Lack of proteolytic processing of alpha-L-fucosidase in human skin fibroblasts.
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 alpha-L-fucosidase was distinguished from that of other acid hydrolases reported when studied in systematic pulse-chase labeling experiments. Only one form of alpha-L-fucosidase, Mr 56,000-57,000, was demonstrated intra- and extracellularly. Under the same conditions, N-acetyl-beta-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. alpha-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-beta-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, Mr 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 alpha-L-fucosidase does not undergo proteolytic processing in human skin fibroblasts in the usual manner described for other acid hydrolases.
Leibold
D M
DM
Department of Pediatrics, University of Pennsylvania Medical School, Philadelphia 19104.
Robinson
C B
CB
Scanlin
T F
TF
Glick
M C
MC
D000118:Acetylglucosaminidase / Q000378:metabolism; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts; D006801:Humans; D008970:Molecular Weight; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D014415:Tunicamycin / Q000494:pharmacology; D005644:alpha-L-Fucosidase / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*
D014415:Tunicamycin; D005644:alpha-L-Fucosidase; D000118:Acetylglucosaminidase