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Originally published as MBC in Press, 10.1091/mbc.E08-05-0459 on July 16, 2008

Vol. 19, Issue 10, 4086-4098, October 2008

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Consequences of Individual N-glycan Deletions and of Proteasomal Inhibition on Secretion of Active BACE

Omar Vanoni*, Paolo Paganetti{dagger}, and Maurizio Molinari*

*Institute for Research in Biomedicine, CH-6500 Bellinzona, Switzerland; and {dagger}Novartis Institutes for Biomedical Research Basel, Neuroscience Research, Novartis Pharma AG, CH-4002 Basel, Switzerland

Submitted May 6, 2008; Revised July 3, 2008; Accepted July 8, 2008
Monitoring Editor: Reid Gilmore

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.

This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08-05-0459) on July 16, 2008.

Address correspondence to: Maurizio Molinari (maurizio.molinari{at}irb.unisi.ch)

Abbreviations used: BACE, Beta-site APP-cleaving enzyme 1; BACEs, soluble form of BACE; bDNJ, N-butyl-deoxynojirimycin; CHO, Chinese hamster ovary cells; DBA, disulfide-bonded aggregates; EndoH, endoglycosidase H; ER, endoplasmic reticulum; ERAD, ER-associated degradation; HEK, human embryonic kidney cells.




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