spermatozoa because of its intracellular localization; 3) extracellular arylsulfatase A is stored in seminal vesicles and thus can interact with spermatozoa during and after ejaculation.
Assuntos
Arilsulfatases/metabolismo , Glicolipídeos/metabolismo , Sêmen/metabolismo , Glândulas Seminais/enzimologia , Animais , Membrana Celular/metabolismo , Cerebrosídeo Sulfatase/metabolismo , Condro-4-Sulfatase/metabolismo , Ejaculação/fisiologia , Masculino , Glândulas Seminais/metabolismo , Espermatozoides/metabolismo , SuínosRESUMO
Previous studies have shown that mature arylsulfatase B purified from human sources is composed of two non-identical chains with apparent molecular masses of 43 kDa and 8 kDa. Arylsulfatase B purified from human placenta in the present study, however, included another 7 kDa component that could be detected only by carbohydrate staining on reducing SDS-PAGE employing the Tris-Tricine system. The 43 kDa and 7 kDa components contained a carbohydrate moiety, but the 8 kDa one did not, as demonstrated by periodic acid-Schiff staining, Con-A lectin blotting, endo-glycosidase treatment and in vitro phosphorylation by UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine 1-phosphotransferase. The purified arylsulfatase B migrated as a single polypeptide of 58 kDa on non-reducing SDS-PAGE, indicating that the three chains are linked by disulfide bonds. In order to determine the origin of the components, N-terminal sequencing of the isolated polypeptides was performed. As a result, the 43, 7 and 8 kDa components were found to commence with Ala-41, Ala-424 and Asp-466, respectively. These results suggest that after removal of the signal peptide, human arylsulfatase B undergoes proteolytic processing on at least two sites during maturation.
Assuntos
Condro-4-Sulfatase/análise , Placenta/enzimologia , Sequência de Aminoácidos , Condro-4-Sulfatase/química , Condro-4-Sulfatase/metabolismo , Feminino , Glicosídeo Hidrolases/farmacologia , Humanos , Dados de Sequência Molecular , GravidezRESUMO
1. Arylsulfatase B (ASB) from lysosomal fraction of rat liver were isolated and purified 260-fold with a recovery of about 5%. 2. The enzyme in gradient PAGE 4-30% followed by immunoelectrophoresis migrated as a single peak of M(r) 84,000. The pI, measured by isoelectrofocusing in agarose followed by immunoelectrophoresis, was equal to 6.7. 3. ASB reacted with Con A, LCA, PSA, LTL, WGA, RCAI and did not react with PHA, SBA, HPA, CAA and PAL in crossed affino-immunoelectrophoresis or rocket immunoelectrophoresis. These results permit of preliminary elucidation of ASB glycan structure.
Assuntos
Condro-4-Sulfatase/isolamento & purificação , Fígado/enzimologia , Lisossomos/enzimologia , Animais , Condro-4-Sulfatase/química , Condro-4-Sulfatase/metabolismo , Eletroforese em Gel de Poliacrilamida , Imunoeletroforese/métodos , Focalização Isoelétrica , Lectinas , RatosRESUMO
Sperm cells and seminal plasma of various mammals contain high levels of arylsulfatase. In the present study, we investigated the composition of soluble AS in these compartments of boar semen by analysing sperm cells and seminal plasma using anion-exchange chromatography. Seminal plasma contained both arylsulfatase B (2.4 units per ml), an enzyme which desulfates sulfoglycosaminoglycans and probably sulfoglycoproteins, and arylsulfatase A (10.2 units per ml), an enzyme which desulfates sulfogalactolipids. Sperm cells contained only arylsulfatase A, which differed biochemically from the extracellular arylsulfatase A of seminal plasma (2.6 units per ml). Both types of arylsulfatase A desulfate seminolipid, the natural sulfolipid substrate in sperm, as well as two brain sulfatides. The possible physiological consequences of the presence of extracellular arylsulfatases in seminal plasma for spermatozoa are discussed.
Assuntos
Arilsulfatases/metabolismo , Sêmen/enzimologia , Espermatozoides/enzimologia , Animais , Arilsulfatases/antagonistas & inibidores , Arilsulfatases/isolamento & purificação , Catecóis , Cerebrosídeo Sulfatase/isolamento & purificação , Cerebrosídeo Sulfatase/metabolismo , Condro-4-Sulfatase/isolamento & purificação , Condro-4-Sulfatase/metabolismo , Cromatografia por Troca Iônica , Concentração de Íons de Hidrogênio , Masculino , Nitrato de Prata/farmacologia , Suínos , Fatores de TempoRESUMO
A full-length human N-acetylgalactosamine-4-sulphatase (4-sulphatase) cDNA clone was constructed and expressed in CHO-DK1 cells under the transcriptional control of the Rous sarcoma virus long terminal repeat. A clonal cell line expressing high activities of human 4-sulphatase was isolated. The maturation and processing of the human enzyme in this transfected CHO cell line showed it to be identical with that seen in normal human skin fibroblasts. The high-uptake precursor form of the recombinant enzyme was purified from the medium of the transfected cells treated with NH4Cl and was shown to be efficiently endocytosed by control fibroblasts and by fibroblasts from a mucopolysaccharidosis type-VI (MPS VI) patient. Enzyme uptake was inhibitable by mannose 6-phosphate. After uptake, the enzyme was processed normally in both normal and MPS VI fibroblasts and was shown both to correct the enzymic defect and to initiate degradation of [35S]sulphated dermatan sulphate in MPS VI fibroblasts. The stabilities of the recombinant enzyme and enzyme from human fibroblasts appeared to be similar after uptake. However, endocytosed enzyme has a significantly shorter half-life than endogenous human enzyme. The purified precursor 4-sulphatase had a similar pH optimum and catalytic parameters to the mature form of 4-sulphatase isolated from human liver.
Assuntos
Condro-4-Sulfatase/genética , Mucopolissacaridose VI/metabolismo , Pele/enzimologia , Fosfatase Alcalina/genética , Animais , Vírus do Sarcoma Aviário/genética , Sequência de Bases , Células CHO , Células Cultivadas , Condro-4-Sulfatase/deficiência , Condro-4-Sulfatase/metabolismo , Clonagem Molecular , Cricetinae , DNA/genética , DNA/isolamento & purificação , Endocitose , Fibroblastos/enzimologia , Glicoconjugados/metabolismo , Humanos , Dados de Sequência Molecular , Peso Molecular , Oligodesoxirribonucleotídeos , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Transcrição Gênica , TransfecçãoRESUMO
Expression of the chondroitin sulfate utilization (csu) genes of Bacterioides thetaiotaomicron is regulated by chondroitin sulfate. We have now found, however, that the csu genes are not all regulated in the same way. In particular, the gene encoding beta-glucuronidase (csuE) is expressed under two different conditions that do not lead to expression of other csu genes.