Human glucosamine-6-sulfatase cDNA reveals homology with steroid sulfatase

doi:10.1016/S0006-291X(88)80035-4

Biochemical and Biophysical Research Communications

Volume 157, Issue 1, 30 November 1988, Pages 218-224

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Glucosamine-6-sulfatase is a lysosomal enzyme which degrades glycosaminoglycans and is deficient in mucopolysaccharidosis type IIID. Human liver contains two major active forms of glucosamine-6-sulfatase, form A which has a single 78 kDa polypeptide and form B which has two polypeptides of 48 kDa and 32 kDa. A 1761 base pair cDNA clone encoding the complete 48 kDa polypeptide of form B was isolated. Form A is shown to be processed to form B with the 48 kDa polypeptide C-terminal to the 32 kDa polypeptide, and it is shown that C-terminal processing is limited to a region of thirty amino acids. The glucosamine-6-sulfatase sequence reveals homology with steroid sulfatase, a microsomal enzyme.

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Previously we developed a multiplex liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay using dried blood spots for all subtypes of mucopolysaccharidoses (MPS) except MPS-IIID. Here we show that the MPS-IIID enzyme N-acetylglucosamine-6-sulfatase (GNS) is inhibited in dried blood spot (DBS) extracts, but activity can be recovered if the extract is diluted to reduce the concentrations of endogenous inhibitors. The new GNS assay displays acceptable characteristics including linearity in product formation with incubation time and amount of enzyme, low variability, and ability to distinguish MPS-IIID-affected from healthy patients using DBS. The assay can be added to the LC-MS/MS multiplex panel for all MPS subtypes requiring ∼2 min per newborn for the LC-MS/MS run.
The mucopolysaccharidoses
2020, Rosenberg’s Molecular and Genetic Basis of Neurological and Psychiatric Disease: Volume 1
The mucopolysaccharidoses (MPSs) are a group of inherited disorders caused by specific enzyme deficiencies in the degradation of glycosaminoglycans (GAGs) (formerly known as mucopolysaccharides). Enzyme deficiencies result in the accumulation of GAGs in lysosomes of various tissues and in the excessive excretion of partially degraded GAGs in the urine. The GAGs that are stored and excreted in the urine include dermatan sulfate, heparan sulfate, keratan sulfate, and chondroitin 4/6 sulfates. Clinical manifestations of the MPSs depend on the specific enzyme deficiency, the end organ affected, and the accumulation of GAGs in the affected organs. Specific degradative lysosomal enzyme deficiencies and gene defects have been identified for all of the MPSs. Currently, treatment for some of the disorders includes hematopoietic stem cell transplant and enzyme replacement therapy.
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2014, Rosenberg's Molecular and Genetic Basis of Neurological and Psychiatric Disease: Fifth Edition
The mucopolysaccharidoses are a group of inherited disorders caused by specific enzyme deficiencies in the degradation of the glycosaminoglycans (mucopolysaccharides). Enzyme deficiencies result in the accumulation of glycosaminoglycans in lysosomes of various tissues and in the excessive excretion of partially degraded glycosaminoglycans in urine. Clinical manifestations of the mucopolysaccharidoses depend on the specific enzyme deficiency, the end organ affected, and the accumulation of glycosaminoglycans in the affected organs. In diseases in which the brain is not involved, there is no mental retardation. On the other hand, if the brain is affected and other somatic manifestations are minimal, the coarse features that are characteristic of the mucopolysaccharidoses are not as prominent. Specific degradative lysosomal enzyme deficiencies have been identified for all the mucopolysaccharidoses. The glycosaminoglycans that are stored and excreted in the urine of the various mucopolysaccharidoses are dermatan sulfate, heparan sulfate, keratan sulfate, and chondroitin 4/6 sulfates.
Identification and characterisation of an 8.7 kb deletion and a novel nonsense mutation in two Italian families with Sanfilippo syndrome type D (mucopolysaccharidosis IIID)
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Sanfilippo syndrome type D is an autosomal recessive lysosomal storage disease that is caused by a deficiency of N-acetylglucosamine-6-sulphatase, one of the enzymes involved in the catabolism of heparan sulphate. Only 15 patients have been described in the literature and just two mutations have been reported to date. We present the clinical, biochemical and molecular analysis of two Italian Sanfilippo D families. Novel homozygous mutations were identified in the affected patients from each family: a large intragenic deletion of 8723 bp encompassing exons 2 and 3 in family 1 and a nonsense mutation, Q272X, in family 2. The deletion is the first large intragenic deletion to be reported in any of the four Sanfilippo subtypes, including Sanfilippo type C in which the gene has recently been identified.
Genomic basis of mucopolysaccharidosis type IIID (MIM 252940) revealed by sequencing of GNS encoding N-acetylglucosamine-6-sulfatase
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Mucopolysaccharidosis type IIID (MPS IIID; Sanfilippo syndrome type D; MIM 252940) is caused by deficiency of the activity of N-acetylglucosamine-6-sulfatase (GNS), which is normally required for degradation of heparan sulfate. The clinical features of MPS IIID include progressive neurodegeneration, with relatively mild somatic symptoms. Biochemical features include accumulation of heparan sulfate and N-acetylglucosamine-6-sulfate in the brain and viscera. To date, diagnosis required a specific lysosomal enzyme assay for GNS activity. From genomic DNA of a subject with MPS IIID, we amplified and sequenced the promoter and 14 exons of GNS. We found a homozygous nonsense mutation in exon 9 (1063C → T), which predicted premature termination of translation (R355X). We also identified two common synonymous coding single-nucleotide polymorphisms and genotyped these in samples from four ethnic groups. This first report of a mutation in GNS resulting in MPS IIID indicates the potential utility of molecular diagnosis for this rare condition.
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X-linked ichthyosis is the result of steroid sulfatase (STS) deficiency. While most affected individuals have extensive deletions of the STS gene, point mutations have been reported in three patients (1). In this study, we identify an additional three point mutations and characterize the effects of all six mutations on STS activity and expression. All six are unique single base pair substitutions. The mutations are located in a 105-amino acid region of the C-terminal half of the polypeptide. Five of the six mutations involve the substitutions of Pro or Arg for Trp³⁷², Arg for His⁴⁴⁴, Tyr for Cys⁴⁴⁶, or Leu for Cys³⁴¹. The other mutation is in a splice junction and results in a frameshift causing premature termination of the polypeptide at residue 427. All the affected residues are conserved to some degree within the sulfatase family. The six mutations were reproduced in normal STS cDNA and transiently expressed in STS-deficient cells. All six mutant vectors direct the expression of STS protein that lacks enzymatic activity. The mutant polypeptides show a shift in mobility on SDS-PAGE and resistance to proteinase K digestion when translated in the presence of dog pancreas microsomes, indicating glycosylation and normal translocation.

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Human glucosamine-6-sulfatase cDNA reveals homology with steroid sulfatase

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Eur. J. Biochem.

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