Profiling of serine protease activities in human stratum corneum and detection of a stratum corneum tryptase‐like enzyme

Abstract

Seasonal variation in stratum corneum (SC) biophysical and biological characteristics has been described previously. In particular, the winter season has been shown to affect more severely the properties of facial skin compared with forearm skin. Moreover, when compromised, such as in dry skin conditions, facial SC has been shown to contain increased inflammatory cytokines and proteases. Nevertheless, there have been no comparative studies of the activities and depth activity of several proteases in the SC on different body sites and at different times of the year. In this study, we examined the distribution of key serine protease activities (kallikrein 5, kallikrein 7, urokinase, plasmin and a tryptase-like enzyme) in different layers of the SC on the cheek and the forearm by analysis of consecutive tape strippings of healthy Caucasian subjects during winter and summer. The protein content of the tape strippings was quantified by absorption measurements with a recently developed and novel infrared densitometer SquameScan 850A while the SC enzyme activities were determined using fluorogenic peptide substrates. Transepidermal water loss (TEWL), skin pH and skin hydration were higher on the cheek than on the forearm. In the same way, the activity of the inflammatory-related proteases plasmin, urokinase and tryptase was approximately five to eight times and the activity of the desquamatory-related proteases kallikrein 5 and kallikrein 7 approximately two to four times higher on the cheek than on the forearm. There were no gender-related differences in these enzyme activities except for the increased kallikrein 7 in the forearm skin of the female subjects in winter. Reduced kallikrein 5 was associated with increased SC cohesion, as judged by increased protein removal, in forearm skin in the winter months of the year although the skin was clinically normal. It can be concluded that (i) protected skin areas show lower TEWL, skin pH and skin hydration and less protease activities than skin areas that are exposed to the environment, possibly indicating subclinical inflammation on these body sites, (ii) in normal healthy forearm skin, the outer SC exhibits greater serine protease activity than its deeper layers, (iii) compared with the forearm, urokinase- and plasmin-like activities are elevated on SC strippings from the cheek, confirming activation of the plasminogen cascade, and (iv) tryptase-like activity in the SC is also elevated in samples from the cheek, possibly indicating involvement of mast cells in these barrier-compromised body sites or the synthesis of a novel tryptase-like enzyme by keratinocytes. Although elevation of the activities of urokinase, plasmin, kallikrein 5, kallikrein 7 and now a tryptase-like enzyme was observed on SC derived from skin of clinically normal cheeks, we anticipate even higher activities in skin conditions where the epidermal barrier is further impaired.

Characterisation of Skin Barrier Function Using Bioengineering and Biophysical Techniques

“…In addition, the face is one of the first body sites affected when atopic dermatitis (AD) develops and the ratio of fragile to mature corneocytes is higher here than on the inner upper arm (21). Voegeli et al (22) have also demonstrated higher levels of desquamation-related proteases on the face. All these features are found in AD and it was highlighted, therefore, that the forearm and forehead skin of healthy volunteers might model the distinction between a competent SC and one pre-predisposed to AD.…”

The Effect of an Emollient Containing Urea, Ceramide NP, and Lactate on Skin Barrier Structure and Function in Older People with Dry Skin

“…Prior to tape-stripping the test sites were cleaned with cotton wool and water, and allowed to reacclimatize for 20 min. Extrapolation of depth reached by tape-stripping was based on the infrared absorbance (SquameScan 850A, CuDerm, Dallas, Tex., USA) of tape strips (to quantify protein) in accordance with published methodology [22,23]. …”

“…Assessment of protease activity was made on samples comprising 3 consecutive tape strips as previously described [22]. Caseinolytic, chymotrypsin-like and trypsin-like activities were determined using EnzCheck® (Life Technologies Ltd., Paisley, UK), MeOSuc-Arg-Pro-Tyr-AMC (Peptide Protein Research Ltd., Funtley, UK), and Boc-Phe-Ser-Arg-AMC (Bachem, Bubendorf, Switzerland) substrates, respectively.…”

The Stratum Corneum and Aging

“…Only KLK5, 8, and 14, however, are capable of degrading Dsg1 [72], but it is known that KLK8 also contributes to desquamation [73]. A depth activity profile of these enzymes are reported [74,66], but Voegeli et al [75] recently demonstrated that premature activation of, and elevated activity levels of KLK5, but not KLK7, on barriercompromised body sites such as the face but not the forearm ( > Fig. 7.7).…”

“…There is a strong association of the . Figure 7.7 Protease activity (mean AE SEM) on tape-stripping pools of forearm and of cheek in function of depth of stratum corneum inhibitor-enzyme complexes at neutral pH, that is, as the SC is formed, and dissociation of the complexes occurs at low pH, within the outermost surface layers of the SC, where higher activities of the kallikreins are actually observed [75]. As at least pro-KLK7 is processed early in the formation of the SC and that LEKTI is secreted before the enzymes [78], presumably the inhibitors diffuse to the activated enzymes to control the later stages of desquamation.…”