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

Xiao-Ping Shi,Elizabeth Chen,Kuo-Chang Yin,Sang Na,Victor M Garsky,Ming-Tain Lai,Yue-Ming Li,Michael Platchek,R Bruce Register,Mohinder K Sardana,Mei-Jy Tang,James Thiebeau,Theresa Wood,Jules A Shafer,Stephen J Gardell

doi:10.1074/jbc.m009200200

Xiao-Ping Shi, Elizabeth Chen + Show 13 more

Open Access

https://doi.org/10.1074/jbc.m009200200

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Abstract

beta-Secretase (BACE) is a membrane-bound aspartyl protease that cleaves the amyloid precursor protein to generate the N terminus of the amyloid beta 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.

Highlights

The amyloid precursor protein (APP)1 is cleaved sequentially by two proteolytic activities, ␤- and ␥-secretase, to generate the N and C termini, respectively, of the amyloid ␤ peptides
SDS-PAGE/immunoblot analysis of conditioned medium (CM) from cells that were infected with BACE460 virus stock revealed the presence of a protein that reacted with antiBACE antibodies raised against the Pro domain (Fig. 2A, lane 2) or protease domain
The ProBACE460-immunodepleted CM still possessed a less abundant species that reacts with the anti-protease antibody and migrates in the vicinity of ProBACE460 (Fig. 2A, lanes 5 and 6)

Summary

Introduction

The amyloid precursor protein (APP) is cleaved sequentially by two proteolytic activities, ␤- and ␥-secretase, to generate the N and C termini, respectively, of the amyloid ␤ peptides. Edman degradation of purified human brain BACE revealed a single N-terminal protein sequence that began at the protease domain [3] (this species is designated mature BACE). N-terminal sequencing of full-length Asp (i.e. BACE), which was expressed in Chinese hamster ovary cells, revealed proteolytic processing at the junction between the putative Pro and protease domains [4]. These results establish that processing of membrane-anchored BACE after Arg-45 to release the Pro segment and generate mature BACE is prevalent in mammalian cells. The studies described show that the Pro domain of ProBACE460, rather than abolishing catalytic activity as in the case of true zymogens, appears to facilitate the proper folding of the BACE protease domain

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