Formation of the corneous layer in the epidermis of the tuatara (Sphenodon punctatus, Sphenodontida, Lepidosauria, Reptilia)

The formation of the stratum corneum in the epidermis of the reptile Sphenodon punctatus has been studied by histochemical, immunohistochemical, and ultrastructural methods. Sulfhydryl groups are present in the mesos and pre-alpha-layer but disappear in the keratinized beta-layer and in most of the mature alpha-layer. This suggests a complete cross-linking of keratin filaments. Tyrosine increases in keratinized layers, especially in the beta-layer. Arginine is present in living epidermal layers, in the presumptive alpha-layer, but decreases in keratinized layers. Histidine is present in corneous layers, especially in the intermediate region between the alpha- and a new beta-layer, but disappears in living layers. It is unknown whether histidine-rich proteins are produced in the intermediate region. Small keratohyalin-like granules are incorporated in the intermediate region. The plane of shedding, as confirmed from the study on molts, is located along the basalmost part of the alpha-layer and may involve the degradation of whole cells or cell junctions of the intermediate region. A specific shedding complex, like that of lizards and snakes, is not formed in tuatara epidermis. AE1-, AE2-, or AE3-positive alpha-keratins are present in different epidermal layers with a pattern similar to that previously described in reptiles. The AE1 antibody stains the basal and, less intensely, the first suprabasal layers. Pre-keratinized, alpha- and beta-layers, and the intermediate region remain unlabeled. The AE2 antibody stains suprabasal and forming alpha- and beta-layers, but does not stain the basal and suprabasal layers. In the mature beta-layer the immunostaining disappears. The AE3 antibody stains all epidermal layers but disappears in alpha- and beta-layers. Immunolocalization for chick scale beta-keratins labels the forming and mature beta-layer, but disappears in the mesos and alpha-layer. This suggests the presence of common epitopes in avian and reptilian beta-keratins. Low molecular weight alpha-keratins present in the basal layer are probably replaced by keratins of higher molecular weight in keratinizing layers (AE2-positive). This keratin pattern was probably established since the beginning of land adaptation in amniotes.     

Ultrastructural localization of alpha-keratins in the regenerating epidermis of the lizard Podarcis muralis during formation of the shedding layer

In the epidermis of lizards, alpha- and beta-keratins are sequentially produced during a shedding cycle. Using pre- and post-embedding immunocytochemistry this study shows the ultrastructural distribution of 3 alpha-keratin antibodies (AE1, AE2, AE3) in the renewing epidermis and in the shedding complex of the regenerating tail of the lizard Podarcis muralis. The AE1 antibody that recognizes acidic low MW keratins is confined to tonofilament bundles in basal and suprabasal cells but is not present in keratinizing beta- and alpha-cells. The AE2 antibody that recognises higher MW keratins weakly stains pre-keratinized cells and intensely keratinized alpha-layers. A weak labeling is present in small electrondense areas within the beta-layer. The AE3 antibody, that recognizes low and high MW basic keratins, immunolabels tonofilament bundles in all epidermal layers but intensely the alpha-keratinizing and keratinized layers (mesos, alpha-, lacunar and clear). Keratohyalin-like granules, present in the clear cells of the shedding layer, are negative to these antibodies so that the cornified clear layer contains keratins mixed with non-keratin material. The AE3 antibody shows that the mature beta-layer and the spinulated folds of the oberhautchen are labeled only in small dense areas among the prevalent electron-pale beta-keratin material. Therefore, some alpha-keratin is still present in the beta-layer, and supports the idea that alpha-keratins (basic) function as scaffold for beta-keratin deposition.     

Immunocytochemical and electrophoretic distribution of cytokeratins in the resting stage epidermis of the lizard Podarcis sicula

The distribution of three anti-cytokeratin (alpha-keratin) antibodies (AE1, AE2, AE3) in the epidermis of a lizard has been studied by immunocytochemistry at light and electron microscope and by immunoblot analysis. This study shows the expression of different keratins in the resting stage epidermis of the lizard Podarcis sicula. In this stage the epidermis has an external beta-layer, an underlying alpha-layer, some layers of living suprabasal cells and a basal stratum germinativum. The AE1 antibody is localized in the basal and suprabasal cells only in the outer scale surface, but is absent from the inner surface, the hinge region and from the keratinized beta- and alpha-layers. The AE2 antibody is mainly localized at the level of the hinge region and of the alpha-layer and gives a lower reaction in the beta-layer. The AE3 antibody is mainly localized in basal and suprabasal cells, lower in the alpha-layer, and absent from the beta-layer. The electron microscope shows that all the three antibodies immunolabel cytoplasmic fibrillar structures in the deep alpha-layers and that AE2 and AE3 antibodies label small electron-dense areas in the external dense beta-layer within the electron-lucid matrix. Immunoblot analysis of the keratins extracted and separated by gel electrophoresis demonstrates the presence of a band of high molecular weight (67-68 kDa) positive to all three antibodies. In addition AE1 antibody recognizes a 44-45 kDa band and a 57-58 kDa band, AE2 recognizes a 60-61 kDa band, and AE3 recognizes a 47 kDa and a 56-57 kDa band. The localization of the keratins identified by immunoblot analysis in the epithelial layers is discussed taking in account the immunolabeling at light and electron microscope. The present study suggests that also in the normal epidermis of this reptiles, in both the alpha- and the beta-layer, the molecular masses of keratins increase from the basal to the keratinized layers, a phenomenon which is generalized to adult and embryonic amniotes epidermis.     

Immunolocalization of keratin-associated beta-proteins (beta-keratins) in scales of the reptiles Sphenodon punctatus indicates that different beta-proteins are present in beta- and alpha-layers

The present ultrastructural immunocytochemical study analyzes the localization of keratin-associated beta-proteins (beta-keratins) in the epidermis of the ancient reptile Sphenodon punctatus, a relict species adapted to mid-cold conditions. The epidermis comprises two main layers, indicated as beta- and alpha-keratin layers. The beta-layer contains small beta-proteins (beta-keratins) identified by using three different antibodies while the alpha-layer is poorly or not labeled for these proteins. Using other two antibodies directed against specific amino acid sequences identified in beta-proteins of lizard it results that a high-glycine beta-protein (HgG5) is specific for the beta-layer. Another antibody that recognizes glycine–cysteine medium-rich beta-proteins (HgGC10) immuno-stains beta- and alpha-layers. This pattern of distribution suggests that both beta- and alpha-layers contain beta-proteins of different types that associate and replace intermediate-filament alpha-keratins during the terminal differentiation of keratinocytes. Therefore the different epidermal layers of the epidermis in S. punctatus, characterized by a specific cytology, material properties and consistency appear to derive from the prevalent type of beta-proteins synthesized in each epidermal layer and not from the alternation between beta- and alpha-keratins. The present observations are discussed in comparison to previous results from lizard epidermis and indicate that beta-keratins correspond to keratin-associated proteins that through their internal beta-pleated region are capable to form filaments in addition to intermediate filaments keratins.     

Immunolocalization of alpha-keratins and associated beta-proteins in lizard epidermis shows that acidic keratins mix with basic keratin-associated beta-proteins

The differentiation of the corneous layers of lizard epidermis has been analyzed by ultrastructural immunocytochemistry using specific antibodies against alpha-keratins and keratin associated beta-proteins (KAbetaPs, formerly indicated as beta-keratins). Both beta-cells and alpha-cells of the corneous layer derive from the same germinal layer. An acidic type I alpha-keratin is present in basal and suprabasal layers, early differentiating clear, oberhautchen, and beta-cells. Type I keratin apparently disappears in differentiated beta- and alpha-layers of the mature corneous layers. Conversely, a basic type II alpha-keratin rich in glycine is absent or very scarce in basal and suprabasal layers and this keratin likely does not pair with type I keratin to form intermediate filaments but is weakly detected in the pre-corneous and corneous alpha-layer. Single and double labeling experiments show that in differentiating beta-cells, basic KAbetaPs are added and replace type-I keratin to form the hard beta-layer. Epidermal alpha-keratins contain scarce cysteine (0.2–1.4 %) that instead represents 4–19 % of amino acids present in KAbetaPs. Possible chemical bonds formed between alpha-keratins and KAbetaPs may derive from electrostatic interactions in addition to cross-linking through disulphide bonds. Both the high content in glycine of keratins and KAbetaPs may also contribute to increase the hydrophobicy of the beta- and alpha-layers and the resistance of the corneous layer. The increase of gly-rich KAbetaPs amount and the bonds to the framework of alpha-keratins give rise to the inflexible beta-layer while the cys-rich KAbetaPs produce a pliable alpha-layer.     

Keratinization in the epidermis of amphibians and the lungfish: comparison with amniote keratinization

Keratinization in the epidermis of amphibians and the lungfish has been studied by electron microscopy, autoradiography and immunocytochemistry to determine whether histidine-rich proteins, filaggrin and loricrin are present. In the lungfish and amphibian tadpoles, anti-keratin antibodies (AE1 and AE3) stain the whole epidermis but not the AE2 antibody, a marker for keratinization. In adult epidermis, the AE2 antibody mainly stains keratinized layers, AE1 mainly stained basal cells, less suprabasal cells and no pre-keratinized and keratinized layers, and AE3 stains all epidermal layers. This staining pattern resembles that of amniote epidermis. Little tritiated histidine is taken up in toad epidermis at 4-6 h post-injection but 24 h after injection the radioactivity is most concentrated in the replacement layer beneath the corneus. This indicates that protein synthesis takes place in the epidermis but, due to the metabolic conversion that takes place in 24 h, it is unlikely that histidine-rich proteins are formed. Neither filaggrin-like nor loricrine-like immunoreactivities are present in amphibian and lungfish epidermis. This indicates absence of histidine-rich matrix proteins and corneous cell envelope proteins and only mucus is present among keratin filaments. Filaggrine-like and loricrin-like proteins are characteristic of amniotes epidermis and might have originated in basic amniotes (cotylosaurs).     

Keratinization of the epidermis of the Australian lungfish Neoceratodus forsteri (dipnoi)

The differentiation of the epidermis in sarcopterigian fish may reveal some trend of keratinization followed by amphibian ancestors to adapt their epidermis to land. Therefore, the process of keratinization of the epidermis of the Australian lungfish Neoceratodus forsteri was studied by histochemistry, electron microscopy, and keratin immunocytochemistry. The epidermis is tri-stratified in a 2-3-month-old tadpole but becomes 6-8 stratified in young adults. Keratin filaments increase from basal to external cells where loose tonofilament bundles are present. This is shown also by the comparison of positivity to sulfhydryl groups and increasing immunoreactivity to alpha-keratins in more external layers of the epidermis. Two broad-spectrum anti alpha-keratin monoclonal antibodies (AE1 and AE3) stain all epidermal layers as they do in actinopterigian fish. In the adult epidermis, but not in that of the larva, the AE2 antibody (a marker of keratinization in mammalian epidermis) often immunolabels more heavily the external keratinized layers where sulfhydryl groups are more abundant. Mucous granules are numerous and concentrate on the external surface of the epidermis to be discharged and contribute to cuticle formation. Keratin is therefore embedded in a mucus matrix, but neither compact keratin masses nor cell corneous envelope were seen in external cells. It is not known whether specific matrix proteins are associated with mucus. There was no immunolocalization of the keratin-associated proteins, filaggrin and loricrin, which suggests that the epidermis of this species lacks the matrix and cell corneus envelope proteins characteristic of that of amniotes. In conclusion, while specific keratins (AE2 positive) are probably produced in the uppermost layers as in amphibian epidermis, no interkeratin, matrix proteins seem to be present in external keratinocytes of the lungfish other than mucus.     

Expression of specific keratin markers by rabbit corneal, conjunctival, and esophageal epithelia during vitamin A deficiency

Using an in vivo rabbit model system, we have studied the morphological and biochemical changes in corneal, conjunctival, and esophageal epithelia during vitamin A deficiency. Light and electron microscopy showed that the three epithelia undergo different degrees of morphological keratinization. Corneal and conjunctival epithelia became heavily keratinized, forming multiple layers of superficial, anucleated cornified cells. In contrast, esophageal epithelium underwent only minor morphological changes. To correlate morphological alterations with the expression of specific keratin molecules, we have analyzed the keratins from these epithelia by the immunoblot technique using the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies. The results indicate that during vitamin A deficiency, all three epithelia express an AE1-reactive, acidic 56.5-kd keratin and an AE3-reactive, basic 65-67-kd keratin. Furthermore, the expression of these two keratins correlated roughly with the degree of morphological keratinization. AE2 antibody (specific for the 56.5- and 65-67-kd keratins) stained keratinized corneal epithelial sections suprabasally, as in the epidermis, suggesting that these two keratins are expressed mainly during advanced stages of keratinization. These two keratins have previously been suggested to represent markers for epidermal keratinization. Our present data indicate that they can also be expressed by other stratified epithelia during vitamin A deficiency-induced keratinization, and suggest the possibility that they may play a role in the formation of the densely packed tonofilament bundles in cornified cells of keratinized tissues.     

Distribution and characterization of keratins in the epidermis of the tuatara (Sphenodon punctatus; Lepidosauria, Reptilia)

Reptilian scales are mainly composed of alpha-and beta-keratins. Epidermis and molts from adult individuals of an ancient reptilian species, the tuatara (Sphenodon punctatus), were analysed by immunocytochemistry, mono- and bi-dimensional electrophoresis, and western blotting for alpha- and beta-keratins. The epidermis of this reptilian species with primitive anatomical traits should represent one of the more ancient amniotic epidermises available. Soft keratins (AE1- and AE3-positive) of 40-63 kDa and with isoelectric points (pI) at 4.0-6.8 were found in molts. The AE3 antibody was diffusely localised over the tonofilaments of keratinocytes. The lack of basic cytokeratins may be due to keratin alteration in molts, following corneification or enzymatic degradation of keratins. Hard (beta-) keratins of 16-18 kDa and pI at 6.8, 8.0, and 9.2 were identified using a beta-1 antibody produced against chick scale beta-keratin. The antibody also labeled filaments of beta-cells and of the mature, compact beta-layer. We have shown that beta-keratins in the tuatara resemble those of lizards and snakes, and that they are mainly basic proteins. These proteins replace cytokeratins in the pre-corneoum beta-layers, from which a hard, mechanically resistant corneoum layer is formed over scales. Beta-keratins may have both a fibrous and a matrix role in forming the hard texture of corneoum scales in this ancient species, as well as in more recently evolved reptiles.     

The use of aIF, AE1, and AE3 monoclonal antibodies for the identification and classification of mammalian epithelial keratins

Recent data have indicated that specific keratin molecules can provide useful markers for studying different types and stages of epithelial differentiation. To utilize these protein markers, however, it is important to establish the keratin nature of the molecules and identify unambiguously the individual keratin species. In this paper, we show that this can be done relatively easily by one- and two-dimensional gel electrophoresis combined with immunoblotting using three monoclonal antibodies (aIF, AE1, and AE3). The aIF antibody has previously been shown to crossreact with all classes of intermediate-filament proteins. Using one- and two-dimensional immunoblotting, we establish that this antibody recognizes all known epithelial keratins of human and rabbit, although the reaction is relatively strong for the larger, basic keratins and is relatively weak for some of the smaller, acidic keratins. In contrast, AE1 and AE3 monoclonal antibodies have previously been shown to be highly specific for the acidic and basic subfamilies of the keratins, respectively. The combined use of the broadly reacting aIF antibody and the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies should facilitate the immunological definition, identification, and classification of mammalian epithelial keratins.     

The epidermis of scales in gecko lizards contains multiple forms of beta-keratins including basic glycine-proline-serine-rich proteins

The epidermis of scales of gecko lizards comprises alpha- and beta-keratins. Using bidimensional electrophoresis and immunoblotting, we have characterized keratins of corneous layers of scales in geckos, especially beta-keratins in digit pad lamellae. In the latter, the formation of thin bristles (setae) allow for the adhesion and climbing vertical or inverted surfaces. alpha-Keratins of 55-66 kDa remain in the acidic and neutral range of pI, while beta-keratins of 13-18 kDa show a broader variation of pI (4-10). Some protein spots for beta-keratins correspond to previously sequenced, basic glycine-proline-serine-rich beta-keratins of 169-191 amino acids. The predicted secondary structure shows that a large part of the molecule has a random-coiled conformation, small alpha helix regions, and a central region with 2-3 strands (beta-folding). The latter, termed core-box, shows homology with feather-scale-claw keratins of birds and is involved in the formation of beta-keratin filaments. Immunolocalization of beta-keratins indicates that these proteins are mainly present in the beta-layer and oberhautchen layer, including setae. The sequenced proteins of setae form bundles of keratins that determine their elongation. This process resembles that of feather-keratin on the elongation of barbule cells in feathers. It is suggested that small proteins rich in glycine, serine, and proline evolved in reptiles and birds to reinforce the mechanical resistance of the cytokeratin cytoskeleton initially present in the epidermis of scales and feathers.     

Immunocytochemistry indicates that glycine‐rich beta‐proteins are present in the beta‐layer,while cysteine‐rich beta‐proteins are present in beta‐ and alpha‐layers of snake epidermis

Immunolocalization of glycine‐rich and cysteine–glycine‐medium‐rich beta‐proteins (Beta‐keratins) in snake epidermis indicates a different distribution between beta‐ and alpha‐layers. Acta Zoologica, Stockholm. The epidermis of snakes consists of hard beta‐keratin layers alternated with softer and pliable alpha‐keratin layers. Using Western blot, light and ultrastructural immunolocalization, we have analyzed the distribution of two specific beta‐proteins (formerly beta‐keratins) in the epidermis of snakes. The study indicates that the antibody HgG5, recognizing glycine‐rich beta‐proteins of 12–15 kDa, is poorly or not reactive with the beta‐layer of snake epidermis. This suggests that glycine‐rich proteins similar to those present in lizards are altered during maturation of the beta‐layer. Conversely, a glycine–cysteine‐medium‐rich beta‐protein (HgGC10) of 10–12 kDa is present in beta‐ and alpha‐layers, but it is reduced or disappears in precorneous and suprabasal cells destined to give rise to beta‐ and alpha‐cells. Together with the previous studies on reptilian epidermis, the present results suggest that beta‐proteins rich in glycine mainly accumulate on a scaffold of alpha‐keratin producing a resistant and hydrophobic beta‐layer. Conversely, beta‐proteins lower in glycine but higher in cysteine accumulate on alpha‐keratin filaments present in beta‐ and alpha‐layers producing resistant but more pliable layers.     

Immunocytochemical observations on the cornification of soft and hard epidermis in the turtle Chrysemys picta

The process of cornification in the shell and non-shelled areas of the epidermis of the turtle Chrysemys picta was analyzed by light and ultrastructural immunohistochemistry for keratins, filaggrin and loricrin. Beta-keratin (hard keratin) was only present in the corneus layer of the plastron and carapace. The use of a beta-keratin antibody, developed against a specific chick scale beta-keratin, demonstrated that avian and reptilian hard keratins share common amino acid sequences. In both, shelled and non-shelled epidermis, acidic alpha keratin (AE1 positive) was limited to tonofilament bundles of the basal and suprabasal layer, while basic keratin (AE3 positive) was present in basal, suprabasal, and less intensely, pre-corneus layers, but tended to disappear in the corneus layer. The AE2 antibody, which in mammalian epidermis recognizes specific keratins of cornification, did not stain turtle shell but only the corneus layer of non-shelled (soft) epidermis. Two and four hours after an injection of tritiated histidine, the labelling was evenly distributed over the whole epidermis of both shelled and non-shelled areas, but was absent from the stratum corneum. In the areas of growth at the margin of the scutes of the shell, the labelling increased in precorneus layers. This suggests that histidine uptake is only related to shell growth and not to the production of a histidine-rich protein involved in keratinization. No filaggrin-like and loricrin-like immunoreactivity was seen in the carapace or plastron epidermis. However, in both proteins, some immunoreactivity was found in the transitional layer and in the lower level of the corneus layer of non-shelled areas. Loricrin- and filaggrin-like labelling was seen in small organelles (0.05-0.3 mum) among keratin bundles, identified with mucous-like granules and vesicular bodies. These organelles, present only in non-shelled epidermis, were more frequent along the border with the corneus layer, and labelling was low to absent in mature keratinocytes. This may be due to epitope masking or degradation. The immunolabelling for filaggrin was seen instead in the extracellular space among mature keratinocytes, over a material previously identified as mucus. The possibility that this labelling identified some epitopes derived from degraded portions of a filaggrin-like molecule is discussed. The present study suggests that proteins with some filaggrin- and loricrin-immunoreactivity are present in alpha-keratinocytes but not in beta-keratin cells of the shell.     

Expression of epidermal keratins and filaggrin during human fetal skin development

The major structural proteins of epithelia, the keratins, and the keratin filament-associated protein, filaggrin, were analyzed in more than 50 samples of human embryonic and fetal skin by one-dimensional SDS PAGE and immunoblotting with monoclonal and polyclonal antibodies. Companion samples were examined by immunohistochemistry and electron microscopy. Based on structural characteristics of the epidermis, four periods of human epidermal development were identified. The first is the embryonic period (before 9 wk estimated gestational age), and the others are within the fetal period: stratification (9-14 wk), follicular keratinization (14-24 wk), and interfollicular keratinization (beginning at approximately 24 wk). Keratin proteins of both the acidic (AE1-reactive, type I) and the basic (AE3-reactive, type II) subfamilies were present throughout development. Keratin intermediate filaments were recognized in the tissue by electron microscopy and immunohistochemical staining. Keratins of 50 and 58 kD were present in the epidermis at all ages studied (8 wk to birth), and those of 56.5 and 67 kD were expressed at the time of stratification and increased in abundance as development proceeded. 40- and 52-kD keratins were present early in development but disappeared with keratinization. Immunohistochemical staining suggested the presence of keratins of 50 and 58 kD in basal cells, 56.5 and 67 kD in intermediate cells, and 40 and 52 kD in the periderm as well as in the basal cells between the time of stratification and birth. Filaggrin was first detected biochemically at approximately 15 wk and was localized immunohistochemically in the keratinizing cells that surround hair follicles. It was identified 8-10 wk later in the granular and cornified cell layers of keratinized interfollicular epidermis. These results demonstrate the following. An intimate relationship exists between expression of structural proteins and morphologic changes during development of the epidermis. The order of expression of individual keratins is consistent with the known expression of keratins in simple vs. stratified vs. keratinized epithelia. Expression of keratins typical of stratified epithelia (50 and 58 kD) precedes stratification, and expression of keratins typical of keratinization (56.5 and 67 kD) precedes keratinization, which suggests that their expression marks the tissue commitment to those processes. Because only keratins that have been demonstrated in various adult tissues are expressed during fetal development, we conclude that there are no "fetal" keratins per se.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alpha- and beta-keratins of the snake epidermis

Snake scales contain specialized hard keratins (beta-keratins) and alpha- or cyto-keratins in their epidermis. The number, isoelectric point, and the evolution of these proteins in snakes and their similarity with those of other vertebrates are not known. In the present study, alpha- and beta-keratins of snake molts and of the whole epidermis have been studied by using two-dimensional electrophoresis and immunocytochemistry. Specific keratins in snake epidermis have been identified by using antibodies that recognize acidic and basic cytokeratins and avian or lizard scale beta-keratin. Alpha keratins of 40-70 kDa and isoelectric point (pI) at 4.5-7.0 are present in molts. The study suggests that cytokeratins in snakes are acidic or neutral, in contrast to mammals and birds where basic keratins are also present. Beta keratins of 10-15 kDa and a pI of 6.5-8.5 are found in molts. Some beta-keratins appear as basic proteins (pI 8.2) comparable to those present in the epidermis of other reptiles. Some basic "beta-keratins" associate with cytokeratins as matrix proteins and replace cytokeratins forming the corneous material of the mature beta-layer of snake scales, as in other reptiles. The study also suggests that more forms of beta-keratins (more than three different types) are present in the epidermis of snakes.     

Review: mapping epidermal beta-protein distribution in the lizard <Emphasis Type="Italic">Anolis carolinensis</Emphasis> shows a specific localization for the formation of scales,pads, and claws

The epidermis of lizards is made of multiple alpha- and beta-layers with different characteristics comprising alpha-keratins and corneous beta-proteins (formerly beta-keratins). Three main modifications of body scales are present in the lizard Anolis carolinensis: gular scales, adhesive pad lamellae, and claws. The 40 corneous beta-proteins present in this specie comprise glycine-rich and glycine-cysteine-rich subfamilies, while the 41 alpha-keratins comprise cysteine-poor and cysteine-rich subfamilies, the latter showing homology to hair keratins. Other genes for corneous proteins are present in the epidermal differentiation complex, the locus where corneous protein genes are located. The review summarizes the main sites of immunolocalization of beta-proteins in different scales and their derivatives producing a unique map of body distribution for these structural proteins. Small glycine-rich beta-proteins participate in the formation of the mechanically resistant beta-layer of most scales. Small glycine-cysteine beta-proteins have a more varied localization in different scales and are also present in the pliable alpha-layer. In claws, cysteine-rich alpha-keratins prevail over cysteine-poor alpha-keratins and mix to glycine-cysteine-rich beta-proteins. The larger beta-proteins with a molecular mass similar to that of alpha-keratins participate in the formation of the fibrous meshwork present in differentiating beta-cells and likely interact with alpha-keratins. The diverse localization of alpha-keratins, beta-proteins, and other proteins of the epidermal differentiation complex gives rise to variably pliable, elastic, or hard corneous layers in different body scales. The corneous layers formed in the softer or harder scales, in the elastic pad lamellae, or in the resistant claws possess peculiar properties depending on the ratio of specific corneous proteins.     

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.     

Wound keratins in the regenerating epidermis of lizard suggest that the wound reaction is similar in the tail and limb

The keratin cytoskeleton of the wound epidermis of lizard limb (which does not regenerate) and tail (which regenerates) hase been studied by qualitative ultrastructural, immunocytochemical, and immunoblotting methods. The process of re-epithelialization is much shorter in the tail than in the limb. In the latter, a massive tissue destruction of bones, and the shrinkage of the old skin over the stump surface, delay wound closure, maintain inflammation, reduce blastemal cell population, resulting in inhibition of regeneration. The expression of special wound keratins found in the newt epidermis (W6) or mammalian epidermis (K6, K16, and K17) is present in the epidermis of both tail and limb of the lizard. These keratins are not immunolocalized in the migrating epithelium or normal (resting) epidermis but only after it has formed the thick wound epithelium, made of lacunar cells. The latter are proliferating keratinocytes produced during the cyclical renewal or regeneration of lizard epidermis. W6-immunolabeled proteic bands mainly at 45-47 kDa are detected by immunoblotting in normal, regenerating, and scarring epidermis of the tail and limb. Immunolabeled proteic bands at 52, 62-67 kDa (with K6), at 44-47, 60, 65 kDa (with K16), and at 44-47 kDa (with K17) were detected in normal and regenerating epidermis. It is suggested that: (1) these keratins constitute normal epidermis, especially where the lacunar layer is still differentiating; (2) the wound epidermis is similar in the limb and tail in terms of morphology and keratin content; (3) the W6 antigen is similar to that of the newt, and is associated with tonofilaments; (4) lizard K6 and K17 have molecular weights similar to mammalian keratins; (5) K16 shows some isoforms or degradative products with different molecular weight from those of mammals; (6) K17 increases in wound keratinocytes and localizes over sparse filaments or small bundles of short filaments, not over tonofilaments joined to desmosomes; and (7) failure of limb regeneration in lizards may not depend on the wound reaction of keratinocytes.     

Presence of putative histidine-rich proteins in the amphibian epidermis

In amphibian epidermis mucus is thought to constitute the matrix material that links keratin filaments present in cells of the corneous layer. As contrast in mammals, and perhaps in all amniotes, histidine-rich proteins form the matrix material. In order to address the study of matrix molecules in the epidermis of the first tetrapods, the amphibians, an autoradiographic and electrophoretic study has been done after administration of tritiated histidine. Histological analysis of amphibian epidermis shows that histidine is taken up in the upper intermediate and replacement layers beneath the corneous layer. Ultrastructural autoradiographic analysis reveals that electron-dense interkeratin material is labeled after administration of tritiated histidine. Electrophoretic analysis of the epidermis shows labeled proteic bands at 58-61, 50-55, 40-45, and some only weakly labeled at 30 and 24-25 kDa at 4-48 hours after injection of tritiated histidine. Keratin markers show that bands at 40-61 kDa contain keratins. Most histidine is probably converted into other amino acids such as glutamate and glutamine that are incorporated into newly synthetized keratins. However, non-keratin histidine-incorporating proteins within the keratin range could also be formed. The bands at 30 and 24-25 kDa suggest that these putative histidine-rich proteins are not keratins. In fact, their molecular weigh is below the range of that for keratins. In contrast with the mammalian condition, but resembling reports for lizard epidermis, putative histidine-rich proteins in amphibians have no high molecular weight precursor. Although filaggrin is not detectable by immunofluorescence in sections of amphibian epidermis, protein extraction, electrophoresis and immunoblotting are more sensitive. In the epidermis of toad and frog, but only occasionally in that of newt, filaggrin cross-reactive proteic bands are seen at 50-55, 40-45, and sometimes at 25 kDa. This suggests that after extraction and unmasking of reactive sites in the epidermis of more terrestrial amphians (anurans), some HRPs with filaggrin-like cross-reactivity are present. The overlap that exists at 50-55 kDa between filaggrin-positive and AE2-positive keratins, but not that at 40-45 kDa further indicate that non-keratin, filaggrin-like proteins may be present in anuran epidermis. The present study suggests for the first time that very small amounts of histidine-rich proteins are produced among keratin filaments in upper intermediate, replacement and corneous layers of amphibian epidermis. Although the molecular composition of these proteins is unknown, precluding understanding of their relationship to those of mammals and reptiles, these cationic proteins might have originated in conjunction with the formation of a horny layer during the adaptation to land during the Carboniferous and were possibly refined later in the epidermis of amniotes.     

Presence of a glycine-cysteine-rich beta-protein in the oberhautchen layer of snake epidermis marks the formation of the shedding layer

The complex differentiation of snake epidermis largely depends on the variation in the production of glycine-cysteine-rich versus glycine-rich beta-proteins (beta-keratins) that are deposited on a framework of alpha-keratins. The knowledge of the amino acid sequences of beta-proteins in the snake Pantherophis guttatus has allowed the localization of a glycine-cysteine-rich beta-protein in the spinulated oberhautchen layer of the differentiating shedding complex before molting takes place. This protein decreases in the beta-layer and disappears in mesos and alpha-layers. Conversely, while the mRNA for a glycine-rich beta-protein is highly expressed in differentiating beta-cells, the immunolocalization for this protein is low in these cells. This discrepancy between expression and localization suggests that the epitope in glycine-rich beta-proteins is cleaved or modified by posttranslational processes that take place during the differentiation and maturation of the beta-layer. The present study suggests that among the numerous beta-proteins coded in the snake genome to produce epidermal layers with different textures, the glycine-cysteine-rich beta-protein marks the shedding complex formed between alpha- and beta-layers that allows for molting while its disappearance between the beta- and alpha-layers (mesos region for scale growth) is connected to the formation of the alpha-layers.