Hard (Beta-)Keratins in the Epidermis of Reptiles: Composition

Aug 18, 2007 - Beta-keratins comprise glycine-proline-rich and cystein-proline-rich proteins forming the hard corneous material of scales. Beta-kerati...
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Hard (Beta-)Keratins in the Epidermis of Reptiles: Composition, Sequence, and Molecular Organization Mattia Toni,† Luisa Dalla Valle,‡ and Lorenzo Alibardi*,† Dipartimento di Biologia evoluzionistica sperimentale, University of Bologna, Italy, and Dipartimento di Biologia, University of Padova, Italy Received May 7, 2007

Beta-keratins form the hard corneous material of reptilian scales. In the present review, the distribution and molecular characteristics of beta-keratins in reptiles are presented. In lepidosaurians immunoreactive, protein bands at 12-18 kDa are generally present with less frequent proteins at higher molecular weight. In chelonians, bands at 13-18 and 22-24 kDa are detected. In crocodilians, bands at 14-20 kDa and weaker bands at 30-32 kDa are seen. Protein bands above 25 kDa are probably polymerized beta-keratins or aggregates. Two-dimensional gel electrophoresis shows that beta-keratins are mainly basic and that acidic-neutral keratins may derive from post-translational modifications. Beta-keratins comprise glycine-proline-rich and cystein-proline-rich proteins of 13-19 kDa. Beta-keratin genes may or may not contain introns and are present in multiple copies with a linear organization as in avian beta-keratin genes. Despite amino acid differences toward N- and C-terminals all beta-keratins share high homology in their central, beta-folded region of 20 amino acids, indicated as core-box. This region is implicated in the formation of beta-keratin filaments of scales, claws, and feathers. The homology of the core-box suggests that these proteins evolved from a progenitor sequence present in the stem of reptiles. Beta-keratins have diversified in their amino acid sequences producing secondary (and tertiary) conformations that suited them for their mechanical role in scales. In birds, a small beta-keratin has allowed the formation of feathers. It is suggested that beta-keratins represent the reptilian counterpart of keratin associated or matrix proteins present in mammalian hairs, claws, and horns. Keywords: reptiles • epidermis • beta-keratins • proteomics • protein sequences • evolution

1. Morphology of Reptilian Scales The skin of most reptiles is hard, coarse, and scaled, and the last of these features is the first characteristic that allow most people to distinguish reptiles from other vertebrates. In fact, differently from birds and mammals, the epidermis of reptiles is subdivided into scales of various pattern, shape, thickness, and degree of overlap (Figure 1). The process of cornification of the epidermis of reptiles is synthetically discussed in the present review. Despite the clear differences in the skin among extant reptiles, birds, and mammals, the basic process of epidermal differentiation may have some common processes since reptiles were the first amniotes of the Carboniferous Period (about 320 million years ago). From stem reptiles, modern reptiles, birds, and mammals later evolved. Despite previous morphological and biochemical studies,1,2 no primary sequences and molecular details on the process of cornification in reptilian scales were known. Immunocytochemical, proteomic, and molecular biology studies conducted in our laboratory in the last 3 years have collected * To whom correspondence should be addressed. Dr. L. Alibardi, Dipartimento di Biologia evoluzionistica sperimentale, University of Bologna, via Selmi 3, 40126, Bologna, Italy. Ph, +39 051 209 4257; E-mail, [email protected]. † University of Bologna. ‡ University of Padova. 10.1021/pr0702619 CCC: $37.00

 2007 American Chemical Society

important cell and molecular information on the process of cornification in reptilian epidermis.3,4-12 The superficial corneous layer of reptilian scales is lost by a periodic and frequent shedding (5-20 moltings per year in squamates), by an occasional shedding (one to five times per year in the tuatara or one-two times in some turtles), or by a gradual superficial wearing (most chelonians and crocodilians).10,13-16 In lizards and snakes, the epidermis undergoes a cyclical process of resting phase and proliferating (renewal) phase that ends with a molt. The latter process occurs with the differentiation of a shedding layer along the shedding plane (SP in Figure 1) after formation of different epidermal layers. In few turtles, the superficial part of the hard corneous layer is shed as thin but compact flakes in Spring-Summer so that the scute is enlarged. This process also occurs through the formation of a shedding plane. From the germinal layer of the epidermis spinosus-like keratinocytes originate alternating hard (beta) and soft (alpha) layers in lepidosaurians (lizards, snakes, and sphenodontids), or variably thick hard (beta)corneous layers in the shell of chelonians and in the scutes of crocodilians. In chelonians, non-shelled epidermis of the body appears as a rough or tuberculate-like epidermis made of soft corneous layers. In the marine leatherback turtle and in the freshwater soft-shelled Journal of Proteome Research 2007, 6, 3377-3392

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Figure 1. Drawing of the histological structure of outer scale surface of scales in reptiles (A-E). In lepidosaurians (lizards-snakes, B), numerous layers form the outer and inner generation of the epidermis, which separate at shedding along the shedding plane (arrow). In the tuatara (Sphenodon, C), the shedding plane (arrow) forms in the intermediate-region between outer and inner epidermal generation. In crocodilians (D), suprabasal cells differentiate into beta-keratin cells that become compacted in the superficial part of the corneous layer. In chelonian scutes (E), a thick corneous layer derives from the compactation of beta-cells of suprabasal layers. In the soft-shelled turtle (F), a hard corneous layer is absent in relation to the likely absence of core-box in the diffuse beta-keratin present in this species, see text. The thick dermis contains 6-10 layers of criss-crossed collagen bundles that form a plywood-like structure that strengthens the whole skin. In the shell of shedding turtle (G), the external part of the corneous layer is lost along an underlying shedding plane. Legend: Ak, alpha-keratin filaments/bundles; EC, external (outer) corneous layer; IC, inner corneous layer; IEG, inner epidermal generation; OEG, outer epidermal generation; SP, shedding plane. Dots in Apalone indicate sparse beta-keratin filaments that do not form beta-keratin packets. The drawing illustrating the core-box indicates the presence of this 20 amino-acid sequence in all reptilian beta-keratins (except in the soft-shelled turtle).

turtle the epidermis of the shell remains soft and deformable, and does not form a hard and inflexible shell like in the remaining chelonians. In the soft-shelled turtle Apalone spinifera, the resistance of the skin is provided by the thick dermis, comprising a plywood-like organization of numerous layers of collagen bundles (Figures 1, 2). The main proteins of the epidermis of reptiles comprise alpha- and beta-keratins.

2. Types of Keratins in Reptilian Epidermis The epidermis of reptiles (and birds) contains relatively large alpha-keratins of 40-68 kDa and beta-keratins of smaller molecular weight (MW) (10-20 kDa) that possess different chemical and physical properties.1,3,4,17-24 Alpha-keratins or cytokeratins constitute the intermediate-filament bundles and are present in most epidermal layers where they have a role in mechanical resistance, impart form to cells, or determine their 3378

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changes in shape.25-28 Alpha-keratins are contained in keratinocytes of the alpha-layer of the epidermis, which mainly functions in permitting epidermal stretching and as a barrier against water-loss.13-15 Alpha-keratins in lizards, snakes, tuatara (Sphenodon punctatus), chelonians, and crocodilians comprise 5-8 low molecular components (40-65 kDa) of acidic type and 5-6 types of neutral-basic type of higher molecular weight (4568 kDa).8,10-12,29-34 Although the general characteristics and components of alpha-keratins in different reptilian groups have been identified, no molecular data on their sequences and genes are presently available. Differently from alpha-keratins, beta-keratins are specific proteins utilized to make hard structures including scales, scutes, and claws (beak and feathers in birds). Beta-keratins are produced in the pre-corneous layer of the specialized

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Figure 2. Immunohistochemical localization with the beta-1 keratin antibody (A-E, M-N) in the epidermis and histological structure of molts (F-K) in representative reptilian epidermis. (A) Wall lizard Podarcis sicula with immunopositive outer beta-layer (arrow). Bar, 10 mm. (B) Python snake (Liasis fuscus) with immuno-positive outer beta-layer (compact, arrow) and inner beta-layer (forming by fusiform cells). Bar, 10 mm. (C) Epidermis of Sphenodon punctatus showing outer (arrow) and inner beta-layer. Bar, 10 mm. (D) Thick immunolabeled corneous layer of carapace scute in Chrysemys picta. Bar, 10 mm. (E) Thick immunofluorescent layer of Crocodylus porosus scale. Bar, 10 mm. (F) Molt of Boa constrictor with pale beta-layer and dark alpha-layer underneath. Bar, 10 mm. (G) Molt of Crotalus atrox. Bar, 10 mm. (H) Undulated shape of molt of the scaleless snake C. atrox costituted mainly by the dark alpha-layer (arrow) and a peripheral pale layer. Bar, 10 mm. (I) Molts of Podarcis muralis with the artefactual detachment of the alpha-layer (curved arrow) from the beta-layer. Bar, 10 mm. (J) Molt of the cameleon Chamaeleo calyptratus with the intensely stained lower part of the alpha-layer. Bar, 10 mm. (K) Molt of Pogona vitticeps showing the thick beta- and alpha-layers. Bar, 10 mm. (L) Section through the carapace of Apalone spinifera showing the thick corneous layer of the epidermis made of dark corneocytes (double arrowhead). The dermis is composed of a superficial and deep cellular parts with in between by 7 layers (1-7) made of collagen bundles with orthogonal orientation. Bar, 10 mm. (M) Detail on beta-1 immunolabeled beta-keratin bundles in differentiating beta-cell of the snake Liasis fuscus. Bar 250 nm. (N) Beta-1 immunolabeling stratum corneum of a carapace scute of the turtle Chrysemys picta. Some cell boundaries of corneocytes are still visible (arrowhead). Bar, 200 nm. (O) Ultrastructural detail of transitional cells of the carapace epidermis of the soft-shelled turtle Apalone spinifera. Only free or associated bundles of keratin are seen (double arrow) but no beta-keratin packets. Also, the dense matrix of cells of corneocytes of the stratum corneoum does not contain beta-keratin. The corneocytes are narrow and irregular as typical alpha-cells and have thickened cornified cell membranes (arrows). Bar, 250 nm. Legends: a, alpha-layer; b, betalayer; bp, beta-keratin packets; c, corneous layer; cco, cross-sectioned collagen bundles; dde, deep dermis; des, desmosomes; e, living cells of the epidermis; ib, inner beta-layer; lco, longitudially sectioned collagen bundles; p, peripheral pale layer; sde, superficial dermis; tr, transitional (pre-corneous) cell. Dashes underline the basal layer of the epidermis. Journal of Proteome Research • Vol. 6, No. 9, 2007 3379

reviews epidermis of these skin appendages. Because of the importance of beta-keratins in reptilian scales and to their relatively small dimensions, these proteins have recently been studied in detail and some of their genes identified, sequenced, and cloned.5-7 The proteomic study of these small proteins in all reptilian orders has also progressed considerably.32,33,35 Beta-keratins associate with alpha-keratins and replace or mask these proteins in cells forming the hard corneous layer of scales, termed beta-layer. Beta-keratins are less extensible proteins than alpha-keratins. They have a beta-sheet conformation, and their filaments show a periodicity of 3-4 nm versus the 8-10 nm of alpha-keratin. The molecular mechanism of ordered aggregation of reptilian beta-keratins is poorly known, but the polymerization of these proteins produces very resistant filament bundles. The above studies have shown that beta-keratin layers of the epidermis react exclusively with an antibody (beta-1) produced against a chicken scale keratin (Figure 2A-E). Cytological studies in lizard and snake epidermis have shown that the biosynthesis of beta-keratins occurs by a progressive process of aggregation of small polymers to form large bundles storing beta-keratin. The bundles are dense and are made by an irregular mass of 3-4 nm thick filaments, which form a homogeneous corneous material at maturation (Figure 2M, N). Only in the soft epidermis of the turtle Apalone spinifera are beta-keratin bundles absent, and beta-keratin appears diffuse among sparse alpha-keratin bundles (Figure 2L-O). The process of maturation of keratinocytes in the corneous layer produces stable molecular aggregations that are difficult to resolved on electrophoretic gels, even under strong dissociating (high urea concentration) and reducing (high thiol solutions) conditions. In our initial study, beta-keratins were studied after extraction and mono- and bi-dimensional electrophoresis separation from fresh epidermis or molts of different lizards and snakes and from the tuatara (Sphenodon punctatus) (Figure 2F-K). Molts (either dry or preserved in ethanol) consist of cornified beta- and alpha-layers and are considered a reliable source of keratins, which are believed to be unaffected by degrading processes other than those naturally occurring during terminal differentiation and cornification. Other studies on fresh or molted scutes of some chelonian and crocodilian species have completed the protein analysis of reptilian beta-keratins (Figures 3 and 4, Table 1). Proteomic analysis has shown the variation in the molecular weight and patterns of beta-keratins among different families of lepidosaurians, chelonians, and crocodilians, using different antibodies against beta-keratins. The first polyclonal antibodies utilized were produced against avian beta-keratins: an antibody against chicken scale beta-keratin (B-1),2 or against a specific epitope present in avian and alligator beta-keratins (BUniversal, the epitope is: SRVVIQPSPVVVTLPGPILS).2,36 Recently, we have utilized polyclonal antibodies directed toward reptilian beta-keratins, a lizard scale beta-keratin of 15-16 kDa (A68b),11 and another antibody directed against a turtle scale beta-keratin of 13-16 kDa (A68 5).31 The antibodies have been used both in immunocytochemistry and in immunoblotting experiments after mono- or two-dimensional gel electrophoretic separation. This study, presented in the following paragraph, has allowed determination of the pattern of variation of beta-keratins in reptiles. 3380

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3. Variation of Beta-Keratins among Reptiles: Mono- and Bi-Dimensional Electrophoretic Patterns 3.1. Mono-Dimensional Beta-1 Patterns in Lizards. Results are summarized in Table 1. In the gila monster, Heloderma suspectum, immunolabeled bands are seen at 20, 35-37, and 43-48 kDa (Figure 3, A1). In the spiny agamid Uromastyx acanthinura, immunolabeled bands are seen at 7-10 kDa, and weaker at 16-18 kDa (Figure 3, A2). In the dragon Pogona vitticeps, numerous immuno-labeled bands around 24, 27, 3536, 40, 47, and 52 kDa are detected (Figure 3, A3). In Iguana iguana, major immunolabeled bands at 13-14 kDa and minor bands at 25-26 and 36-40 kDa are seen (Figure 3, A4). Strong immunolabeled bands at 11-12, 16, 18, and above 50 kDa are seen for Hemidactylus turcicus (Figure 3, A5), whereas in Tarentola mauritanica, bands at 12, 14, 44, and 47 kDa are present (Figure 3, A6). From molts of the gecko Nephrurus milii, immunolabeled bands at 14-15, 18-20, and 24 kDa are seen (Figure 3, A7). In another sample taken from another individual of the same species, strong bands at 18 and 19 kDa are seen (Figure 3, A8). In the lizard Podarcis sicula, immunolabeled bands at 10, 15, 18-20, and 50 kDa are seen (Figure 3, A9-10), whereas in the chameleon Chamaeleo calyptratus labeled bands are present at 8 and 33 kDa (Figure 3, A11). The analysis of the legless lizard Lialis burtonis shows the presence of extensive bands at 10, 13-14, and 17 kDa (Figure 3, A12), whereas in the legless lizard Pygopus lepidopodus, immunolabeled bands at 10, 13-15, 1718, and 30-35 kDa are seen (Figure 3, A13). Results from the epidermis of the large scincid Trachydosaurus rugosus reveal immunolabeled bands at 13-18 and 26 kDa (Figure 3, A14). The study on the epidermis of Tiliqua scincoides shows the presence of immunolabeled bands at 18, 24-26, 28-30, and 40-50 kDa (Figure 3, A15). From the molt of the goanna Varanus timorensis a main labeled band at 13-14 kDa, and weaker ones at 10 and 24-25 kDa are obtained (Figure 3, A16), whereas in Varanus gilleni, labeled bands are seen at 7, 1415, and 27-29 kDa (Figure 3, A17). 3.2. Mono-Dimensional Beta-1 Patterns in Snakes. Results are summarized in Table 1. In the molt extract of two specimens of Morelia spilota, weak immunolabeled bands are seen at 8-9 and 45 kDa, with stronger bands at 15-16 kDa (Figure 3, B1 and B2). In a specimen of Liasis maculosa, weak labeled bands at 16-17 and45 kDa are seen (Figure 3, B3). A specimen of Liasis childreni shows labeled bands at 8-9, 1516, 18, and 45 kDa (Figure 3, B4). From the python Morelia bredli, labeled bands at 15-18 kDa and weaker bands at 29 kDa are present (Figure 3, B5). In Natrix natrix, labeled bands at 15-18 and 29 kDa are present (Figure 3, B6); in Elaphe situla, labeled bands at 15, 17-18, 23, 29-35, and 45 kDa are seen (Figure 3, B7); in Elaphe guttata, strong labeled bands at 13-15, 29-30, and 50 kDa are seen (Figure 3, B8); in Crotalus atrox, weak bands at 15, 17-18, and above 50 kDa are present (Figure 3, B9). From the protein patterns of a specimen of Crotalus durissus, labeled bands at 7-10 are seen (Figure 3, B10). From a specimen of Boa constrictor, labeled bands in the range 1417, 28-30, and 50 kDa are seen (Figure 3, B11). In Cerastes cerastes, a strong-labeled band at 15 kDa and a weak band at 50 kDa are present (Figure 3, B12). From Bitis gabonica, an immunolabeled band at 10 kDa was seen (Figure 3, B13). From Trimeresurus albolabris, strong labeled bands at 16-18, and at 30-40 kDa are seen (Figure 3, B14). In another specimen of

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Figure 3. Monodimensional gel electrophoretic immunoblots for beta-1 antibody (A-C), beta-universal antibody (D), lizard beta-K antibody (E), and turtle beta-K antibody (F). See Text and Table 1 for details. Species: Heloderma suspectum (A1, E2); Uromastyx acanthinura (A2); Pogona vitticeps (A3); Iguana iguana (A4, E1, F3); Hemidactylus turcicus (A5); Tarentola mauritanica (A6, D2, E4); Nephrurus milii (A7, A8); Podarcis sicula (A9-10, D1); Chamaeleo calyptratus (A11); Lialis burtonis (A12); Pygopus lepidopodus (A13); Trachysosaurus rugosus (A14); Tiliqua scincoides (A15, F4); Varanus timorensis (A16); Varanus gilleni (A17); Morelia spilota spilota (B1, D3); Morelia spilota McDowelli (B2), Liasis maculosa (B3); Liasis childreni (B4); Morelia bredli (B5); Natrix natrix (B6); Elaphe situla (B7); Elaphe guttata (B8), Crotalus atrox (B9); Crotalus durissus (B10); Agkistrodon contortrix (B11); Cerastes cerastes (B12); Bitis gabonica (B13), Trimerosurus albolensis (B14. E3); Naja naja (B15); Acanthophis antarcticus (B16); Oxyuranus scutellatus (B17); Notechis scutatus (B18), Pseudechis porphyriacus (B19); normal Crotalus atrox (C1), scaleless mutant Crotalus atrox (C2-C4); Sphenodon punctatus (C5-7, D4), Testudo hermanni (C8); Chrysemys picta (C9-10, molt E5, F1 F2); Pelomedusa subrufa (C11); Alligator mississippiensis epidermis (C12-C13); Crocodylus niloticus (C14).

Naja naja, a weak labeled band at 7 kDa and stronger 16 kDa are seen (Figure 3, B15). In Acanthophis antarcticus, a band at 16 kDa is seen (Figure 3, B16). From Oxyuranus scutellatus, a labeled band at 14-15 is observed (Figure 3, B17). From one specimen of Notechis scutatus, an immunolabeled band at 16 kDa is seen (Figure 3, B18). From a specimen of Pseudechis porphyriacus, a labeled band at 16 kDa is seen (Figure 3, B19). The mono-dimensional immunoblotting analysis by using beta-1 antibody on molts of normally scaled Crotalus atrox shows strong-labeled bands at 12-14, 17, and 37 kDa (Figure 3, C1). In comparison, from molts of differently scaled body regions of scaleless C. atrox, weak labeled bands at 25-33 kDa are seen, but no bands are seen at 17-18 kDa, and faint or no bands appeared in the 12-13 kDa range (Figure 3, C2-C4). 3.3. Mono-Dimensional Beta-1 Patterns in Tuatara, Chelonian, and Crocodilian Epidermis. A summary of the western blotting is presented in Table 1. In the tuatara, Sphenodon punctatus, using a concentration of 15% polyacrylamide gel, labeled bands are seen in 3 main bands at 7, 11, and 16 kDa (Figure 3, C5). In another sample taken from another individual, immunolabeled bands at 13-14 and 25-30 kDa are present (Figure 3, C6). Finally, in another sample, bands at 9-11 kDa

are seen in gels at lower concentration of polyacrylamide (12%, see Figure 3, C7). In Testudo hermanni, labeled bands at 15-20, 25, and 36 kDa are detected (Figure 3, C8). From Chrysemys picta, immunolabeled bands in the range 15-20 kDa are produced in one sample (Figure 3, C9). In another sample of shed scutes of C. picta, immunolabeled bands in the range 12-24 and 33-36 kDa are seen (Figure 3, C10). From Pelomedusa subrufa, immunolabeled bands at 10 and 18-20 kDa are seen (Figure 3, C11). From the epidermis of nearly hatching embryo of Alligator mississippiensis, immunolabeled bands at 12-14, 18 kDa and a weak band at 32-34 kDa are seen (Figure 3, C12). From the epidermis of a preserved alligator embryo at stage 25-26, only bands at 20-23, 30-34, and above 40 kDa are present (Figure 3, C13). In juveniles of Crocodilus niloticus, bands at 20, 21-22 kDa and weak bands at 36-37 kDa are seen (Figure 3, C14). 3.4. Mono-Dimensional Beta-Universal, Beta-Lizard, and Beta-Turtle Immunoblotting Patterns. The reactivity of epidermal proteins with the beta-universal antibody shows immunopositive bands in all reptiles (Figure 3 D). In Podarcis sicula, strong bands at 13 and 15-18 kDa are seen (Figure 3, Journal of Proteome Research • Vol. 6, No. 9, 2007 3381

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Figure 4. Bi-dimensional gel electrophoretic patterns (Ponceau-red, 1-12), Beta-1 patterns (13-19), and Beta-universal patterns (2024) of keratins in the epidermis of different reptiles. The schematic drawing (25) indicates the main fractions, as alpha-keratin range, intermediate range, and beta-keratin range. The arrowheads indicate protein spots of likely known amino acid sequences and cloned beta-keratins.5-7 On the ordinate are the values of molecular weight in kDa, and on the abscissa are the pI values. The species analyzed are indicated in the left-bottom corner of each bi-dimensional pattern. 3382

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Hard (Beta-)Keratins in the Epidermis of Reptiles Table 1. Molts of the Species Analyzed by Using Beta-1 Antibody and Their Taxonomic Position Are Reported

* indicates lane numbers in Figure 4. + indicates not shown (see Figure 8).

D1). In one sample of the gecko Tarentola mauritanica, very strong bands at 13-14 and 15-18 kDa are detected (Figure 3, D2). In Morelia spilota, bands at 14-15 kDa are seen (Figure 3, D3). In one sample of Sphenodon punctatus, a band at 15 kDa is seen (Figure 4, D4). The lizard beta-keratin antibodies show immunoreactive bands in epidermal proteins of most lizards and numerous snake species, but there was limited or no reactivity with those of chelonians and crocodilians (Figure 3 E). In a specimen of Iguana iguana, a band at 15-16 kDa is seen (Figure 3, E1). In Heloderma suspectum, strong bands at 16-19 kDa are seen (Figure 3, E2). In the snake Trimeresurus albolabris, bands at 13, 16, and 24-30 kDa are seen (Figure 3, E3). In a specimen of gecko Tarentola mauritanica, no significant bands are detected (Figure 3, E4). In a sample of Chrysemys picta, molts bands at 12-14 and 15-16 kDa were detected (Figure 3. E5). This is also the case for the turtle antibody that does not cross-react with most lizards, snake, and crocodilian epidermal proteins (Figure 3 F). In a sample from C. picta, molts strong bands at 6-10, 14-16, and 30 kDa are seen (Figure 3, F1). In another sample from C. picta, bands at 7, 15, and 30 kDa are seen (Figure 3, F2). In Iguana iguana, bands at 8-10

and 15-16 kDa are seen (Figure 3, F3). In Tiliqua scincoides, a strong band at 15 kDa is seen (Figure 3, F4). 3.5. Two-Dimensional-Gel Patterns. Recent studies have detailed the alpha- and beta-keratin patterns of reptilian species representing all orders of reptiles.10-12,29-33,35 These studies have shown for the first time that reptilian alpha-keratins resemble those previously analyzed in avian and mammalian epidermis2,25,37 but that basic keratins are uncommon or absent. Betakeratins instead are basic, whereas the acidic fractions may be altered or processed forms of keratins with an acidic pI.35 The two-dimensional separation of epidermal proteins has shown that proteins are distributed in two main areas, one with a MW of 45-70 kDa and pI of 4-6 and the other with a MW of 14-23 kDa and pI of 6-9 (Figure 4, 1-12). In Hemidactylus turcicus, spots from 6 to 18 kDa with a pI of 4.3-8.8 and from 24 to 60 kDa with a pI of 4.3-6 are seen (Figure 4, 1). In Pogona vitticeps, spots from 7 to 18 kDa with a pI of 4-6.5, weaker spots with a pI of 7-9, and spots from 24 to 50 kDa with a pI of 4.5-5.5 are detected (Figure 4, 2). In Tarentola mauritanica, spots with a MW of 14-24 kDa and pI of 6-9.3 and spots from 45 to 66 kDa with a pI of 4.5-5.8 are detected (Figure 4, 3). In Journal of Proteome Research • Vol. 6, No. 9, 2007 3383

reviews the lizard Podarcis muralis, some spots distributed in a large area from 6.5 to 36 kDa and with a pI of 4.5-9 are visualized. Moreover, spots of 50-60 kDa with a pI of 4.5-7 are also detected (Figure 4, 4). In the snake Crotalus atrox, spots in the range 14-18 kDa with a pI of 7.5-9.5 and spots with a MW from 50 to 65 kDa with a pI of 4.5-5.5 are detected (Figure 4, 5). In Morelia spilota, few spots at 14-15 kDa with a pI of 7-9.3 and more intense spots with a MW from 40 to 60 kDa with a pI of 4.5-6.5 are detected (Figure 4, 6). In the tuatara (Sphenodon punctatus), several spots with a low MW (6-14 kDa) and a pI from 6 to 9.5 are detected (Figure 4, 7). In the turtle Testudo hermanni, several spots from 10 to 18 kDa with a pI of 5-9.5 are detected (Figure 4, 8). Moreover, spots characterized by an acid pI (3.5-4.5) with a MW of 22 and 45 kDa are stained (Figure 4, 8). In Pseudemys nelsonii, spots from 14 to 18 kDa with a pI of 6-9 are detected (Figure 4, 9). In the softshelled turtle Apalone spinifera, spots at low MW (14-20 kDa) with a pI of 3.5-4.5, 6-6.5, and 7.5 are detected (Figure 4, 10). Other spots with a pI of 4-6 and MW of 24, 36-38, and 55-70 kDa are detected (Figure 4, 10). In the crocodile Crocodilus niloticus, spots at 16-20 kDa with a pI from 6 to 8.3 and weak spots at 45-65 kDa and pI from 4 to 6 are detected (Figure 4, 11). In the alligator Alligator mississippiensis, spots with a MW from 14 to 24 kDa and pI of 5-9 are detected (Figure 4, 12). Moreover, a spot at 30 kDa with a pI of 4.5 and some weak spots at 45-66 kDa with pI of 4-6.5 are detected (Figure 4, 12). The immunoblotting using beta-1 antibodies is shown in Figure 4, 13-19. The beta-1 antibody in Podarcis muralis immunostains a main spot at 14-15 kDa with a pI of 6.5 (Figure 4, 13). Two weaker immunolabeled spots at 15-16 kDa show pI at 4.6 and 6.5. More immunolabeled spots at 17-18 kDa show pI at 6.2 and 8.2-8.4 (Figure 4, 13). In Tarentola mauritanica, several spots at 16-23 kDa with a pI of 4-9 are immunolabeled by beta-1 antibody (Figure 4, 14). In Elaphe guttata, spots at 14-15 kDa with a pI of 8.8, 7.2, 6.2, 5.2 and an intense spot with a pI of 4.2 are detected (Figure 4, 15). Other spots with a MW of 28-29 kDa and with a pI of 4.2 and 4.8 are detected (Figure 4, 15). In Crocodilus niloticus, two strong spots at 17-18 kDa with a pI of 8-8.5 and 7.3-7.6 are detected (Figure 4, 16). Three weaker spots at 19 kDa with a pI of 7.5, 8.0, and 8.5 are immunolabeled. Two ill-defined spots at low molecular weight with a pI of 4.6 and 3.6 are detected (Figure 4, 16). In Testudo hermanni, numerous spots with MW in the range 15-17 kDa and with a pI of 5.0-8.8 are detected (Figure 4, 17). Other weak spots at 31-33 kDa with pI of 6.0-6.5 are detected (Figure 4, 17). In the soft epidermis of Apalone spinifera, two spots at 20 kDa with a pI of 7.8 and 7.3, a weak spot at 22 kDa with a pI of 6.0 and a spot at 36 kDa with a pI of 7.3 are detected (Figure 4, 18). In the carapace of T. spiniferus, spots at 19-21 kDa with pI of 7.7, 7.2, 6.2, and 5.2 are detected (Figure 4, 19). The immunoblotting using beta-universal antibody is shown in Figure 4, 20-24. In Tarentola mauritanica, several weak spots are presents in the range 10-20 kDa with a pI from 6.2 to 8.0 and other spots with a pI from 3.5 to 4.5 (Figure 4, 20). In Testudo hermanni, spots at 17 kDa with pI of 5.2-9.0 are immunolabeled (Figure 4, 21). In Crocodilus niloticus, two strong spots at 17 kDa with pI of 7.8 and 6.9 and three weaker spots at 19-20 kDa with a pI of 6.7, 7.4, and 8.2 are immunolabeled (Figure 4, 22). In Morelia spilota, one intense spot at 16 kDa with a pI of 7.2 and some spots at 29-30 kDa with a pI of 4.0-7.2 are found (Figure 4, 23). In Chrysemys picta, two 3384

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intense spots at 15 kDa with a pI of 6.2 and 6.4 and spots not well resolved at 24-29 kDa with a pI 6.0-6.5 are detected (Figure 4, 24). In conclusion, three main groups of proteins can be found in reptile epidermis (summarized in Figure 4, 25). Spots with a MW from 10 to 20 kDa with a pI of 3.5-9.0 (mainly concentrated at basic pI) represent beta keratins, spots from 20 to 30 kDa with pI from 4.3 to 9.0 represent an intermediate region probably constituted by KAPs or HRPs, and spots from 45 to 66 kDa with a pI from 3.5 to 7.5 (mainly concentrated from 5.0 to 6.0) represent alpha keratins.

4. Genomic and Proteomic Organization of Beta-Keratins The first molecular biology studies on reptilian beta-keratins have indicated that genes coding for these relatively small proteins are linked in the genome and organized in a linear array like the corresponding avian beta-keratin genes (Figure 5A, B).5-7 Beta-keratin genes in reptiles, as in birds, probably derive from tandem-gene duplication and diversification.22 So far, no introns have been found in the studied lizards genes, whereas one intron in the 5′-non coding region is present in beta-keratin genes of snakes. The latter organization is similar to that present in feather and scale beta-keratin genes.38-42 Preliminary observations, however, suggest that introns are also present in other beta-keratin genes in the lizard genome, but further studies are needed to confirm this initial indication (Dalla Valle et al., unpublished). Beta-keratins deduced from the sequenced genes of reptilian epidermis are represented by glycine-proline-serine-rich proteins of 13-19 kDa with basic pI (7.5-9.4) that presents variable homology, in particular in their central region indicated as corebox (Figure 5C, D). The latter is made of 20-24 amino acids and also has 70-85% homology with the core-box of avian beta-keratins (Figure 5D). Some of these deduced proteins have been mapped in two-dimensional gels of epidermal proteins extracted from the epidermis in the relative species (see arrowheads in Figure 4, 13 and 15) and have confirmed that they are basic proteins. Among different amino acids present in the beta-keratins initially sequenced, a high amount of glycine, proline, serine, alanine, and leucine, low-medium content in cystein, and low content in tyrosine, are present (Table 2). Histidine, methionine, and tryptophane are low to absent in these beta-keratins. Only a specialized keratin from the claw of a varanus lizard shows numerous differences from the above cloned scale betakeratins (Figure 5C).43 Among other differences, the claw keratin shows a high content of cysteine (above 15%, see Table 2) and has an acidic isoelectric point (pI 5.6). The claw keratin is, however, rich in glycine, and it shows amino acid sequences with some homology to avian or mammalian proteins. The predicted secondary structure of the claw beta-keratin also shows that the protein contains a core-box with two double strands (Figures 5-7). The claw keratin may represent a member of a different category of sulfur-rich beta-keratins that is more specific to claws or specialized scales. In fact, recent studies have shown that this class of sulfur-rich beta-keratins is present in specialized digital scales and claws of gecko lizards (Hallahan et al., unpublished observations). The new data indicate that glycine-rich and cysteine-rich beta-keratins are present in the hard epidermis of reptilian scales and claws. The comparative analysis of the sequences of some beta-keratins of reptiles and birds shows which regions of these proteins have

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Figure 5. Schematic representation of beta-keratin genes in lizard (A) and snake (B). The comparison of some scale beta-keratin amino acid sequences among reptiles (C) and between reptiles and the chick (D) were assayed using the ClustalW software at http:// www.ebi.ac.uk/clustalw/. The non-homologous structure of claw keratin in a Varanus is shown in (E). In (C), the comparison among seven species of reptiles is presented (for the alligator, crocodile, and turtle, only a central sequence is known). Two regions of high homology (stars, dots) are underlined, the N-terminal region and the central region(core-box). In (D), the comparison is extended to chick scale, claw, and feather keratin, and the homology mainly remains in the core-box (boxed sequences). In (E), the claw keratin of a lizard is represented where the boxed region present less than 43% of identity with the core-box of the other beta-keratins (see text for further details). Journal of Proteome Research • Vol. 6, No. 9, 2007 3385

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Table 2. Percentage of Key Amino Acids in Known Beta-Keratins of Reptiles

species

sequence name

accession number or references

%age aa

MW (Da)

pI

Cys

Tyr

Ser

Pro

Gly

13137 15490 15501 15830 15860 16513 16790

5.63 8.18 8.18 8.18 8.17 8.17 8.86

15.5 4.3 4.3 4.2 4.2 4.0 2.2

2.1 4.9 4.9 4.8 5.4 5.7 2.2

9.2 7.4 7.4 7.2 7.2 6.9 17.3

7.7 8.0 8.0 7.8 7.8 7.5 10.6

28.9 29.4 29.4 29.3 29.3 30.5 20.7

16847 17997 16196

8.75 9.49 9.34

2.8 2.6 4.1

1.7 2.6 0.6

18.1 16.2 14.2

10.7 8.4 14.8

18.6 23.0 17.8

13067 13037

7.49 7.49

5.8 5.8

1.5 1.5

10.9 10.2

12.4 12.4

18.2 19

12573 13581

7.53 8.16

5.3 5.8 6.8 6.6

0.8 2.2

10.5 13.7 13.3 14.7

12.0 12.9 10.7 15.9

19.5 17.3 16.2 18.6

9.9

9.5

29.7

Lizards V. gouldii (claw) P. sicula

T. mauritanica H. turcicus

Li-gprp-1 Li-gprp-2 Li-gprp-3 Li-gprp-4 Li-gprp-5 Ge-gprp-1-2 Ge-gprp-3 Ge-gprp-3 Ge-gprp-4 Ge-gprp-5

Inglis et al., 1987 CAI67601 AM259048 AM259049 AM259050 AM259051 AM162665 AM263204 AM263205 AM258989 AM263206 Snakes

E. guttata

Sn-gprp-1 Sn-gprp-2-3 Sn-gprp-4 Sn-gprp-5

AM404184 AM404185 AM404186 AM404187 AM407413 17

C. constrictor B. constrictor

54

2.4

Sphenodontids 17

S. punctatus

4.0 Chelonians

17

C. picta C. mydas G. agassizii C. niloticus

54 55

Crocodilians Dalla Valle et al., unpublished 17449

more conservative amino acid sequences and the localization of key-amino acids such as glycine, proline, serine, and cysteine (Figure 6). The core-box is quite conserved (70-95% among different species) and contains most of the proline residues responsible for bending the amino acid chain. The presence of valine, leucine, and isoleucine contributes to the formation of betasheet in this region, the region that likely allows the polymerization among beta-keratins monomers into beta-keratin filaments.22,24,42 Glycine is mainly localized between the core-box and the N- or C-terminals. Serine may be the site of phosphorylation of beta-keratins, a post-translational process of unknown physiological consequences, but that lowers the pI of beta-keratins.2,35,37 Cysteine is mainly localized toward the extremities of these proteins (C- and N-terminals), and this amino acid is probably involved in intra- and intermolecular bonding responsible for forming hard corneous material. The recent knowledge of the amino acid sequences of reptilian beta-keratins has also allowed the prevision of their secondary conformations using computer programs.44 To date, over 30 different proteins have been deduced from numerous genes isolated from four species of lepidosaurian reptiles, three species of lizards, and one species of snake.5-7,9 The sequencing of more beta-keratins from crocodile and turtle scales is mostly completed (Dalla Valle et al., ongoing studies). Some examples of the deduced beta-keratins from the lizard P. sicula, geckos T. mauritanica and H. turcicus, and the snake E. guttata, and for the claw keratin of Varanus, are shown (Figure 7). The cross-reactivity with the beta-keratin universal antibody, observed in both immunocytochemical and immunoblotting studies (Figures 2-4), indicates that crocodilian and chelonian beta-keratins also contain the core-box. In fact, although only partial amino acid-sequences are available for the alligator,2 crocodile, and turtle (Dalla Valle et al., unpublished), a core3386

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7.97

6.2 5.5 10.9

10.4 12.7 10.0

6.7 5.6 5.6

8.3 10.1 8.4

32.8 30.3 22.8

6.1

10.1

9.5

8.9

30.2

box has recently been found in all these proteins. The corebox shows a high homology with that of chick scale and feather keratins (Figures 5C and 6). These beta-keratins show the constant presence of two strand regions (beta-sheets) in the core box region, each strand spanning between 4 and 6 amino acids. A similar secondary conformation in the core-box region is present in the acidic beta-keratin of the lizard claw, the alligator scale, and scale and feather keratins. Despite the different sequences, the Varanus claw protein also shows a core-box-like central region where three strand regions are present (Figures 5 and 7). Finally, a core-box region is also present in cysteine-rich betakeratins of digital scales of geckos (Hallahan et al., unpublished observations). This indicates that the core-box region is present in all betakeratins where it probably has a universal role in the folding, compaction and, in particular, in the formation of beta-keratin filaments in the epidermis of reptiles and birds (Figure 8A).

5. Secondary Structures and Formation of Beta-Keratin Filaments Classically, beta-keratins were named “keratins” as they were major proteins accumulated in epidermal cells that gave origin to protein bundles constitutive of the packets or tonofilaments formed in differentiating beta-cells of reptilian scales or feathers.19,22,37,45-48 Over the years, the term “keratin” has been refined by the accumulation of numerous biochemical and molecular biology data that have identified (alpha-)keratins as a type of intermediate filament, now universally termed cytokeratins.26-28 The latter form fibrous filaments by progressive stages of assembling of the monomers into filaments of increasing size that eventually form tonofilaments. This process also occurs in specialized keratins of skin derivatives, the best known of which are the trichocytic keratins.49,50-52

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Figure 6. Alignment of the known reptilian with some avian beta-keratins that highlights the position of four key amino acids (glycine, proline, serine, and cysteine). The central, boxed-area represents the core-box and shows the main concentration of prolines (blue), which are organized in a universal triplet (PGP, arrow), a high-frequent triplet (arrowhead), and frequent duet (double arrowhead). Glycine-rich regions (in red) are present in the lateral regions toward the N- and C-terminals (except in avian beta-keratins where the N-region is absent). Most serine is also present outside the core-box in the glycine-rich regions. Finally, cysteins tend to be more frequent toward the N- and C-regions. The core-box is the epitope mainly recognized by the Beta-universal antibody while the glycinerich regions are likely targets of the beta-1, beta-lizard antibodies (A68-1 and A68-b), and beta-turtle antibody (A68-5). Glycine-rich regions present in some high molecular weight alpha-keratins can be responsible of some cross-reactivity with those antibodies, especially the beta-1. This sometimes occurs when alpha-keratins isolated in immunoblots are quantitatively abundant. The specific fK epitope present in feather-keratin is recognized by the FBK antibody (see text for details). 1, Ge-prp-1-2 (AC AM162665, AC AM263204); 2, Ge-gprp-3 (AC AM 263205); 3, Ge-gprp-4 (AC AM 258989); 4, Ge-gprp-5 (AC 263206); 5, Li-gprp-1 (AC AJ890445); 6, Li-gprp-2 (AC AM259048); 7, Li-gprp-3 (AC AM259049); 8, Li-gprp-4 (AC AM259050); 9, Li-gprp-5 (AC AM259051); 10, Sn-gprp-1 (AC AM 404184); 11, Sn-gprp-2-3 (AC AM 404185, AM 404186); 12, Sn-gprp-4 (AC AM 404187); 13, Sn-gprp-5 (AC AM 407413); 14, AC NP_001001310; 15, chick scale (AC P04459); 16, chick claw (AC AAA62730); 17, chick feather I (AC P02450); 18, chick feather II (AC P04458); 19, chick feather III (AC P20307); 20, chick feather IV (AC P20308); 21, turkey vulture feather (AC Q98U06); 22, varanus claw.43

However, despite their X-ray alpha-keratin pattern, the mechanical deformation of these proteins by stretching determines the change of the alpha-pattern and the acquisition of a partial or complete X-ray beta-pattern.19,24 Molecular models for beta-keratins indicated that the central part of the molecule presents classical secondary conformation of amino acid chain into beta-pleated sheets.19,22,24,42 Also, the prediction analysis suggests that beta-keratins, aside their X-ray and irregular 3-4 nm ultrastructural pattern, contain beta-sheets in the core box and few other regions of these proteins. A strand conformation represents a beta-turn region in which the protein bends into a rigid steric motif.22,24,42 Most of the molecule of beta-keratins is occupied by amino acids forming random-coiled chains (53.5-81.0%) whereas R helix regions are limited (7.2-25.4%) (Figure 7). The different properties of each type of beta-keratin remain unknown at the present time and require specific chemical-physical studies. For instance, a type of beta-keratin found in lizard (Li-gprp-1, AC AJ890445; Figure 7) may assume a more compacted conformation due to its 4 beta-folds and shorter alpha-helical regions than in another type of beta-keratin with more extended R helix regions (Li-gprp-3, AC AM259049; Figure 8). Another example indicates that the 5 beta-folds of gecko beta-keratins (Ge-gprp5; Figure 8) may determine a higher packing of the protein than in Ge-gprp-1-3 beta-keratins (compared with the gecko sequence Ge-gprp-3 shown in Figure 7). It remains unexplained how these beta-keratins form the elongated, prevalently parallel bundles that constitute most of the long filaments of betakeratin in gecko adhesive setae. The latter are responsible for

the formation of 30-120 mm long bristles (setae) in the digit adhesive pads that allow geckos to climb vertical walls. Another example suggests that the 7 beta-turns predicted in the small snake beta-keratins 2-3 (Sn-gprp-2-3, AC AM404185; Figure 8) or the 6 in Sn-gprp-4 (Figure 8) may determine more compact aggregates than snake beta-keratin-5 with only four strands (Sn-gprp-5, AC AM407413; Figure 7) where only 4 betaturns are present. These theoretical considerations would require a three-dimensional crystallographic study on different beta-keratins for verification, a task that is not presently feasible. Other examples of variation of secondary structures could be mentioned for more beta-keratins, but the different properties of these proteins are not known yet. The clarification of the molecular modality of packing of beta-keratins will be possible after more beta-keratin sequences and the prevision of their tertiary structure are obtained (ongoing studies). This knowledge will help to explain possible mechanisms of interactions among beta-keratins that produce the dense and resistant corneous material of scales. Further, amino acid sequences of beta-keratins will be used to study their possible evolutionary relationship with beta-keratins of birds and with hard-keratins of mammalian hairs, claws and horns (Keratin Associated Proteins, KAPs).49,53 The beta-strands of the core-box region of the protein is believed to interact via hydrogen or polar bonds with the same region present in other beta-keratins. This antiparallel interaction origins a central beta-keratin framework and produces the aggregation of these proteins (monomers) into beta-keratin filaments (polymers) (Figure 9A). Journal of Proteome Research • Vol. 6, No. 9, 2007 3387

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Figure 7. Prediction of the secondary structure of lizard, gecko, snake, and Varanus beta-keratin proteins using the program “PSIPRED Protein Structure Prediction Server.”44 The theoretical MW and pI are indicated (see Text for details). Also, the partial known sequence of the core-box for the alligator beta-keratin is shown for comparison (the indicated molecular weigh and pI refer to unpublished proteonic data on these proteins). 3388

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Figure 8. Prediction of the secondary structure of other lizards, geckos. and snakes to show the variation of regions within these proteins using the program “PSIPRED Protein Structure Prediction Server.”44 The theoretical MW and pI are indicated (see text for details). Journal of Proteome Research • Vol. 6, No. 9, 2007 3389

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Figure 9. Importance of beta-keratin and in particular of the core-box for the formation of the hard beta-keratin layer of reptilian scales is shown in this figure. (A) Schematic representation of the molecular organization of beta-keratin monomers (1), to form the framework by antiparallel polymerization in the core-boxes (2), the consequent formation of beta-keratin filaments of 3-4 nm (3), their aggregation among the initial alpha-keratin filaments (4), and the compaction into larger beta-keratin aggregated (beta-packets) while alpha-keratin is degraded or disappears (5). In (B), different parts of the mutant scaleless snake (Crotalus atrox) are analyzed for the presence of beta-keratin. The smooth region, completely scaleless (1, 1′, 1′′), does not present beta-keratin bands (both beta-1 and beta-universal negative) in gels. The scaled areas (ventral gastrosteges) present beta-1 and beta-universal positive bands (2, 2′, 2′′). Finally, the intermediate region also shows these bands, although the intensity is somewhat lower (3, 3′, 3′′). (C) Soft-shelled turtle (Apalone spinifera) in which all areas of the epidermis contain a similar pattern with soft keratin (1-3). Bands at 20-22 and weaker at 30-40 kDa are reactive with the Beta-1 antibody (1ı´-3ı´) indicating presence of beta-keratins epitopes. In contrast, no bands are detected using the beta-universal antibody (1′′-3′′), indicating that beta-keratins may lack the core-box. This is also indicated by the lack of beta-keratin packets and filaments in the epidermis of the soft-shelled turtle (Figure 2O).

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6. Effects of the Lack of Beta-Keratins on Skin Phenotypes Two examples further suggest the fundamental role of betakeratins, in particular of the core-box, in the origin of the resistant beta-layer in reptilian scales (Figure 9B, C). In the scaleless snake (a mutant of Crotalus atrox), the complete or partial absence of scaling is due to the absence of beta-keratin and of the beta-layer.34 Beta-keratins are completely missing in areas devoid of scales and are decreased in regions where scales are reduced (Figure 9B). In the latter regions, the epidermis has the same structure of hinge regions of normal scales and the skin can form wrinkles like mammalian epidermis. Beta-keratins are, however, present in the scaled areas of some individuals, especially in the large ventral scales, the gastrosteges, which allow the movement of these mutant snakes. The immunocytochemical and immunoblotting study of the latter areas show that they contain beta-keratins and also the epitope recognized by the universal antibody (SRVVIQPSPVVVTLPGPILS),2,36 suggesting that the core-box is present in beta-keratins of these large, functional scales (Figure 9B). The case of the soft-shelled turtle (Apalone spinifera, previously indicated Trionyx spiniferus)12 brings other considerations to the role of the core-box in beta-keratin for the origin of scale toughness.12,16 In the soft skin of the carapace and plastron and in the remaining skin of limbs, neck, and tail, scales are basically absent (Figure 9C). A type of general beta-keratin seems, however, to be present in the epidermis of this species as indicated by immunocytochemical and immunoblotting analysis. However, these beta-keratins have a higher molecular weight (18-20 and 32 kDa) than those found in other chelonians with hard shells (13-15 kDa). Besides, the electron microscopic and immunocytochemical study of the corneous layer of the carapace and plastron shows that the beta-keratins are diffuse among cytokeratin filaments of the pre-corneous and corneous layer, and therefore, no dense beta-packets or larger bundles of beta-keratin filaments are formed in this species. The lack of reactivity to the Beta-universal antibody suggests that this beta-keratin is missing of the core-box epitope (recognized by the latter antibody) although it conserves the other epitopes recognized by the beta-1 antibody (Figures 1 and 9C). The lack of the core-box probably impedes the polymerization of beta-keratin to form the framework of beta-keratin filaments as illustrated in Figure 9A, and therefore the formation of the typical beta-packets cannot occur (Figure 2O). This in turn, determines the deposition of separated betakeratin molecules that cannot merge into the dense betakeratin hard material present in normal scales. The above, preliminary studies will only be confirmed after the sequencing of keratins from this species (ongoing studies). Aside from the type of beta-keratins, also the presence of a specific ratio of different beta-keratins in different scales (ventral scales versus dorsal scales, claws versus body scales, or scales with thick corneous layers or possessing keels, tubercles, spines, bristles, etc.) can affect their mechanical properties.1,20 Only future studies on the complete variety of beta-keratins will explain the different mechanical properties of reptilian scales, claws, ramphotheca and other skin derivatives.

Acknowledgment. Antibodies against beta-keratins (Beta-1 and beta-universal) were donated by Profs. R. H. Sawyer and L. W. Knapp (University of South Carolina, Columbia).

Materials of Australian species were kindly supplied by Dr. J. Weigel (Australian Reptilian Park, Gosford). Molts from gila monster was supplied by Dr. R. Myers (Rattlesnake Museum, Albuquerque, NM). The collection of Sphenodon molts was made possible by Dr. B. Gill (Auckland Museum, New Zealand) and Dr. N. Nelson (Victoria University, Wellington, New Zealand). Most of the remaining material was kindly supplied by Mr. M. Ghidotti, Mr. A. Faggin (scaleless snake), Mr. A. Zerbini (Serpico, Bologna, Italy), Dr. E. Moretto (Butterfly Arc, Montegrotto Terme, Padova, Italy), and Prof. M. Marini (University of Bologna, Italy). Prof. M. Starck (University of Munich II, Germany) supplied crocodilian skin samples. The study was funded by a University of Bologna Grant “60%” and largely by self-support (L.A.).

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