Vivienne Muller

Enzyme replacement therapy in a feline model of Maroteaux-Lamy syndrome.

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 approximately 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.

A fluorometric assay using 4-methylumbelliferyl α-l-iduronide for the estimation of α-l-iduronidase activity and the detection of Hurler and Scheie syndromes

Abstract Incubation of 4-methylumbelliferyl α-L-iduronide with whole cell homogenates prepared from cultured skin fibroblasts and amniotic cells, and peripheral blood leukocytes gave 4-methylumbelliferone which was easily measured fluorometrically. This reaction, presumably due to the action of α- l -iduronidase, has a maximum hydrolytic activity at pH 3.25. The apparent K M value of α- l -iduronidase in leukocyte whole cell homogenates for this substrate was 179 μmol/l compared to 353, 41 and 116 μmol/l for other α- l -iduronidase substrates phenyl α-L-iduronide, iduronosyl anhydro [l- 3 H]mannitol 6-sulfate and iduronosyl anhydro [l- 3 H] mannitol respectively; the corresponding V max values were 617, 394, 158 and 10 pmol/min/mg protein respectively. Incubation of the 4-methylumbelliferyl α-L-iduronide with whole cell homogenates prepared from cultured skin fibroblasts and leukocytes from a Hurler patient gave 4-methylumbelliferone at a rate more than 20 times less than found for control normal preparations. 4-Methylumbelliferyl α-L-iduronide is a sensitive, convenient and superior substrate to phenyl α-L-iduronide for the assay of α-L-iduronidase activity, but is not a suitable replacement for the radiolabelled substrate iduronosyl anhydro [1−su3H] mannitol 6-sulfate.

Feline mucopolysaccharidosis type VI. Characterization of recombinant N-acetylgalactosamine 4-sulfatase and identification of a mutation causing the disease.

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.

TWO MUTATIONS WITHIN A FELINE MUCOPOLYSACCHARIDOSIS TYPE VI COLONY CAUSE THREE DIFFERENT CLINICAL PHENOTYPES

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.

Molecular heterogeneity in mucopolysaccharidosis IVA in Australia and Northern Ireland: Nine novel mutations including T312S, a common allele that confers a mild phenotype

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive lysosomal storage disorder caused by a genetic defect in N‐acetylgalactosamine‐6‐sulfate sulfatase (GALNS). Previous studies of patients from a British–Irish population showed that the I113F mutation is the most common single mutation among MPS IVA patients and produces a severe clinical phenotype. We studied mutations in the GALNS gene from 23 additional MPS IVA patients (15 from Australia, 8 from Northern Ireland), with various clinical phenotypes (severe, 16 cases; intermediate, 4 cases; mild, 3 cases). We found two common mutations that together accounted for 32% of the 44 unrelated alleles in these patients. One is the T312S mutation, a novel mutation found exclusively in milder patients. The other is the previously described I113F that produces a severe phenotype. The I113F and T312S mutations accounted for 8 (18%) and 6 (14%) of 44 unrelated alleles, respectively. The relatively high residual GALNS activity seen when the T312S mutant cDNA is overexpressed in mutant cells provides an explanation for the mild phenotype in patients with this mutation. The distribution and relative frequencies of the I113F and T312S mutations in Australia corresponded to those observed in Northern Ireland and are unique to these two populations, suggesting that both mutations were probably introduced to Australia by Irish migrants during the 19th century. Haplotype analysis using 6 RFLPs provides additional data that the I113F mutation originated from a common ancestor. The other 9 novel mutations identified in these 23 patients were each limited to a single family. These data provide further evidence for extensive allelic heterogeneity in MPS IVA in British–Irish patients and provide evidence for their transmission to Australia by British–Irish migrants. Hum Mutat 11:202–208, 1998.

α‐L‐iduronidase deficiency in mucopolysaccharidosis type I against a radio‐labelled sulfated disaccharide substrate derived from dermatan sulfate

α‐L‐Iduronidase activity was assayed by incubation of a radiolabeled disaccharide, 0‐(α‐L‐idopyranosyluronic acid)‐(l → 3)‐2,5 anhydro‐D‐[I,3H]‐talitol 4‐sulfate (IdoA‐anT4S) derived from dermatan sulfate, with homogenates of leucocytes, cultured amniotic cells and skin fibroblasts from normal individuals and patients affected with an α‐L‐iduronidase‐deficiency disorder (mucopolysaccharidosis type I, MPS I), parents of such patients and patients affected with other mucopolysaccharidoses. The assay clearly distinguished affected homozygotes from normal controls, heterozygotes and other mucopolysaccharidosis types. Preliminary results show that fibroblast homogenates from patients with the MPS I Hurler phenotype were virtually unable to hydrolyse IdoA‐anT4S, whereas fibroblast homogenates from a patient with a relatively mild (Scheie) phenotype exhibited a residual activity with Vmax value of 2.5 pmol/min/mg protein and an apparent Km of 21 /anol/1 compared to a range of 1020–2105 pmol/min/mg for Vmax and 12–35 /rniol/1 for Km for fibroblasts from normal controls.

Regulation of N-acetylgalactosamine 4-sulfatase expression in retrovirus-transduced feline mucopolysaccharidosis type VI muscle cells.

As a preliminary step toward muscle-mediated gene therapy in the mucopolysaccharidosis (MPS) type VI cat, we have analyzed the transcriptional regulation of feline N-acetylgalactosamine 4-sulfatase (f4S) gene expression from various retroviral constructs in primary cultures of muscle cells. Two retroviral constructs were made containing the f4S cDNA under the transcriptional control of the human polypeptide chain-elongation factor 1alpha (EF1alpha) gene promoter or the cytomegalovirus (CMV) immediate-early promoter. Two further retroviral constructs were made with the murine muscle creatine kinase (mck) enhancer sequence upstream of the internal promoter. Virus made from each construct was used to transduce feline MPS VI myoblasts. The mck enhancer significantly upregulated f4S gene expression from both the EF1alpha promoter and the CMV promoter in transduced myoblasts and in differentiated myofibers. The highest level of 4S activity was observed in myoblasts and myofibers transduced with the retroviral construct Lmckcmv4S, in which the f4S gene is under the transcriptional regulation of the mck enhancer and CMV immediate-early promoter. Lmckcmv4S-transduced myofibers demonstrated correction of glycosaminoglycan storage and contained a 58-fold elevated level of 4S activity compared with normal myofibers. Recombinant f4S secreted from Lmckcmv4S-transduced myofibers was endocytosed by feline MPS VI myofibers, leading to correction of the biochemical storage phenotype.

Selective depolymerisation of dermatan sulfate: Production of radiolabelled substrates for α-l-iduronidase, sulfoiduronate sulfatase, and β-d-glucuronidase

Abstract Radiolabelled disaccharide substrates for α- l -iduronidase, β- d -glucuronidase, and sulfoiduronate sulfatase have been prepared from dermatan sulfate by application in sequence of N -deacetylation, deaminative cleavage, and reduction with NaBT 4 . The yield of disaccharides was ∼87% of the total oligosaccharide fraction. Five disaccharides were isolated and tentatively identified. The major disaccharide, O -(α- l -idopyranosyluronic acid)-(1→3)-2,5-anhydro- d -[1- 3 H]talitol 4-sulfate (IdoA-anT4S), represented ∼75% of the total disaccharide fraction. The other disaccharides were O -(α- l -idopyranosyluronic acid 2-sulfate)-(1→3)-2,5-anhydro- d -[1- 3 H]talitol 4-sulfate (IdoA2S-anT4S), O -(β- d -glucopyranosyluronic acid)-(1→3)-2,5-anhydro- d -[1- 3 H]talitol 4-sulfate (GlcA-anT4S), O -(β- d -glucopyranosyluronic acid)-(1→3)-2,5-anhydro- d -[1- 3 H]talitol 6-sulfate (GlcA-anT6S), and O -(α- l -idopyranosyluronic acid)-(1→3)-2,5-anhydro- d -[1- 3 H]talitol (IdoA-anT), which represented ∼4.5, 11.2, 1.0, and 1.8%, respectively, of the total disaccharide fraction. When incubated with cultured skin-fibroblasts from normal controls, IdoA-anT4S was shown to be a sensitive substrate for α- l -iduronidase to produce 2,5-anhydro- d -talitol 4-sulfate (anT4S). Activity toward IdoA-anT4S was not observed with fibroblast homogenates from α- l -iduronidase-deficient patients (Mucopolysaccharidosis Type I). Similarly, normal-fibroblast homogenates degraded GlcA-anT6S to anT6S, and GlcA-anT4S to anT4S, at a rate 6 to 8 times greater than found for fibroblasts from β- d -glucuronidase-deficient patients (Mucopolysaccharidosis Type VII). IdoA-anT4S was hydrolysed by α- l -iduronidase at a rate 365 times greater than that for IdoA-anT. Sulfation of the anhydro- d -[1- 3 H]talitol residues is an important structural determinant in the mechanism of action of α- l -iduronidase on disaccharide substrates. IdoA2S-anT4S was degraded to IdoA-anT4S and then to anT4S by normal-fibroblast homogenates, whereas fibroblasts from α- l -iduronidase-deficient and sulfoiduronate sulfatase-deficient (Mucopolysaccharidosis Type II) patients produced considerably decreased levels of anT4s and IdoA-anT4S (and anT4S), respectively.

Isolation and use of a new natural substrate for the diagnosis of MPS VII

Mucopolysaccharidosis Type VII is characterized by a severe deficiency of the enzyme β-D-glucuronidase. As with other mucopolysaccharidoses, this inherited enzyme disorder results in a wide range of phenotypes. from extremely mild to severe. Diagnosis of the enzyme deficiency has usually been made using artificial substrates such as 4-methylumbelliferyl-β-D-glucuronide (MuGlcUA), and p-nitrophenyl-β-D-glucuronide. We now report the isolation of a tritium-labelled disaccharide, namely glucuronosyl anhydromannitol 6-sulphate (GMs) derived from the controlled degradation of heparin. Under optimal assay conditions for normal skin fibroblast homogenates, this natural substrate gives β-D-glucuronidase activities of 0-2.4, 9 17 and 38 56xa0pmol/min/mg protein for homozygote affected, heterozygote and normal cell lines, respectively. Compared with the artificial substrate MuGlcUA, normal activity measured with GMs demonstrates a similar pH profile and time course, but a changed response to protein levels, greater heat sensitivity, and also lower K m and V max values. GMs has a similar structure to iduronosyl anhydromannitol 6-sulphate (IMs), a natural substrate of α-L-iduronidase. However, in contrast to α-L-iduronidase, β-D-glucuromdase is inhibited only slightly by SO 4 2 or Cu 2+ ions; while NaCl is a much more effective inhibitor at lower pH values. This suggests that β-D-glucuronidase may-lack the specific SO 4 2 and Cu 2+ binding sites postulated for α-L-iduronidase.

Biochemical discrimination of hurler and scheie syndromes

1. Homogenates of cultured skin fibroblasts derived from patients with alpha-L-iduronidase-deficiency disorders (Hurler and Scheie syndromes) were capable of hydrolysing iduronosyl anhydro-[1-3H]mannitol 6-sulphate although at considerably reduced rates compared with normal controls. 2. The Vmax. values of alpha-L-iduronidase from patients with Hurler or Scheie syndromes and from normal controls were 11, 12 and 833 pmol min-1 mg-1 of protein respectively; the corresponding apparent Km values were 656, 50 and 53 mumol/l respectively. The alpha-L-iduronidases from normal and Scheie fibroblast homogenates were shown to exhibit pH optima at 3.6 and 4.1 and were competitively inhibited by both chloride and sulphate ions: Hurler alpha-L-iduronidase activity exhibited the pH optimum at 3.8 and was also inhibited by chloride and to a lesser extent by sulphate ions. 3. The thermal stability of Hurler, Scheie and normal alpha-L-iduronidase activities at 55 degrees C gave half-lives of approximately 1.0, 2.5 and 1.0 h respectively. 4. These biochemical findings clearly demonstrate enzyme differences for these two clinically distinct phenotypes and provide biochemical evidence that the Hurler and Scheie syndromes result from different allelic mutations.