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PubMed2003

Construction of a high efficiency retroviral vector for gene therapy of Hunter's syndrome.

BACKGROUND: As an alternative method to the conventional therapies for Hunter's syndrome, which is a lethal lysosomal storage disorder, we have developed gene delivery vehicles using a series of retroviral vectors. The objective of this study was to develop a safe and efficient retroviral vector and to optimize conditions for efficient transduction of human bone marrow CD34+ stem cells using our vector. METHODS: We constructed three types of MLV-based retroviral vectors expressing iduronate-2-sulfatase (IDS) which is deficient in patients suffering from Hunter's syndrome: MIN-IDS and MIM-IDS, which express IDS along with bacterial neo and human MDR genes, respectively, and MT-IDS lacking any selectable marker. Respective producer lines were derived from the packaging line, PG13, and compared for viral titer and levels of gene expression. After comparing, the retroviral vector, MT-IDS, was used to transduce human bone marrow CD34+ stem cells on fibronectin under various MOIs. RESULTS: In comparison, MT-IDS not only produced the highest viral titer (close to 10(7) cfu/ml), but also showed the highest level of gene expression in various transduction assays and RNA analysis. When 1.5 x 10(5) human CD34+ bone marrow cells were transduced with MT-IDS under the most optimal MOIs, about 80% of total colony forming units were shown to contain the IDS cDNA. CONCLUSIONS: Minimum-sized retroviral vector that contains no selective marker as well as a viral coding sequences could drive a high level of gene expression, be produced efficiently from the producer line, and enter hematopoietic cells at a high frequency. Our data suggest the great potential for using MT-based vector(s) in a gene therapy trial for Hunter's syndrome utilizing human CD34+ stem cells as target cells.

Bone Marrow Cells↗
PubMed2005

Sulfamates and their therapeutic potential.

Starting from the very simple molecule sulfamic acid, O-substituted-, N-substituted-, or di-/tri-substituted sulfamates may be obtained, which show specific biological activities which were or started to be exploited for the design of many types of therapeutic agents. Among them, sulfamate inhibitors of aminoacyl-tRNA synthetases (aaRSs) were recently reported, constituting completely new classes of antibiotics, useful in the fight of drug-resistant infections. Anti-viral agents incorporating sulfamate moieties have also been obtained, with at least two types of such derivatives investigated: the nucleoside/nucleotide human immunodeficiency virus (HIV) reverse transcriptase inhibitors, and the HIV protease inhibitors (PIs). In the increasing armamentarium of anti-cancer drugs, the sulfamates occupy a special position, with at least two important targets evidenced so far: the steroid sulfatases (STSs) and the carbonic anhydrases (CAs). An impressing number of inhibitors of STSs of the sulfamate type have been reported in the last years, with several compounds, such as 667COUMATE among others, progressing to clinical trials for the treatment of hormone-dependent tumors (breast and prostate cancers). This field is rapidly evolving, with many types of new inhibitors being constantly reported and designed in such a way as to increase their anti-tumor properties, and decrease undesired features (for example, estrogenicity, a problem encountered with the first generation such inhibitors, such as EMATE). Among the many isozymes of CAs, at least two, CA IX and CA XII, are highly overexpressed in tumors, being generally absent in the normal tissues. Inhibition of tumor-associated CAs was hypothesized to lead to novel therapeutic approaches for the treatment of cancer. Many sulfamates act as very potent (low nanomolar) CA inhibitors. The X-ray crystal structure of the best-studied isozyme, CA II, with three sulfamates (sulfamic acid, topiramate, and EMATE) has recently been reported, which allowed for a rationale drug design of new inhibitors. Indeed, low nanomolar CA IX inhibitors of the sulfamate type have been reported, although such compounds also act as efficient inhibitors of isozymes CA I and II, which are not associated with tumors. A large number of anti-convulsant sulfamates have been described, with one such compound, topiramate, being widely used clinically as anti-epileptic drug. By taking into consideration a side effect of topiramate, an anti-epileptic drug leading to weight loss in some patients, it has recently been proposed to use this drug and related sulfamates for the treatment of obesity. The rationale of this use is based on the inhibition of the mitochondrial CA isozyme, CA V, involved in lipogenesis. Some sulfamates were also shown to possess potent inhibitory activity against acyl coenzyme A:cholesterol acyltransferase, an enzyme involved in cholesterol metabolism. One such agent, avasimibe, is in advanced clinical trials for the treatment of hyperlipidemia and atherosclerosis. Thus, the sulfamate moiety offers very attractive possibilities for the drug design of various pharmacological agents, which are on one hand due to the relative ease with which such compounds are synthesized, and on the other one, due to the fact that biological activity of most of them is impressive.

Amino Acyl-tRNA Synthetases↗
PubMedDate not supplied

Prenatal diagnosis of Hunter syndrome using chorionic villi.

A case of the Hunter syndrome diagnosed prenatally using chorionic villi is presented. Chorion biopsy was performed in the 10th week of pregnancy. The sample was examined for karyotype and for iduronate sulfatase activity. The fetus was male and the enzyme activity reduced to 4 per cent of normal. Termination of pregnancy occurred at the 11th week.

Adult↗
PubMed2000

Estradiol-17beta sulfotransferase activity in canine osteosarcoma D17 cells.

Estrogen sulfatase and sulfotransferase (EST) activities are present in breast cancer tissues but there are no reports on EST in cancerous bone cells. We incubated [(3)H]estradiol-17beta with cells from a canine osteosarcoma D17 line for periods up to 24 h. Radioactive steroids were recovered from the media and separated into unconjugated and conjugated fractions using Sep-Pak C18 cartridges. The conjugate fraction was solvolyzed and the resulting free steroids were obtained from a second C18 cartridge. Little metabolism was apparent in 4 h of incubation, but by 24 h as much as one half of the radioactivity was seen in the conjugate fraction. Most of the conjugates were recovered as sulfates in all three experiments. HPLC profiles showed a limited metabolism of estradiol to other compounds except for estrone, which was clearly present in both free and sulfate fractions. These results suggest that EST may have a role in the local metabolism of estrogens in bone.

Animals↗
PubMed1997

IDS transfer from overexpressing cells to IDS-deficient cells.

Iduronate sulfatase (IDS) is responsible for mucopolysaccharidosis type II, a rare recessive X-linked lysosomal storage disease. The aim of this work was to test the ability of overexpressing cells to transfer IDS to deficient cells. In the first part of our work, IDS processing steps were compared in fibroblasts, COS cells, and lymphoblastoid cell lines and shown to be identical: the two precursor forms (76 and 90 kDa) were processed by a series of intermediate forms to the 55- and 45-kDa mature polypeptides. Then IDS transfer to IDS-deficient cells was tested either by incubation with cell-free medium of overexpressing cells or by coculture. Endocytosis and coculture experiments between transfected L beta and deleted fibroblasts showed that IDS transfer occurred preferentially by cell-to-cell contact as IDS precursors are poorly secreted by transfected L beta. The 76- and 62-kDa IDS polypeptides transferred to deleted fibroblasts were correctly processed to the mature 55- and 45-kDa forms. L beta were not able to internalize the 90-kDa phosphorylated precursor forms excreted in large amounts in the medium of overexpressing fibroblasts. Enzyme transfer occurred only by cell-to-cell contact, but the precursor forms transferred in L beta after cell-to-cell contact were not processed. This absence of maturation was probably due to a mistargeting of IDS precursors in these cells.

Animals↗
PubMed1997

Two distinct deletions in the IDS gene and the gene W: a novel type of mutation associated with the Hunter syndrome.

A novel mutation has been identified in a patient with the Hunter syndrome (mucopolysaccharidosis type II), in whom the disorder is associated with two distinct deletions separated by 30 kb. The deletions were characterized by Southern blot and PCR analyses, and the nucleotide sequences at both junctions were determined. The first deletion, corresponding to a loss of 3152 bp of DNA, included exons 5 and 6 of the iduronate-2-sulfatase (IDS) gene. The second deletion was 3603 bp long and included exons 3 and 4 of gene W, which is located in the DXS466 locus telomeric of the IDS gene. Both deletions are the result of nonhomologous (illegitimate) recombination events between short direct repeats at the deletion breakpoints. An interesting finding was the presence of the heptamer sequence 5'-TACTCTA-3' present at both deletion junctions, suggesting that this motif might be a hot spot for recombination. We propose that the double deletion is the result of homology-associated nonhomologous recombinations caused by the presence of large duplicated regions in Xq27.3-q28.

Base Sequence↗
PubMed2001

Crystal structure of an enzyme-substrate complex provides insight into the interaction between human arylsulfatase A and its substrates during catalysis.

Arylsulfatase A (ASA) belongs to the sulfatase family whose members carry a C(alpha)-formylglycine that is post-translationally generated by oxidation of a conserved cysteine or serine residue. The crystal structures of two arylsulfatases, ASA and ASB, and kinetic studies on ASA mutants led to different proposals for the catalytic mechanism in the hydrolysis of sulfate esters. The structures of two ASA mutants that lack the functional C(alpha)-formylglycine residue 69, in complex with a synthetic substrate, have been determined in order to unravel the reaction mechanism. The crystal structure of the inactive mutant C69A-ASA in complex with p-nitrocatechol sulfate (pNCS) mimics a reaction intermediate during sulfate ester hydrolysis by the active enzyme, without the covalent bond to the key side-chain FGly69. The structure shows that the side-chains of lysine 123, lysine 302, serine 150, histidine 229, the main-chain of the key residue 69 and the divalent cation in the active center are involved in sulfate binding. It is proposed that histidine 229 protonates the leaving alcoholate after hydrolysis.C69S-ASA is able to bind covalently to the substrate and hydrolyze it, but is unable to release the resulting sulfate. Nevertheless, the resulting sulfation is low. The structure of C69S-ASA shows the serine side-chain in a single conformation, turned away from the position a substrate occupies in the complex. This suggests that the double conformation observed in the structure of wild-type ASA is more likely to correspond to a formylglycine hydrate than to a twofold disordered aldehyde oxo group, and accounts for the relative inertness of the C69S-ASA mutant. In the C69S-ASA-pNCS complex, the substrate occupies the same position as in the C69A-ASA-pNCS complex, which corresponds to the non-covalently bonded substrate. Based on the structural data, a detailed mechanism for sulfate ester cleavage is proposed, involving an aldehyde hydrate as the functional group.

Alanine↗
PubMed1992

Structural gene aberrations in mucopolysaccharidosis II (Hunter).

A total of 14 unrelated German patients with X-linked iduronate-2-sulfatase (IDS) deficiency (Hunter syndrome, MPS II) showing variable clinical manifestations was screened for structural gene aberrations by Southern analysis. Using the IDS cDNA clone c2S15 as a probe, no Southern fragments could be detected in blots in the severely affected patient G-65 with respect to DNA digested by HindIII, PstI and TaqI, suggesting a total loss of the IDS structural gene. In this patient, the flanking loci DXS 297, DXS 296 and DXS 466 were tested. The locus DXS 466 is involved in the deletion, whereas both of the other loci are present. A normal 9.0-kb fragment disappeared and an aberrant fragment of 3.5 kb occurred in the HindIII blot of patient G-117. A normal Southern pattern was found in PstI and TaqI blots of this patient. This result can be interpreted as the generation of an additional HindIII restriction site by point mutation in an IDS gene intron.

Blotting, Southern↗
PubMed1995

Mucopolysaccharidosis IVA: structural gene alterations identified by Southern blot analysis and identification of racial differences.

Ninety-six alleles (36 alleles of Japanese and 60 of Caucasian origin) from forty-eight patients with mucopolysaccharidosis IVA were investigated for structural gene alterations using Southern blot analysis. All patients had a previously demonstrated deficiency of N-acetyl-galactosamine-6-sulfate-sulfatase and exhibited a wide spectrum of clinical severity. Initially, using the full-length cDNA as a probe, five of 36 chromosomes from the Japanese patients revealed similar rearrangements with respect to DNA digested with BamHI, SacI, and XhoI. Subsequent analysis using seven genomic fragments, covering the entire gene, enhanced the detection of aberrant fragments produced by the above restriction enzymes. Conversely, the 60 chromosomes of Caucasian origin revealed no evidence of large structural rearrangements when analyzed by these methods. There was a statistically significant difference between the two populations (P < 0.01). A severely affected Japanese patient showed structural rearrangements on both chromosomes by means of BamHI blots. An 8.0-kb fragment and a highly polymorphic 7.0-kb to 11.0-kb fragment present in normal individuals disappeared and two aberrant fragments of 11.5 kb and 12.0 kb were observed. Three other Japanese patients also showed these two aberrant fragments, in addition to the normal fragment pattern, and were thus heterozygous for this rearrangement. Interpretation of Southern blots was difficult because of the complexity of polymorphic bands resulting from variable number of tandem repeat elements. However, by utilizing these aberrant fragments or polymorphic bands, carrier detection was effective, even in families with poorly characterized mutations. Hybridization with probe MG-A (5'-end genomic probe in intron 1) showed a 8.4-kb fragment in BamHI blots of one Japanese and one Caucasian patient; XhoI, SacI, and EcoRI blots were normal. Since this BamHI alteration was also observed in one normal control, it appears to be a rare nonpathological polymorphism.

Alleles↗
PubMed1994

Four novel mutant alleles of the arylsulfatase B gene in two patients with intermediate form of mucopolysaccharidosis VI (Maroteaux-Lamy syndrome).

Mucopolysaccharidosis type VI (MPSVI, Maroteaux-Lamy syndrome) is a lysosomal storage disease for which multiple clinical phenotypes have been described. A deficiency of the enzyme arylsulfatase B (ASB, N-acetylgalactosamine-4-sulfatase) is the cause of this autosomal recessively inherited disorder. The genotypes of two patients with an intermediate form of MPSVI have been determined by polymerase chain reaction (PCR) amplification of the entire open reading frame of the ASB gene and subsequent direct sequencing of both strands of the PCR fragments by an automated nonradioactive approach. In patient A, a C to T transition in allele I resulting in an exchange of the Arg codon 160 for a premature stop codon (R160*, exon 2), and a G to A transition in allele II leading to a Gln to Arg160 substitution (R160Q, exon 2) were detected. Patient B exhibited a 7-bp deletion in exon 1 of allele I resulting in a frame shift and a premature stop codon 33 triplets 3' of the site of deletion (delta G237-C243), and a C to T transition in allele II giving rise to a Trp to Arg152 substitution (R152W, exon 2). None of these four mutant alleles was present among 60 alleles of the ASB gene in unrelated controls, indicating that the former are not polymorphisms. These results emphasize the broad molecular heterogeneity of Maroteaux-Lamy syndrome and contribute to the establishment of a genotype/phenotype correlation in this disease.

Alleles↗
PubMed1993

Mucopolysaccharidosis type II (Hunter disease): 13 gene mutations in 52 Japanese patients and carrier detection in four families.

Southern blot analysis of the iduronate sulfatase (IDS) gene in 52 unrelated Japanese patients with mucopolysaccharidosis type II was carried out using a cDNA probe, and mutations in 13 patients (25%) were identified. Of these, 3 had partial gene deletions (in 2 the normal 9.4-kb fragment was absent and in 1 the normal 7.4-kb fragment was absent, as determined by Southern blot analysis using EcoRI-digested DNA, respectively), 2 had gene insertions (in 1 there was a unique 11.2-kb fragment and in the other there was a unique 5-kb fragment, determined by Southern blot analysis using EcoRI-digested DNA), and 8 had rearrangements (in 6 the normal 9.4-kb and 7.0-kb fragments were absent and a unique 11.2-kb fragment was present; in the remaining 2 patients there were different rearrangements). In these 13 patients, the similar Southern blot patterns were indicative of structural alterations of the IDS gene, as revealed when their DNA was digested with HindIII or PstI and probed with IDS cDNA. All patients with these structural alterations were in a clinically severe state, except for 1 with an intermediate clinical phenotype. Our analyses of four families among those of the 13 patients revealed that all four mothers were carriers. The detection of structural abnormalities led to a precise identification of Hunter heterozygotes and revealed one de novo rearrangement in a germ cell of one of the maternal grandparents.

Adolescent↗
PubMed1993

Molecular analysis in patients with mucopolysaccharidosis type II suggests that DXS466 maps within the Hunter gene.

Hunter disease is an X-linked mucopolysaccharidosis caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). Using the IDS cDNA and DNA probes corresponding to loci flanking the IDS locus, we performed molecular genetic studies in two patients with Hunter syndrome. An interstitial deletion spanning the middle part of the IDS gene was found in the first patient. The second patient carries a gross gene rearrangement that can be detected after HindIII or EcoRI digestion of genomic DNA, and is similar to that found recently in seven unrelated Hunter patients. Our data suggest that the structural aberration observed is a partial intragenic inversion. As the same altered hybridization pattern is also revealed by the recently described anonymous DNA probe II-10, which recognizes a frequent TaqI restriction fragment length polymorphism at the DXS466 locus, we conclude that DXS466 maps within the IDS gene, probably in an intron.

Blotting, Southern↗
PubMed1995

ANT3 and STS are autosomal in prosimian lemurs: implications for the evolution of the pseudoautosomal region.

Comparative in situ hybridization in various primate species has revealed a pseudoautosomal location for the human ANT3 gene and an X-specific location for the steroid sulfatase (STS) gene throughout the higher primate species up to the New World monkeys. However, ANT3 and STS map together on an autosome of two prosimian species of the genus Lemur and Eulemur. These results suggest an autosome-to-X/Y translocation after the simians radiated from the prosimians, resulting in a pseudoautosomal location of genes such as ANT3 and STS. In simian primates, STS then became X-specific by a pericentric inversion in the Y chromosome followed by mutational inactivation of the Y allele.

Animals↗
PubMed1975

Electron microscopie and enzyme cytochemical studies on the guinia pig metaphysis with special reference to the lysosomal system of different cell types.

A transmission electron microscopic study of demineralized, methaphyseal bone of the young guinea pig is presented. Special attention is paid to the lysosomal system of the different cell types. Visualization of acid phosphatase and aryl sulfatase activity was used to identify tissue components as belonging thereto. The distribution of alkaline phosphatase activity, a plasma membrane marker, was also examined. Osteoblasts were distinguished by a marked development of the granular endoplasmic reticulum and the Golgi complex. Perivascular cells type A, morphologically resembled the osteoblasts, and are believed to represent an early stage in the specialization of the latter. A few lysosomes were normally found in the osteoblasts; they were less common in the type A cells. In contrasts to their regular occurrence in guinea pig epiphyseal cartilage, dense bodies of lysosomal nature ("type I vesicles") were only rarely seen in the bone matrix. Structures analogous to the type II vesicles in cartilage were, however, normally present. Their membrane showed activity of alkaline phosphatase. Possible functions of lysosomes and matrix vesicles in osteogenesis are discussed. Perivascular cells type B and chondroclasts both contained a prominent Golgi complex and large numbers of free ribosomes, mitochondria and lysosomes. In the type B cells, inclusion material of varying appearance often occurred in the lysosomes and in endocytic vesicles. The chondroclasts sometimes presented a ruffled border, with associated vacuoles and lysosomes in the subjacent cytoplasm. It is suggested that both cell types participate in the resorption of the epiphyseal cartilage. Chondroclasts presumably arise by fusion of type B cells and/or monocytic precursors from the peripheral blood.

Acid Phosphatase↗
PubMed1989

Localization of Y chromosome sequences and X chromosomal replication studies in XX males.

By in situ hybridization, Y-specific DNA sequences were localized on Xp22.3-Xpter of one of the two X chromosomes in all of eleven XX males studied. In nine of the cases the presence of the Y-specific DNA did not affect random X inactivation in fibroblasts. Fibroblasts of the other two cases showed a preferential inactivation of the Y DNA-carrying X chromosome. In only one of these two exceptions blood lymphocytes could also be studied, and here, random inactivation of the Y DNA-carrying X chromosome occurred. Furthermore, the gene dosage of steroid sulfatase (STS) was examined by Southern blot analysis. In ten of the cases including the one showing random X-inactivation in lymphocytes but not in fibroblasts, a double dosage of the STS gene is present. The remaining case with non-random inactivation shows a single STS gene dosage. This case was reported previously to have STS enzyme activity in the male range. It is assumed that, as a consequence DNA sequences may result in the preferential inactivation of the Y DNA-carrying X chromosome.

Arylsulfatases↗
PubMed1983

Clinical heterogeneity in Sanfilippo disease (mucopolysaccharidosis III) type D: presentation of two new cases.

Clinical, radiological and biochemical findings of two new cases of Sanfilippo disease, type D are reported. A high percentage of heparan sulfate was found in the urinary glycosaminoglycan pattern and a severe deficiency of N-acetylglucosamine-6-sulfate sulfatase was demonstrated in skin cultured fibroblasts from the patients. One of the patients presented mild intellectual impairment which differentiates him from the other cases described to date.

Child↗
PubMed1976

Distribution and possible function of lysosomal enzymes in the inner ear under normal and pathophysiological conditions.

The normal distribution of several lysosomal enzymes was studied in 20 guinea pigs. In the outer hair cells lysosomal enzymes are mainly localized at the apical cell pole, while in inner hair cells the distribution was uniform. Nonlysosomal enzymes like alcaline phosphatase are of predominantly basal localization. The concentration of some lysosomal enzymes like N-acetyl-beta-glucosaminidase was higher in outer than in inner hair cells while others like acid phosphatase, beta-glucuronidase and sulfatase showed a stronger reaction in the inner hair cells. After 10 days of sound overstimulation with 120 dB for 1 h a day, there was an increase of lysosomal enzyme content namely in the outer hair cells. There was no change of non-lysosomal enzymes. Under these conditions there might be a partial destruction of cellular organelles eliminated by lysosomal activity without loss of a total cell. In addition the distribution and possible function of lysosomal enzymes in other labyrinthine tissues was discussed.

Acetylglucosaminidase↗
PubMed1975

Sensitivity of glucose 6-phosphatase activity to glutaraldehyde.

The effect of glutaraldehyde fixation on glucose 6-phosphatase activity in mouse liver was investigated. After transparenchymal perfusion with 2% glutaraldehyde for 1.5 minutes, the activity of the recovered enzyme was higher than those reported for acid phosphatase and aryl sulfatase activities after fixation under similar condition, and an abundant deposition of reaction product was observed in hepatocytes. Subsequent immersion in the same fixative solution for 30 minutes after 4 degrees C resulted in only a slight decrease in the activity. However, the activity was almost completely destroyed after 3 hours of immersion fixation at 4 degrees C following the perfusion. Therefore, the enzyme can be said to be aldehyde-sensitive when a long fixation time is used, but not aldehyde-sensitive during a short fixation time.

Acid Phosphatase↗