Consequences of individual N-glycan deletions and of proteasomal inhibition on secretion of active BACE.
BACE is an aspartic protease involved in the production of a toxic peptide accumulating in the brain of Alzheimer's disease patients. After attainment of the native structure in the endoplasmic reticulum (ER), BACE is released into the secretory pathway. To better understand the mechanisms regulating protein biogenesis in the mammalian ER, we determined the fate of five variants of soluble BACE with 4, 3, 2, 1, or 0 N-linked glycans. The number of N-glycans displayed on BACE correlated directly with folding and secretion rates and with the yield of active BACE harvested from the cell culture media. Addition of a single N-glycan was sufficient to recruit the calnexin chaperone system and/or for oligosaccharide de-glucosylation by the ER-resident alpha-glucosidase II. Addition of 1-4 N-glycans progressively enhanced the dissociation rate from BiP and reduced the propensity of newly synthesized BACE to enter aberrant soluble and insoluble aggregates. Finally, inhibition of the proteasome increased the yield of active BACE. This shows that active protein normally targeted for destruction can be diverted for secretion, as if for BACE the quality control system would be acting too stringently in the ER lumen, thus causing loss of functional polypeptides.
Vanoni
Omar
O
Institute for Research in Biomedicine, CH-6500 Bellinzona, Switzerland.
Paganetti
Paolo
P
Molinari
Maurizio
M
D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism*; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D010455:Peptides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D000520:alpha-Glucosidases / Q000737:chemistry
D010455:Peptides; D011134:Polysaccharides; D006026:Glycoside Hydrolases; D000520:alpha-Glucosidases; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; D046988:Proteasome Endopeptidase Complex