Iduronate 2-sulfatase (IDS, EC 3.1.6.13) is required for the lysosomal degradation of heparan sulfate and dermatan sulfate. Mutations causing IDS deficiency in humans result in the lysosomal storage of these glycosaminoglycans and Hunter syndrome, an X chromosome-linked disease. We have isolated and sequenced a 2.3-kilobase cDNA clone coding for the entire sequence of human IDS. Analysis of the deduced 550-amino acid IDS precursor sequence indicates that IDS has a 25-amino acid amino-terminal signal sequence, followed by 8 amino acids that are removed from the proprotein. An internal proteolytic cleavage occurs to produce the mature IDS present in human liver shown to contain a 42-kDa polypeptide N-terminal to a 14-kDa polypeptide. The IDS sequence has strong sequence homology with other sulfatases (such as sea urchin arylsulfatase, human arylsulfatases A, B, and C, and human glucosamine 6-sulfatase), suggesting that the sulfatases comprise an evolutionarily related family of genes that arose by gene duplication and divergent evolution. The arylsulfatases have a greater homology with each other than with the nonarylsulfatases (IDS and glucosamine 6-sulfatase). The IDS cDNA detected RNA species of 5.7, 5.4, 2.1, and 1.4 kilobases in human placental RNA and revealed structural alterations and gross deletions of the IDS gene in many of the clinically severe Hunter syndrome patients studied.Iduronate 2-sulfatase (IDS, EC 3.1.6.13) acts as an exosulfatase in lysosomes to hydrolyze the C2-sulfate ester bond from non-reducing-terminal iduronic acid residues in the glycosaminoglycans heparan sulfate and dermatan sulfate (1). IDS is one of a family of at least nine sulfatases that hydrolyze sulfate esters in human cells. They are all lysosomal enzymes that act on sulfated monosaccharide residues in a variety of complex substrates with the exception of microsomal steroid sulfatase (or arylsulfatase C), which acts on sulfated 3,8-hydroxysteroids (1, 2). Each sulfatase displays absolute substrate specificity, making the sulfatase family an attractive model to investigate the molecular requirements for substrate binding and the catalysis of sulfate ester hydrolysis.A deficiency in the activity of IDS in humans leads to the lysosomal accumulation of heparan sulfate and dermatan sulfate fragments and their excretion in urine (1). This storage results in the clinical disorder Hunter syndrome (mucopolysaccharidosis type II, MPS-II) in which patients may present with variable phenotypes from severe mental retardation, skeletal deformities, and stiff joints to a relatively mild course (1). It has been postulated that this clinical heterogeneity reflects different mutations at the IDS locus affecting enzyme expression, stability, or function. MPS-II is one of the most common mucopolysaccharidoses and is the only one that is X chromosome-linked (1).We have reported (3) the purification of IDS from human liver and other tissues and in this communication we report the nucleotide sequence for a full-length cDNA clone for IDSt f...

We report studies that suggest enzyme replacement therapy will result in a significant reduction in disease progression and tissue pathology in patients with Maroteaux-Lamy syndrome (Mucopolysaccharidosis type VI, MPS VI). A feline model for MPS VI was used to evaluate tissue distribution and clinical efficacy of three forms of recombinant human N -acetylgalactosamine-4-sulfatase (rh4S, EC 3.1.6.1). Intravenously administered rh4S was rapidly cleared from circulation. The majority of rh4S was distributed to liver, but was also detected in most other tissues. Tissue half-life was ‫ف‬ 2-4 d. Three MPS VI cats given regular intravenous infusions of rh4S for up to 20 mo showed variable reduction of storage vacuoles in Kupffer cells and connective tissues, however cartilage chondrocytes remained vacuolated. Vertebral bone mineral volume was improved in two MPS VI cats in which therapy was initiated before skeletal maturity, and increased bone volume appeared to correlate with earlier age of onset of therapy. One cat showed greater mobility in response to therapy. (

Human iduronate-2-sulphatase (EC 3.1.6.13), which is involved in the lysosomal degradation of the glycosaminoglycans heparan sulphate and dermatan sulphate, was purified more than 500,000-fold in 5% yield from liver with a six-step column procedure, which consisted of a concanavalin A-Sepharose-Blue A-agarose coupled step, chromatofocusing, gel filtration on TSK HW 50S-Fractogel, hydrophobic separation on phenyl-Sepharose CL-4B and size separation on TSK G3000SW Ultrapac. Two major forms were identified. Form A and form B, with pI values of 4.5 and less than 4.0 respectively, separated at the chromatofocusing step in approximately equal amounts of recovered enzyme activity. By gel-filtration methods form A had a native molecular mass in the range 42-65 kDa. When analysed by SDS/PAGE, dithioerythritol-reduced and non-reduced form A and form B consistently contained polypeptides of molecular masses 42 kDa and 14 kDa. Iduronate-2-sulphatase was purified from human kidney, placenta and lung, and form A was shown to have similar native molecular mass and subunit components to those observed for liver enzyme. Both forms of liver iduronate-2-sulphatase were active towards a variety of substrates derived from heparin and dermatan sulphate. Kinetic parameters (Km and Kcat) of form A were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo, namely heparan sulphate, heparin and dermatan sulphate. Substrate with 6-sulphate esters on the aglycone residue adjacent to the iduronic acid 2-sulphate residue being attack were hydrolysed with catalytic efficiencies up to 200 times above that observed for the simplest disaccharide substrate without a 6-sulphated aglycone residue. The effect of incubation pH on enzyme activity towards the variety of substrates evaluated was complex and dependent on substrate aglycone structure, substrate concentration, buffer type and the presence of other proteins. Sulphate and phosphate ions and a number of substrate and product analogues were potent inhibitor of form A and form B enzyme activities.

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.

Apha-LFucosidases (EC 3.2.1.51), the only members of the CAZy family GH-29, are widespread glycosidases involved in many biological processes including inflammation, metastasis, and the lysosomal storage disease fucosidosis. Despite their biological significance, information concerning the mechanism of alpha-Lfucosidases has only recently become available. In an attempt to obtain further data concerning their mechanism, we have investigated the hydrolytic and transglycosylation properties of a canine and a mollusk (Pecten maximus) alpha-Lfucosidase. Our results show that, despite the evolutionary distance between these two species, both enzymes have similar hydrolysis and transglycosylation properties. Surprisingly, we found that, starting from monosaccharides, these exoglycosidases were able to catalyze efficiently the synthesis of highly branched fuco-oligosaccharides as large as tetrasaccharides, a unique feature for a wild-type exoglycosidase. The structural analysis of the compounds formed revealed that the regioselectivity of alpha-Lfucosidases is strongly influenced by the structure of the acceptor. This leads us to propose an enzymatic approach for the preparative synthesis of fuco-oligosaccharides. This will not only allow the synthesis of biological determinants containing fucose but also of new fucose-containing oligosaccharides as alpha-glycosynthases appear to be difficult to obtain.