Kallikrein-related Peptidase-8 (KLK8) Is an Active Serine Protease in Human Epidermis and Sweat and Is Involved in a Skin Barrier Proteolytic Cascade

Abstract

Kallikrein-related peptidase-8 (KLK8) is a relatively uncharacterized epidermal protease. Although proposed to regulate skin-barrier desquamation and recovery, the catalytic activity of KLK8 was never demonstrated in human epidermis, and its regulators and targets remain unknown. Herein, we elucidated for the first time KLK8 activity in human non-palmoplantar stratum corneum and sweat ex vivo. The majority of stratum corneum and sweat KLK8 was catalytically active, displaying optimal activity at pH 8.5 and considerable activity at pH 5. We also showed that KLK8 is a keratinocyte-specific protease, not secreted by human melanocytes or dermal fibroblasts. KLK8 secretion increased significantly upon calcium induction of terminal keratinocyte differentiation, suggesting an active role for this protease in upper epidermis. Potential activators, regulators, and targets of KLK8 activity were identified by in vitro kinetic assays using pro-KLK8 and mature KLK8 recombinant proteins produced in Pichia pastoris. Mature KLK8 activity was enhanced by calcium and magnesium ions and attenuated by zinc ions and by autocleavage after Arg 164 . Upon screening KLK8 cleavage of a library of FRET-quenched peptides, trypsin-like specificity was observed with the highest preference for (R/K)(S/T)(A/V) at P1-P1-P2. We also demonstrated that KLK5 and lysyl endopeptidase activate latent pro-KLK8, whereas active KLK8 targets pro-KLK11, pro-KLK1, and LL-37 antimicrobial peptide activation in vitro. Together, our data identify KLK8 as a new active serine protease in human stratum corneum and sweat, and we propose regulators and targets that augment its involvement in a skin barrier proteolytic cascade. The implications of KLK8 elevation and hyperactivity in desquamatory and inflammatory skin disease conditions remain to be studied.

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

“…Indeed, presence of a PI-6⅐KLK8 complex has been reported in the cytoplasm of differentiated keratinocyte (28,29). Furthermore, pro-KLK8 is activated by the cleavage at Lys 32 (28). They also suggested Lys-specific enterokinase could be involved in this process, because it is expressed at the SG/SC boundary area (30).…”

“…Although proKLKs are secreted into lamellar granules and further into the intercellular space of the SC (26), this might not be the case for all the KLKs. Indeed, presence of a PI-6⅐KLK8 complex has been reported in the cytoplasm of differentiated keratinocyte (28,29). Furthermore, pro-KLK8 is activated by the cleavage at Lys 32 (28).…”

False Biomarker Discovery due to Reactivity of a Commercial ELISA for CUZD1 with Cancer Antigen CA125

“…A stable clone was selected from the GS115 strain according to the manufacturer's recommendations (Invitrogen). Protein was produced by growing the stable yeast clone in methanol-containing media following our previously reported protocols (10 ). CUZD1 expression and identity were confirmed with MS analysis.…”

Sweetened kallikrein-related peptidases (KLKs): glycan trees as potential regulators of activation and activity

“…Besides occurring in skin, expression of mammalian KLK8 appears to be mostly restricted to the hippocampus, where it contributes to long term potentiation (LTP) and memory acquisition by rebuilding of synaptic connections (Tamura et al, 2006;Eissa et al, 2011). In line with this proposed function, genetic variations of KLK8 in the 3′ regulatory region of the gene have been observed in patients with manic-depressive disorder and cognitive impairment (Izumi et al, 2008).…”