Regulated Expression of Human Filaggrin in Keratinocytes Results in Cytoskeletal Disruption, Loss of Cell–Cell Adhesion, and Cell Cycle Arrest

Filaggrin is an intermediate filament (IF)-associated protein that aggregates keratin IFs in vitro and is thought to perform a similar function during the terminal differentiation of epidermal keratinocytes. To further explore the role of filaggrin in the cytoskeletal rearrangement that accompanies epidermal differentiation, we generated keratinocyte cell lines that express human filaggrin using a tetracycline-inducible promoter system. Filaggrin expression resulted in reduced keratinocyte proliferation and caused an alteration in cell cycle distribution consistent with a post-G1 phase arrest. Keratin filament distribution was disrupted in filaggrin-expressing lines, while the organization of actin microfilaments and microtubules was more mildly affected. Evidence for direct interaction of filaggrin and keratin IFs was seen by overlay assays of GFP-filaggrin with keratin proteins in vitro and by filamentous filaggrin distribution in cells with low levels of expression. Cells expressing moderate to high levels of filaggrin showed a rounded cell morphology, loss of cell-cell adhesion, and compacted cytoplasm. There was also partial or complete loss of the desmosomal proteins desmoplakin, plakoglobin, and desmogleins from cell-cell borders, while the distribution of the adherens junction protein E-cadherin was not affected. No alterations in keratin cytoskeleton, desmosomal protein distribution, or cell shape were observed in control cell lines expressing beta-galactosidase. Filaggrin altered the cell shape and disrupted the actin filament distribution in IF-deficient SW13 cells, demonstrating that filaggrin can affect cell morphology independent of the presence of a cytoplasmic IF network. These studies demonstrate that filaggrin, in addition to its known effects on IF organization, can affect the distribution of other cytoskeletal elements including actin microfilaments, which can occur in the absence of a cytoplasmic IF network. Further, filaggrin can disrupt the distribution of desmosome proteins, suggesting an additional role(s) for this protein in the cytoskeletal and desmosomal reorganization that occurs at the granular to cornified cell transition during terminal differentiation of epidermal keratinocytes.     

Histidine-rich proteins (filaggrins): structural and functional heterogeneity during epidermal differentiation

The urea-soluble protein profiles of guinea pig, rat, mouse and human epidermis have been compared by non-equilibrium pH gradient/sodium dodecyl sulphate two-dimensional gel electrophoresis. The histidine-rich proteins (filaggrins) were identified firstly by their characteristic specificity and kinetics of labelling with [3H]histidine and [32P]phosphate, and secondly by their ability in vitro to aggregate keratin filaments specifically into bundles. In all species the phosphorylated filaggrin precursor, profilaggrin, is resolved as a single or doublet band with an apparent molecular weight greater than 300,000 and a neutral or slightly acidic iso-electric point. In striking contrast, the strongly basic filaggrins produced from similar profilaggrins form molecular weight families that are clearly species specific. In rat and man there is a single, principal molecular weight form of filaggrin (Mr 45,000 and 38,000, respectively), while mouse and guinea pig have heterogeneous families, including high molecular weight variants (Mr greater than 200,000). Even filaggrins of a particular molecular weight are not homogeneous proteins, but consist of a number of iso-electric variants, some of which are considerably less basic than the bulk of the filaggrins. Incorporation studies using [3H]arginine and [32P]phosphate indicate that the iso-electric variance is not due to residual phosphate, following profilaggrin breakdown, but rather to a conversion of basic arginine residues into neutral citrulline residues. Filaggrins of all the molecular weights from all the species studied share the ability to aggregate keratin filaments into large, insoluble macrofibrils. However, the more acidic iso-electric variants have lower affinities for keratin, particularly in man and guinea pig where the most acidic filaggrins have completely lost the ability to aggregate keratins. We discuss the possibility that a loss of keratin binding ability, resulting in a loosening of the keratin fibre/filaggrin matrix is necessary before the normal complete proteolysis of the filaggrins can occur.     

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.     

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.     

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.     

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.     

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.     

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.     

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)     

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.     

Formation of a Normal Epidermis Supported by Increased Stability of Keratins 5 and 14 in Keratin 10 Null Mice

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.     

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.     

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.     

Filaggrin breakdown to water binding compounds during development of the rat stratum corneum is controlled by the water activity of the environment

Filaggrin is a specific epidermal protein which is the precursor of the free amino acids, urocanic acid and pyrrolidone carboxylic acid which are largely responsible for the ability of the stratum corneum of the skin to remain hydrated at low environmental humidity. The distribution of filaggrin shown by immunofluorescence in the stratum corneum of the rat changed dramatically during the first hours of postnatal life. During late foetal development, filaggrin accumulated through the entire thickness of the stratum corneum, indicating that there was a block on the subsequent processing of the protein which normally would convert it to free amino acids. Immediately after birth this block was lifted and normal proteolysis of the filaggrin took place in the outer part of the stratum corneum, leaving the normal adult pattern of a thin zone of cells containing filaggrin at the bottom of the stratum corneum. This activation of filaggrin proteolysis was dependent on the drop in external water activity caused by the transition from an aqueous environment in utero to a dryer environment after birth and it could be blocked by maintaining a 100% humidity atmosphere around the newborn rat after birth. In isolated stratum corneum in vitro, filaggrin proteolysis took place only between 80 and 95% relative humidity, both higher and lower relative humidity blocked the proteolysis. Application of occlusive patches to adult rats prevented the normal proteolysis of filaggrin, indicating that this mechanism controls not only the massive filaggrin proteolysis occurring after birth but also the proteolysis occurring during normal stratum corneum maturation. The stratum corneum therefore has the ability to respond to changes in external humidity by altering the level of the stratum corneum where it converts its reserves of filaggrin into water binding amino acids, such that under humid conditions water binding components will be produced in only the most superficial stratum corneum, or even not produced at all.     

Cornification in reptilian epidermis occurs through the deposition of keratin‐associated beta‐proteins (beta‐keratins) onto a scaffold of intermediate filament keratins

The isolation of genes for alpha‐keratins and keratin‐associated beta‐proteins (formerly beta‐keratins) has allowed the production of epitope‐specific antibodies for localizing these proteins during the process of cornification epidermis of reptilian sauropsids. The antibodies are directed toward proteins in the alpha‐keratin range (40–70 kDa) or beta‐protein range (10–30 kDa) of most reptilian sauropsids. The ultrastructural immunogold study shows the localization of acidic alpha‐proteins in suprabasal and precorneous epidermal layers in lizard, snake, tuatara, crocodile, and turtle while keratin‐associated beta‐proteins are localized in precorneous and corneous layers. This late activation of the synthesis of keratin‐associated beta‐proteins is typical for keratin‐associated and corneous proteins in mammalian epidermis (involucrin, filaggrin, loricrin) or hair (tyrosine‐rich or sulfur‐rich proteins). In turtles and crocodilians epidermis, keratin‐associated beta‐proteins are synthesized in upper spinosus and precorneous layers and accumulate in the corneous layer. The complex stratification of lepidosaurian epidermis derives from the deposition of specific glycine‐rich versus cysteine‐glycine‐rich keratin‐associated beta‐proteins in cells sequentially produced from the basal layer and not from the alternation of beta‐ with alpha‐keratins. The process gives rise to Oberhäutchen, beta‐, mesos‐, and alpha‐layers during the shedding cycle of lizards and snakes. Differently from fish, amphibian, and mammalian keratin‐associated proteins (KAPs) of the epidermis, the keratin‐associated beta‐proteins of sauropsids are capable to form filaments of 3–4 nm which give rise to an X‐ray beta‐pattern as a consequence of the presence of a beta‐pleated central region of high homology, which seems to be absent in KAPs of the other vertebrates. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

Phosphorylation of microtubule-associated proteins regulates their interaction with actin filaments

We have determined the absolute phosphate content of microtubule-associated proteins (MAPs) and established that phosphorylation inhibits the actin filament cross-linking activity of MAPs and both of the major MAP components, MAP-2 and tau. Similar results were obtained with actin from rabbit muscle, hog brain, and Acanthamoeba castellanii. We used the endogenous phosphatases and kinases in hog brain microtubule protein to modulate MAP phosphate level before isolating heat-stable MAPs. MAPs isolated directly from twice-cycled microtubule protein contain 7.1 +/- 0.1 (S.E.) mol of phosphate/300,000 g protein. After incubating microtubule protein without ATP, MAPs, had 4.9 +/- 0.6 phosphates. After incubating microtubule protein with 1 mM ATP and 5 microM cAMP in 2 mM EGTA, MAPs had 8.6 +/- 0.5 phosphates but there was also exchange of three more [32P]phosphates from gamma-labeled ATP for preexisting MAP phosphate. Incubation of microtubule protein with ATP and cAMP in 5 mM CaCl2 resulted in exchange but no net addition of phosphate to MAPs. We fractionated the MAP preparations by gel filtration and obtained MAP-2 with 4.3 to 7.5 and tau with 1.5 to 2.2 mol of phosphate/mol of protein depending on how we treated the microtubule protein prior to MAP isolation. The actin filament cross-linking activity of whole MAPs, MAP-2, and tau depended on the MAP-phosphate content. In all cases, phosphorylation of MAPs inhibited actin filament cross-linking activity. The concentration of high phosphate MAPs required to form a high viscosity solution with actin filaments was 2 to 4 times more than that of low phosphate. MAPs. During incubation of microtubule protein with [gamma-32P]ATP, only MAP peptides are labeled. Treatment of these MAPs with either acid or alkaline phosphatase removes phosphate mainly from MAP-2, with an increase in actin filament cross-linking activity. Thus, both MAP phosphorylation and the effect of phosphorylation on actin cross-linking activity of MAPs are reversible.     

Effect of chronic ethanol ingestion on phosphate content of neurofilament proteins and neurofilament associated protein phosphatase in rat spinal cord

Rats were trained to drink alcohol solution by gradually increasing the ethanol content [2.5–15% (v/v)] in drinking water. After 11 months of alcohol (15% v/v) ingestion, animals were guillotined and the spinal cords were used for the preparation of neurofilaments (NF). NF triplet proteins were separated by SDS-PAGE and the phosphate contents of individual components were estimated. Results indicated a significant increase in phosphate content of 200 KD protein in alcohol fed rats (30.19±4.12 mol of phosphate/mole of protein: p<0.001) compared to control group (18.42 ±3.91 mol of phosphate/mole of protein). No significant change in the phosphate content of 150KD and 68KD components of NF were seen in experimental group. Further, the studies on NF associated protein phosphatase activity indicated a significant decrease in phosphatase activity among the alcohol fed rats (14.10±2.5 mU; p<0.001) against NF rich fraction as a substrate, as compared to control (20.15±2.15 mU). While the observed decrease in NF associated protein phosphatase would possibly explain the increase in phosphate content of NF proteins in alcohol fed rats, the precise mechanism of decrease in enzyme activity remains to be elucidated. Nevertheless, the change seen in phosphate content and NF associated protein phosphatase activity as a result of ethanol ingestion would possibly form the biochemical basis of some of the neuropathological changes seen in alcoholics.     

Biosynthetic pathways of filaggrin and loricrin--two major proteins expressed by terminally differentiated epidermal keratinocytes

We have used immunoelectron microscopy to map the biosynthetic pathways of loricrin and filaggrin in epidermal keratinocytes at successive stages of differentiation in newborn mouse skin. The filaggrin epitope is first detected in large, irregularly shaped, keratohyalin granules (F-granules) in the stratum granulosum, and then distributed throughout the cytoplasms of the innermost layers of stratum corneum cells. We conclude that the poly-protein filaggrin precursor is first accumulated in F-granules, from which it is subsequently released and processed into filaggrin, and becomes associated with the densely packed bundles of keratin filaments inside stratum corneum cells. Its diminished visibility in the outer layers correlates with the known degradation of filaggrin to free amino acids. Loricrin is first detected in small round keratohyalin granules (L-granules), and subsequently at the periphery of cells throughout the stratum corneum. Labeling of purified keratinocyte envelopes establishes that this loricrin epitope is exposed only at their inner (cytoplasmic) surface. Thus loricrin is initially accumulated in L-granules, to be released at a specifically programmed stage of keratinocyte maturation, and incorporated into the covalently cross-linked lining of the cell envelope. Since loricrin is rich in cysteine, L-granules account for the sulfur-rich keratohyalin granules described earlier. Proposals are made to rationalize why, subsequent to synthesis, filaggrin precursor and loricrin should be segregated both from each other and from the rest of the cytoplasm.     

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.