Biodegradation of exogenously applied silk peptides by stratum corneum cysteine proteases
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Presented by: Miyuki Omoi
Background
The stratum corneum (SC), the outermost layer of the skin, is responsible for barrier and water-retaining functions to maintain the health of the skin. Its critical components are natural moisturizing factors (NMFs) and intercellular lipids. Amino acids constituting the majority of the NMFs are produced by the degradation of filaggrin, which is expressed as a precursor protein, profilaggrin, in the granular layer, followed by degradation into filaggrin in the process of moving to the SC. It has been clarified that filaggrin is degraded by various SC proteases. Bleomycin hydrolase (BH) is a type of aminopeptidase of the cysteine protease family that degrades filaggrin-derived peptides into free amino acids. Additionally, SC proteases such as KLK5 and KLK7 degrade corneodesmosomes for exfoliation of the SC and play pivotal roles in maintaining skin homeostasis. Thus, various proteases are active in the SC.
Various peptides are included in skincare and haircare cosmetics for the purposes of adhesion, repair, and moisturization. However, no study has reported the action of SC proteases towards exogenous peptides applied to the SC. Therefore, we hypothesized that exogenous peptides can be hydrolyzed into amino acids by SC proteases and that these amino acids may serve as NMFs. We focused on hydrolyzed silk protein (silk peptides), which includes various peptides and has been used as a cosmetic ingredient. In this study, we aimed to clarify whether silk peptides can be hydrolyzed by SC proteases and determine the characteristics of SC proteases involved in silk peptide degradation.
Methods
Samples of the outermost SC were collected non-invasively from the cheek of healthy volunteers by stripping with adhesive tape, and were incubated in the presence of silk peptide solution at 37℃ for 48 h. Degradation of peptides was monitored by the increase of amino groups quantified by the ninhydrin method, which was corrected by the quantified protein content of the tape-stripped SC. Aminopeptidase activity of the tape-stripped SC was detected using a fluorogenic substrate, Arg-4-methylcoumaryl-7-amide.
Results and Discussion
Incubation of silk peptides with a tape-stripped SC resulted in an increase in the quantity of amino groups in a time-dependent manner, clearly showing that silk peptides can be degraded by SC proteases into smaller peptides or amino acids. The pH dependence and protease inhibitor profiles of the silk peptide degradation by SC proteases showed the involvement of cysteine proteases that function optimally at neutral pH. As we also detected the activity of aminopeptidase in the tape-stripped SC using a fluorogenic substrate, it was suggested that BH is involved, at least in part, in the degradation of silk peptides by SC proteases. Interestingly, we obtained evidence suggesting that not only cysteine proteases but also other proteases are present in the SC and these proteases may also degrade silk peptides. These results suggest that exogenously applied silk peptides are degraded by multiple SC proteases.
Conclusion
We found that exogenously applied silk peptides are degraded by cysteine proteases such as BH. This concept of peptide degradation upon application to the SC provides a novel approach to skin moisturization.
The stratum corneum (SC), the outermost layer of the skin, is responsible for barrier and water-retaining functions to maintain the health of the skin. Its critical components are natural moisturizing factors (NMFs) and intercellular lipids. Amino acids constituting the majority of the NMFs are produced by the degradation of filaggrin, which is expressed as a precursor protein, profilaggrin, in the granular layer, followed by degradation into filaggrin in the process of moving to the SC. It has been clarified that filaggrin is degraded by various SC proteases. Bleomycin hydrolase (BH) is a type of aminopeptidase of the cysteine protease family that degrades filaggrin-derived peptides into free amino acids. Additionally, SC proteases such as KLK5 and KLK7 degrade corneodesmosomes for exfoliation of the SC and play pivotal roles in maintaining skin homeostasis. Thus, various proteases are active in the SC.
Various peptides are included in skincare and haircare cosmetics for the purposes of adhesion, repair, and moisturization. However, no study has reported the action of SC proteases towards exogenous peptides applied to the SC. Therefore, we hypothesized that exogenous peptides can be hydrolyzed into amino acids by SC proteases and that these amino acids may serve as NMFs. We focused on hydrolyzed silk protein (silk peptides), which includes various peptides and has been used as a cosmetic ingredient. In this study, we aimed to clarify whether silk peptides can be hydrolyzed by SC proteases and determine the characteristics of SC proteases involved in silk peptide degradation.
Methods
Samples of the outermost SC were collected non-invasively from the cheek of healthy volunteers by stripping with adhesive tape, and were incubated in the presence of silk peptide solution at 37℃ for 48 h. Degradation of peptides was monitored by the increase of amino groups quantified by the ninhydrin method, which was corrected by the quantified protein content of the tape-stripped SC. Aminopeptidase activity of the tape-stripped SC was detected using a fluorogenic substrate, Arg-4-methylcoumaryl-7-amide.
Results and Discussion
Incubation of silk peptides with a tape-stripped SC resulted in an increase in the quantity of amino groups in a time-dependent manner, clearly showing that silk peptides can be degraded by SC proteases into smaller peptides or amino acids. The pH dependence and protease inhibitor profiles of the silk peptide degradation by SC proteases showed the involvement of cysteine proteases that function optimally at neutral pH. As we also detected the activity of aminopeptidase in the tape-stripped SC using a fluorogenic substrate, it was suggested that BH is involved, at least in part, in the degradation of silk peptides by SC proteases. Interestingly, we obtained evidence suggesting that not only cysteine proteases but also other proteases are present in the SC and these proteases may also degrade silk peptides. These results suggest that exogenously applied silk peptides are degraded by multiple SC proteases.
Conclusion
We found that exogenously applied silk peptides are degraded by cysteine proteases such as BH. This concept of peptide degradation upon application to the SC provides a novel approach to skin moisturization.