Purification and characterization of active caspase‐14 from human epidermis and development of the cleavage site‐directed antibody

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.

Kallikrein-related Peptidase-7 Regulates Caspase-14 Maturation during Keratinocyte Terminal Differentiation by Generating an Intermediate Form

“…h14Y178 Ab staining was confined to the cytoplasm of granular cells just below the SC. The mature form stained with h14D146 Ab was localized in the SC, whereas the staining of the proform started from the upper spinous cells, as previously reported (12). Transient appearance of the intermediate form just prior to corneocyte formation strongly supports the involvement of the proposed activation cascade of procaspase-14 in vivo.…”

“…We confirmed the formation of the intermediate form by raising a cleavage site-directed antibody, h14Y178 Ab. This antibody was prepared in the same manner as h14D146, an antibody specific to the active, mature form of caspase-14 (12). h14Y178 Ab specifically recognizes p20, the large subunit of the intermediate form, and does not react with procaspase-14.…”

“…Construction of Constitutively Active Intermediate Form (revC14-Y178)-Human caspase-14 was purified from cornified cells according to our previous work, using successive chromatographies (12). Based on the sequence information, a constitutively active intermediate form (revC14-Y178) was prepared by reversing the large and small subunits, ending at Tyr 178 (see Fig.…”

“…The C-terminal of the large subunit was identified as Asp-146. This is identical with the granzyme B-cleaved activation site, although granzyme B is not the physiological activation enzyme of caspase-14 (12,13). A cleavage site-directed antibody, h14D146, was prepared and found to react only with active caspase-14.…”

“…We have recently purified active caspase-14 from extract of human cornified cells (12), and found that it consists of a large subunit (p17) and a small subunit (p11) generated by the removal of a 6-amino acid linker peptide. The C-terminal of the large subunit was identified as Asp-146.…”