Immunolocalization of corneous proteins including a serine-tyrosine-rich beta-protein in the adhesive pads in the tokay gecko.

Adhesive pads of geckos contain many thousands of nanoscale spatulae for the adhesion and movement along vertical or inverted surfaces. Setae are composed of interlaced corneous bundles made of small cysteine-glycine-rich corneous beta proteins (CBPs, formerly indicated as beta-keratins), embedded in a matrix material composed of cytoskeletal proteins and lipids. Negatively charged intermediate filament keratins (IFKs) and positively charged CBPs likely interact within setae, aside disulphide bonds, giving rise to a flexible and resistant corneous material. Using differernt antibodies against CBPs and IFKs an updated model of the composition of setae and spatulae is presented. Immunofluorescence and ultrastructural immunogold labeling reveal that one type of neutral serine-tyrosine-rich CBP is weakly localized in the setae while it is absent from the spatula. This uncharged protein is mainly present in the thin Oberhautchen layer sustaining the setae, although with a much lower intensity with respect to the cysteine-rich CBPs. These proteins in the spatula likely originate a positively charged or neutral contact surface with the substrate but the influence of lipids and cytoskeletal proteins present in setae on the mechanism of adhesion is not known. In the spatula, protein-lipid complexes may impart the pliability for the attachment and adapt to irregular surfaces. The presence of cysteine-glycine medium rich CBPs and softer IFKs in alpha-layers sustaining the setae forms a flexible base for compliance of the setae to substrate and improved adhesion.

Immunoreactivity to the pre-core box antibody shows that most glycine-rich beta-proteins accumulate in lepidosaurian beta-layer and in the corneous layer of crocodilian and turtle epidermis.

The differentiation of the corneous layers of reptilian epidermis has been analyzed by ultrastructural immunocytochemistry using specific antibodies against the conserved pre-core box region of their keratin-associated beta-proteins (KAbetaPs, formerly indicated as beta-keratins) and silver-intensification. The epitope analysis in the sequences of different reptilian KAbetaPs indicates that this antibody recognizes mainly glycine-rich beta-proteins in lizards and snakes. The immunoreactivity of the beta-layer of the tuatara to this antibody also suggests that a similar epitope is present in beta-proteins of this relict species. In crocodilians the antibody recognizes glycine-rich beta-proteins, so far representing all the known crocodilian KAbetaPs. In hard-shelled turtle the antibody labels mainly type 1 KAbetaPs that represent most types found in this turtle. The antibody does not label the corneous layer of the soft-shelled turtle that contains exclusively type 2 KAbetaPs, with a low identity to the epitope recognized by the antibody. The prevalent labeling of the beta-layers in lepidosaurian epidermis and of the corneous layer in turtle and crocodilian epidermis suggest that this antibody is mainly directed toward KAbetaPs rich in glycine. The latter are main constituents of the corneous layer in turtles and crocodilians and of the beta-layer in lizards, snakes and the tuatara. These proteins are largely responsible for the inflexibility, mechanical resistance, chromophobicity and relative hydrophobicity of the reptilian corneous layer.

Ultrastructural immunocytochemistry for the central region of keratin associated-beta-proteins (beta-keratins) shows the epitope is constantly expressed in reptilian epidermis.

The presence of beta-proteins containing a core-box region in specific regions of reptilian epidermis has been studied by immunological methods. Alpha-keratins are detected by the antibody AK2 that recognizes a sequence toward the C-terminal of acidic alpha-keratins of 48-52kDa. Beta-proteins are recognized by an antibody directed to the core-box region specific for these proteins of 18-37kDa. The AK2 antibody labels with variable intensity alpha-keratin bundles in basal and suprabasal keratinocytes in the epidermis of representative species of reptiles but immunolabeling decreases or disappears in pre-corneous and corneous cells. As opposite, the core-box antibody only labels with variable intensity the dense beta-corneous material formed in pre-corneous and corneous layers of crocodilian and turtle epidermis. In lepidosaurian epidermis the core-box antibody labels the beta-layer while the mesos and alpha-layers are poorly or not labeled. The immunological evidence indicates that beta-proteins are synthesized in the upper spinosus and pre-corneous layers of the epidermis and replace or mask the initial alpha-keratin framework present in keratinocytes as they differentiate into cells of the beta-layer. In the specialized pad lamellae of gecko and anoline lizards charged beta-proteins accumulate in the adhesive setae and may affect the mechanism of adhesion that allows these lizards to walk vertical surfaces. The addition of beta-proteins to the alpha-keratins in upper cell layers of the epidermis recalls the process of cornification of mammalian epidermis where specific keratin-associated proteins (involucrin, loricrin and filaggrin) associate with the keratin framework in terminally differentiating keratinocytes of the stratum corneum.

Characterization of keratins and associated proteins involved in the corneification of crocodilian epidermis.

Crocodilian keratinocytes accumulate keratin and form a corneous cell envelope of which the composition is poorly known. The present immunological study characterizes the molecular weight, isoelectric point (pI) and the protein pattern of alpha- and beta-keratins in the epidermis of crocodilians. Some acidic alpha-keratins of 47-68 kDa are present. Cross-reactive bands for loricrin (70, 66, 55 kDa), sciellin (66, 55-57 kDa), and filaggrin-AE2-positive keratins (67, 55 kDa) are detected while caveolin is absent. These proteins may participate in the formation of the cornified cell membranes, especially in hinge regions among scales. Beta-keratins of 17-20 kDa and of prevalent basic pI (7.0-8.4) are also present. Acidic beta-keratins of 10-16 kDa are scarce and may represent altered forms of the original basic proteins. Crocodilian beta-keratins are not recognized by a lizard beta-keratin antibody (A68B), and by a turtle beta-keratin antibody (A685). This result indicates that these antibodies recognize specific epitopes in different reptiles. Conversely, crocodilian beta-keratins cross-react with the Beta-universal antibody indicating they share a specific 20 amino acid epitope with avian beta-keratins. Although crocodilian beta-keratins are larger proteins than those present in birds our results indicate presence of shared epitopes between avian and crocodilian beta-keratins which give good indication for the future determination of the sequence of these proteins.

Characterization of beta-keratins in lizard epidermis: electrophoresis, immunocytochemical and in situ-hybridization study.

Lizard scales are composed of alpha-(cyto-) keratins and beta-keratins. The characterization of the molecular weight and isoelectric point (pI) of alpha- and beta-keratins of lizard epidermis (Podarcis sicula) has been done by using two-dimensional electrophoresis, immunoblotting, and immunocytochemistry. Antibodies against cytokeratins, against a chicken scale beta-keratin or against lizard beta-keratin bands of 15-16kDa, have been used to recognize alpha- and beta-keratins. Acid and basic cytokeratins of 42-67kDa show a pI from 5.0 to 8.9. This indicates the presence of specific keratins for the formation of the stratum corneum. Main protein spots of beta-keratin at 15-17kDa, and pI at 8.5, 8.2, and 6.7, and one spot at 10kDa and pI at 7.3 were recognized. Therefore, beta-keratins are mainly basic proteins, and are used for the formation of the hard corneous layer of the epidermis. Ultrastructural immunocytochemistry confirms that beta-keratin is packed into large and dense bundles of beta-keratin cells of lizard epidermis. The use of a probe against a lizard beta-keratin in situ-hybridization studies confirms that the mRNA for beta-keratins is present in beta-cells and is localized around or even associated with beta-keratin filaments.

Immunological characterization of a newly developed antibody for localization of a beta-keratin in turtle epidermis.

Turtle scutes are made of hard (beta)-keratins. In order to study size and localization of beta-keratins in turtle shell, we produced a rat polyclonal antiserum against a turtle scute beta-keratin of 13-16 kDa, which allowed the immunolocalization of the protein in the epidermis. In immunoblots the antiserum recognized turtle beta-keratins but showed variable cross-reactivity with lizard, snake, and avian beta-keratins. The turtle antiserum appears less cross-reactive than a chicken scale antiserum (Beta-1). In bidimensional immunoblots, three main protein spots at 15-16 kDa with pI at 7.3, 6.8, 6.4, and an unresolved large spot at 40-45 kDa with pI around 5 were more constantly obtained. The latter may result from the aggregation of the smaller beta-keratin protein. The corneous layer of the carapace and plastron of various species of chelonians appeared immunofluorescent. The ultrastructural immunolocalization showed sparse labeling over beta-keratin filaments of cells of the horny layer of both carapace and plastron. The study for the first time shows that the isolated protein band derived from a component of the beta-keratin filaments of the corneous layer of turtles. This antibody can be used for further studies on beta-keratin expression and sequencing in chelonian shell. No labeling was present over other cell organelles or layers of turtle epidermis and it was absent in non-epidermal cells. The specificity for turtle beta-keratin suggests that the antiserum recognizes some epitope/s specific for chelonians beta-keratins, and that it also variably recognizes other reptilian and avian beta-keratins.

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.