Two
beta-glucuronidase-deficient Mennonite siblings were found to be homozygous for a mutation in exon 3 of the
beta-glucuronidase gene that produces a
Leu-->Phe substitution (L176F). The siblings also have the previously described benign polymorphism, P649L. Although their cultured fibroblasts contained 1.5-2.2% of normal
beta-glucuronidase activity, transient expression of the L176F/P649L
cDNA in COS cells produced nearly as much
enzyme activity as the wild-type control
cDNA. The L176F/P649L
enzyme was as stable as wild-type
enzyme following endocytosis by fibroblasts and delivery to lysosomes, but was more labile to heat inactivation at 65 degrees C. To study the mutant
enzyme at lower levels of expression, we stably transfected mouse
mucopolysaccharidosis type VII cells with the L176F/P649L
cDNA and selected single-copy cell lines. Metabolic labeling with [35S]
methionine revealed that cell lines expressing the mutant
enzyme activity at low levels (7-10% of the wild type) actually produced the same amount of
enzyme protein as the cell lines expressing the more active wild-type
enzyme. However, the cell lines expressing four times this much mutant
enzyme protein produced 150-200% as much
enzyme activity as the cell line expressing the single-copy wild-type
cDNA. These data suggest that overexpression can drive the folding reaction or the self-association of mutant monomers to form active tetramers and, at least partially, correct the
beta-glucuronidase deficiency seen at low levels of expression with certain missense mutations.