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Background: Deimination (also known as citrullination), the conversion of arginine in a protein to citrulline, is catalyzed by a family of enzymes called peptidylarginine deiminases (PADs). Three PADs are expressed in the epidermis, one of their targets being filaggrin. Filaggrin plays a central role in atopic dermatitis and is a key protein for the epidermal barrier. It aggregates keratins and is cross-linked to cornified envelopes. Following its deimination, it is totally degraded to release free amino acids, contributing to the natural moisturizing factor (NMF). The mechanisms controlling this multistep catabolism in human are unknown., Objective: To test whether external humidity plays a role, and investigate the molecular mechanisms involved., Methods: Specimens of reconstructed human epidermis (RHEs) produced in humid or dry conditions (>95% or 30-50% relative humidity) were compared., Results: RHEs produced in the dry condition presented structural changes, including a thicker stratum corneum and a larger amount of keratohyalin granules. The transepidermal water loss and the stratum corneum pH were decreased whereas the quantity of NMF was greater. This highly suggested that filaggrin proteolysis was up-regulated. The expression/activity of the proteases involved in filaggrin breakdown did not increase while PAD1 expression and the deimination rate of proteins, including filaggrin, were drastically enhanced. Partial inhibition of PADs with Cl-amidine reversed the effect of dryness on filaggrin breakdown., Conclusion: These results demonstrate the importance of external humidity in the control of human filaggrin metabolism, and suggest that deimination plays a major role in this regulation., (Copyright © 2017 Japanese Society for Investigative Dermatology. All rights reserved.)

Background: Peptidylarginine deiminases (PADs) catalyze deimination (or citrullination), a calcium-dependent post-translational modification involved in several physiological processes and human diseases, such as rheumatoid arthritis and cancer. Deimination of filaggrin (FLG) by PAD1 and PAD3 during the last steps of keratinocyte differentiation is a crucial event for the epidermis function and homeostasis. This allows the complete degradation of FLG, leading to the production of free amino acids and their derivatives that are essential for epidermal photoprotection and moisturizing of the stratum corneum., Objective: To increase the flux of this catabolic pathway, we searched for activators of PADs., Methods: A large chemical library was screened first in silico and then by using an automated assay based on an indirect colorimetric measurement of recombinant human PAD activity. Potential activators were then confirmed using a recombinant human FLG as a substrate, and secondly after topical application at the surface of three-dimensional reconstructed human epidermis., Results: The data obtained after the library screening pointed to xanthine derivatives as potential PAD activators. Among seven xanthine derivatives tested at 50-300μM, caffeine, theobromine and acefylline proved to be the most potent enhancers of in vitro deimination of FLG by PAD1 and PAD3. After topical application of a gel formulation containing 3% acefylline at the surface of reconstructed epidermis, immunoblotting analysis showed an increase in the total amount of deiminated proteins, and confocal microscopy showed an enhanced deimination in the stratum corneum. This demonstrated the activation of PADs in living cells., Conclusion: As a PAD activator, acefylline will be useful to study the role of deimination and could be proposed to increase or correct the hydration of the cornified layers of the epidermis., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: The tight junction (TJ) barrier is located in the granular layer of the epidermis. Filaggrin deficiency predisposes patients to atopic dermatitis (AD) by impairing stratum corneum (SC) barrier function. Altered TJ barrier function has been observed in the skin of patients with AD; however, it remains unclear whether TJ function is influenced by filaggrin deficiency directly or secondarily via skin inflammation., Objective: To investigate the in vivo effects of filaggrin deficiency and skin inflammation on epidermal TJ function., Methods: Morphological changes in the TJ were investigated in filaggrin knockout mice and mice with hapten-induced dermatitis using en face visualization of epidermal sheets, and functional changes in the TJ were assessed with an in vivo permeation assay using tracers of various sizes., Results: In filaggrin knockout mice, there was no apparent change in the honeycomb morphology of the TJ, TJ component mRNA expression, or TJ barrier function in neonates and adults, indicating that filaggrin-deficiency had no direct effects on the TJ. By contrast, in mice with hapten-induced dermatitis, the mRNA expression of TJ components was decreased markedly and the TJ barrier function was size-dependently impaired: the TJ leaked small tracers (<5 kDa), but not large tracers (>30 kDa)., Conclusion: Filaggrin deficiency did not affect the epidermal TJ barrier directly, but once dermatitis occurred, the skin inflammation induced TJ dysfunction. Since TJ dysfunction induces the SC barrier impairment, skin inflammation will enhance skin permeability to external antigens and result in a vicious cycle of barrier dysfunction and skin inflammation., (Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

The S100 fused-type proteins (SFTPs) are thought to be involved in the barrier formation and function of the skin. Mutations in the profilaggrin gene, one of the best investigated members of this family, are known to be the major risk factors for ichthyosis vulgaris and atopic dermatitis. Recently, we identified human filaggrin-2 as a new member of the SFTP family. To achieve further insight into its function, here the murine filaggrin-2 was analysed as a possible orthologue. The 5' and 3' ends of the mouse filaggrin-2 cDNA of the BALB/c strain were sequenced and confirmed an organization typical for SFTPs. Murine filaggrin-2 showed an expression pattern mainly in keratinizing epithelia in the upper cell layers on both mRNA and protein levels. The expression in cultured mouse keratinocytes was increased upon elevated Ca(2+) levels. Immunoblotting experiments indicated an intraepidermal processing of the 250-kDa full-length protein. In metabolically (essential fatty acid-deficient diet) induced skin barrier dysfunction, filaggrin-2 expression was significantly reduced, whereas filaggrin expression was up-regulated. In contrast, mechanical barrier disruption with acetone treatment did not affect filaggrin-2 mRNA expression. These results suggest that filaggrin-2 may contribute to epidermal barrier function and its regulation differs, at least in parts, from that of filaggrin., (© 2012 John Wiley & Sons A/S.)

Filaggrin-2 (FLG2), a member of the S100-fused type protein family, shares numerous features with filaggrin (FLG), a key protein implicated in the epidermal barrier functions. Both display a related structural organization, an identical pattern of expression and localization in human epidermis, and proteolytic processing of a large precursor. Here, we tested whether FLG2 was a substrate of calpain 1, a calcium-dependent protease directly involved in FLG catabolism. In addition, deimination being critical for FLG degradation, we analyzed whether FLG2 deimination interfered with its proteolytic processing. With this aim, we first produced a recombinant form of FLG2 corresponding to subunits B7 to B10 fused to a COOH-terminal His tag. Incubation with calpain 1 in the presence of calcium induced a rapid degradation of the recombinant protein and the production of several peptides, as shown by Coomassie Blue-stained gels and Western blotting with anti-FLG2 or anti-His antibodies. MALDI-TOF mass spectrometry confirmed this result and further evidenced the production of non-immunoreactive smaller peptides. The degradation was not observed when a calpain 1-specific inhibitor was added. The calpain cleavage sites identified by Edman degradation were regularly present in the B-type repeats of FLG2. Moreover, immunohistochemical analysis of normal human skin revealed colocalization of FLG2 and calpain 1 in the upper epidermis. Finally, the FLG2 deiminated by human peptidylarginine deiminases was shown to be more susceptible to calpain 1 than the unmodified protein. Altogether, these data demonstrate that calpain 1 is essential for the proteolytic processing of FLG2 and that deimination accelerates this process.

Mutations in the gene-encoding filaggrin (FLG), a key molecule involved in skin barrier function, have been shown to be a major predisposing factor for atopic dermatitis (AD; eczema). To elucidate the pathomechanisms underlying filaggrin-related AD, we investigated stratum corneum (SC) hydration and transepidermal water loss (TEWL) as parameters of barrier function in AD patients harboring FLG mutations compared to AD patients without any FLG mutation. In filaggrin-related AD, SC hydration was both significantly reduced (P<0.01-0.05) and thicker (P<0.01-0.05) than that in healthy controls. TEWL was demonstrably increased in non-filaggrin AD compared to healthy controls (P<0.01-0.05). The objective score of atopic dermatitis (OSCORAD), a disease clinical severity index, significantly correlated with TEWL (r=0.81, P<0.005), SC hydration (r=-0.65, P<0.05), and SC thickness (r=0.59, P<0.05) in filaggrin-related AD. On the contrary, there was no correlation between these parameters and the OSCORAD in non-filaggrin AD. Furthermore, a significant correlation was obtained between the OSCORAD and specific IgE for house dust (r=0.66, P<0.05), mite allergen (r=0.53, P<0.05), and cat dander (r=0.64, P<0.05) in filaggrin-related AD, but not in non-filaggrin AD. All these data suggest that experimentally demonstrable skin barrier defects due to FLG mutations may play a crucial role in the pathogenesis of AD.

The outermost layer of the skin, the stratum corneum (SC), is composed of corneocytes and an intercellular lipid matrix. The matrix acts as both the main barrier and also as the pathway of water, drugs, etc. across the SC. In the mammalian SC, the longitudinal arrangement of the lipid molecules, consisting of long and short lamellar structures with repeat distances of about 13 nm and 6 nm, respectively, has been observed by small-angle X-ray diffraction. In the lateral arrangement of the lipid molecules, hexagonal and orthorhombic hydrocarbon-chain packing has been observed by wide-angle X-ray diffraction. From the systematic study of the temperature dependence of simultaneous small- and wide-angle X-ray diffraction patterns, we demonstrate that the intercellular lipid matrix forms two domains, which consist at room temperature of a long lamellar structure with hexagonal hydrocarbon-chain packing and a short lamellar structure with orthorhombic hydrocarbon-chain packing.

Little is known about proteins involved in the formation of the stratum corneum in the avian apteric epidermis. The present immunocytochemical, autoradiographic and electrophoretic study shows that antibodies against characteristic proteins of mammalian cornification (alpha-keratins, loricrin, sciellin, filaggrin, transglutaminase) recognize avian epidermal proteins. This suggests the presence of avian protein with epitopes common to related mammalian proteins. These proteins may also be involved in the formation of the cornified core and cell envelope of mature avian corneocytes. The immunoblotting study suggests that protein bands, cross-reactive for antibodies against loricrin (45, 52-57 kDa), sciellin (54, 84 kDa), filaggrin (32, 38, 45-48 kDa), and transglutaminase (40, 50, 58 kDa), are present in the avian epidermis. Immunocytochemistry shows that immunoreactivity for the above proteins is localized in the transitional and lowermost corneous layer of apteric epidermis. Their epitopes are rapidly masked/altered in cornifying cells and are no longer detectable in mature corneocytes. In scaled epidermis a thick layer made of beta-keratins of 14-18, 20-22, and 33 kDa is formed. Only in feathered epidermis (not in scale epidermis), an antifeather chicken beta-keratin antibody recognized a protein band at 8-12 kDa. This small beta-keratin is probably suitable for the formation of long, axial filaments in elongated barb, barbule and calamus cells. Conversely, the larger beta-keratins in scales are irregularly deposited forming flat plates. Tritiated histidine coupled to autoradiography show an absence of both keratohyalin and histidine-rich proteins in adult feathered and scaled epidermis. Most of the labeling appears in proteins within the range of beta- and alpha-keratins. These data on apteric epidermis support the hypothesis of an evolution of the apteric and interfollicular epidermis from the expansion of hinge regions of protoavian archosaurians., (Copyright 2004 S. Karger AG, Basel)

Filaggrin is a keratin filament associated protein that is expressed in granular layer keratinocytes and derived by sequential proteolysis from a polyprotein precursor termed profilaggrin. Depending on the species, each profilaggrin molecule contains between 10 and 20 filaggrin subunits organized as tandem repeats with a calcium-binding domain at the N- terminal end. We now report the characterization of the complete mouse gene. The structural organization of the mouse gene is identical to the human profilaggrin gene and consists of three exons with a 4 kb intron within the 5' noncoding region and a 1.7 kb intron separating the sequences encoding the calcium-binding EF-hand motifs. A processed pseudogene was found embedded within the second intron. The third and largest exon encodes the second EF-hand, a basic domain (designated the B-domain) followed by 12 filaggrin repeats and a unique C-terminal tail domain. A polyclonal antibody raised against the conceptually translated sequence of the B-domain specifically stained keratohyalin granules and colocalized with a filaggrin antibody in granular layer cells. In upper granular layer cells, B-domain containing keratohyalin granules were in close apposition to the nucleus and, in some cells, appeared to be completely engulfed by the nucleus. In transition layer cells, B-domain staining was evident in the nucleus whereas filaggrin staining remained cytoplasmic. Nuclear staining of the B-domain was also observed in primary mouse keratinocytes induced to differentiate. This study has also revealed significant sequence homology between the mouse and human promoter sequences and in the calcium-binding domain but the remainder of the protein-coding region shows substantial divergence.

The expression of distinct keratin pairs during epidermal differentiation is assumed to fulfill specific and essential cytoskeletal functions. This is supported by a great variety of genodermatoses exhibiting tissue fragility because of keratin mutations. Here, we show that the loss of K10, the most prominent epidermal protein, allowed the formation of a normal epidermis in neonatal mice without signs of fragility or wound-healing response. However, there were profound changes in the composition of suprabasal keratin filaments. K5/14 persisted suprabasally at elevated protein levels, whereas their mRNAs remained restricted to the basal keratinocytes. This indicated a novel mechanism regulating keratin turnover. Moreover, the amount of K1 was reduced. In the absence of its natural partner we observed the formation of a minor amount of novel K1/14/15 filaments as revealed by immunogold electron microscopy. We suggest that these changes maintained epidermal integrity. Furthermore, suprabasal keratinocytes contained larger keratohyalin granules similar to our previous K10T mice. A comparison of profilaggrin processing in K10T and K10(-/-) mice revealed an accumulation of filaggrin precursors in the former but not in the latter, suggesting a requirement of intact keratin filaments for the processing. The mild phenotype of K10(-/-) mice suggests that there is a considerable redundancy in the keratin gene family.

Specific proteolysis plays an important role in the terminal differentiation of keratinocytes in the epidermis and several types of proteases have been implicated in this process. The proprotein convertases (PCs) are a family of Ca2+-dependent serine proteases involved in processing and activation of several types of substrates. In this study we examined the expression and some potential substrates of PCs in epidermis. Four PCs are expressed in epidermis: furin, PACE4, PC5/6 and PC7/8. Furin is detected in two forms, either with or without the transmembrane domain, suggesting occurrence of post-translational cleavage to produce a soluble enzyme. In addition the furin active site has differential accessibility in the granular layer of the epidermis relative to the basal layer, whereas antibodies to the transmembrane domain stain both layers. These findings suggest that furin has access to different types of substrates in granular cells as opposed to basal cells. PC7/8, in contrast, is detected throughout the epidermis with antibodies to both the transmembrane and active site and no soluble form observed. A peptide PC inhibitor (dec-RVKR-CMK) inhibits cleavage of Notch-1, a receptor important in cell fate determination that is found throughout the epidermis. Profilaggrin, found in the granular layer, is specifically cleaved by furin and PACE4 in vitro at a site between the amino terminus and the first filaggrin repeat. This work suggests that the PCs play multiple roles during epidermal differentiation.

In the stratum granulosum of mammalian epidermis, keratohyalin contains the histidine-rich protein filagrin which determines the aggregation of keratin bundles in terminally differentiating keratinocytes. Among reptiles, the lizard epidermis possesses keratohyalin-like granules during a period preceding molting. Tritiated histidine is taken up at 3-22 h post-injection mainly in keratohyalin-like granules of the clear layer, and less intensely in oberhautchen cells, in pre-keratinizing cells of wound epidermis and in the alpha-layer. No labelling was observed in beta-cells and mesos cells. Single and double immunogold localization of filagrin and keratin, using mammalian antibodies, show that some filagrin-like immunoreactivity is localized in 0.15-0.40 microm diameter roundish keratohyalin-like granules in cells of wound epidermis, and lacunar and clear layers (soft alpha-layers). Pro-filagrin was not immuno-localized in these layers with the mammalian antibodies. The small granules merge with keratin bundles into mature keratinocytes where immunopositive keratin filaments predominate and the filagrin-like immunolabelling rapidly disappears. Little or no labelling is observed in the large keratohyaline-like granules of the clear layer. This may be due to lack of filagrin-like immunoreactivity but may also be due to epitope-masking or chemical degradation of filagrin-like molecules. No immunoreactivity is present in the beta-layer and mesos-layer but the immunolabelling reappears in the maturing alpha-layer and lacunar layer. This study suggests that histidin-rich protein with some filagrin-like immunoreactivity is initially present in those alpha-layers of lizard epidermis where keratohyalin-like granules are present, such as lacunar and clear cells, and that a filagrin-like molecule is degraded or altered in mature keratinocytes.

Two novel cytoplasmic intermediate filament (IF) proteins (C and D) from the tunicate (urochordate) Styela are characterised as putative keratin orthologs. The coexpression of C and D in all epidermal cells and the obligatory heteropolymeric IF assembly of the recombinant proteins argue for keratin orthologs, but the sequences do not directly reveal which protein behaves as a keratin I or II ortholog. This problem is solved by the finding that keratin 8, a type II keratin from man or Xenopus, forms chimeric IF when mixed with Styela D. Mutant proteins of Styela D and keratin 8 with a single cysteine in equivalent positions show that these chimeric IF are, like vertebrate keratin filaments, based on the hetero coiled coil. We propose that Styela D retains, in spite of its strong sequence drift, important molecular features of type I keratins. By inference Styela C reflects a type II ortholog. We discuss that type I to III IF proteins are expressed along the chordate branch of metazoa.

The upper layers of mammalian epidermis contain citrulline-containing proteins formed by enzymatic deimination of arginine residues. To study the role of protein deimination in epidermal differentiation, we identified deiminated proteins extracted from human epidermis. Major deiminated proteins were identified as partially degraded keratin K1, while those from keratin K10 and a highly heterogeneous mixture of deiminated filaggrin isomers were detected as minor components. Deiminated keratins were recovered in a fraction enriched with keratins from the cornified layers. The subsequent immunohistochemical study showed that deiminated proteins were localized mainly in the lowermost cornified layer, but not in the granular layer. These data suggested that partially degraded/disulfide-cross-linked keratin K1 was preferentially deiminated during the terminal stages of epidermal differentiation. We therefore speculated that the protein deimination might influence the interaction of basic K1 with its acidic partner K10, pre-existent K5/K14 networks or keratin-associated protein filaggrin.

Filaggrin is the product of posttranslational processing of the large, epidermal protein profilaggrin, which consists of 10 or more tandem filaggrin domains plus an amino and a carboxyl domain. According to fragmentary cDNA sequences, the filaggrin domains in the human protein vary at 40% of the amino acid positions; hence, mature filaggrin is a population of homologous but heterogeneous proteins, even within one individual. Available gene sequences give only a limited picture of the heterogeneity of human filaggrin protein because no complete human profilaggrin gene has been sequenced. Questions about the extent of heterogeneity of filaggrin within and between individuals have not been answered, nor have questions concerning the limited proteolytic cleavage of human profilaggrin that generates filaggrin in vivo. In order to address these questions and to provide an analysis of the primary structure of human filaggrins, we employed various methods of mass spectrometry. The intact protein and a tryptic digest of the mixture of human filaggrins were examined by matrix-assisted laser desorption time-of-flight mass spectrometry. Tryptic digests of human filaggrin from single individuals were also separated and analyzed by liquid chromatography/mass spectrometry (LC/MS) (using electrospray mass spectrometry), and specific peptides were identified by tandem mass spectrometry (MS/MS). A robust data analysis program, Sherpa, was developed to facilitate the interpretation of both LC/MS and MS/MS. These experiments show that human filaggrin includes heterogeneity not yet seen in cDNA sequences, but that much structure is highly conserved. Interestingly, we found that the heterogeneity is conserved among individuals. An approximation of the regions linking filaggrins in human profilaggrin is developed. These investigations provide a unique test of the limits of tryptic mapping of complex mixtures using mass spectrometry.

Profilaggrin, an insoluble precursor of the intermediate filament-associated protein filaggrin, contains multiple internal repeats (PIRs). At terminal differentiation of epidermis, proteolytic processing within a "linker" region of each PIR releases soluble filaggrin in a two-stage process. The first stage endoproteinase (PEP1, profilaggrin endoproteinase 1) cleaves mouse profilaggrin at a subset of the linkers, yielding processing intermediates consisting of several filaggrin repeats. An epidermal endoproteinase that cleaves the requisite linker subset has been purified 4,966-fold from mouse epidermal extracts. SDS-polyacrylamide gel electrophoresis demonstrated a band of molecular mass of 29.5 kDa that correlated with the activity. Labeling with [3H]diisopropylfluorophosphate identified PEP1 as a serine protease; inhibitor studies suggest that it is similar to chymotrypsin, as expected from previous in vivo studies. The purified PEP1 cleaved a peptide derived from profilaggrin (P1) at three residues within and adjacent to a multiple tyrosine sequence, consistent with the in vivo processing sites. No exopeptidase activity was detected. PEP1 is only active toward insoluble profilaggrin, resulting in partial solubilization, consistent with a role in dispersal of profilaggrin during terminal differentiation. In contrast to the specific cleavage of mouse profilaggrin, PEP1 cleaved all linker regions of rat profilaggrin. Studies with phosphorylated P1 suggest that PEP1 specificity may be partly regulated by profilaggrin phosphorylation.

To improve understanding of human profilaggrin processing to filaggrin, we produced seven monoclonal antibodies against epidermal filaggrin (AHF1-7). They were characterized on human epidermis by indirect immunofluorescence, immunogold labeling, and immunoblotting and found to be directed against seven different epitopes of (pro)filaggrin. AHF1-5 labeled the keratohyalin granules and the fibrous matrix of the lower corneocytes, and recognized filaggrin and profilaggrin. AHF6 also labeled the keratohyalin granules and the corneocyte matrix, but only recognized filaggrin. In addition to this reactivity within the upper epidermis, AHF4-6 stained the cytoplasm of the basal cells, and cross-reactivity of AHF5 and AHF6 with cytokeratin K14 was revealed on immunoblots. It is interesting that AHF7 recognized filaggrin, but not profilaggrin, and labeled only the corneocyte matrix and not the keratohyalin granules. This indicates that filaggrin and cytokeratins share several antigenic determinants and that filaggrin bears at least one epitope absent from its precursor. The original series of monoclonal antibodies described here appears to be a powerful tool for studying human profilaggrin processing in normal conditions and in the keratinization disorders in which processing is altered.

The cornified cell envelope (CE) is a 15-nm thick layer of insoluble protein deposited on the intracellular side of the cell membrane of terminally differentiated stratified squamous epithelia. The CE is thought to consist of a complex amalgam of proteins cross-linked by isodipeptide bonds formed by the action of transglutaminases, but little is known about how or in which order the several putative proteins are cross-linked together. In this paper, CEs purified from human foreskin epidermis were digested in two steps by proteinase K, which released as soluble peptides about 30% and then another 35% of CE protein mass, corresponding to approximately the outer third (cytoplasmic surface) and middle third, respectively. Following fractionation, 145 unique peptides containing two or more sequences cross-linked by isodipeptide bond(s) were sequenced. Based on these data, most (94% molar mass) of the outer third of CE structure consists of intra- and interchain cross-linked loricrin, admixed with SPR1 and SPR2 proteins as bridging cross-links between loricrin. Likewise, the middle third of CE structure consists largely of cross-linked loricrin and SPR proteins, but is mixed with the novel protein elafin which also forms cross-bridges between loricrin. In addition, cross-links involving loricrin and keratins 1, 2e, and 10 or filaggrin were recovered in both levels. The data establish for the first time that these several proteins are indeed cross-linked protein components of the CE structure. In addition, the data support a model for the intermediate to final stages of CE assembly: the proteins elafin, SPR1 and SPR2, and loricrin begin to be deposited on a preformed scaffold; later, elafin deposition decreases as loricrin and SPR accumulation continues to effect final assembly. The recovery of cross-links involving keratins further suggests that the subjacent cytoplasmic keratin intermediate filament-filaggrin network is anchored to the developing CE during these events.

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-benzyloxy-carbonyl-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 Km value for Z-Arg-Arg MCA was 0.34 mM and pH optimum 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.

Profilaggrin is an intermediate filament-associated protein of cornified epithelia. It consists of multiple copies of similar filaggrin domains joined by peptide linker regions; during terminal differentiation of the epidermis, the linker regions are processed away in a regulated manner. In order to characterize the sites of proteolysis in rat profilaggrin, tryptic peptides of filaggrin and profilaggrin were fractionated by reverse-phase HPLC, and the HPLC fractions were analyzed by nebulization-assisted electrospray ionization mass spectrometry. Peptide sequences were confirmed or corrected by tandem mass spectrometry; in several cases, this was achieved by collisional activation of multiply charged precursor ions of peptides exceeding 3 kDa in mass. The tryptic peptides accounted for all of the sequence predicted by a partial cDNA sequence, with the exception of six arginines or dipeptides. Although the cDNA sequence predicted eight sites of heterogeneity among the filaggrin domains, only one of these was observed. An additional unpredicted site of heterogeneity was also seen. Comparison of the peptides from filaggrin with those of profilaggrin revealed several peptides unique to filaggrin, specifically at the new amino- and carboxyl-termini, that result from proteolytic processing of the linker region of profilaggrin. Both the amino- and carboxyl-termini were "ragged", suggesting that processing may involve exopeptidase action after an initial endopeptidase cleavage. The average mass of this mixture of filaggrins was determined by electrospray mass spectrometry to be 42 452 Da, in reasonable agreement with that predicted from the mass spectrometric analysis of the terminal sequences. The linker peptide of rat profilaggrin was found in two forms, which differed only in the phosphorylation state of serine 22.

Filaggrin and trichohyalin are the two major intermediate filament associated proteins which interact with keratin filaments in the skin. These two proteins initially accumulate in cytoplasmic granules called keratohyalin or trichohyalin granules which provide prominent morphological hallmarks of differentiation in the epidermis and the inner root sheath of hair follicles, respectively. The contents of each of these granules are modified and subsequently released into the cytoplasm of the fully mature cells where they function in the role of aggregating keratin filament bundles. We are beginning to identify several important aspects relative to the unique biological functions of both filaggrin and trichohyalin during the late stages of keratinocyte differentiation. This overview summarizes recent work on these proteins and will also highlight the existence of novel cytoplasmic granules, keratohyalin-trichohyalin hybrid granules, in dorsal tongue epithelia.

In the staphylococcal scalded skin syndrome, spontaneous intraepithelial cleavages are due to the exfoliative toxins A or B (ETA or ETB). Until now, these toxins have been studied either on epidermis or on organotypic skin cultures. In the present study, we compare the effects of these toxins on human keratinocyte cell cultures to those on human and mouse organotypic skin cultures. With concentrations of ETA or ETB of 1 mg/ml for 3 hours, spontaneous intraepithelial cleavages were noted in both cell and organotypic cultures. Keratinocyte cell cultures were as sensitive as organotypic skin cultures to these toxins. Since keratohyaline granules may represent a possible binding site for ETA or ETB, we tried to correlate the expression of keratohyaline granules with the appearance of intraepithelial clefts due to the toxins. However, when cultured in liquid medium, epithelia were not differentiated enough to allow the detection of the binding site of ETA-ETB.