Purification and characterization of active caspase-14 from human epidermis and development of the cleavage site-directed antibody.
J Cell Biochem
; 109(3): 487-97, 2010 Feb 15.
Article
en En
| MEDLINE
| ID: mdl-19960512
ABSTRACT
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Epidermis
/
Caspasa 14
Límite:
Humans
Idioma:
En
Revista:
J Cell Biochem
Año:
2010
Tipo del documento:
Article
País de afiliación:
Japón