Identification of a BACE dimer and characterization of its biochemical and enzymatic properties

The deposition amyloid β peptide in the brains of patients is a
hallmark of Alzheimer’s disease and is thought to play a major
pathogenetic role in the development of the demential symptoms of
this severe illness. The amyloid β peptide is generated from the
β-amyloid precursor protein (APP) by cleavage of the “β-site
APP-cleaving enzyme” (BACE) followed by cleavage of the
"gamma-secretase”. Whereas it has recently been discovered that the
gamma-secretase is a multi-protein complex, it has not yet been
investigated whether under native conditions, BACE functions in
association with other proteins. The present work thus studied BACE
by means of blue native gel electrophoresis and found that native
BACE has a molecular weight of 140 kDa, whereas BACE under
denaturing conditions has a molecular weight of 70 kDa which is
only half of its native mass. Co-immunoprecipitation experiments
with differently tagged full-length BACE constructs subsequently
showed that this higher molecular weight species of BACE
corresponds to a BACE homodimer. In contrast, a BACE ectodomain,
lacking the C-terminus and the transmembrane domain, is a monomer.
A consecutive domain analysis revealed that both the C-terminus and
the transmembrane domain of BACE are dispensable for dimerization.
In line with this, it could be shown that the ectodomain of BACE
can dimerize if it is attached to the membrane by a GPI anchor. In
terms of the cellular localization of the dimerization process, it
could furthermore be demonstrated that retention of BACE in the ER
by addition of a KKXX-motif does not prevent dimerization. This
suggests that dimerization can occur prior to full maturation of
BACE which takes place in the Golgi apparatus. In addition, kinetic
analyses of the purified native BACE dimer revealed a higher
affinity and turnover rate for an APP-like substrate in comparison
to the monomeric soluble BACE ectodomain. This suggests a putative
function of dimerization in improving enzymatic efficiency. The
implication of these findings for our understanding of the
Amyloid-β synthesis as well as for a putatively alternative
therapeutic strategy are discussed.