Biosynthesis of the lysosomal enzyme glucocerebrosidase.
Multiple biosynthetic forms of glucocerebrosidase were immunoprecipitated after synthesis in vitro using cell-free translation or in vivo using pulse-chase conditions in porcine kidney cells or human fibroblasts. The initial product in vitro was a 52-kDa polypeptide. When canine pancreatic microsomes were present during translation, the nascent polypeptide crossed the microsomal membrane and increased its mass to 60 kDa. Treatment of the 60-kDa polypeptide with endoglycosidase H to remove high mannose carbohydrate yielded a 51-kDa polypeptide. Thus, the membrane-translocated molecule was apparently a high mannose glycoprotein from which a signal peptide had been cleaved, as observed for the lysosomal protease cathepsin D (Erickson, A. H., and Blobel, G. (1979) J. Biol. Chem. 254, 11771-11774). Treatment of pancreatic microsomes or microsomes from porcine kidney cells with protease did not decrease the size of the polypeptide, which shows that this form is not a transmembrane protein bearing a cytoplasmic domain susceptible to digestion. The in vitro product synthesized in the presence of microsomal membranes was indistinguishable from the in vivo product synthesized during pulse-labeling of cultured porcine kidney cells. Following a 2-h chase period, the 60-kDa product was converted to a 59-kDa polypeptide. The major form of glucocerebrosidase detected after a 24-h chase period was a 56-kDa polypeptide, which in turn was converted to a 55-kDa polypeptide by 72 h. The same forms were precipitated from human fibroblasts but the rate of processing was accelerated in this cell type. Limited treatment of the 60-kDa form of glucocerebrosidase with endoglycosidase H suggested that high mannose carbohydrate is added to at least four sites on the polypeptide chain. By 24 h after synthesis, conversion to endoglycosidase H-resistant complex carbohydrate had occurred. Thus, both polypeptide and carbohydrate processing steps are involved in the biosynthesis of glucocerebrosidase.
D000118:Acetylglucosaminidase / Q000378:metabolism; D000818:Animals; D050260:Carbohydrate Metabolism; D002460:Cell Line; D002474:Cell-Free System; D003593:Cytoplasm / Q000201:enzymology; D004285:Dogs; D005347:Fibroblasts / Q000201:enzymology; D005959:Glucosidases / Q000096:biosynthesis*; D005962:Glucosylceramidase / Q000096:biosynthesis*; D006801:Humans; D007668:Kidney / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology*; D008358:Mannose / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology; D010447:Peptide Hydrolases / Q000378:metabolism; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000378:metabolism; D013552:Swine
D021382:Protein Sorting Signals; D005959:Glucosidases; D005962:Glucosylceramidase; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D010447:Peptide Hydrolases; D008358:Mannose