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We describe two common single-base polymorphisms of the N-acetylgalactosamine-6-sulfate sulfatase gene after StyI or StuI restriction sites. These polymorphisms were readily detected by single-strand conformation polymorphism (SSCP), using the polymerase chain reaction (PCR).
We report here a novel splice site mutation in intron 4 of the gene for N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in an Afghanistan girl with severe mucopolysaccharidosis IVA (classical Morquio disease). Direct sequencing revealed a homozygous G to A transition in the conserved splice acceptor site in intron 4 (cagG-->caaG: designated IVS 4(-I) G-->A) which eliminates 144 nucleotides of exon 5 in her GALNS transcript and introduces an immediate premature termination codon (at Trp 141 of exon 4). The IVS 4(-1) G-->A has not been seen in other populations and this is the first report of the molecular basis of classical Morquio disease in an Afghanistan patient.
We generated anti-human E1-STS monoclonal antibodies to localize estrone sulfatase (E1-STS) in human breast carcinomas. In particular, we examined the MCF-7 clone E3, ZR-75-1, MDA-MB 231, and MDA-MB-468 breast cancer cell lines and 25 breast carcinomas by either immunohistochemistry or Western blotting analysis. Simultaneously, we analyzed histological data, estrogen receptor (ER) status, progesterone receptor (PgR) status and epidermal growth factor receptor (EGFR) in breast tissue. All were surgical specimens from female patients. Nine of 25 carcinomas were obtained from premenopausal women, and 16 carcinomas were obtained frompostmenopausal women. All cell lines demonstrated positive staining for E1-STS.Interestingly, fine granulated staining of E1-STS on the cell membrane was observed. In addition, Western blotting analysis detected a 65 kD protein with an E1-STS specific band in all breast cancer cell lines regardless of the presence orabsence of E2. Twenty-two of 25 (88.0%) carcinomas showed positive staining forE1-STS, whereas negative staining was observed in the interstitial tissue surrounding tumors. In the premenopausal patients, 8 of 10 carcinomas (80.0%) showed positive staining for E1-STS, whereas 14 of 15 carcinomas (93.3%) revealed positive staining in the postmenopausal patients. The frequency of E1-STS expression was relatively higher in postmenopausal patients than in premenopausal patients but not statistically significant. The intensity of immunostaining for E1-STS depended upon the size of the tumor (NS). There was no correlation between E1-STS expression and other parameters. This evidence suggests E1-STS expression may beinvolved in the development of breast cancer. Further studies are necessary to clarify the relationship between E1-STS expression and prognostic factors. Immunoreactive E1-STS may be localized in cancer cells but not in surrounding tissuesin breast cancer.
Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive lysosomal storage disorder caused by a genetic defect in N-acetylgalactosamine-6-sulfate sulfatase (GALNS). In previous studies, we have found two common mutations in Caucasians and Japanese, respectively. To characterize the mutational spectrum in various ethnic groups, mutations in the GALNS gene in Colombian MPS IVA patients were investigated, and genetic backgrounds were extensively analyzed to identify racial origin, based on mitochondrial DNA (mtDNA) lineages. Three novel missense mutations never identified previously in other populations and found in 16 out of 19 Colombian MPS IVA unrelated alleles account for 84.2% of the alleles in this study. The G301C and S162F mutations account for 68.4% and 10.5% of mutations, respectively, whereas the remaining F69V is limited to a single allele. The skewed prevalence of G301C in only Colombian patients and haplotype analysis by restriction fragment length polymorphisms in the GALNS gene suggest that G301C originated from a common ancestor. Investigation of the genetic background by means of mtDNA lineages indicate that all our patients are probably of native American descent.
1. Human N-acetylgalactosamine-6-sulfate sulfatase (EC 3.1.6.-) from human placenta has been purified more than 3000-fold by gel filtration, ion-exchange and substrate affinity chromatography. The enzyme has a molecular weight of 90 000 by gel filtration chromatography and 85 000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Enzyme purified from cultured human skin fibroblasts has similar properties. 2. The tritium-labeled chrondroitin 6-sulfate trisaccharide N-acetylgalactosamine 6-sulfate-(beta, 1-4)-glucuronic acid-(beta, 1-3(-N-acetyl[1-3H]galactosaminitol 6-sulfate as substrate demonstrated a Km of 0.12 mM at pH 4.5. Sulfate was hydrolyzed only from the non-reducing terminal of this disulfated trisaccharide. Hyaluronic acid, dermatan sulfate, chondroitin 4-sulfate, heparin and chondroitin 6-sulfate tetrasaccharide were slightly inhibitory, whereas 6-sulfated pentasaccharides and heptasaccharides were strongly inhibitory. The enzyme dose not hydrolyze sulfate from N-acetylglucosamine 6-sulfate.
Keratan sulfate (KS) was N-deacetylated with anhydrous hydrazine and then degraded with nitrous acid, and the products were reduced with NaBT4. Radiolabelled disaccharides constituted at least 76% of the total oligosaccharide fraction. Three major disaccharides were isolated and identified. Of the total disaccharide isolated from bovine intervertebral-disc and human costal-cartilage, 91 and 79%, respectively, was identified as a disulfated disaccharide, O-(beta-D-galactopyranosyl 6-sulfate)-(1 leads to 4)-2,5-anhydro-D-[1-3H]mannitol 6-sulfate (Gal6S-anM6S). The disaccharide fraction isolated from bovine-cornea KS contained only 14% of Gal6S-anM6S. The yield of monosulfated disaccharide, identified as O-beta-D-galactopyranosyl-(1 leads to 4)-2,5-anhydro-D-[1-3H]mannitol 6-sulfate, was 9, 17, and 84% of the total KS-disaccharide fraction isolated from intervertebral disc, costal cartilage, and cornea, respectively. For each of the KS type studied, the yield of unsulfated disaccharide was less than 4% of the total disaccharide-fraction. The tetrasaccharides were fractionated, on the basis of their sulfate content, into at least four species by paper electrophoresis, and some tentative structures are proposed. Disaccharide and tetrasaccharide species were evaluated as substrates for beta-D-galactosidase and 6-O-sulfogalactose sulfatase.
Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency in N-acetylgalactosamine-6-sulfatase (GALNS). We found two separate deletions of nearly 8.0 and 6.0 kb in the GALNS gene, including some exons. There are Alu repetitive elements near the breakpoints of the 8.0-kb deletion, and this deletion resulted from an Alu-Alu recombination. The other 6.0-kb deletion involved illegitimate recombinational events between incomplete short direct repeats of 8 bp at deletion breakpoints. The same rearrangement has been observed in a heteroallelic state in four unrelated patients. This is the first documentation of a common double deletion a gene that is not a member of a gene cluster.
BACKGROUND: Hunter syndrome (mucopolysaccharidosis type II) is an X-linked recessive lysosomal storage disease caused by a defect of the iduronate-2-sulfatase (IDS) gene. The result is impaired IDS enzyme function. METHODS: To characterize the biochemical and molecular defects in IDS-deficient patients and their families, we measured IDS enzyme activity by fluorimetric enzyme assay and identified the IDS gene mutations in 14 unrelated Taiwanese patients with varying clinical phenotypes. In addition, haplotype analysis was also performed. RESULTS: Three novel (IVS2+1G>C, 1055del12, and G489D) and 7 previously reported (N63K, P228L, K347E, R468Q, R468W, I485R, and 1241delAG) mutations were found. Together R468Q and R468W account for 42.8% mutations found in our patients. Haplotype analysis using IDS flanking markers DXS1113 and DXS1123 revealed that the unrelated R468Q alleles were independent in origin whereas the unrelated R468W alleles are probably of the same origin. The R468Q mutation in patient 1150 and I485R mutation in patient 710 occurred de novo in male meioses. Once the mutation in a family was identified, restriction analysis was also performed for rapid diagnosis of female carriers in 8 families. Leukocyte IDS measurement revealed significantly wide range of IDS activity in normal controls and MPS II carriers (19.2 - 70.6 vs. 8.4 - 26.6 nmol/h/mg cell protein). The average leukocyte IDS activity of normal controls (n=43) was 43.9+/-13.3 nmol/h/mg protein, whereas patients with MPS II (n=14) had <5% of mean normal IDS activity (0.9+/-0.6 nmol/h/mg protein), and carriers (n=13) had a mean activity of 17.5 (+/-5.7) nmol/h/mg protein. The mean leukocyte IDS activity in female carriers was less than a half of the normal level. CONCLUSION: Due to a small overlapping range of normal and carriers, the level of enzyme activity cannot be used alone for carrier detection.
To address the question of the origin of glial cells and the mechanisms leading to their specification, we have sought to identify novel genes expressed in glial progenitors. We adopted suppression subtractive hybridization (SSH) to establish a chick cDNA library enriched for genes specifically expressed at 6 days of incubation (E6) in the ventral neuroepithelium, a tissue previously shown to contain glial progenitors. Screens were then undertaken to select differentially expressed cDNAs, and out of 82 unique SSH clones, 21 were confirmed to display a regionalized expression along the dorsoventral axis of the E6 ventral neuroepithelium. Among these, we identified a transcript coding for the chick orthologue of Sulf1, a recently identified cell surface sulfatase, as a new, early marker of oligodendrocyte (OL) precursors in the chick embryonic spinal cord. This study provides groundwork for the further identification of genes involved in glial specification.
Mucopolysaccharidosis type IVA (MPS IVA; OMIM #253000) or Morquio A syndrome is an autosomal recessive inborn error resulting from the deficient activity of the lysosomal enzyme, N-acetylgalactosamine-6-sulfatase (GALNS), and the progressive lysosomal accumulation of sulfated glycosaminoglycans. Clinically, the severe form of this lysosomal storage disease is characterized by a characteristic severe bone dysplasia and normal intelligence. To date, a variety of mutations have been associated with the severe MPS IVA phenotype. Here, we report the GALNS mutations in six severe MPS IVA patients from four unrelated Tunisian families. For mutation detection, each of the 14 exons and adjacent intron-exon junctions of the GALNS gene were sequenced after PCR-amplification from genomic DNA. Two novel mutations were identified: a G to A transition in the conserved 5' donor splice site of intron 1 (GACgt-->GACat: designated IVS1(+1g-->a)) and a G to C transversion in codon 66 of exon 2 predicting a glycine to arginine substitution (G66R). The IVS1(+1g-->a) mutation was homozygous in five similarly affected patients from three presumably unrelated families, but haplotype analysis suggested a common ancestor. The affected patient in the fourth family was homozygous for the G66R mutation. These are the first GALNS mutations causing severe MPS IVA disease identified in Tunisia. These molecular findings provide genotype/phenotype correlations, and permit accurate carrier detection, prenatal diagnosis, and counseling for MPS IVA disease in Tunisia where first cousin consanguineous mating remains frequent.
Deficiency of lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS) leads to mucopolysaccharidosis IV A (MPS IV A), for which there is no definitive treatment so far. Although a number of mutations of the GALNS gene of MPS IV A patients have been described, pathogenesis of the disorder still remains elusive. In order to facilitate in vivo studies using model animals for MPS IV A, we isolated and performed molecular characterization of the mouse homolog of human GALNS. The 2.3-kb cDNA contains a 1560-bp open reading frame encoding 520 amino acid residues. The coding region has 84% similarity to the human GALNS cDNA at amino acid level. The mouse Galns gene was mapped by interspecific backcross analysis to the distal region of chromosome 8 where it co-segregates with Aprt. Northern blot analysis showed a wide expression of a single-copy gene, being higher especially in liver and kidney. The Galns gene was isolated from S129vJ genomic library and its genomic organization was characterized. The mouse Galns gene was about 50-kb long and organized into 14 exons and 13 introns. All intron-exon splice junctions conformed to the GT/AG consensus sequence except exon 8/intron 8 junction. Primer extension shows multiple transcription initiation sites between -44 and -75 although major transcription initiation site was observed at -90 bp from the ATG codon. The 5'-flanking region lacks canonical TATA and CAAT box sequences, but is G+C rich with 10 GC boxes (potential Sp1 binding sites), characteristic of a housekeeping gene promoter.
Mucopolysaccharidosis IVA (MPS IVA) is caused by pathogenic variants in the GALNS gene encoding N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme, leading to glycosaminoglycan (GAG) accumulation in multiple tissues, resulting in progressive skeletal dysplasia and poor quality of life. There is currently no effective treatment for this skeletal disease. This study proposes a novel lentiviral vector (LV)-based gene therapy that produces and secretes the active GALNS enzyme at supraphysiologic levels within the cells. LVs carrying the native GALNS encoding sequence (cDNA) were made under three different promoters: CBh, COL2A1, and CD11b. Moreover, we designed LVs carrying the native GALNS cDNA tagged with D8 octapeptide under the CD11b promoter and a human codon-optimized GALNS cDNA under the CBh promoter, respectively. Transduced HEK293 cells, HepG2 cells, and MPS IVA fibroblasts and chondrocytes were cultured for 8 and 30 days, and the media were collected every three days. The enzyme activity, GAG levels, and vector copy numbers (VCNs) in these cells and media were analyzed. LV with the COL2A1 promoter produced the highest enzyme activity in HEK293, HepG2, MPS IVA fibroblasts, and chondrocytes, followed by LV with the CBh promoter. VCNs were higher in MPS IVA fibroblasts treated with LV-CBh-hGALNS and in HepG2 cells treated with LV-CD11b-hGALNS than in HEK293 cells. Accumulated GAGs were normalized to wild-type levels by the LV gene therapy, especially with CBh and COL2A1 promoters. These findings, if further validated, could significantly impact the treatment of MPS IVA, offering a more effective and feasible treatment option.
Steroid sulfatase (STS) catalyzes the hydrolyis of steroidal sulfates such as estrone sulfate (ES1) and is considered to be an attractive target in the treatment of steroid dependent cancers. A non-hydrolyzable estrone sulfate (ES1) analogue bearing an alpha,alpha-difluorosulfonamide moiety at the 3-position on the A-ring, compound , was synthesized. Key to the success of this synthesis was the first use of the allyl group as a sulfonamide protecting group. The pK(a) of this ES1 mimic in 0.1 M bis-tris propane, 10% DMSO was determined to be 8.05 using 19F NMR. Compound is a reversible inhibitor with a K(i) similar to that of its sulfonate analogue at pH 7.0. It is more potent than its non-fluorinated sulfonamide analogue and, its inhibitory potency increases with increasing pH, a trend opposite to that of other STS inhibitors. Possible reasons for this are presented.
BACKGROUND AND AIMS: The heparin-binding growth factors fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) are potent mitogens for hepatocellular carcinomas (HCCs). Heparin-binding growth factor signaling is regulated by sulfation of cell-surface heparan sulfate proteoglycans (HSPGs). We hypothesized that hSulf1, a recently described sulfatase, regulates growth signaling in HCCs. METHODS: Expression of hSulf1 in human HCC tumors was determined by real-time PCR. Down-regulation of hSulf1 expression was investigated by analyzing loss of heterozygosity (LOH) at the hSulf1 locus and the effect of the DNA methylation inhibitor 5-aza-deoxycytidine on hSulf1 expression. The subcellular location of hSulf1 and sulfation state of cell-surface HSPGs were assessed by immunocytochemistry. FGF and HGF signaling was examined by phospho-specific immunoblot analysis. Cell growth was measured by trypan blue exclusion, and the MTT assay and apoptosis were quantitated by fluorescence microscopy. RESULTS: hSulf1 expression was decreased in 29% of HCCs and 82% of HCC cell lines. There was LOH at the hSulf1 locus in 42% of HCCs. Treatment with 5-aza-deoxycytidine reactivated hSulf1 expression in hSulf1-negative cell lines. Low hSulf1-expressing cells showed increased sulfation of cell-surface HSPGs, enhanced FGF and HGF-mediated signaling, and increased HCC cell growth. Conversely, forced expression of hSulf1 decreased sulfation of cell-surface HSPGs and abrogated growth signaling. HCC cells with high-level hSulf1 expression were sensitive to staurosporine- or cisplatin-induced apoptosis, whereas low expressing cells were resistant. Transfection of hSulf1 into hSulf1-negative cells restored staurosporine and cisplatin sensitivity. CONCLUSIONS: Down-regulation of hSulf1 contributes to hepatocarcinogenesis by enhancing heparin-binding growth factor signaling and resistance to apoptosis.
N-Acetylgalactosamine-6-sulfate sulfatase (GALNS) catalyzes the first step of intralysosomal keratan sulfate (KS) catabolism. In Morquio type A syndrome GALNS deficiency causes the accumulation of KS in tissues and results in generalized skeletal dysplasia in affected patients. We show that in normal cells GALNS is in a 1.27-MDa complex with three other lysosomal hydrolases: beta-galactosidase, alpha-neuraminidase, and cathepsin A (protective protein). GALNS copurifies with the complex by different chromatography techniques: affinity chromatography on both cathepsin A-binding and beta-galactosidase-binding columns, gel filtration, and chromatofocusing. Anti-human cathepsin A rabbit antiserum coprecipitates GALNS together with cathepsin A, beta-galactosidase, and alpha-neuraminidase in both a purified preparation of the 1. 27-MDa complex and crude glycoprotein fraction from human placenta extract. Gel filtration analysis of fibroblast extracts of patients deficient in either beta-galactosidase (beta-galactosidosis) or cathepsin A (galactosialidosis), which accumulate KS, demonstrates that the 1.27-MDa complex is disrupted and that GALNS is present only in free homodimeric form. The GALNS activity and cross-reacting material are reduced in the fibroblasts of patients affected with galactosialidosis, indicating that the complex with cathepsin A may protect GALNS in the lysosome. We suggest that the 1.27-MDa complex of lysosomal hydrolases is essential for KS catabolism and that the disruption of this complex may be responsible for the KS accumulation in beta-galactosidosis and galactosialidosis patients.
Mucopolysaccharidosis type IVA (Morquio A syndrome, MPS IVA) is a rare, autosomal recessive disorder with a prevalence of 1 in 170,000 live births. It is caused by a deficiency of N-acetylgalactosamine 6-sulfatase (GALNS), a lysosomal hydrolase encoded by a gene on human chromosome 16q24.3. Mucopolysaccharidosis type IVA is the only known MPS that is associated with structural defects in dental enamel. GALNS cleaves the sulfate group from N-acetylgalactosamine 6-sulfate and galactose 6-sulfate, which are specifically found in keratan sulfate and chondroitin 6-sulfate. A pathologic absence of GALNS activity results in the accumulation of these glycosaminoaglycans in the urine and in the lysosomes of tissues that turn them over. There is currently no animal model for MPS IVA. To learn more about how a GALNS deficit could lead to enamel defects, we have cloned and characterized a full-length pig GALNS cDNA. GALNS mRNA was localized in developing teeth by in situ hybridization, Northern blot, and reverse-transcription polymerase chain reaction analyses, while GALNS substrates were localized using immunohistochemistry. We report that secretory ameloblasts were positive for GALNS mRNA, as well as for keratan sulfate and chondroitin 6-sulfate. We conclude that enamel defects associated with the loss of GALNS activity in persons with MPS IVA are likely to result from the pathological accumulation of keratan sulfate and chondroitin 6-sulfate in the lysosomes of secretory stage ameloblasts.
Gene therapy may provide a long-term approach to the treatment of mucopolysaccharidoses. As a first step toward the development of an effective gene therapy for mucopolysaccharidosis type IVA (Morquio syndrome), a recombinant retroviral vector, LGSN, derived from the LXSN vector, containing a full-length human wildtype N-acetylgalactosamine-6-sulfate sulfatase (GALNS) cDNA, was produced. Severe Morquio and normal donor fibroblasts were transduced by LGSN. GALNS activity in both Morquio and normal transduced cells was several fold higher than normal values. To measure the variability of GALNS expression among different transduced cells, we transduced normal and Morquio lymphoblastoid B cells and PBLs, human keratinocytes, murine myoblasts C2C12, and rabbit synoviocytes HIG-82 with LGSN. In all cases, an increase of GALNS activity after transduction was measured. In Morquio cells co-cultivated with enzyme-deficient transduced cells, we demonstrated enzyme uptake and persistence of GALNS activity above normal levels for up to 6 days. The uptake was mannose-6-phosphate dependent. Furthermore, we achieved clear evidence that LGSN transduction of Morquio fibroblasts led to correction of the metabolic defect. These results provide the first evidence that GALNS may be delivered either locally or systematically by various cells in an ex vivo gene therapy of MPS IVA.
Mutations causing mucopolysaccharidosis IVA in 15 Japanese and one Caucasian patient were characterized. To screen these mutations, we used a combination of single strand conformation polymorphism analysis and heteroduplex analysis for PCR products of targeted cDNA or genomic DNA. Various small mutations were identified in 23 of 26 alleles, while the other six alleles had large rearrangements. Cycle sequencing of PCR products revealed 15 different mutations, including 12 missense, one nonsense, one frame shift (2 bp deletion) and one splice site mutation, in accord with the broad range of clinical phenotypes. Two alleles have different mutations in the same nucleotide position of exon 3 (R94C, CGC-->TGC; R94G, CGC-->GGC), diagnosed by sequencing and by allelic-specific oligohybridization (ASO). One allele had two amino acid changes, E450V in exon 12 and V488M in exon 13, thereby indicating a double point mutation. All 16 mutations reported were confirmed by restriction enzyme assay or by allelic-specific oligohybridization. Transfection of mutagenized cDNAs into patients' fibroblasts showed that all mutations caused completely deficient or markedly decreased N-acetylgalactosamine-6-sulfate sulfatase (GALNS) activity, thereby indicating that these mutations were responsible for the enzyme deficiency.