The Pro Domain of β-Secretase Does Not Confer Strict Zymogen-like Properties but Does Assist Proper Folding of the Protease Domain

Abstract

␤-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid ␤ peptide. BACE is expressed as a precursor protein containing Pre, Pro, protease, transmembrane, and cytosolic domains. A soluble BACE derivative (PreProBACE460) that is truncated between the protease and transmembrane domains was produced by baculovirus-mediated expression. ProBACE460 was purified from conditioned media of infected insect cells using immobilized concanavalin A and immobilized BACE inhibitor, P10-P4 Stat(Val). Furin cleaves ProBACE460 between the Pro and protease regions to generate mature BACE460. The k cat /K m of ProBACE460 when assayed with a polypeptide substrate is only 2.3-fold less than that of BACE460. This finding and the similar inhibitory potency of P10-P4 Stat(Val) for ProBACE460 and BACE460 suggest that the Pro domain has little effect on the BACE active site. Exposure of ProBACE460 to guanidine denaturation/ renaturation results in a 7-fold higher recovery of BACE activity than when BACE460 is similarly treated. The presence of free BACE Pro peptide during renaturation of BACE460 but not ProBACE460 increases recovery of activity. These findings show that the Pro domain in ProBACE460 does not suppress activity as in a strict zymogen but does appear to facilitate proper folding of an active protease domain.

Dimerization of β-Site β-Amyloid Precursor Protein-cleaving Enzyme

“…On the corresponding Western blot, the pro-domain-specific antibody GM190 (9) demonstrated that BACE-NT-onco and BACE-NT produced in our laboratory still carried the pro-domain (Fig. 5A), which is in agreement with earlier reports (9,11,14,60). The Michaelis-Menten kinetics obtained for the quenched fluorogenic substrate harboring the Swedish APP cleavage site (62) revealed a higher affinity and catalytic activity for dimeric BACE-FL compared with monomeric BACE-NT and the commercial BACE-NT-onco (Fig.…”

“…For full maturation, BACE is transported through the secretory pathway, subjected to complex glycosylation, and processed by a furin-like pro-protein convertase (9 -13). The pro-domain of BACE assists proper folding of BACE but does not confer strict zymogen-like activity (14). On a cellular level BACE is found mainly in acidic compartments such as the trans-Golgi network and endosomes (9,10,13) where it is associated with rafts by palmitoylation (15,16).…”

The Transmembrane Domain of the Alzheimer's β-Secretase (BACE1) Determines Its Late Golgi Localization and Access to β-Amyloid Precursor Protein (APP) Substrate

“…A small fraction of BACE1 is shed near its transmembrane domain on the luminal side of the membrane to release a soluble BACE1, which cleaves APP, although at a much lower efficiency compared with full-length BACE1 (17). The pro-BACE1 in the ER has also been shown to have an enzymatic activity that cleaves APP at the ␤-site (17,18), consistent with the observation that a small percentage of A␤42 is generated in the ER whereas the majority of A␤ is made in the TGN (19 -22). In addition, although BACE1 catalytic domains have been defined, the targeting signal(s) required for correct localization remain unclear.…”

Pharmacological applications of a novel neoepitope antibody to a modified amyloid precursor protein-derived beta-secretase product

“…Briefly, baculovirus expressed and purified truncated BACE1 (aa 1-460) (Shi et al, 2001) at 0.3 nmol/L was incubated at a range of concentrations with a previously characterized BACE1 inhibitor MRK-3 (Stachel et al, 2004;Sankaranarayanan et al, 2008;Wu et al, 2011) for 30 min at RT. After this preincubation, the BACE1 substrate (15 amino acid peptide with C-terminal biotin) was added at a final concentration of 300 nmol/L to the reaction buffer (50 mmol/L NaOAc, 0.01% BSA, 15 mmol/L EDTA, 0.2% CHAPS, 1 mmol/L Deferoxamine Mesylate and 10 μmol/L pepstatin A at pH 4.5) and incubated for 2 h at 37°C in a shaker at 40 rpm .…”

“…The endogenous APP site for BACE cleavage (KMDA) shows very low cleavage activity under normal physiological conditions (Shi et al, 2001;Yang et al, 2003). This observation led to numerous peptide screening efforts to find an optimal cleavage site for BACE inhibitor screening (Tomasselli et al, 2003;Turner et al, 2001).…”