Purification and characterization of active caspase-14 from human epidermis and development of the cleavage site-directed antibody.
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.
Hibino
Toshihiko
T
Shiseido Life Science Research Center, 2-12-1 Fukuura, Kanazawa-ku, Yokohama 236-8643, Japan.
Fujita
Eriko
E
Tsuji
Yumiko
Y
Nakanishi
Jotaro
J
Iwaki
Haruhi
H
Katagiri
Chika
C
Momoi
Takashi
T
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D001665:Binding Sites; D053456:Caspase 14 / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D002454:Cell Differentiation; D004817:Epidermis / Q000737:chemistry / Q000201:enzymology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D015603:Keratinocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D000906:Antibodies; D011994:Recombinant Proteins; D053456:Caspase 14