Asparagine-linked oligosaccharides protect Lamp-1 and Lamp-2 from intracellular proteolysis.
Lysosomes contain several integral membrane proteins (termed Lamps and Limps) that are extensively glycosylated with asparagine-linked oligosaccharides. It has been postulated that these glycans protect the underlying polypeptides from the proteolytic environment of the lysosome. Previous attempts to test this hypothesis have been inconclusive because they utilized approaches that prevent initial glycosylation and thereby impair protein folding. We have used endoglycosidase H to remove the Asn-linked glycans from fully folded lysosomal membrane proteins in living cells. Deglycosylation of Lamp-1 and Lamp-2 resulted in their rapid degradation, whereas Limp-2 was relatively stable in the lysosome in the absence of high mannose Asn-linked oligosaccharides. Depletion of Lamp-1 and Lamp-2 had no measurable effect on endosomal/lysosomal pH, osmotic stability, or density, and cell viability was maintained. Transport of endocytosed material to dense lysosomes was delayed in endoglycosidase H treated cells, but the rate of degradation of internalized bovine serum albumin was unchanged. These data provide direct evidence that Asn-linked oligosaccharides protect a subset of lysosomal membrane proteins from proteolytic digestion in intact cells.
Kundra
R
R
Washington University School of Medicine, Division of Hematology, St. Louis, Missouri 63110, USA.
Kornfeld
S
S
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D001216:Asparagine / Q000494:pharmacology*; D016466:CHO Cells; D002417:Cattle; D002460:Cell Line; D006224:Cricetinae; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D005966:Glucuronidase / Q000378:metabolism; D006596:Hexosaminidases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism* / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009844:Oligosaccharides / Q000494:pharmacology*; D051381:Rats; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014617:Vacuoles / Q000648:ultrastructure
D015703:Antigens, CD; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D009844:Oligosaccharides; D012710:Serum Albumin, Bovine; D001216:Asparagine; D006596:Hexosaminidases; D005966:Glucuronidase