Epidermal Cathepsin Research Articles

Ethnic skin types: are there differences in skin structure and function?1

People of skin of colour comprise the majority of the world's population and Asian subjects comprise more than half of the total population of the earth. Even so, the literature on the characteristics of the subjects with skin of colour is limited. Several groups over the past decades have attempted to decipher the underlying differences in skin structure and function in different ethnic skin types. However, most of these studies have been of small scale and in some studies interindividual differences in skin quality overwhelm any racial differences. There has been a recent call for more studies to address genetic together with phenotypic differences among different racial groups and in this respect several large-scale studies have been conducted recently. The most obvious ethnic skin difference relates to skin colour which is dominated by the presence of melanin. The photoprotection derived from this polymer influences the rate of the skin aging changes between the different racial groups. However, all racial groups are eventually subjected to the photoaging process. Generally Caucasians have an earlier onset and greater skin wrinkling and sagging signs than other skin types and in general increased pigmentary problems are seen in skin of colour although one large study reported that East Asians living in the U.S.A. had the least pigment spots. Induction of a hyperpigmentary response is thought to be through signaling by the protease-activated receptor-2 which together with its activating protease is increased in the epidermis of subjects with skin of colour. Changes in skin biophysical properties with age demonstrate that the more darkly pigmented subjects retaining younger skin properties compared with the more lightly pigmented groups. However, despite having a more compact stratum corneum (SC) there are conflicting reports on barrier function in these subjects. Nevertheless, upon a chemical or mechanical challenge the SC barrier function is reported to be stronger in subjects with darker skin despite having the reported lowest ceramide levels. One has to remember that barrier function relates to the total architecture of the SC and not just its lipid levels. Asian skin is reported to possess a similar basal transepidermal water loss (TEWL) to Caucasian skin and similar ceramide levels but upon mechanical challenge it has the weakest barrier function. Differences in intercellular cohesion are obviously apparent. In contrast reduced SC natural moisturizing factor levels have been reported compared with Caucasian and African American skin. These differences will contribute to differences in desquamation but few data are available. One recent study has shown reduced epidermal Cathepsin L2 levels in darker skin types which if also occurs in the SC could contribute to the known skin ashing problems these subjects experience. In very general terms as the desquamatory enzymes are extruded with the lamellar granules subjects with lowered SC lipid levels are expected to have lowered desquamatory enzyme levels. Increased pores size, sebum secretion and skin surface microflora occur in Negroid subjects. Equally increased mast cell granule size occurs in these subjects. The frequency of skin sensitivity is quite similar across different racial groups but the stimuli for its induction shows subtle differences. Nevertheless, several studies indicate that Asian skin maybe more sensitive to exogenous chemicals probably due to a thinner SC and higher eccrine gland density. In conclusion, we know more of the biophysical and somatosensory characteristics of ethnic skin types but clearly, there is still more to learn and especially about the inherent underlying biological differences in ethnic skin types.

Precursor of rat epidermal cathepsin L: purification and immunohistochemical localization

Immunoblot study using anti-rat cathepsin L antibody revealed almost only a precursor present in a crude extract of homogenized lower epidermis of rat skin, while the precursor and mature form were detected in the upper epidermis. To elucidate mechanisms of synthesis of cathepsin L, we have purified a precursor of cathepsin L from rat epidermis and investigated its localization in the skin. The precursor was purified after separation from the mature form in the final purification step of active fraction for N-benzyloxycarbonyl- l-phenylalanyl- l-arginine-7-amido-4-methylcoumarin. The precursor showed a single protein band with Mr 39 kDa on SDS/polyacrylamide gel electrophoresis and was immunoreacted with the anti-rat cathepsin L antibody. Two types of NH 2-terminal sequences obtained were identical to the amino acid residues from −96 to −86 and those from −93 to −87 deduced from cDNA of the precursor of cathepsin L. The precursor was processed to mature form of the enzyme and the enzyme activity remarkably increased after 48-h incubation of the whole epidermis in 1 M acetate buffer (pH 3.5) at 20°C. In histological sections of the skin, a thin and diffuse staining pattern was present in the spinous layer and a dense and linear staining in the granular layer of the epidermis. In contrast, rat liver showed more mature form than the precursor by immunohistological findings. These results suggest that cathepsin L may have some roles in the terminal differentiation.

Lysis of pathogenic bacteria by epidermal cathepsins L and B in the Japanese eel

Cysteine proteases, cathepsins L and B, in Japanese eel epidermis are suspected bacteriolysins against aquatic pathogens. This work examines the lysis of three species of gram-negative pathogenic bacteria by the eel epidermal extract according to the assay method for cysteine proteases. The optimum pH for the lysis of the three species of bacteria were commonly determined to be 5.0, where both of eel epidermal cathepsins L and B caused active proteolysis. Four kinds of specific inhibitors for cysteine proteases strongly inhibited these catheptic proteolyses and bacteriolyses. The activities for the lysis of three types of bacteria were at a similar level, but the effects on the bacterium which infects predominantly eel skin largely varied with individual fish. These results suggest that epidermal cathepsins L and B are responsible for lysis of any pathogenic bacteria in the nonspecific defense of Japanese eel.

Epidermal response of the Japanese eel to environmental stress

Nonspecific responses of Japanese eels to environmental stress were monitored by assaying various lytic activities in eel epidermal extract. In fish maintained at 10 and 30 °C for up to 10 days, epidermal proteolytic activities due to serine protease and aminopeptidase and hemolytic activity varied within a 2-fold value range. Other proteolytic activities, due to cathepsins B and L, in the fish at 30 °C increased for up to 8 days and were 3.4 and 2.9-fold over those in fish maintained at 10 °C, respectively. This was accompanied by a 3.0-fold increase in bacteriolytic activity. Other forms of stress were exerted on the fishes by immersing them in a suspension of Flavobacterium columnare or giving them intraperitoneal injections of Edwardsiella tarda over 72 h. Although serine protease and aminopeptidase activities and hemolytic activity in the fishes exposed to F. columnare changed marginally, and were similar to those in the control fish, cathepsins B and L activities in the infected fishes increased more than 1.5-fold over their initial values over a 48 h period, along with a 4.5-fold increase in bacteriolytic activity. No marked change was detected in any of the lytic activities of the fishes exposed to E. tarda. These findings indicate that epidermal cathepsins B and L probably participate in bacteriolysis associated with Japanese eel skin and that their activities are elicited by environmental stimuli and may be an important nonspecific response of eels. Abbreviations: Cbz – carbobenzoxy; MCA – 4-methylcoumaryl-7-amide.

Fluorescence localization of epidermal cathepsins L and B in the Japanese eel

Histochemical localization of proteolytic activities in the dorsal epidermis of Japanese eel was demonstrated by fluorescent microscopy utilizing 4-methoxy-2-naphthylamide (4M$\beta$NA) derivatives as substrates and 5-nitrosalicylaldehyde as a trapping agent. Carbobenzoxy-L-phenylalanyl-L-arginyl-4M$\beta$NA (Cbz-Phe-Arg-4M$\beta$NA) and Cbz-Arg-Arg-4M$\beta$NA were used for direct detection of cathepsins L and B activities, respectively, in fresh frozen sections and unfixed cells of the eel epidermis. The fluorescing areas, where Cbz-Phe-Arg-4M$\beta$NA was hydrolyzed by cathepsin L, were shown in mucus secretory cells and club cells and broadly around skin surface. The fluorescing areas due to Cbz-Arg-Arg-4M$\beta$NA hydrolysis by cathepsin B were localized similarly in these tissues. The fluorescing intensity for both catheptic activities in mucus secretory cells was higher than that in club cells, where small fluorescing granules were distributed. These results indicate that eel cathepsins L and B are stored in epidermal secretory cells at different levels and probably serve as defense factors before or after secretion by these cells. Abbreviations: Cbz – carbobenzoxy; 4M$\beta$NA – 4-methoxy-2-naphthylamide; NSA – 5-nitrosalicylaldehyde.

112 Rat epidermal cathepsin L-like proteinase: Purification and some hydrolytic properties toward filaggrin and synthetic substrates

112 Rat epidermal cathepsin L-like proteinase: Purification and some hydrolytic properties toward filaggrin and synthetic substrates

Rat Epidermal Cathepsin L-Like Proteinase: Purification and Some Hydrolytic Properties toward Filaggrin and Synthetic Substrates

We have purified cathepsin L-like proteinase from rat epidermis, determined its NH2-terminal amino acid sequence, and investigated its proteolytic activities on an intermediate filament-associated protein filaggrin and several synthetic substrates. The amino acid sequence of its NH2-terminus was determined to be Val-Pro-Asn-Ser-Leu-Asp-Trp-Arg-Glu-Lys-Gly-Tyr-Val-Thr-Pro-, which differed from that of rat cathepsin L and was not found in the amino acid sequence data bank. The enzyme consisted of a single-chain form with M(r) 30,000. Its hydrolytic properties toward synthetic substrates were similar to those of cathepsin L in other tissues. The enzyme effectively proteolyzed rat epidermal filaggrin into small fragments at pH 4.0-6.0 and was inhibited by a specific cysteine proteinase inhibitor, N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)L-leucyl]-agmatin. However, cathepsins D and E from rat epidermis did not hydrolyze filaggrin. This study demonstrated that filaggrin was susceptible to degradation by rat epidermal cathepsin L-like proteinase, suggesting that this proteolytic activity may have relevance to skin differentiation, in which acid proteases are thought to participate.

052 Rat epidermal cathepsin B: Purification and characterization of proteolytic properties toward filaggrin and synthetic substrates

052 Rat epidermal cathepsin B: Purification and characterization of proteolytic properties toward filaggrin and synthetic substrates

Rat epidermal cathepsin B: Purification and characterization of proteolytic properties toward filaggrin and synthetic substrates

The aim of this study was to purify epidermal cathepsin B from rat skin and investigate its proteolytic activities on filaggrin and several synthetic substrates. The molecular weight of purified monomeric cathepsin B was estimated to be 30 kDa by SDS-polyacrylamide gel electrophoresis. The amino acid composition, similar to that of liver cathepsin B, indicated the enzyme to be an acidic protease. The enzyme had strong hydrolytic activity toward N-benzyloxycarbonyl- l-arginyl- l-arginine-7-amido-4-methylcoumarin (Z-Arg-Arg-MCA) (152 mU/mg) and N-benzyloxycarbonyl- l-phenylalanyl- l-arginine-7-amido-4-methylcoumarin (424 mU/mg), but had no proteolytic activity toward l-arginine-7-amido-4-methylcoumarin. The K m value for Z-Arg-Arg MCA was 0.34 mM and pH optimun was 5.5. Cathepsin B degraded rat epidermal filaggrin into small fragments at pH 4.0 and 5.5, and was inhibited by a specific cysteine proteinase inhibitor, N-[ N-( l-3-trans-carboxyoxirane-2-carbonyl) l-leucyl]-agmatin. This study demonstrated that filaggrin was susceptible to degradation by cathepsin B. Such an action may have relevance to skin differentiation in which acid proteases are thought to participate.

Purification and Immunohistochemical Localization of Aspartic Proteinases in Rat Epidermis

Investigation of skin cathepsin E (EC 3.4.23.-) has been totally ignored compared to skin cathepsin D (ED 3.4.23.5). In this study both cathepsins E and D were simultaneously purified to homogeneity up to 370 and 640 times, respectively, from 2-day-old rat epidermis. The total aspartic proteinase activity of rat epidermis detected after Q-Sepharose column chromatography was attributed to 27% cathepsin E, 63% cathepsin D, and 10% other enzymes. The purified enzymes showed that cathepsin E (90 kDa) is a dimer of 45 kDa subunits whereas cathepsin D is a monomer of 42 kDa. Other biochemical properties of epidermal cathepsins E and D were similar to those reported from other tissue sources. Immunologically cathepsins E and D were distinct from each other and localization of the two enzymes differed in both rat and human skin by immunohistochemistry. Cathepsin E was distributed diffusely in the cytoplasm of almost all epidermal cells, though its concentration increased above suprabasal cells, whereas cathepsin D appeared in particulate form only in cells of the granular layer. The findings indicate that two aspartic proteinases that have similar enzymatic properties exist in the epidermis. They are, however, differentially distributed in the organ, presumably for different functions during the process of keratinization.

Changes of lysosomal proteinase activities and their expression in rat cultured keratinocytes during differentiation

The cathepsins B, H and L, lysosomal cysteine proteinases, play a major role in intracellular protein degradation. These proteinase activities and expressions were examined in a Ca 2+ regulated epidermal culture system which consists of two morphological cell types: undifferentiated cells grown in low Ca 2+ (0.1 mM concentration) and differentiated cells grown in high Ca 2+ (1.8 mM concentration), respectively. Cathepsin B and L activities of the differentiated cells showed a several-fold increase compared to that of the undifferentiated cells. In addition, by using CM-cellulose column chromatography, cathepsin B and L were separated and the level of cathepsin L activity increased significantly. Cathepsin B, L and H were also detected by using an immunoblotting procedure in which their bands were expressed after differentiation was induced by the increasing calcium concentration. Cathepsin L activity and immunostaining intensity reached a maximum at 1 or 2 days of differentiation. In contrast, cystatin α (an endogenous inhibitor of cysteine-dependent cathepsins) appeared in the final stage of differentiation. These results indicate that the expression of epidermal cathepsins and their endogenous inhibitor are involved in part of the program of cell differentiation and the terminal differentiation process in cultured rat keratinocytes.

Separation and Identification of Cathepsins in Newborn Rat Epidermis

Cathepsins B, D, H, and L were identified in the extract of 2-day-old rat epidermis and separated by gel filtration from aminoendopeptidase with a Mr of 400,000 and from the low-molecular-weight cysteine proteinase inhibitor. They were further purified by ion exchange column chromatography. The final separation for cathepsins B and H was performed by gel filtration, while cathepsin D was purified by pepstatin affinity chromatography and cathepsin L by fast protein liquid chromatography (FPLC). Substrate specificity, inhibitor susceptibility, and apparent molecular weights of the separated proteinases were determined and values compared to rat liver enzymes. Apparent molecular weights for epidermal cathepsins B, H, and L were higher than those for comparable liver enzymes of adult rats. The cysteine proteinase inhibitor in epidermis was found to inhibit cathepsins B, H, and L but not cathepsin D and aminoendopeptidase of rat epidermis. This study demonstrates the presence of cathepsin L in the epidermis and describes simultaneous separation and comparison of epidermal catheptic proteinases.