90
fi bers embedded in fi laggrin to form macrofi brils. They also gain a
reinforced, cytoplasmic membrane called CE, wherein omega-
hydroxyceramides are covalently attached to cover the interstices
of the cornifi ed cells [ 5 ], and special structural proteins, such as
involucrin and loricrin, are cross-linked to the membrane by trans-
glutaminase [ 6 , 7 ]. Internally, the cells lose their nuclei and cyto-
plasmic organelles, becoming fi lled with keratin fi laments that are
bound with an amorphous material, fi laggrin. Caspase-14 is acti-
vated during this transition phase.
Previously we purifi ed active caspase-14 from the extract of
human cornifi ed cells [ 8 ]. The primary structure of the purifi ed
caspase-14 consists of large and small subunits generated by the
removal of a 6-amino-acid linker peptide. The C-terminal of the
large subunit was identifi ed as D^146 , indicating that the activation-
dependent cleavage occurs at this site. A cleavage-site-directed
antibody, h14D146, was prepared and found to react only with
active caspase-14. Immunohistochemical study using this antibody
showed strong positive staining in the cornifi ed layer, as well as of
some of the granular cells.
We also constructed a constitutively active caspase-14 (revC14)
based on the sequence information of purifi ed human caspase-14.
Large subunit and small subunit was reversely fused with an
N-terminal His-tag. Recombinant revC14 demonstrated similar
enzymatic activity with that of the purifi ed caspase-14 from the
corneocyte extracts.
For the quantitative analysis of caspase-14, we established two
ELISA assays to quantify the total amount of caspase-14 and that
of active caspase-14. We produced four clones of monoclonal anti-
bodies to caspase-14 and used clone 3 as the immobilizing anti-
body. A commercial antibody, H99, and cleavage-site-directed
antibody, h14D146 [ 8 ], were used for the quantifi cation of total
and active caspase-14 in extracts of tape-stripped corneocytes,
respectively. Caspase-14 was found mostly in active form (71–94 %)
in the extracts from healthy individuals. In patients with atopic
dermatitis, active caspase-14 was markedly downregulated com-
pared to age-matched controls in both lesional (7.5 %) and non-
lesional skin (10.6 %) [ 9 ].
The maturation and activation mechanisms of caspases are
generally well understood, except for those of caspase-14, which is
activated at the onset of keratinocyte terminal differentiation. We
found that the chymotrypsin-like serine protease kallikrein-related
peptidase 7 (KLK7) cleaved procaspase-14 at Y^178 , generating an
intermediate form consisting of a large (20 kDa) and a small sub-
unit (8 kDa) [ 10 ]. We confi rmed the formation of the intermediate
form by raising a cleavage-site-directed antibody, h14Y178. This
antibody was prepared in the same manner as h14D146, an anti-
body specifi c to the active, mature form of caspase-14. We also
constructed a constitutively active form of the intermediate,
revC14-Y178. The substrate specifi city of revC14-Y178 wasMami Yamamoto-Tanaka and Toshihiko Hibinohttp://www.ebook3000.com