Filaggrin, an intermediate filament-associated protein: structural and functional implications from the sequence of a cDNA from rat.

Filaggrin is an intermediate filament-associated protein that is involved in aggregation of keratin filaments in fully cornified cells of the mammalian epidermis, and is an important marker for epidermal differentiation. In this report, the sequence of a rat cDNA clone coding for a portion of the polymeric precursor, profilaggrin, is presented. The cDNA is 2,314 bp long with 1,875 bp of coding region ending with an A-T-rich 3' noncoding region. Genomic analysis indicates that the profilaggrin gene consists of 20 +/- 2 repeats of 1,218 bp of sequence coding for 406 amino acids, making the mRNA at least 25-27 kb in length. Each repeat consists of a filaggrin domain and a linker sequence with an estimated size of 380 and 26 amino acids, respectively. High levels of profilaggrin mRNA are found only in keratinizing epithelia. Comparison of the rat filaggrin sequence with that of mouse and human filaggrin and with the sequence of phosphorylated peptides from mouse profilaggrin indicates that the proteins share extensive amino acid sequence similarities, especially in the two phosphorylated regions. Proteolytic processing sites are also quite similar in rat and mouse. The three species show blocks of sequence that are similar in length and composition which alternate with sequences that are variable in length. This analysis suggests that the evolution of the present-day filaggrins has been constrained by maintenance of phosphorylation sites and overall amino acid composition. The cDNAs for the profilaggrins are similar in structure, reflecting genes that have simple repeating structures and lack introns within their coding regions. Mouse and rat profilaggrin terminate with a nonpolar sequence atypical of the rest of the coding region, and have similar 3' noncoding regions. To explain these observations, a novel evolutionary model is proposed.     

The repetitive structure of the profilaggrin gene as demonstrated using epidermal profilaggrin cDNA

Filaggrin is the histidine-rich basic protein that aggregates keratin filaments in fully differentiated cells of the epidermis. Filaggrin is synthesized in the granular cell layer as a high molecular weight precursor protein (profilaggrin) that consists of multiple repeated copies of filaggrin. cDNA clones for rat and mouse epidermal profilaggrin have been constructed from sucrose gradient-enriched RNA in order to study the repetitive structure of profilaggrin. These clones hybridize to high molecular weight epidermal mRNA (23 kilobase pairs, rat and 19 kilobase pairs, mouse) and exhibit limited cross-hybridization between species. Several rat clones direct the synthesis of a portion of rat profilaggrin in bacteria. One of these, rat profilaggrin cDNA clone R4D6, is 2400 base pairs in length. The R4D6 cDNA is shown to contain repetitive sequence by restriction mapping and southern hybridization analysis of restriction digests of this plasmid, using subfragments of the plasmid as hybridization probes. Southern hybridization analysis of rat genomic DNA, digested to completion with several restriction enzymes, reveals a simple hybridization pattern of fragments equal in size to those of the cDNA. Partial digestion of rat genomic DNA results in a ladder of bands based on a 1200-base pair repeat, equal to the size of the repeating unit of the cDNA clone, and consistent with the expected repeating size of profilaggrin. Together, these results show that the profilaggrin mRNA and gene have repetitive structure and that the gene apparently lacks introns in the coding region.     

High-molecular-weight precursor of epidermal filaggrin and hypothesis for its tandem repeating structure

Filaggrin is a histidine-rich protein that is intimately involved in mammalian epidermal keratinization. Using a combination of immunologic and in vivo pulse-chase studies with radiolabeled histidine and phosphate, we show that the phosphorylated precursor of both rat and mouse filaggrin has an apparent molecular weight much higher than previously realized (6 X 10(5) and 3.9 X 10(5), respectively). These high-molecular-weight filaggrin precursors can be rapidly labeled with histidine and extracted from the epidermis under denaturing conditions. More than half of the label incorporated in the precursor at 2 h is found in filaggrin at 24 h after injection, even though filaggrin is less than 10% of the size of the precursor. Limited proteolytic digestion of the precursor in vitro results in the formation of an oligomeric series of peptides based on a phosphorylated fragment slightly larger than filaggrin itself. More extensive digestion of this fragment shows that it is composed of filaggrin with few or no additional unrelated peptides, suggesting that the major part of the high-molecular-weight filaggrin precursor must be composed of repeated domains of filaggrin. Because the primary translation product of filaggrin mRNA is large, we propose that these domains are repeated in tandem. In addition, from molecular weight computations and peptide map analyses, we suggest that the filaggrins are themselves composed of multiple repeating units of an unidentified peptide of approximately Mr 8600. This value is derived from the molecular weights of filaggrin from several mammalian species that differ by integral multiples of 8600. A model for the structure of the high-molecular-weight precursor of filaggrin is presented.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of two intermediates during processing of profilaggrin to filaggrin in neonatal mouse epidermis

A major event in the keratinization of epidermis is the production of the histidine-rich protein filaggrin (26,000 mol wt) from its high molecular weight (greater than 350,000) phosphorylated precursor (profilaggrin). We have identified two nonphosphorylated intermediates (60,000 and 90,000 mol wt) in NaSCN extracts of epidermis from C57/Bl6 mice by in vivo pulse-chase studies. Results of peptide mapping using a two-dimensional technique suggest that these intermediates consist of either two or three copies of filaggrin domains. Each of the intermediates has been purified. The ratios of amino acids in the purified components are unusual and essentially identical. The data are discussed in terms of a precursor containing tandem repeats of similar domains. In vivo pulse-chase experiments demonstrate that the processing of the high molecular weight phosphorylated precursor involves dephosphorylation and proteolytic steps through three-domain and two-domain intermediates to filaggrin. These processing steps appear to occur as the cell goes through the transition cell stage to form a cornified cell.     

Identification of proteolytic cleavage sites in the conversion of profilaggrin to filaggrin in mammalian epidermis

Profilaggrin consists of multiple filaggrin domains joined by linker segments which are removed during proteolytic conversion to filaggrin. Analysis of tryptic peptides of filaggrin defined a 26-residue linker segment when aligned on the amino acid sequence of one repeat unit of mouse profilaggrin deduced from a cDNA sequence (Rothnagel, J. A., Mehrel, T., Idler, W. W., Roop, D. R., and Steinert, P. M. (1987) J. Biol. Chem. 262, 15643-15648). Two types of linker segments were distinguished by their different susceptibility to thermolysin and by the presence of a Phe-Tyr-Pro-Val sequence in only one type. These data led to a model of profilaggrin in which the two types of linker segments alternate along the length of profilaggrin. This model provides a structural basis for the two stages of proteolytic processing seen in vivo. In the first stage intermediates accumulate which have several filaggrin domains still joined by linker segments lacking Phe-Tyr-Pro-Val. In the second stage, the other linker segments are cleaved and mature filaggrin domains are released. Proteolytic activity with specificity consistent with first stage cleavage was partially purified from rat epidermis. Chymostatin inhibited both the in vitro enzymatic activity and the processing of profilaggrin in a cultured rat keratinocyte cell line. The products formed in vitro were 3-5 kDa larger than intermediates produced in vivo, suggesting that the linker segments are cleaved at one end only. This implies the existence of a third protease which completes the removal of the linker segments.     

Filaggrin production by cultured human epidermal keratinocytes and its regulation by retinoic acid

Filaggrin is a basic protein normally present in the stratum corneum of epidermis. It derives from a high-molecular-weight precursor synthesized in the stratum granulosum of epidermis. This precursor, called profilaggrin, is thought to be associated with the keratohyaline granules of granular cells. It is known that profilaggrin, but not filaggrin, is present in conventional cultures of human keratinocytes grown on plastic petri dishes. In this study, we show that cultured human keratinocytes can convert profilaggrin into filaggrin, when they are grown on a collagen matrix and raised at the liquid-air interface in order to induce terminal differentiation. Moreover, the presence of terminally differentiating keratinocytes above the granular layer is necessary, but not sufficient, for the accumulation of filaggrin. Finally, we show that the accumulation of filaggrin in the outermost layers of submerged cultured human keratinocytes can be triggered by extensive removal (double delipidization) of retinoids from the serum supplement and inhibited when small concentrations (10(-11)-10(-10) M) of retinoic acid are readded to the culture medium. Altogether, the data reported suggest that not only the synthesis of profilaggrin, but also the conversion of profilaggrin into filaggrin are negatively controlled by retinoic acid. Further, it seems that retinoic acid acts directly on the conversion of profilaggrin into filaggrin rather than on the production of terminally differentiating cells capable of accumulating this protein.     

Characterization of the human epidermal profilaggrin gene. Genomic organization and identification of an S-100-like calcium binding domain at the amino terminus.

Filaggrin is an intermediate filament-associated protein which functions to aggregate keratin intermediate filaments in the stratum corneum of mammalian epidermis. It is synthesized as a large precursor protein, profilaggrin, that consists of multiple filaggrin units and is localized in keratohyalin granules. In this report, we describe the characterization of cosmid genomic clones containing the human profilaggrin gene coding for 11 complete filaggrin repeats of 324 amino acids each. At the amino- and carboxyl-terminal ends of human profilaggrin are leader and tail peptide sequences of 293 and 157 amino acids, respectively, which differ from filaggrin. The leader peptide is composed of two distinct domains: an 81-residue segment which shows significant homology to the S-100 family of EF hand-containing calcium-binding proteins, and a hydrophilic second domain of 212 residues. The gene is divided into three exons, with one intron (approximately 9.6 kilobase pairs) in the 5' noncoding region and a second one of 570 base pairs between the EF hands. The position of intron 2 is identical to that of other members of the S-100-like family. The presence of an S-100-like domain suggests that profilaggrin binds calcium and that the calcium binding domain is functionally significant in the formation of keratohyalin and/or the subsequent processing of profilaggrin to filaggrin, both of which may be calcium-dependent events.     

The structure of the gene for mouse filaggrin and a comparison of the repeating units

Filaggrins are an important class of intermediate filament-associated proteins that are involved in the organization of keratin filaments in the terminal stages of mammalian epidermal differentiation. Filaggrins are initially synthesized as very large polyprotein precursors consisting of many tandemly arranged repeats that are later liberated by proteolytic processes to yield many copies of the functional protein. We have recently characterized a cDNA clone to mouse filaggrin (Rothnagel, J. A., Mehrel. T., Idler, W. W., Roop, D. R., and Steinert, P. M. (1987) J. Biol. Chem. 262, 15643-15648) which encodes a 750-base pair (250-amino acid) repeating element having properties consistent with a filaggrin molecule. Southern blot analysis of total mouse DNA and the mouse gene isolated from a cosmid library (cosmid clone cFM6.1A2) has also revealed a repeat length of about 750 base pairs. The cosmid clone contains most of the mouse filaggrin gene, but it is missing the 5'-noncoding sequences and possibly some coding sequences as well. We report here that cosmid clone cFM6.1A2 contains 20 filaggrin repeats and 15,213 base pairs of coding sequences. Sequence analysis of this clone has revealed at least two different types of repeating element. Type B has a repeat length of 750 base pairs (250 amino acids), whereas type A is 765 base pairs (255 amino acids) long and contains an additional five amino acids inserted next to an acidic sequence that delineates the amino and carboxyl termini of the filaggrin repeats. It is supposed that these additional five amino acids may alter the proteolytic sensitivity of the acidic linker sequence, thereby affecting the processing of the precursor. The random distribution of the two types of repeats in the precursor indicates that the mouse filaggrin gene arose by a complicated series of duplications and/or rearrangements.     

Loss of proteolytically processed filaggrin caused by epidermal deletion of Matriptase/MT-SP1

Profilaggrin is a large epidermal polyprotein that is proteolytically processed during keratinocyte differentiation to release multiple filaggrin monomer units as well as a calcium-binding regulatory NH2-terminal filaggrin S-100 protein. We show that epidermal deficiency of the transmembrane serine protease Matriptase/MT-SP1 perturbs lipid matrix formation, cornified envelope morphogenesis, and stratum corneum desquamation. Surprisingly, proteomic analysis of Matriptase/MT-SP1-deficient epidermis revealed the selective loss of both proteolytically processed filaggrin monomer units and the NH2-terminal filaggrin S-100 regulatory protein. This was associated with a profound accumulation of profilaggrin and aberrant profilaggrin-processing products in the stratum corneum. The data identify keratinocyte Matriptase/MT-SP1 as an essential component of the profilaggrin-processing pathway and a key regulator of terminal epidermal differentiation.     

Microheterogeneity of human filaggrin: analysis of a complex peptide mixture using mass spectrometry.

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.     

Neutral Cysteine Protease Bleomycin Hydrolase Is Essential for the Breakdown of Deiminated Filaggrin into Amino Acids

Filaggrin is a component of the cornified cell envelope and the precursor of free amino acids acting as a natural moisturizing factor in the stratum corneum. Deimination is critical for the degradation of filaggrin into free amino acids. In this study, we tried to identify the enzyme(s) responsible for the cleavage of deiminated filaggrin in vitro. First, we investigated citrulline aminopeptidase activity in the extract of newborn rat epidermis by double layer fluorescent zymography and detected strong activity at neutral pH. Monitoring the citrulline-releasing activity, we purified an enzyme of 280 kDa, comprised of six identical subunits of 48 kDa. The NH₂ terminus of representative tryptic peptides perfectly matched the sequence of rat bleomycin hydrolase (BH). The enzyme released various amino acids except Pro from β-naphthylamide derivatives and hydrolyzed citrulline-β-naphthylamide most effectively. Thus, to break down deiminated filaggrin, another protease would be required. Among proteases tested, calpain I degraded the deiminated filaggrin effectively into many peptides of different mass on the matrix-assisted laser desorption/ionization-time of flight mass spectrum. We confirmed that various amino acids including citrulline were released by BH from those peptides. On the other hand, caspase 14 degraded deiminated filaggrin into a few peptides of limited mass. Immunohistochemical analysis of normal human skin revealed co-localization of BH and filaggrin in the granular layer. Collectively, our results suggest that BH is essential for the synthesis of natural moisturizing factors and that calpain I would play a role as an upstream protease in the degradation of filaggrin.The mammalian epidermal keratinocytes arise from proliferating basal cells and move outward through a series of distinct differentiation events to form the stratum corneum (1, 2). During this progressive epidermal differentiation, keratinocytes express different proteins such as keratins, profilaggrin/filaggrin, involucrin, small proline-rich proteins, loricrin, cystatin A, and elafin, which form the cornified envelope of mature corneocytes (3–7). Profilaggrin is synthesized as a large, extremely insoluble phosphoprotein that consists of a unique NH₂-terminal Ca²⁺-binding protein of the S-100 family, linked to 10–20 tandem filaggrin monomer repeats (8–10). Each individual filaggrin repeat is completely removed by proteolysis to generate the mature filaggrin monomer (a molecular mass of 37 kDa in human). Then, filaggrin is completely degraded in the uppermost layer of the stratum corneum to produce a mixture of free and modified hygroscopic amino acids that are important for maintaining epidermal hydration (2, 11–13). In addition, a number of proteins are subjected to various post-translational modifications such as disulfide bonding, N-(γ-glutamyl)-lysine isopeptide cross-linking, and deimination during the terminal differentiation of epidermal keratinocytes (4, 6, 14, 15). Deimination is catalyzed by peptidylarginine deiminase (PAD),² which converts arginine to citrulline in proteins (17–19). The modification seems essential for the processing into free amino acids including citrulline.Several proteases reportedly participate in the processing of profilaggrin. Furin, a member of the proprotein convertase family, has been proposed to cleave the NH₂ terminus of profilaggrin, facilitating the release of the NH₂-terminal S-100 protein (20, 21). In contrast, calpain I and profilaggrin endopeptidase I (PEP-I) were implicated in the processing of the linker regions between the filaggrin monomer repeats to generate the filaggrin monomer (22–25). Recently, significant results regarding the conversion of profilaggrin to filaggrin have been obtained with the knock-out of matriptase/MT-SP1, prostasin/channel-activating serine protease 1/Prss 8, and caspase 14 in mice (26–28). These proteases were a key component of the profilaggrin-processing pathway in terminal epidermal differentiation. However, although the signal initiating the degradation of profilaggrin at a defined stage of the maturation of the stratum corneum was found to be the water gradient within the stratum corneum itself (11), the proteases for the processing of filaggrin and/or the deiminated form into peptides following the breakdown of these peptides to amino acids including citrulline remain unknown.In this study, we have purified a novel aminopeptidase using a deiminated substrate from rat skin homogenate and identified it as a neutral cysteine protease, bleomycin hydrolase (BH). Furthermore, we investigated the processing of the deiminated filaggrin by calpain I or caspase 14. Based on these results, we proposed that calpain I participated preferentially in the processing of deiminated filaggrin into peptides and then BH appeared essential for the breakdown of the peptides into amino acids.     

Interaction of filaggrin with keratin filaments during advanced stages of normal human epidermal differentiation and in Ichthyosis vulgaris

Filaggrin is a histidine-rich, basic protein whose name was first proposed based on its ability to aggregate intermediate filaments in vitro. Based on this in vitro observation, it has generally been assumed that filaggrin functions in vivo as a matrix protein which causes keratin filaments to become densely packed in the terminally differentiated cornified cells. Inconsistent with this view however, is the well-known observation that keratin aggregation appears to proceed normally in the affected epidermis of ichthyosis vulgaris patients despite a greatly reduced quantity of filaggrin. To address this issue, we used immuno-electron microscopy to localize filaggrin and its cross-reactive precursor, profilaggrin, in human and mouse epidermis, as well as in ichthyosis vulgaris epidermis. We found that the localization of filaggrin in lower cornified cells correlates precisely with the formation of aggregated keratin filaments, and the disappearance of filaggrin in upper cornified cells correlates precisely with the loosening of keratin filaments. Furthermore, we showed that, even in ichthyosis vulgaris, small amounts of filaggrin/profilaggrin are present as electron-dense deposits associated with keratin filaments in the granular cells, and that the localization of this small amount of antigen again correlates with the aggregation state of keratin filaments. These data strongly suggest that filaggrin is indeed involved in filament aggregation in vivo.     

Hyaluronan suppresses epidermal differentiation in organotypic cultures of rat keratinocytes

Hyaluronan (hyaluronic acid, HA) is an abundant matrix component between keratinocytes of the epidermis in vivo, but its function there remains unclear. We used a lift culture model, in which rat epidermal keratinocytes (REKs) stratify at an air-liquid interface, to ask whether HA may regulate epidermal proliferation and/or differentiation. In this model, early markers of differentiation (keratin 10), and later markers (profilaggrin, keratohyalin granules, cornified layers) are faithfully expressed, both temporally and spatially. HA, measured using two different analytical techniques, accumulated to high levels only in the presence of an intact basement membrane that seals the epidermal compartment. To test whether HA has a functional role in differentiation, Streptomyces hyaluronidase (StrepH, 1 U/ml; digests >95% of HA within 4 h) was added daily to lift cultures during stratification time-course experiments over 5 days. In StrepH-treated cultures, the expression of profilaggrin and the number and size of keratohyalin granules were significantly increased relative to controls using semiquantitative histological analyses. The StrepH-related accumulation of K10 protein and profilaggrin/filaggrin were confirmed by Western analyses. Thus, it appears that the presence of intercellular HA in the epidermis acts as a brake upon intracellular events that occur during keratinocyte differentiation.     

Sequence-specific recognition of the internalization motif of the Alzheimer's amyloid precursor protein by the X11 PTB domain.

The crystal structure of the phosphotyrosine-binding domain (PTB) of the X11 protein has been determined, in complex with unphosphorylated peptides corresponding to a region of beta-amyloid precursor protein (betaAPP) that is required for receptor internalization. The mode of binding to X11 of the unphosphorylated peptides, which contain an NPxY motif, resembles that of phosphorylated peptides bound to the Shc and IRS-1 PTB domains. Eight peptide residues make specific contacts with the X11 PTB domain, and they collectively achieve high affinity (KD = 0.32 microM) and specificity. These results suggest that, in contrast to the SH2 domains, the PTB domains are primarily peptide-binding domains that have, in some cases, acquired specificity for phosphorylated tyrosines.     

Kallikrein-related Peptidase 5 Functions in Proteolytic Processing of Profilaggrin in Cultured Human Keratinocytes

Filaggrin protein is synthesized in the stratum granulosum of the skin and contributes to the formation of the human skin barrier. Profilaggrin is cleaved by proteolytic enzymes and converted to functional filaggrin, but its processing mechanism remains not fully elucidated. Kallikrein-related peptidase 5 (KLK5) is a major serine protease found in the skin, which is secreted from lamellar granules following its expression in the stratum granulosum and activated in the extracellular space of the stratum corneum. Here, we searched for profilaggrin-processing protease(s) by partial purification of epidermal extracts and found KLK5 as a possible candidate. We used high performance liquid chromatography coupled with electrospray tandem mass spectrometry to show that KLK5 cleaves profilaggrin. Furthermore, based on a proximity ligation assay, immunohistochemistry, and immunoelectron microscopy analysis, we reveal that KLK5 and profilaggrin co-localize in the stratum granulosum in human epidermis. KLK5 knockdown in normal cultured human epidermal keratinocytes resulted in higher levels of profilaggrin, indicating that KLK5 potentially functions in profilaggrin cleavage.     

Inducible expression of filaggrin increases keratinocyte susceptibility to apoptotic cell death

Filaggrin is an intermediate filament associated protein that aids the packing of keratin filaments during terminal differentiation of keratinocytes. Premature aggregation of keratin filaments is prevented by filaggrin expression as the inactive precursor, profilaggrin, which is localized in keratohyalin granules in vivo. We have previously shown that filaggrin constructs, when transiently transfected into epithelial cells, lead to a collapsed keratin cytoskeletal network and dysmorphic nuclei with features of apoptosis. The apparent transfection rate is low with filaggrin constructs, supporting their disruptive role but hindering further study. To bypass this problem, we generated stable keratinocyte cell lines that express mature human filaggrin using a tetracycline-inducible promoter system. We found that cell lines expressing filaggrin, but not control cell lines, exhibited increased sensitivity to multiple apoptotic stimuli as measured by morphologic and biochemical criteria. None of the cell lines showed an increase in endogenous expression of filaggrin in response to the same stimuli. Filaggrin expression alone was insufficient to induce apoptosis in these keratinocyte cell lines. We conclude that filaggrin, due to its keratin binding ability, primes cells for apoptosis. Because filaggrin is expressed at a level of the epidermis where keratinocytes are in transition between the nucleated granular and the anucleate cornified layers, we hypothesize that filaggrin aids in the terminal differentiation process by facilitating apoptotic machinery.     

Normal human epidermal keratinocytes express in vitro specific molecular forms of (pro)filaggrin recognized by rheumatoid arthritis-associated antifilaggrin autoantibodies.

BACKGROUND: The so-called antikeratin antibodies and the antiperinuclear factor are the most specific serological markers of rheumatoid arthritis (RA). They were recently shown to be largely the same autoantibodies and to recognize human epidermal filaggrins and profilaggrin-related proteins of buccal epithelial cells (collectively referred to as (pro)filaggrin). MATERIALS AND METHODS: To further characterize the target antigens, we investigated their expression by normal human epidermal keratinocytes cultured in differentiating conditions, using immunofluorescence and immunoblotting with RA sera and three different monoclonal antibodies to (pro)filaggrin. RESULTS: On the cornified, stratified epithelial sheets obtained in vitro, RA sera with anti(pro)filaggrin autoantibodies (AFA) produced granular staining of the stratum granulosum and diffuse staining of the stratum corneum. The antigens recognized by RA sera strictly colocalized with (pro)filaggrin in keratohyalin granules. Following sequential extraction of the proteins from the epithelial sheets, the RA sera and the three monoclonal antibodies to (pro)filaggrin, recognized a series of low-salt-soluble molecules, including a neutral/acidic isoform of filaggrin and several proteins with sizes and pI intermediates between this isoform and profilaggrin. They also recognized urea-soluble high-molecular-weight profilaggrin-related molecules. CONCLUSIONS: These results show that in vitro epidermal keratinocytes express various molecular forms of (pro) filaggrin that bear epitopes targeted by AFA of RA sera, and that some of these are absent from epidermis. Moreover, these epitopes, which are present on the keratohyalin granules of buccal epithelial cells but not on those of epidermal cells, are present on the granules of the cultured keratinocytes. This work completes the molecular characterization of the proteins targeted by AFA.     

Organization, structure, and polymorphisms of the human profilaggrin gene

Profilaggrin is a major protein component of the keratohyalin granules of mammalian epidermis. It is initially expressed as a large polyprotein precursor and is subsequently proteolytically processed into individual functional filaggrin molecules. We have isolated genomic DNA and cDNA clones encoding the 5'- and 3'-ends of the human gene and mRNA. The data reveal the presence of likely "CAT" and "TATA" sequences, an intron in the 5'-untranslated region, and several potential regulatory sequences. While all repeats are of the same length (972 bp, 324 amino acids), sequences display considerable variation (10-15%) between repeats on the same clone and between different clones. Most variations are attributable to single-base changes, but many also involve changes in charge. Thus, human filaggrin consists of a heterogeneous population of molecules of different sizes, charges, and sequences. However, amino acid sequences encoding the amino and carboxyl termini are more conserved, as are the 5' and 3' DNA sequences flanking the coding portions of the gene. The presence of unique restriction enzyme sites in these conserved flanking sequences has enabled calculations on the size of the full-length gene and the numbers of repeats in it: depending on the source of genomic DNA, the gene contains 10, 11, or 12 filaggrin repeats that segregate in kindred families by normal Mendelian genetic mechanisms. This means that the human profilaggrin gene system is also polymorphic with respect to size due to simple allelic differences between different individuals. The amino- and carboxyl-terminal sequences of profilaggrin contain partial or truncated repeats with unusual un-filaggrin-like sequences on the termini.(ABSTRACT TRUNCATED AT 250 WORDS)