Biomedical Engineering Reference
In-Depth Information
Furthermore, cathepsin B cleaves the internally quenched fluorescent peptide
substrates that contain the WT b-secretase site within the RE(Edans)
EVKM
k
DAEFK(Dabcl)R-NH
2
substrate.
43
These long peptide substrates are
utilized to evaluate cleavage of the b-secretase site by b-secretases.
15,17,18
Table 6.1 Selectivity of cathepsin B for cleaving the wild-type b-secretase site,
rather than the Swedish mutant b-secretase site of APP.
Cathepsin B: b-secretase
activity
Wild-type b-secretase site: Z-Val-Lys-Met-
k
MCA 547
Swedish mutant b-secretase site: Z-Val-Asn-Leu-
k
MCA 0.2
A cleavage-site-specific protease assaywas designed to detect cleavage at the wild-type (WT) b-secretase
site utilizing the Z-Val-Lys-Met-
k
MCA (Z-V-K-M-MCA) substrate that mimics the wild-type
cleavage site. In parallel, the substrate Z-Val-
Asn-Leu-
k
MCA was utilized to assay protease cleavage
at the Swedish mutant site (mutant residues are underlined). These substrates provided
cleavage-site-specific protease assays for cleavage of the WT or Swedish mutant sites. Assay
of cathepsin B with these substrates provided comparison of its high specific activity (pmol AMCmin
-1
mg enzyme
-1
) for the WT substrate, and essentially no activity with the Swedish mutant substrate.
42
Substrate
Figure 6.3
Cathepsin B is identified as a b-secretase in regulated secretory vesicles. (a)
Purification of proteolytic activity cleaving the wild-type (WT) b-secretase
cleavage site substrate Z-Val-Lys-Met-MCA: active-site directed probe
labeling. Regulated secretory vesicles of neuronal-like chroman cells
(from the sympathetic nervous system) produce and secrete Ab pep-
tides.
15,41
Purification of proteolytic activity that cleaves the WT
b-secretase site, present in the majority of Alzheimer's patients, was con-
ducted using isolated secretory vesicles of the regulated secretory pathway.
The b-site cleaving protease was labeled with the active-site directed a
-
nity probe DCG-04 (shown by the arrow) as a protein band of
31 kDa
(lane 1). The selective inhibitor of cathepsin B, CA074, blocked the DCG-
04 probe labeling, suggesting that the protease may be represented by
cathepsin B (lane 2). (b) Inhibition of Z-Val-Lys-Met-MCA cleaving
activity by E64c and CA074. The Z-Val-Lys-Met-MCA cleaving activity
purified from Ab-containing regulated secretory vesicles (chroman
secretory vesicles) was inhibited by the cysteine protease inhibitor E64c
and by the selective inhibitor of cathepsin B, CA074.
41
(c) Peptide
sequencing identifies cathepsin B as b-secretase. The 31 kDa band (shown
in part 'a') was indicated as the responsible enzyme for b-secretase activity
in regulated secretory vesicles. The 31 kDa band was subjected to peptide
sequencing by tryptic digestion and tandem mass spectrometry (MS/MS)
of peptide fragments. The determined sequences of tryptic peptides are
underlined within the complete primary sequence of cathepsin B. (d)
Colocalization of cathepsin B with Ab peptides in regulated secretory
vesicles: analyses by immunoelectron microscopy. Cathepsin B (Cat. B,
15 nm gold particles) was colocalized with Ab40 (6 nm gold particles)
within regulated secretory vesicles (i). Cathepsin B (15 nm gold particles)
was also colocalized with Ab42 (6 nm gold particles) in regulated secretory
vesicles (ii).
B
