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Purification and characterization of active caspase‐14 from human epidermis and development of the cleavage site‐directed antibody
Author(s) -
Hibino Toshihiko,
Fujita Eriko,
Tsuji Yumiko,
Nakanishi Jotaro,
Iwaki Haruhi,
Katagiri Chika,
Momoi Takashi
Publication year - 2009
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.22425
Subject(s) - protein subunit , epidermis (zoology) , caspase , cleavage (geology) , tunel assay , microbiology and biotechnology , caspase 3 , chemistry , caspase 9 , biology , biochemistry , apoptosis , programmed cell death , anatomy , paleontology , fracture (geology) , gene
Restricted expression of caspase‐14 in differentiating keratinocytes suggests the involvement of caspase‐14 in terminal differentiation. We purified active caspase‐14 from human cornified cells with sequential chromatographic procedures. Specific activity increased 764‐fold with a yield of 9.1%. Purified caspase‐14 revealed the highest activity on WEHD‐methylcoumaryl‐amide (MCA), although YVAD‐MCA, another caspase‐1 substrate, was poorly hydrolyzed. The purified protein was a heterodimer with 17 and 11 kDa subunits. N‐terminal and C‐terminal analyses demonstrated that the large subunit consisted of Ser 6 ‐Asp 146 and N‐terminal of small subunit was identified as Lys 153 . We successfully developed an antiserum (anti‐h14D146) directed against the Asp 146 cleavage site, which reacted only with active caspase‐14 but not with procaspase‐14. Furthermore we confirmed that anti‐h14D146 did not show any reactivity to the active forms of other caspases. Immunohistochemical analysis demonstrated that anti‐h14D146 staining was mostly restricted to the cornified layer and co‐localized with some of the TUNEL positive‐granular cells in the normal human epidermis. UV radiation study demonstrated that caspase‐3 was activated and co‐localized with TUNEL‐positive cells in the middle layer of human epidermis. In contrast, we could not detect caspase‐14 activation in response to UV. Our study revealed tightly regulated action of caspase‐14, in which only the terminal differentiation of keratinocytes controls its activation process. J. Cell. Biochem. 109: 487–497, 2010. © 2009 Wiley‐Liss, Inc.