Function of oligosaccharide modification in glucocerebrosidase, a membrane-associated lysosomal hydrolase.
The nature and function of oligosaccharide modification in glucocerebrosidase, a membrane-associated lysosomal hydrolase, have been investigated in cultured human skin fibroblasts. Glucocerebrosidase is synthesised as a 62.5-kDa precursor with high-mannose-type oligosaccharide chains and an apparent native isoelectric point of 6.0-7.0. Subsequent processing of the oligosaccharide moieties to sialylated complex-type structures results in formation of 65-68-kDa forms of the enzyme with apparent native isoelectric points of 4.3-5.0. These forms are transported to lysosomes and subsequently modified by the sequential action of lysosomal exoglycosidases, finally resulting in a 59-kDa form with an isoelectric point near neutrality. The existence of oligosaccharide modification of the enzyme in the lysosomes is illustrated by the accumulation of different intermediate forms of glucocerebrosidase in mutant cell lines deficient in lysosomal exoglycosidases. The enzyme does not undergo proteolytic modification during maturation. The possible physiological relevance of the oligosaccharide modification of glucocerebrosidase in the lysosomes was investigated by studying the properties of the enzyme in fibroblasts deficient in lysosomal exoglycosidases, and also the properties of homogeneous pure glucocerebrosidase from placenta, modified in the oligosaccharide moieties by digestion in vitro with glycosidases. Modification of the oligosaccharide moieties of glucocerebrosidase had no significant effect on the catalytic activity of the enzyme as measured with either artificial or natural substrates in the presence of artificial or natural activators. There was also no effect of modification of the oligosaccharide chains on the intracellular stability of the enzyme or on its apparent hydrophobicity. We conclude that oligosaccharide modification of glucocerebrosidase in the lysosomes simply reflects further maturation of the enzyme in the lysosome and is of no importance to its function.
Van Weely
S
S
E. C. Slater Institute for Biochemical Research, University of Amsterdam, The Netherlands.
Aerts
J M
JM
Van Leeuwen
M B
MB
Heikoop
J C
JC
Donker-Koopman
W E
WE
Barranger
J A
JA
Tager
J M
JM
Schram
A W
AW
D000118:Acetylglucosaminidase / Q000378:metabolism; D002384:Catalysis; D002402:Cathepsin D / Q000378:metabolism; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004795:Enzyme Stability; D005347:Fibroblasts / Q000201:enzymology; D005959:Glucosidases / Q000378:metabolism*; D005962:Glucosylceramidase / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D007526:Isoelectric Point; D008247:Lysosomes / Q000201:enzymology*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D009439:Neuraminidase / Q000172:deficiency / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D010920:Placenta / Q000201:enzymology; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship; D001616:beta-Galactosidase / Q000172:deficiency / Q000378:metabolism; D001619:beta-N-Acetylhexosaminidases / Q000172:deficiency / Q000378:metabolism
D009844:Oligosaccharides; D011498:Protein Precursors; D005959:Glucosidases; D006026:Glycoside Hydrolases; D009439:Neuraminidase; D001616:beta-Galactosidase; D005962:Glucosylceramidase; D000118:Acetylglucosaminidase; D001619:beta-N-Acetylhexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D002402:Cathepsin D