Preservation of corneous ß-proteins in Mesozoic feathers.

Fossil proteins are valuable tools in evolutionary biology. Recent technological advances and better integration of experimental methods have confirmed the feasibility of biomolecular preservation in deep time, yielding new insights into the timing of key evolutionary transitions. Keratins (formerly α-keratins) and corneous ß-proteins (CBPs, formerly ß-keratins) are of particular interest as they define tissue structures that underpin fundamental physiological and ecological strategies and have the potential to inform on the molecular evolution of the vertebrate integument. Reports of CBPs in Mesozoic fossils, however, appear to conflict with experimental evidence for CBP degradation during fossilization. Further, the recent model for molecular modification of feather chemistry during the dinosaur-bird transition does not consider the relative preservation potential of different feather proteins. Here we use controlled taphonomic experiments coupled with infrared and sulfur X-ray spectroscopy to show that the dominant ß-sheet structure of CBPs is progressively altered to α-helices with increasing temperature, suggesting that (α-)keratins and α-helices in fossil feathers are most likely artefacts of fossilization. Our analyses of fossil feathers shows that this process is independent of geological age, as even Cenozoic feathers can comprise primarily α-helices and disordered structures. Critically, our experiments show that feather CBPs can survive moderate thermal maturation. As predicted by our experiments, analyses of Mesozoic feathers confirm that evidence of feather CBPs can persist through deep time.

Insights of keratin geometry from agro-industrial wastes: A comparative computational and experimental assessment.

Understanding the structural properties of keratin is of great importance to managing their potential application in keratin-inspired biomaterials and its management of wastes. In this work, the molecular structure of chicken feather keratin 1 was characterized by AlphaFold2 and quantum chemistry calculation. The predicted IR spectrum of the N-terminal region of feather keratin 1, consisting of 28 amino acid residues, was used to assign the Raman frequencies of the extracted keratin. The MW of experimental samples were 6 & 1 kDa while the predicted MW (∼10 kDa) of ß-keratin. Experimental analysis shows the magnetic field treatment could affect the functional and surface structural properties of keratin. The particle size distribution curve illustrates the dispersion of particle size concentration, while TEM analysis demonstrates the reduction of particle diameter to 23.71 ± 1.1 nm following treatment. High-resolution XPS analysis confirmed the displacement of molecular elements from their orbital.

Molecular Structure and Dynamics in Wet Gecko ß-Keratin.

Molecular dynamics simulations are performed to investigate the molecular picture of water sorption in gecko keratin and the influence of relative humidity (RH) on the local structure and dynamics in water-swollen keratin. At low RHs, water sorption occurs through hydrogen bonding of water with the hydrophilic groups of keratin. At high RHs (>80%), additional water molecules connect to the first "layer" of amide-connected water molecules (multimolecular sorption) through hydrogen bonds, giving rise to a sigmoidal shape of the sorption isotherm. This causes the formation of large chain-like clusters surrounding the hydrophilic groups of keratin, which upon a further increase of the RH form a percolating water network. An examination of the dynamics of water molecules sorbed in keratin demonstrates that there are two states, bound and free, for water. The dynamics of water in these states depends on the RH. At low RHs, large-scale translational motions of tightly bound water molecules to keratin are needed to remake the entire hydration shell of the keratin. At high RHs (>80%), the water molecules more quickly exchange between the two states. The center-of-mass mean-square displacement of water molecules indicates a hopping motion of water molecules in the keratin solvation shell. The hopping mechanism is more pronounced at RHs < 80%. At higher RHs, water translation through water clusters (water network) dominates. We have observed two regimes for the dependence of dynamical properties on the RH: a regime of gradual increase of the dynamics over 10% < RH < 80% and a regime of drastic dynamic acceleration at RH > 80%. The latter regime begins exactly where the water uptake and the volume swelling also increase much more and where a drastic change in the elastic properties of gecko keratin has been observed. A nearly linear relation between the relaxation times for all dynamical processes and the water content of gecko keratin is observed.

Electron microscopic analysis in the gecko Lygodactylus reveals variations in micro-ornamentation and sensory organs distribution in the epidermis that indicate regional functions.

Possible pattern variations of micro-ornamentation in different areas of the skin in the gecko Lygodactylus have been analyzed by scanning and transmission electron microscopy. A map of micro-ornamentation present in various areas of the skin has been obtained. Differences in micro-ornamentation pattern and sensory organ distribution were detected. The "spinulated pattern" consists of shorter spinulae in dorsal versus ventral scales, and spinules are shorter in inner scale surface and hinge regions with respect to the outer scale surface. The spines derive from the accumulation of struts of corneous material mainly composed of corneous beta proteins (CBPs, formerly indicated as beta-keratins) that merge into pointed micro-ornamentation. The 3D-accumulation of CBPs within Oberhautchen cells can vary in some regions of different scales during Oberhautchen-beta cell differentiation, perhaps also under physical tensile forces derived from continuous scale growth. Three other main patterns of micro-ornamentation were detected and indicated as "corneous belts," "corneous dendritic ramification," and "serpentine-pit and groove." These variations from the typical spinulated pattern present in gecko epidermis are interpreted as transitional regions where the accumulation of corneous material in Oberhautchen cells that merges with underlying beta-cells gives rise to nonspinulated surfaces. Spinulated sensory organs with bristles and lenticular-shaped or knob-like tactile corpuscles are more numerous in ventral scales of the tail tip close to adhesive pads and near the digital pads. These regions are likely those most involved in the fine control of movements and response to vibrational stimuli derived from air and objects movements, including potential preys or predators.

Regional specific differentiation of integumentary organs: SATB2 is involved in α- and ß-keratin gene cluster switching in the chicken.

BACKGROUND: Animals develop skin regional specificities to best adapt to their environments. Birds are excellent models in which to study the epigenetic mechanisms that facilitate these adaptions. Patients suffering from SATB2 mutations exhibit multiple defects including ectodermal dysplasia-like changes. The preferential expression of SATB2, a chromatin regulator, in feather-forming compared to scale-forming regions, suggests it functions in regional specification of chicken skin appendages by acting on either differentiation or morphogenesis. RESULTS: Retrovirus mediated SATB2 misexpression in developing feathers, beaks, and claws causes epidermal differentiation abnormalities (e.g. knobs, plaques) with few organ morphology alterations. Chicken ß-keratins are encoded in 5 sub-clusters (Claw, Feather, Feather-like, Scale, and Keratinocyte) on Chromosome 25 and a large Feather keratin cluster on Chromosome 27. Type I and II α-keratin clusters are located on Chromosomes 27 and 33, respectively. Transcriptome analyses showed these keratins (1) are often tuned up or down collectively as a sub-cluster, and (2) these changes occur in a temporo-spatial specific manner. CONCLUSIONS: These results suggest an organizing role of SATB2 in cluster-level gene co-regulation during skin regional specification.

Theoretical approaches to study the optical response of the red-legged honeycreeper's plumage (Cyanerpes cyaneus).

In this paper, we investigate the unusual color effect exhibited by the plumage of the heads of Cyanerpes cyaneus males, whose color turns from green to turquoise as the angle between the illumination and observation directions is increased. This singular color effect is characteristic of species that have quasi-ordered nanostructures of short-range order within the feather barbs. However, among species of the same family and even within feather patches of the same individual, one can find barbs with different characteristics, both macroscopic (curvature, shape, cross-sectional area) and in their internal microstructure. We apply the Korringa-Kohn-Rostoker method with the averaging technique to model the reflectance spectra for different angles of incidence and explain the dependence of the observed color with the incidence-collection angle. To investigate the influence of the disorder in the optical response of the spongy matrix, we apply the integral method for a two-dimensional cylinder system that simulates the distribution of air cavities within the $ \beta $ß-keratin medium. The experimental reflectance was interpreted as the result of multiple reflections in the internal interfaces generated by large air voids present within the spongy matrix. The application of rigorous methods to the study of natural photonic structures is of fundamental relevance for the design of efficient bioinspired artificial materials.

The feather degradation mechanisms of a new Streptomyces sp. isolate SCUT-3.

Feather waste is the highest protein-containing resource in nature and is poorly reused. Bioconversion is widely accepted as a low-cost and environmentally benign process, but limited by the availability of safe and highly efficient feather degrading bacteria (FDB) for its industrial-scale fermentation. Excessive focuses on keratinase and limited knowledge of other factors have hindered complete understanding of the mechanisms employed by FDB to utilize feathers and feather cycling in the biosphere. Streptomyces sp. SCUT-3 can efficiently degrade feather to products with high amino acid content, useful as a nutrition source for animals, plants and microorganisms. Using multiple omics and other techniques, we reveal how SCUT-3 turns on its feather utilization machinery, including its colonization, reducing agent and protease secretion, peptide/amino acid importation and metabolism, oxygen consumption and iron uptake, spore formation and resuscitation, and so on. This study would shed light on the feather utilization mechanisms of FDBs.

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.

Corneous beta proteins of the epidermal differentiation complex (EDC) form large part of the corneous material of claws and rhamphothecae in turtles.

The presence of specific protein types in claws and beaks of turtles is poorly known. The present immunological study describes the localization of some of the main corneous beta proteins (CBPs) coded in the epidermal differentiation complex of turtles. Three antibodies here utilized revealed that glycine-, cysteine-, tyrosine-, and valine-rich CBPs are present in differentiating keratinocytes of the beak and of the dorsal (unguis) and ventral (sub-unguis) sides of the claw in different species, semi-aquatic and terrestrial. These proteins provide mechanical resilience to the horny material of claws and beaks through the formation of numerous -S-S- bonds and also hydrophobicity that contributes to preserve wearing of the horny material. The thicker corneous layer of the unguis is made of elongated and partially merged corneocytes, and no or few cells desquamate superficially. Unknown junctional proteins may contribute to maintain corneocytes connected one to another. In contrast, corneocytes of the sub-unguis show an elongated but lenticular shape and form a looser corneous layer whose cells remain separate and desquamate superficially. This suggests that other specific corneous proteins are present in the unguis in comparison with the sub-unguis to determine this different compaction. The wearing process present in the sub-unguis creates a loss of tissue that may favor the slow by continuous apical migration of corneocytes from the unguis into the initial part of the sub-unguis. Beak corneocytes form a compact corneous layer like the unguis but numerous superficial cells desquamate on both outer (epidermal) and inner (oral) sides.

Alpha-keratin and corneous beta protein in the parakeratinized epithelium of the tongue in the domestic goose (Anser anser f. domestica).

The parakeratinized epithelium is a common epithelium in the oral cavity in birds and is characterized by the presence of cell nuclei in the cells of the cornified layer. This epithelium covers almost the entire dorsal surface of the tongue in the domestic goose apart of the lingual nail and conical papillae. So far no study has identified the molecular proteins alpha-keratin (IF-keratin) and/or corneous beta protein (CBP), which are responsible for keratinization or cornification processes in the parakeratinized epithelium of domestic geese. The study was performed using immunohistochemical (IHC) methods to identify alpha-keratin. The innovative method of Raman microspectroscopy was used to determine the presence of CBP and specify their percentage in epithelial layers of the parakeratinized epithelium. The results revealed that alpha-keratin is present in the whole parakeratinized epithelium. A strong staining reaction was detected in the basal and intermediate layers and a less strong staining reaction in the cornified layer. Raman microspectroscopy analysis confirmed the presence of alpha-keratin and demonstrated that its percentage decreases from the basal layer to the cornified layer. The Raman microspectroscopy technique revealed the occurrence of CBP in the parakeratinized epithelium and demonstrated that the percentage of this protein increases from the basal layer to the cornified layer. Performed analysis determines that parakeratinized epithelium undergoes cornification. However, the lower percentage of CBP in the cornified layer of parakeratinized epithelium than in orthokeratinized epithelium points to the fact that parakeratinized epithelium has a weaker protective function.

A comprehensive and comparative study of the internal structure and dynamics of natural ß-keratin and regeneratedß-keratin by solid state NMR spectroscopy.

Structure and dynamics of natural and regenerated chicken feather ß-keratin were investigated by 13C cross-polarization (CP) magic angle spinning (MAS) solid state nuclear magnetic resonance (SSNMR) spectral analysis, 13C and 1H spin-lattice relaxation time measurements, and 13C two dimensional phase adjusted spinning sidebands (2DPASS) MAS SSNMR measurements. Chemical shift anisotropy (CSA) parameters of both natural and regenerated chicken feather ß-keratin were extracted by using 2DPASS MAS SSNMR experiment. The beauty of 2DPASS MAS SSNMR experiment is it can correlate the isotropic and anisotropic dimension with the help of shearing transformation and two dimensional Fourier Transformation. Molecular correlation time at each and every magnetically inequivalent carbon site of both natural and regenerated chicken feather ß-keratin were also determined. The change in molecular dynamics of structural protein after pretreatment was monitored by 2DPASS MAS SSNMR and 13C relaxation measurement. This type of comprehensive study will provide the information about the interrelation between the structure and dynamics of structural protein and will also shed light in the way of developing methods for conversion of animal by-products to novel product.

Duplications in Corneous Beta Protein Genes and the Evolution of Gecko Adhesion.

Corneous proteins are an important component of the tetrapod integument. Duplication and diversification of keratins and associated proteins are linked with the origin of most novel integumentary structures like mammalian hair, avian feathers, and scutes covering turtle shells. Accordingly, the loss of integumentary structures often coincides with the loss of genes encoding keratin and associated proteins. For example, many hair keratins in dolphins and whales have become pseudogenes. The adhesive setae of geckos and anoles are composed of both intermediate filament keratins (IF-keratins, formerly known as alpha-keratins) and corneous beta-proteins (CBPs, formerly known as beta-keratins) and recent whole genome assemblies of two gecko species and an anole uncovered duplications in seta-specific CBPs in each of these lineages. While anoles evolved adhesive toepads just once, there are two competing hypotheses about the origin(s) of digital adhesion in geckos involving either a single origin or multiple origins. Using data from three published gecko genomes, I examine CBP gene evolution in geckos and find support for a hypothesis where CBP gene duplications are associated with the repeated evolution of digital adhesion. Although these results are preliminary, I discuss how additional gecko genome assemblies, combined with phylogenies of keratin and associated protein genes and gene duplication models, can provide rigorous tests of several hypotheses related to gecko CBP evolution. This includes a taxon sampling strategy for sequencing and assembly of gecko genomes that could help resolve competing hypotheses surrounding the origin(s) of digital adhesion.

Multiple Regulatory Modules Are Required for Scale-to-Feather Conversion.

The origin of feathers is an important question in Evo-Devo studies, with the eventual evolution of vaned feathers which are aerodynamic, allowing feathered dinosaurs and early birds to fly and venture into new ecological niches. Studying how feathers and scales are developmentally specified provides insight into how a new organ may evolve. We identified feather-associated genes using genomic analyses. The candidate genes were tested by expressing them in chicken and alligator scale forming regions. Ectopic expression of these genes induced intermediate morphotypes between scales and feathers which revealed several major morphogenetic events along this path: Localized growth zone formation, follicle invagination, epithelial branching, feather keratin differentiation, and dermal papilla formation. In addition to molecules known to induce feathers on scales (retinoic acid, ß-catenin), we identified novel scale-feather converters (Sox2, Zic1, Grem1, Spry2, Sox18) which induce one or more regulatory modules guiding these morphogenetic events. Some morphotypes resemble filamentous appendages found in feathered dinosaur fossils, whereas others exhibit characteristics of modern avian feathers. We propose these morpho-regulatory modules were used to diversify archosaur scales and to initiate feather evolution. The regulatory combination and hierarchical integration may have led to the formation of extant feather forms. Our study highlights the importance of integrating discoveries between developmental biology and paleontology.

Disulfide-bond-mediated cross-linking of corneous beta-proteins in lepidosaurian epidermis.

Corneous beta-proteins (CBPs), formerly referred to as beta-keratins, are major protein components of the epidermis in lepidosaurian reptiles and are largely responsible for their material properties. These proteins have been suggested to form filaments of 3.4nm in thickness and to interact with themselves or with other proteins, including intermediate filament (IF) keratins. Here, we performed immunocytochemical labeling of CBPs in the epidermis of different lizards and snakes and investigated by immunoblotting analysis whether the reduction of disulfide bonds or protein oxidation affects the solubility and mobility of these CBPs. Immunogold labeling suggested that CBPs are partly co-localized with IF-keratins in differentiating and mature beta-cells. The chemical reduction of epidermal proteins from lizard and snake epidermis increased the abundance of CBP-immunoreactive bands in the size range of CBP monomers on Western blots. Conversely, in vitro oxidation of epidermal proteins reduced the abundance of putative CBP monomers. Some modifications in the IF-keratin range were also noted. These results strongly indicate that CBPs associate with IF-keratins and other proteins via disulfide bonds in the epidermis of lizards and snakes, which likely contributes to the resilience of the cornified beta- and alpha-layers of the lepidosaurian epidermis in live animals and after shedding.

Review: Evolution and diversification of corneous beta-proteins, the characteristic epidermal proteins of reptiles and birds.

In all amniotes specialized intermediate filament keratins (IF-keratins), in addition to keratin-associated and corneous proteins form the outermost cornified layer of the epidermis. Only in reptiles and birds (sauropsids) the epidermis of scales, claws, beaks, and feathers, largely comprises small proteins formerly indicated as "beta-keratins" but here identified as corneous beta-proteins (CBPs) to avoid confusion with true keratins. Genes coding for CBPs have evolved within the epidermal differentiation complex (EDC), a locus with no relationship with those of IF-keratins. CBP genes have the same exon-intron structure as EDC genes encoding other corneous proteins of sauropsids and mammals, but they are unique by encoding a peculiar internal amino acid sequence motif beta-sheet region that allows formation of CBP filaments in the epidermis and epidermal appendages of reptiles and birds. In contrast, skin appendages of mammals, like hairs, claws, horns and nails, contain keratin-associated proteins that, like IF-keratin genes, are encoded by genes in loci different from the EDC. Phylogenetic analysis shows that lepidosaurian (lizards and snakes) and nonlepidosaurian (crocodilians, birds, and turtles) CBPs form two separate clades that likely originated after the divergence of these groups of sauropsids in the Permian Period. Clade-specific CBPs evolved to make most of the corneous material of feathers in birds and of the shell in turtles. Based on the recent identification of the complete sets of CBPs in all major phylogenetic clades of sauropsids, this review provides a comprehensive overview of the molecular evolution of CBPs.

Localization of Alpha-Keratin and Beta-Keratin (Corneous Beta Protein) in the Epithelium on the Ventral Surface of the Lingual Apex and Its Lingual Nail in the Domestic Goose (Anser Anser f. domestica) by Using Immunohistochemistry and Raman Microspectroscopy Analysis.

The epithelium of the ventral surface of the apex of the tongue in most birds is specified by the presence of the special superficial layer called lingual nail. The aim of the present study is to determine the localization of the alpha-keratin and beta-keratin (corneous beta protein) in this special epithelium in the domestic goose by using immunohistochemistry staining and the Raman spectroscopy analysis. Due to lack of commercially available antibodies to detect beta-keratin (corneous beta protein), the Raman spectroscopy was used as a specific tool to detect and describe the secondary structure of proteins. The immunohistochemical (IHC) detections reveal the presence of alpha-keratin in all layers of the epithelium, but significant differences in the distribution of the alpha-keratin in the epithelial layers appear. The staining reaction is stronger from the basal layer to the upper zone of the intermediate layer. The unique result is weak staining for the alpha-keratin in the lingual nail. Applications of the Raman spectroscopy as a complementary method not only confirmed results of IHC staining for alpha-keratin, but showed that this technique could be used to demonstrate the presence of beta-keratin (corneous beta protein). Functionally, the localization of alpha-keratin in the epithelium of the ventral surface of the lingual apex provides a proper scaffold for epithelial cells and promotes structural integrity, whereas the presence of beta-keratin (corneous beta protein) in the lingual nail, described also as exoskeleton of the ventral surface of the apex, endures mechanical stress. Anat Rec, 300:1361-1368, 2017. © 2017 Wiley Periodicals, Inc.

Sauropsids Cornification is Based on Corneous Beta-Proteins, a Special Type of Keratin-Associated Corneous Proteins of the Epidermis.

The evolution of the process of cornification in amniote epidermis from the general process of keratinization present in simple epithelia of anamniotes took place through the evolution of specialized intermediate filament (α) keratins, keratin-associated proteins (KAPs) and corneous proteins (CPs). The scanty information on the three-dimensional conformation of known KAPs and CPs indicate these proteins contain α-helix, random coiled, or beta sheets with different lengths and organizations. CP genes originated in a chromosome locus indicated as epidermal differentiation complex (EDC), and transformed the epidermal keratinization of anamniotes into the cornified epidermis and skin appendages of amniotes (claws, beaks, and feathers). In particular, peculiar genes encoding for small proteins with a central region of 34 amino acids conformed as beta sheets were originated in the EDC of sauropsids (reptiles and birds). These proteins were traditionally indicated as beta-keratins because they form filaments of 3-4 nm in diameter and show an X-ray beta pattern. Different from other proteins of the EDC, dimers of these corneous beta-proteins associate into long polymers of filamentous proteins utilized in sauropsids skin appendages, such as scales and feathers. Future challenges in this area of research will be the study on gene regulation and expression for these proteins, their origin and evolution in different lineages of sauropsids, and their role in determining the material properties of sauropsid scales and other skin appendages.

Regulatory Divergence among Beta-Keratin Genes during Bird Evolution.

Feathers, which are mainly composed of α- and ß-keratins, are highly diversified, largely owing to duplication and diversification of ß-keratin genes during bird evolution. However, little is known about the regulatory changes that contributed to the expressional diversification of ß-keratin genes. To address this issue, we studied transcriptomes from five different parts of chicken contour and flight feathers. From these transcriptomes we inferred ß-keratin enriched co-expression modules of genes and predicted transcription factors (TFs) of ß-keratin genes. In total, we predicted 262 TF-target gene relationships in which 56 TFs regulate 91 ß-keratin genes; we validated 14 of them by in vitro tests. A dual criterion of TF enrichment and "TF-target gene" expression correlation identified 26 TFs as the major regulators of ß-keratin genes. According to our predictions, the ancestral scale and claw ß-keratin genes have common and unique regulators, whereas most feather ß-keratin genes show chromosome-wise regulation, distinct from scale and claw ß-keratin genes. Thus, after expansion from the ß-keratin gene on Chr7 to other chromosomes, which still shares a TF with scale and claw ß-keratin genes, most feather ß-keratin genes have recruited distinct or chromosome-specific regulators. Moreover, our data showed correlated gene expression profiles, positive or negative, between predicted TFs and their target genes over the five studied feather regions. Therefore, regulatory divergences among feather ß-keratin genes have contributed to structural differences among different parts of feathers. Our study sheds light on how feather ß-keratin genes have diverged in regulation from scale and claw ß-keratin genes and among themselves.

Review: mapping epidermal beta-protein distribution in the lizard Anolis carolinensis 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.

Immunolocalization of large corneous beta-proteins in the green anole lizard (Anolis carolinensis) suggests that they form filaments that associate to the smaller beta-proteins in the beta-layer of the epidermis.

The distribution of large corneous beta-proteins of 18-43 kDa (Ac37, 39, and 40) in the epidermis of the lizard Anolis carolinensis is unknown. This study analyses the localization of these beta-proteins in different body scales during regeneration. Western blot analysis indicates most protein bands at 40-50 kDa suggesting they mix with alpha-keratin of intermediate filament keratin proteins. Ac37 is present in mature alpha-layers of most scales and in beta-cells of the outer scale surface in some scales but is absent in the Oberhäutchen, in the setae and beta-layer of adhesive pads and in mesos cells. In differentiating beta-keratinocytes Ac37 is present over 3-4 nm thick filaments located around the amorphous beta-packets and in alpha-cells, but is scarce in precorneous and corneous layers of the claw. Ac37 forms long filaments and, therefore, resembles alpha-keratins to which it probably associates. Ac39 is seen in the beta-layer of tail and digital scales, in beta-cells of regenerating scales but not in the Oberhäutchen (and adhesive setae) or in beta- and alpha-layers of the other scales. Ac40 is present in the mature beta-layer of most scales and dewlap, in differentiating beta-cells of regenerating scales, but is absent in all the other epidermal layers. The large beta-proteins are accumulated among forming beta-packets of beta-cells and are packed in the beta-corneous material of mature beta-layer. Together alpha-keratins, large beta-proteins form the denser areas of mature beta-layer that may have a different consistence that the electron-paler areas.