Caspase-14 protocols.

Unlike other caspase family members, caspase-14 shows restricted expression, being found mostly in epidermis and its appendages. It has been suggested that caspase-14 is not involved in apoptosis or inflammation, but participates in keratinocyte terminal differentiation. Its activation occurs at the corneocyte formation. In previous work, we have purified active caspase-14 from human corneocyte extracts. In addition, we have clarified activation mechanism of caspase-14, where kallikrein-related peptidase 7 (KLK7) generates an intermediate form from procaspase-14 and this form finally converts procaspase-14 to active, mature caspase-14. Here we describe techniques for measurement of caspase-14 activity using synthetic substrate, purification of caspase-14 from corneocyte extract, preparation of constitutively active caspase-14 and specific antibody, quantification of total and active caspase-14 in corneocyte extracts using ELISA, as well as methods for caspase-14 activation and its visualization by immunohistochemistry.

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.