Cross-Linking of SPINK6 by Transglutaminases Protects from Epidermal Proteases

Extracellular kallikrein-related peptidases (KLKs) are involved in the desquamation process and the initiation of epidermal inflammation by different mechanisms. Their action is tightly controlled by specific protease inhibitors. Recently, we have identified the serine protease inhibitor of Kazal-type (SPINK) 6 as a selective inhibitor of KLKs in human stratum comeum extracts. As SPINK6 is expressed in the same localization as transglutaminases (TGM) and contains TGM substrate motifs, SPINK6 was tested to be cross-linked in the epidermis. Recombinant SPINK6 was shown to be cross-linked to fibronectin (FN) by TGM1 by western blot analyses. Moreover, SPINK6 was crosslinked in epidermal extracts and cultured keratinocytes by innmunoblotting analyses. The use of TGM1 and TGM3 resulted in different immunoreactivities in western blot analyses of SPINK6 and epidermal extracts, suggesting substrate specifities of different TGMs for SPINK6 cross-linking in the epidermis. Conjugated SPINK6 exhibited protease inhibitory activity in keratinocytes and stratum comeum extracts; cross-linked SPINK6 protected FN from KLK5-mediated cleavage, whereas a lower KLK-inhibiting SPINK6-GM mutation did not. In conclusion, we demonstrated that SPINK6 is cross-linked in keratinocytes and human epidermis and remains inhibitory active. Thus, cross-linked SPINK6 might protect specific substrates such as FN from KLK cleavage and contributes to the regulation of proteases in the epidermis.