Elevated Cystine Levels in Cultured Skin Fibroblasts From Patients With I-Cell Disease

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Abstract
Summary: Cultured skin fibroblasts from patients with I-cell disease (mucolipidosis II) exhibit multiple deficiency of acid hydrolase activities associated with a defect in the mechanism of packaging of these enzymes into lysosomes. The authors have examined such cells to ascertain whether the impairment of lysosomal function is of so broad a nature as to result in the storage of the amino acid cystine in a manner similar to that seen in cells derived from patients with cystinosis, an unrelated lysosomal storage disease of unknown etiology. Of 10 I-cell lines examined by automated amino acid analysis, seven were found to possess abnormally high levels of total free cyst(e)ine (i.e., greater than 1 nmole 1/2 Cys/mg protein). The mean half-cystine content of those I-cell lines subjected to multiple analysis ranged from 3–10 nmole/mg protein, levels which are comparable to those seen in homozygous cystinotic cells. The cystine content of several of these lines appeared to increase with subculture. Cultured fibroblasts from two patients with the biochemically similar, but clinically less severe, mucolipidosis III (pseudo-Hurler polydystrophy) exhibited normal to marginally elevated levels of cystine, whereas cells from individuals with three different muco-polysaccharide storage disorders contained normal levels of the amino acid. It was concluded that cystine, and not cysteine, was the predominant form of this amino acid in these cells because previous reaction of I-cell extracts with N-ethylmaleimide did not alter the observed cystine levels. The further identification of excess cystine in these cells was corroborated by analytical results obtained with a highly specific cystine-binding protein method as well as by high-voltage electrophoresis of extracts from cells pulsed with 35Scystine. Comparative analysis of intracellular amino acids in normal and I-cell fibroblasts indicated that the elevation of cystine seen in the latter was unique to this amino acid and did not reflect a generalized increase in the total free amino acid content of these mutant cells. Speculation: Cystinosis has been classified as a lysosomal storage disorder on the basis of cytologic and other evidence pointing to the intralysosomal localization of the stored cystine in cells from affected individuals. The fact that the principal enzyme function in lysosomes, viz., hydrolysis, is not known to play a role in the metabolic disposition of cystine sets this abnormality apart from the more typical lysosomal storage disorder and raises questions concerning the precise relation of lysosomal function to the pathogenesis of cystinosis. The present finding that I-cell fibroblasts, which are characterized by a broad spectrum of acid hydrolase deficiencies, also exhibit abnormally elevated cystine levels, appears to strengthen this relationship. However, further experiments will be necessary to determine whether the abnormal cystine levels in these two disorders arise from a common or related defect.