The defect in Hurler's and Hunter's syndromes: faulty degradation of mucopolysaccharide.

Abstract

Hurler's syndrome is a genetically transmitted disorder of mucopolysaccharide metabolism, distinguished chemically by excessive intracellular accumulation and urinary excretion of chondroitin sulfate B and heparitin monosulfate, and clinically, by mental retardation, skeletal deformities, corneal clouding, and early death.1 While Hurler's syndrome follows autosomal recessive inheritance, there also exists a sex-linked variant, Hunter's syndrome, which is chemically similar, but clinically less severe. The study of these disorders has been greatly facilitated by the discovery that fibroblasts cultured from the skin of affected individuals also manifest the disease, in that they store an abnormal amount of mucopolysaccharide as determined by histological observation, chemical analysis, and measurement of isotope incorporation.2, We have undertaken a study of the kinetics of this accumulation in fibroblasts to determine whether it is a result of excessive synthesis, insufficient degradation, or defective secretion of mucopolysaccharide. Although accumulation of mucopolysaccharide has been attributed to overproduction,3 our results suggest that the lesion(s) in both Hurler's and Hunter's syndromes is faulty degradation.4 Materials and Methods.-Culture of fibroblasts: Fibroblasts were grown from skin biopsies or from infant foreskin obtained at circumcision. Tissue was obtained from two normal infants, a normal adult, a Hurler infant, and a Hunter adult, and cultured for 2-8 months prior to use. Cultures were maintained as monolayers at 37? in 100-mm Falcon plastic Petri dishes in an atmosphere of 5% C02-95% air. They were fed three times per week with Eagle's minimal essential medium (in which MgCl2 was substituted for MgSO4) supplemented with nonessential amino acids, 10% fetal calf serum, penicillin, streptomycin, and, in some cases, Mycostatin. Inorganic sulfate, derived primarily from the streptomycin, was about 0.4 mM. When used, the Hurler and Hunter cells displayed strong metachromasia in toluidine blue (a histological marker for mucopolysaccharide). They were stained essentially as described by Danes and Bearn, except that methanol fixation was omitted and the cover slips were immersed for 8 min in 0.1% toluidine blue in 60% ethanol. This modification was introduced because, in some cultures, mucopolysaccharide appeared to be leached out in the 30% methanolic solution of dye originally recommended. Incorporation of S304 into mucopolysaccharide: A simple assay was devised to measure the incorporation of S304 into mucopolysaccharides, based on the fact that the latter are the only macromolecules of the fibroblast to become labeled with inorganic sulfate (mammalian cells in general do not introduce sulfate into cystine or methionine residues of proteins'). Cells were incubated in the medium described above to which had been added 3-15 X 106 cpm of carrier-free H2S3()4 (New England Nuclear Corp.) per ml. Petri dishes routinely contained 1-3 million cells (0.5-1.5 mg protein). At specified intervals, the labeled medium was removed and dialyzed for 4 hr against 0.1 M (NH4)2SO4, followed by running tap water for 20 hr. The cells were washed with isotonic saline, detached from the plate by treatment with 0.4% trypsin (Microbiological Associates) in saline at 37? for 15 min, centrifuged, and extracted four times with boiling 80% ethanol (I min boiling