Use of a monoclonal antibody to distinguish between precursor and mature forms of human lysosomal α‐glucosidase

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Abstract
The maturation of lysosomal α-glucosidase in cultured human skin fibroblasts was studied using a monoclonal antibody that distinguishes between the precursor and mature forms of the enzyme. 1. Monoclonal antibodies against α-glucosidase isolated from placenta were produced by the hybridoma technique [Hilkens et al. (1981) Biochim. Biophys. Acta 678, 7–11]. One of these monoclonal antibodies, that synthesized by clone 43G8, reacts with the mature forms, but not with the precursor form of α-glucosidase isolated from urine. 2. By means of pulse-labelling studies, it could be shown that monoclonal antibody 43G8 does not react with either the intracellular or the secreted precursor of α-glucosidase from cultured human skin fibroblasts. However, the antibody does react with the intermediate and mature forms of α-glucosidase. 3. Endocytosis of the precursor of α-glucosidase from urine by fibroblasts is followed by its conversion to a form with lower molecular mass. 4. After endocytosis urinary precursor α-glucosidase is converted to a form that binds to monoclonal antibody 43G8. The t1/2 for this conversion is 2 h. The conversion is inhibited by addition of leupeptin to the culture medium. 5. It is concluded that a thiol proteinase is involved in the maturation of α-glucosidase in fibroblasts and the appearance of the antigenic determinant for 43G8.