Biochemical characterization and structural modeling of human cathepsin E variant 2 in comparison to the wild-type protein.
Cathepsin E splice variant 2 appears in a number of gastric carcinomas. Here we report detecting this variant in HeLa cells using polyclonal antibodies and biotinylated inhibitor pepstatin A. An overexpression of GFP fusion proteins of cathepsin E and its splice variant within HEK-293T cells was performed to show their localization. Their distribution under a fluorescence microscope showed that they are colocalized. We also expressed variants 1 and 2 of cathepsins E, with propeptide and without it, in Escherichia coli. After refolding from the inclusion bodies, the enzymatic activity and circular dichroism spectra of the splice variant 2 were compared to those of the wild-type mature active cathepsins E. While full-length cathepsin E variant 1 is activated at acid pH, the splice variant remains inactive. In contrast to the active cathepsin E, the splice variant 2 predominantly assumes β-sheet structure, prone to oligomerization, at least under in vitro conditions, as shown by atomic force microscopy as shallow disk-like particles. A comparative structure model of splice variant 2 was computed based on its alignment to the known structure of cathepsin E intermediate (Protein Data Bank code 1TZS) and used to rationalize its conformational properties and loss of activity.
Department of Biochemistry and Molecular and Structural Biology, Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia.
Vida
V
Puizdar
Tajana
T
Zajc
Eva
E
Zerovnik
Miha
M
Renko
Ursula
U
Pieper
Narayanan
N
Eswar
Andrej
A
Sali
Iztok
I
Dolenc
Vito
V
Turk
D000595:Amino Acid Sequence; D020059:Cathepsin E / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D020033:Protein Isoforms; D011994:Recombinant Proteins; D020059:Cathepsin E