Arvan L. Fluharty

Molecular basis of different forms of metachromatic leukodystrophy

BACKGROUND Metachromatic leukodystrophy is an autosomal recessive inherited lysosomal storage disorder caused by a deficiency of arylsulfatase A. Three forms of the disease can be distinguished according to severity and the age at onset: late infantile (1 to 2 years), juvenile (3 to 16), and adult (greater than 16). METHODS AND RESULTS To understand the molecular basis of the different forms of the disease, we analyzed arylsulfatase A alleles associated with metachromatic leukodystrophy. Two alleles (termed I and A) were identified and accounted for about half of all arylsulfatase A alleles among 68 patients with metachromatic leukodystrophy whom we examined. Sufficient information was available for 66 of the patients to allow classification of their disease. Of the six instances of homozygosity for allele I, all were associated with the late-infantile form of the disease; of the eight instances of homozygosity for allele A, five were associated with the adult form and three with the juvenile form. When both alleles were present, the juvenile form resulted (seven of seven instances). Heterozygosity for allele I (with the other allele unknown) is usually associated with late-infantile disease, and heterozygosity for allele A with a later onset of the disease. The clinical variability can be explained by the different levels of residual arylsulfatase A activity associated with these genotypes. CONCLUSIONS Like many lysosomal storage disorders, metachromatic leukodystrophy shows clinical heterogeneity that seems to reflect genetic heterogeneity. One of the known alleles (allele I) is associated with earlier and more severe disease than the other (allele A).

Crystal structure of saposin B reveals a dimeric shell for lipid binding

Saposin B is a small, nonenzymatic glycosphingolipid activator protein required for the breakdown of cerebroside sulfates (sulfatides) within the lysosome. The protein can extract target lipids from membranes, forming soluble protein-lipid complexes that are recognized by arylsulfatase A. The crystal structure of human saposin B reveals an unusual shell-like dimer consisting of a monolayer of α-helices enclosing a large hydrophobic cavity. Although the secondary structure of saposin B is similar to that of the known monomeric members of the saposin-like superfamily, the helices are repacked into a different tertiary arrangement to form the homodimer. A comparison of the two forms of the saposin B dimer suggests that extraction of target lipids from membranes involves a conformational change that facilitates access to the inner cavity.

METACHROMATIC LEUKODYSTROPHY WITHOUT ARYLSULFATASE A DEFICIENCY

Summary: Two siblings of consanguinous parents were noted to have a neurologic syndrome marked by developmental delay, regression of psychomotor performance, marked spasticity and progressive central nervous system degeneration. Markedly delayed nerve conduction times and a sural nerve biopsy which demonstrated changes typical of metachromatic leukodystrophy (MLD) were evident. Impairment of sulfated glycolipid metabolism was documented by analysis of glycospingolipid in urinary sediment. In spite of these findings, activities of arylsulfatase A and cerebroside sulfatidase in white blood cells and cultured skin fibroblasts were near normal. However, when intact growing fibroblasts were loaded with 35SO4-sulfatide a clear defect in sulfatide cleavage, comparable to that seen in MLD patients, was observed. Thus, these patients represent a new form of sulfatide storage disease – MLD characterized by intact enzyme activity in cell homogenates but defective sulfolipid metabolism in vivo and in intact fibroblasts.Speculation: Since cell homogenates from these patients can cleave sulfatide in the presence of detergents while the patients themselves and their intact cells cannot, soire explanation other than decreased activity of the relevant lysosomal enzyme must be invoked to explain this storage disease. The two most plausible hypotheses are that either these patients have a defect which prevents enzyme and substrate interaction in the proper subcellular location, or that these patients are missing the putative glycoprotein ″activating factor″ necessary for sulfolipid hydrolysis in vivo.

A correlation of intracellular cerebroside sulfatase activity in fibroblasts with latency in metachromatic leukodystrophy

Summary Despite the absence of cerebroside sulfatase activity in cellfree preparations, fibroblasts in culture derived from patients with metachromatic leukodystrophy were capable of hydrolyzing exogenous cerebroside sulfate. Moreover, the degree of whole-cell sulfatase activity was directly correlated to the age of onset of clinical symptoms in the patients from whom the fibroblasts were derived. Intact fibroblasts from patients with the earliest manifesting form, late infantile metachromatic leukodystrophy, did not hydrolyze any cerebroside sulfate, while fibroblasts from patients with later manifesting forms hydrolyzed appreciable amounts of the sulfolipid.

Steroid Sulfatase Deficiency

Summary: Placental steroid sulfatase deficiency is a genetic disorder only recently reported in the medical literature. Most documented cases of placental sulfatase deficiency have been marked by delay in onset of labor, lack of cervical dilatation, and relative refractoriness of oxytocic agents and amniotomy. We have studied the placenta, cultured fibroblasts, and amniotic fluid cells from an affected patient. The activities of estrone sulfatase, pregnenolone sulfatase, dehydrocpiandrosterone sulfatase, and arylsulfatase C in the placenta from the patient were severely deficient. Arylsulfatases A and B were present at levels within the normal range for this tissue.Fibroblast dehydroepiandrosterone sulfatase activity was virtually absent in the patients cells and present at normal levels in individuals with a variety of lysosomal disorders. It would thus appear that the mutation responsible for steroid sulfatase deficiency is genetically and biochemically distinct from those involved in the lysosomal sulfatase deficiency states. The cell culture studies further suggest that the defect is a generalized one which should be detectable in midtrimester of pregnancy and may have phenotypic consequences in later postnatal life.Speculation: Further clinical and in vitro studies of patients with this rare inborn error of metabolism should yield information regarding the role of steroid sulfatase in normal metabolism, and in the progress of normal parturition. Once the mechanism by which the level of activity of this enzyme exerts an effect upon labor and delivery is known, appropriate pharmacologic invervention might provide a means for the inhibition of premature labor.

Correction of Abnormal Cerebroside Sulfate Metabolism in Cultured Metachromatic Leukodystrophy Fibroblasts

Cultured fibroblasts derived from patients with late infantile metachromatic leukodystrophy incorporated arylsulfatase A from the growth medium. Upon exposure to cerebroside sulfate, they exhibited patterns of uptake and hydrolysis indistinguishable from cells derived from control subjects. Furthermore, inclusion granules formed in the metachromatic leukodystrophy fibroblasts upon exposure to sulfatides were cleared by subsequent supplementation of the growth medium with arylsulfatase A.

Prenatal Diagnosis of Metachromatic Leukodystrophy in a Family with Pseudo Arylsulfatase A Deficiency by the Cerebroside Sulfate Loading Test

Summary: Prenatal diagnosis was requested by a family at risk for metachromatic leukodystrophy (MLD). An examination of the family leukocyte arylsulfatase A profile revealed that the mother had pseudo arylsulfatase A deficiency. Cultured amniotic fluid cells were deficient in arylsulfatase A, so two possibilities were indicated: the fetus was affected with MLD or had the pseudodeficiency phenotype. The only known biochemical test to differentiate the two enzyme deficient phenotypes is cerebroside sulfate loading of growing fibroblasts. The pseudodeficient cells hydrolyze the incorporated sulfatide as efficiently as control cells, whereas MLD cells show no hydrolysis. Application of this test to the at risk cultured amniotic fluid cells resulted in appreciable uptake of the sulfolipid, but no hydrolysis. Control amniotic fluid cell cultures hydrolyzed 82 to 95% of the incorporated sulfatide. Therefore, an affected fetus was indicated. Fibroblasts derived from the aborted fetus showed a deficiency of arylsulfatase A and a similar inability to hydrolyze cerebroside sulfate in the loading test. The loading technique allowed the prenatal diagnosis of MLD when the arylsulfatase A analysis was equivocal.Speculation: In metachromatic leukodystrophy families with pseudo arylsulfatase A deficiency, the usual enzyme assays on cultured amniotic fluid cell extracts fail to differentiate between the fetus with the affected phenotype and the fetus with the pseudodeflciency phenotype. The cerebroside sulfate loading test in growing cultured amniotic fluid cells allowed this discrimination. It is important to examine the family enzyme profile for the pseudodeficiency phenotype as a prerequisite hi the prenatal diagnosis of metachromatic leukodystrophy to avoid the erroneous identification of a pseudo-deficient fetus as a metachromatic leukodystrophy fetus.

Arylsulfatase B deficiency in maroteaux-lamy syndrome cultured fibroblasts

Summary Fibroblasts derived from patients with Maroteaux-Lamy syndrome (mucopolysaccharidosis VI) contained about 10% of normal arylsulfatase B activity, other lysosomal enzymes being unaltered. The properties of residual arylsulfatase B in Maroteaux-Lamy fibroblasts were identical to the enzyme derived from control fibroblasts. The decreased mucopolysaccharide turnover in Maroteaux-Lamy fibroblasts was normalized by supplementation with an arylsulfatase B fraction from normal fibroblasts. This data is consistent with the suggestion that the primary biochemical defect in Maroteaux-Lamy syndrome is a deficiency of arylsulfatase B.

Uridine diphospho-N-acetylgalactosamine-4-sulfate sulfohydrolase activity of human arylsulfatase B and its deficiency in the Maroteaux-Lamy syndrome

Abstract The hydrolysis of UDP-N-acetylgalactosamine-4-sulfate by human arylsulfatase B has been demonstrated with an enzyme preparation purified 200-fold from placenta. No hydrolysis was observed with arylsulfatase A. UDP-N-acetylgalactosamine-4-sulfate is the first fully characterized physiological compound shown to be a substrate for arylsulfatase B, confirming that arylsulfatase B is an N-acetylgalactosamine-4-sulfate sulfohydrolase. Cultured fibroblasts derived from patients with Maroteaux-Lamy syndrome were deficient in UDP-N-acetylgalactosamine-4-sulfate sulfohydrolase to the same extent that they were deficient in arylsulfatase B.

Cerebroside sulfatase determination in cultured human fibroblasts

A procedure for the determination of cerebroside sulfatase activity in extracts of cultured human fibroblasts is described. Cerebroside [35S]sulfate prepared from developing rat brain serves as substrate and enzyme activity is estimated by measurement of released inorganic [35S]sulfate. The reaction has a sharp optimum at pH 4.5, shows an absolute requirement for bile salts, and is stimulated by Mn2+ and Cl−. The Km for cerebroside sulfate is 0.1–0.2 mM. Inorganic sulfate and 4-nitrocatechol sulfate are inhibitory.