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.     

The characterization of human epidermal filaggrin. A histidine-rich, keratin filament-aggregating protein

Filaggrin is a histidine-rich, cationic protein that aggregates with keratin filaments in vitro and may function as the keratin matrix protein in the terminally differentiated cells of the epidermis. This protein has been previously isolated from rodent epidermis. In this investigation, a similar protein from human skin was identified, isolated and characterized by biochemical and immunologic techniques. Indirect immunofluorescence of human skin using antiserum to rat filaggrin gave positive immunofluorescence of keratohyalin granules and the stratum corneum. This indicated the presence of a human filaggrin in the epidermis in a localization similar to that of the rodent. The protein was isolated from human epidermis and purified by ion-exchange chromatography and preparative gel electrophoresis. The purified protein crossreacts with antibody to rat filaggrin and migrates as a doublet of molecular weight (Mr) approximately 35 000 on SDS-polyacrylamide gels. It is relatively rich in polar amino acids such as histidine, arginine, serine and glycine, but is poor in nonpolar amino acids. Unlike rodent filaggrin, the human protein contains ornithine. This protein aggregates with human keratin filaments, forming compact macrofibrils in a manner analogous to that of rodent filaggrin. Thus, a human epidermal protein has been isolated which has many of the characteristics of rodent filaggrin and may function as the human keratin matrix protein.     

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.     

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)     

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.     

Multiple copies of phosphorylated filaggrin in epidermal profilaggrin demonstrated by analysis of tryptic peptides

The precursor of mouse (c57/B16) epidermal filaggrin (profilaggrin) is a very large (ca. 500 000 daltons), highly phosphorylated protein containing multiple copies of filaggrin (26 000 daltons). The conversion of profilaggrin to filaggrin late in epidermal cell differentiation involves dephosphorylation and proteolysis to yield the unphosphorylated filaggrin, which polymerizes with keratin filaments into macrofibrils. In order to gain insight in the nature of these processes, we compared tryptic digests of profilaggrin with those of filaggrin by reverse-phase liquid chromatography. Approximately 80% of the profilaggrin mass consists of multiple copies of filaggrin. Twenty peptides purified in good yield from both profilaggrin and filaggrin accounted for most of the filaggrin sequence. A detailed analysis of the yield of several peptides provided an estimate of the size and frequency of the repeat unit within profilaggrin. These data indicate that the repeating substructure of profilaggrin contains about 265 amino acids and that about 50 residues are removed per filaggrin domain as the precursor is processed to filaggrin. Assuming a molecular weight of 500 000 (as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis), this indicates there are 16 repeats. Analysis of phosphopeptides isolated from profilaggrin showed that 66% of the phosphate was located on peptides that are unphosphorylated in filaggrin. Analysis of peptide recoveries confirmed the repeat size and showed that every copy of filaggrin was phosphorylated in profilaggrin.     

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.     

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)     

Mouse "protective protein". cDNA cloning, sequence comparison, and expression

The "protective protein" is the glycoprotein that forms a complex with the lysosomal enzymes beta-galactosidase and neuraminidase. Its deficiency in man leads to the metabolic storage disorder galactosialidosis. The primary structure of human protective protein, deduced from its cloned cDNA, shows homology to yeast serine carboxypeptidases. We have isolated a full-length cDNA encoding murine protective protein. The nucleotide sequences as well as the predicted amino acid sequences are highly conserved between man and mouse. Domains important for the protease function are completely identical in the two proteins. Both human and mouse mature protective proteins covalently bind radiolabeled diisopropyl fluorophosphate. Transient expression of the murine cDNA in COS-1 cells yields a protective protein precursor of 54 kDa, a size characteristic of the glycosylated form. This cDNA-encoded precursor, endocytosed by human galactosialidosis fibroblasts, is processed into a 32- and a 20-kDa heterodimer and corrects beta-galactosidase and neuraminidase activities. A tissue-specific expression of protective protein mRNA is observed when total RNA from different mouse organs is analyzed on Northern blots.     

Molecular isolation and sequence determination of the cDNA for the mouse sperm-specific lactate dehydrogenase-X gene

Several cDNA clones for the mouse lactate dehydrogenase-X (LDH-X), a sperm-specific glycolytic enzyme, were isolated from mouse testicular cDNA libraries constructed in the bacteriophage vectors, lambda gt11 and gt10. The largest cDNA clone contains an insert of 1135 base pairs in length and an open reading frame that encodes a 332 amino acid polypeptide with a molecular weight of 35.89 kD. The deduced amino acid sequence of this protein is in close agreement with the published sequence of mouse LDH-X obtained by direct protein sequencing. Northern analysis of RNA isolated from different tissues detected a single size mRNA of 1.5 kilobases in mouse testis but not in brain or liver. The Ldh-x structural gene was estimated to be about 12 kb in size as demonstrated by Southern hybridization analysis of mouse genomic DNA using the full-length cDNA as a probe.     

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.     

A single WAP domain-containing protein from Litopenaeus vannamei hemocytes

A cDNA clone coding for a single WAP domain (SWD) protein was isolated from a hemocyte cDNA library of Litopenaeus vannamei. The full-length cDNA sequence is 0.4kb long and encodes a 93-amino acid protein. Using this sequence as a probe a similar clone coding for a 92-amino acids protein was found in a cDNA library from Penaeus monodon hemocytes. The mRNA size was confirmed by Northern blot as well as that gene is expressed in hemocytes, but not in hepatopancreas. mRNA levels of the shrimp SWD protein were modified after injection of Vibrio alginolyticus, indicating the probable role of this protein in the immune response. Although amino acid sequence seems to be similar to those of other WAP domain-containing proteins, shrimp SWD protein does not have any other functional domain, similar to a mouse single WAP motif (SWAM) protein reported in mouse; however, the phylogenetic analysis shows that shrimp SWD is more related to other WAP proteins than to mouse SWAM.     

Hornerin, a novel profilaggrin-like protein and differentiation-specific marker isolated from mouse skin

A novel mouse cDNA named hornerin was isolated by RNA differential display applied to developing mouse skin. Hornerin, which has 2,496 amino acids, comprises EF-hand domains at the N terminus followed by a spacer sequence and a large repetitive domain, indicating that hornerin is a novel member of the "fused gene"-type cornified envelope precursor protein family. The repetitive domain of hornerin was found to be rich in glycine, serine, and glutamine. Hornerin was expressed in the tongue, esophagus, forestomach, and skin among the adult mouse tissues examined, all of them cornifying stratified epithelium. In the embryonic mouse skin, hornerin mRNA was first detected on gestational day 15.5 in the epidermis coincidentally with the formation of a granular layer. In accordance with this, hornerin was detected in the granular and cornified layers of the mature epidermis. In the granular cells of the epidermis, the hornerin protein was detected in keratohyalin granules together with profilaggrin. Furthermore, Western blot analysis of the mouse skin showed that the hornerin protein was cleaved during the process of epidermal differentiation, indicating possible posttranslational proteolytic processing as is observed in profilaggrin. Differentiation of primary mouse epidermal keratinocytes with 0.12 mm Ca(2+) resulted in the induction of hornerin. These results indicate that hornerin is structurally as well as functionally most similar to profilaggrin among the family members and possibly plays pleiotropic roles, including a role in cornification.