Enzymic properties of recombinant BACE2

Abstract

BACE2 (Memapsin 1) is a membrane‐bound aspartic protease that is highly homologous with BACE1 (Memapsin 2). While BACE1 processes the amyloid precursor protein (APP) at a key step in generating the β‐amyloid peptide and presumably causes Alzheimer's disease (AD), BACE2 has not been demonstrated to be directly involved in APP processing, and its physiological functions remain to be determined. In vivo, BACE2 is expressed as a precursor protein containing pre‐, pro‐, protease, transmembrane, and cytosolic domains/peptides. To determine the enzymatic properties of BACE2, two variants of its pro‐protease domain, pro‐BACE2‐T1 (PB2‐T1) and pro‐BACE2‐T2 (PB2‐T2), were constructed. They have been expressed in Escherichia coli as inclusion bodies, refolded and purified. These two recombinant proteins have the same N terminus but differ at their C‐terminal ends: PB2‐T1 ends at Pro466, on the boundary of the postulated transmembrane domain, and PB2‐T2 ends at Ser431, close to the homologous ends of other aspartic proteases such as pepsin. While PB2‐T1 shares similar substrate specificities with BACE1 and other ‘general’ aspartic proteases, the specificity of PB2‐T2 is more constrained, apparently preferring to cleave at the NH2‐terminal side of paired basic residues. Unlike other ‘typical’ aspartic proteases, which are active only under acidic conditions, the recombinant BACE2, PB2‐T1, was active at a broad pH range. In addition, pro‐BACE2 can be processed at its in vivo maturation site by BACE1.

Crystal Structure of Human BACE2 in Complex with a Hydroxyethylamine Transition-state Inhibitor

“…The exact activation mechanism is, however, still unclear: Pro-BACE2, expressed in COS-7 cells, autoactivates intramolecularly, resulting in cleavage between Leu62p and Ala1, 26 while pro-BACE2 expressed in E. coli and refolded from inclusion bodies was reported not to autoactivate in a pH range of 4.5-12, but to be activated by BACE1. 27 Our studies show, in agreement with autoactivation of pro-BACE2 derived from COS-7 cells, that refolded pro-BACE2 can also be autoactivated in vitro by a pH shift to 3.4, resulting in the same cleavage between Leu62p and Ala1. Mature BACE2 cleaves a designed peptide comprising the reported activation site between Leu62p and Ala1 of BACE2 with a catalytic efficiency (k cat /K M ) of 4 !10 4 M K1 s K1 .…”

Bace2 Is a β Cell-Enriched Protease that Regulates Pancreatic β Cell Function and Mass

“…The fidelity of Tmem27 toward Bace2 rather than Bace1 might in part be explained by the enrichment of hydrophobic moieties in the cleavage site that has been found in other Bace2 substrates and distinctively in domains in which Bace1 did not cleave those proteins (Fluhrer et al, 2002;Gruninger-Leitch et al, 2002;Sun et al, 2006;Yan et al, 2001). Another way by which the specificity of interaction of Tmem27 with Bace2 rather than Bace1 could be conferred is by differential compartmentalization: Bace1 is known to be resident primarily in the Golgi-TGN compartments (Gandhi et al, 2004), while Bace2 is localized to the endosomal compartments (Fluhrer et al, 2002;Walter, 2006) and active at less acidic pH than Bace1 (Kim et al, 2002).…”

Identification of Human Islet Amyloid Polypeptide as a BACE2 Substrate

“…Similar peak results were detected using commercial recombinant mouse BACE1 and BACE2 (data not shown). hIAPP digestion was more pronounced under acidic conditions (data not shown); which is both optimal for BACE2 and BACE1 enzymatic activity [ 31 ] and more analogous to the acidic environment of secretory vesicles [ 5 ].…”