Mucopolysaccharidosis type III A (MPS III A, Sanfilippo syndrome) is a rare, autosomal recessive, lysosomal storage disease characterized by accumulation of heparan sulfate secondary to defective function of the lysosomal enzyme heparan N- sulfatase (sulfamidase). Here we describe a spontaneous mouse mutant that replicates many of the features found in MPS III A in children. Brain sections revealed neurons with distended lysosomes filled with membranous and floccular materials with some having a classical zebra body morphology. Storage materials were also present in lysosomes of cells of many other tissues, and these often stained positively with periodic-acid Schiff reagent. Affected mice usually died at 7-10 months of age exhibiting a distended bladder and hepatosplenomegaly. Heparan sulfate isolated from urine and brain had nonreducing end glucosamine- N -sulfate residues that were digested with recombinant human sulfamidase. Enzyme assays of liver and brain extracts revealed a dramatic reduction in sulfamidase activity. Other lysosomal hydrolases that degrade heparan sulfate or other glycans and glycosaminoglycans were either normal, or were somewhat increased in specific activity. The MPS III A mouse provides an excellent model for evaluating pathogenic mechanisms of disease and for testing treatment strategies, including enzyme or cell replacement and gene therapy.

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. (

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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.

The kinetic parameters (Km and V) of human arylsulphatase B (4-sulpho-N-acetylgalactosamine sulphatase) activity in cultured skin fibroblasts were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo chondroitin 4-sulphate and dermatan sulphate. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, were desulphated up to 4400 times faster than the minimum arylsulphatase-B-specific substrate, namely the monosaccharide N-acetylgalactosamine 4-sulphate. Aglycone structures that influence substrate binding and/or enzyme activity were an adjacent-residue C-6 carboxy group and a second but internal N-acetylgalactosamine 4-sulphate residue. Arylsulphatase B activity in fibroblast homogenates assayed with O-(beta-N-acetylgalactosamine 4-sulphate)-(1----4)-O-D-(beta-glucuronic acid)-(1----3)-O-D-N-acetyl[1-3H] galactosaminitol 4-sulphate derived from chondroitin 4-sulphate as substrate clearly distinguished Maroteaux-Lamy-syndrome patients from normal controls and other mucopolysaccharidosis patients. We recommend the use of the above trisaccharide substrate for both postnatal and prenatal diagnosis of Maroteaux-Lamy syndrome.

Mucopolysaccharidosis type VI (MPS VI)is an autosomal recessive disease caused by a deficiency of N-acetylgalactosamine 4-sulfatase (4S) leading to the lysosomal accumulation and urinary excretion of dermatan sulfate. MPS VI has also been described in the Siamese cat. As an initial step toward enzyme replacement therapy with recombinant feline 4S (rf4S) in MPS VI cats, the feline 4S cDNA was isolated and expressed in CHO-KI cells and rf4S was immunopurified from the culture medium. SDS-polyacrylamide gel electrophoresis analysis showed that the precursor form of immunopurified rf4S was a 66-kDa polypeptide that underwent maturation to a 43-44-kDa polypeptide. Endocytosis of rf4S by cultured feline MPS VI myoblasts was predominantly mediated by a mannose 6-phosphate receptor and resulted in the correction of dermatan sulfate storage. The mutation causing feline MPS VI was identified as a base substitution at codon 476, altering a leucine codon to a proline (L476P). The L476P allele displayed no detectable 4S activity when expressed in CHO-KI cells and was observed only as a "precursor" polypeptide that was not secreted into the medium. Identification of the mutation has allowed the development of a rapid PCR-based screening method to genotype individuals within the cat colony.

1. The kinetic parameters of human liver a-L-iduronidase were determined with three disaccharide substrates: a-L-iduronosyl(l+4)2,5-anhydro-~-[l-~H]mannitol 6-sulphate, a-i,-iduronosyl(1+4)2,5-anhydro-~-[l-~H]mannitol and a-L-iduronosyl(1 + 3)2,5-anhydro-u-[l -3H]talitol 4-sulphate, derived from the natural substrates heparin and dermatan sulphate and one synthetic, fluorogenic substrate, 4-methylumbelliferyl a-L-iduronide.2. The enzyme activity with all four substrates was optimal at about pH 4.5. The K, values derived using the disaccharide substrates were elevated up to 10-fold with u p to a 6.5-fold increase in ionic strength whereas that for the synthetic substrate was only increased by 1.7-fold. The V values for all substrates were unaffected. The inhibitory effect of NaCl, Na2S04, NaH2P04 or CuCI, on enzyme activity was more pronounced with the disaccharide substrates than with the synthetic substrate.3. The moiety which is most important in binding is the idopyranosyl residue. While the aglycone residue adds to the nett affinity for the enzyme, it is the substituent groups of both residues which appear to control catalysis. Specifically the carboxyl moiety of the a-L-iduronic acid residue is essential for catalysis while the presence of sulphate on the C4 or C6 position of the aglycone residue has a major influence on catalysis rather than binding. a-L-Idosyl(l-+4)2,5-anhydro-~-[l-~H]mannitol6-sulphate did not undergo catalysis and was a potent inhibitor of enzyme activity, whereas /?-glucuronosyl(1+4)2,5-anhydro-u-[1 -3H]mannitol 6-sulphate, ELiduronosyl-2-sulphate(l+4)2,5-anhydro-~-[l-~H]-mannitol 6-sulphate and 4-methylumbelliferyl a-L-idoside did not undergo catalysis and were not inhibitory. . A model of the catalytic requirements of a-L-iduronidase is proposed.sr-L-Iduronidase (a-L-iduronide iduronohydrolase) is one of many lysosomal hydrolases involved in the degradation of the sulphated glycosaminoglycans heparan sulphate (HS) and dermatan sulphate (DS) [l]. Deficiency of this enzyme activity in humans leads to the accumulation of HS and DS fragments with non-reducing-end a-I,-iduronosyl residues and leads to the disease state described as mucopolysaccharidosis 1 (MPS the I ) [l]. The disease can range in clinical symptoms from severe (MPS, IH, Hurler Syndrome) to mild (MPS IS, Scheie syndrome) and all shades in between. This phenotypic variation cannot yet be predicted from biochemical data and it has not yet been established whether the variation results from an absencc of enzyme protein or inactive or partially active 6-sulphate; Ido-anM6S, a-I.-idosyl(l-t4)2,5-anhydro-u-[I -3 H]mannitol6-sulphate; IdoA-4MeUmb, 4-methylumbelliferyl a-1.-iduronide; Ido-4MeUmb, 4-methylumbelliferyl a-i-idoside; GlcNAc, N-acctyl-mglucosamine; GlcNAc6S, N-acctyl-u-glucosamine 6-sulphate; NaMe2Glt, sodium dimethylglutarate; HS, heparan sulphate; DS, dermatan sulphate. ~-Enzjrne. a-1,-Iduronidase (EC 3.2.1.76).protein. Understanding the catalytic properties of a-Liduronidase toward its natural substrates is an impor...