Lack of proteolytic processing of ?-L-fucosidase in human skin fibroblasts

Abstract

Acid hydrolases are synthesized as precursors that undergo several posttranslational modifications including proteolytic processing to a smaller mature enzyme. The amount of proteolytic processing varies for different acid hydrolases, and many details of the intracellular pathways are not known. The processing of α-L-fucosidase was distinguished from that of other acid hydrolases reported when studied in systematic pulse-chase labeling experiments. Only one form of α-L-fucosidase, M_r 56,000-57,000, was demonstrated intra- and extracellularly. Under the same conditions, N-acetyl-β-D-glucosaminidase was shown to be processed with several forms, as previously reported by Hasilik and Neufeld (1980a). To obtain these results, human skin fibroblasts were labeled metabolically with L-[³H]leucine for periods of 20 min to 8 hr with varying periods of chase from 1 to 96 hr with nonradioactive L-leucine. α-L-Fucosidase was immunoprecipitated by a polyclonal antibody from material extracted from cells and ammonium sulfate precipitated medium and was examined by polyacrylamide gel electrophoresis under denaturing conditions. N-Acetyl-β-D-glucosaminidase was examined with similar procedures and served as a control for the methods. Tunicamycin treatment of the cells was used to show that glycosylation did not obscure proteolytic processing because, again, only one form of the intra- and extracellular enzyme was observed, although of smaller size, M_r 52,000-53,000. In addition, separation of the cells into prelysosomal and lysosomal fractions showed only one form of the enzyme. It is concluded that α-L-fucosidase does not undergo proteolytic processing in human skin fibroblasts in the usual manner described for other acid hydrolases.