The Epidermis of Scales in Gecko Lizards Contains Multiple Forms of

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The Epidermis of Scales in Gecko Lizards Contains Multiple Forms of β-Keratins Including Basic Glycine-Proline-Serine-Rich Proteins M. Toni,† L. Dalla Valle,‡ and L. Alibardi*,† Dipartimento di Biologia evoluzionistica sperimentale, University of Bologna, Italy, and Dipartimento di Biologia, University of Padova, Italy Received November 24, 2006

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

Introduction The epidermis of lepidosaurian reptiles (lizards and snakes) is made of alternating generations of keratinocytes that form an outer, hard β-layer, and an inner softer R-layer.1 An epidermal generation is made of six different epidermal layers termed oberhautchen, β, mesos, R, lacunar, and clear layer. Cells of the oberhautchen and β layers synthesize a hard type of keratin, termed β-keratin, which is deposited after the initial accumulation of cytokeratin (R-keratin). A new (inner) generation of epidermal layers is formed beneath the old (outer) generation, and the latter is desquamated to produce the molt. The last formed layer of the outer generation, the clear layer, is initially joined with the first layer of the inner generation, the oberhautchen layer, forming the shedding complex. Cell junctions between clear and oberhautchen cells are later degraded before shedding. The oberhautchen layer of the inner generation produces 1-3 µm long spinules directed toward the clear layer of the outer generation. However, in some lizards (geckos, anolids, scincids), some digital scales form lamellar surfaces where the spinulae of the oberhautchen layer grow into 20-100 µm long setae with a diameter of 1-2 µm (Figures * To whom correspondence should be addressed. L. Alibardi, Dipartimento di Biologia evoluzionistica sperimentale, University of Bologna, via Selmi 3, 40126, Bologna, Italy. † University of Bologna. ‡ University of Padova.

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1-3).2 Both during embryonic development and epidermal renewal, the setae are surrounded by the cytoplasm of clear cells, and the latter are eventually shed with the other epidermal layers during molting (Figures 2-4).3-6 Therefore oberhautchen setae and the clear layer constitute a specialized form of shedding complex. Geckos utilize the setae to form an efficient adhesive device for climbing smooth and vertical surfaces. The structure and mechanism of adhesion of climbing setae in geckos are inspiring studies aiming to the production of a new generation of dry adhesives.7-10 The knowledge on the proteins involved in the production of setae is important for understanding how they are organized to produce 0.5-2.5 µm thick setae and how they are assembled in the terminal part of setae (spatula), which is the effective site of adhesion. Whether the variation of adhesive properties may vary with the state of hydration of the substrates on which setae adhere is not known.9 The humidity may influence the proteins of setae, since keratins can absorb water and become hydrated.1,11,12 Ultrastructural studies have indicated that setae grow by the addition of keratin at their base (Figure 3).6,13 Biochemical analyses have indicated that scales and setae of geckos are largely made of β-keratins of 10-18 kDa.14-16 β-Keratins form a resistant corneous material for sustaining adhesion: this corneous material is deposited as long bundles oriented along the main axis of setae. β-Keratins are present in reptilian and avian scales, claws, beak, and feathers.17-21 The molecular 10.1021/pr060626+ CCC: $37.00

 2007 American Chemical Society

Epidermis of Scales in Gecko Lizards Contains β-Keratins

characteristics of β-keratins of setae, as those of reptilian scales in general, are poorly known, but recent molecular studies have sequenced for the first time some of these β-keratins.22-24 The proteins in lizard scales contain a high amount of glycine and over 8% of proline, and they have been indicated as glycineproline-rich proteins. The present study deals with the characterization, localization, and amino acid sequentiation of β-keratins from gecko scales and setae. The study represents part of a broader survey on corneous proteins of the reptilian epidermis with the final goal to determine the molecular evolution of these specialized proteins from more generalized cytoskeletal proteins.

Materials and Methods Tissue Collection and Fixation. Eight adults of small Mediterranean gecko Hemidactylus turcicus and 14 adults of a larger species, the Moorish gecko Tarentola mauritanica were used in the present study. About three-fourth of the tail was amputated by pulling the tail in order to determine the release of the tail by autotomy, a natural and harmless mechanism of autoamputation of the tail that is commonly used in these species to escape predators. The released tail was immediately frozen in liquid nitrogen and stored at -80 °C (first sampling). The tail, at summer temperature and photoperiods (25-35 °C and at over 10 h of daily light), regenerated within 4-7 weeks. When regenerating tails of 0.6-1.0 cm in length began to form new scales (3-4 weeks postamputation), the tail was reamputated, and the regenerating epidermis (second sampling) was used for analysis. To collect enough epidermis (third sampling), the whole procedure was repeated another time, within a total period of about 2.5 months at summer room temperatures (25-35 °C; third sampling). Geckos regenerated their tail twice in sequence (within a total period of 2.5 month) with no apparent difference. The animals were killed by decapitation (final third sampling), and the skin, including the digits with the pad lamellae, was collected. Other sampled tissues consisted in molts from six geckos (T. mauritanica). When the animals started the process of shedding, the bodies appeared covered by pale epidermis (indicating detachment of the outer from the inner epidermal generation). The molt was collected with tweezers by pealing off the shed which was divided into body molt, and digit molt, the latter mainly containing setae. The two groups were then analyzed independently using bidimensional gel electrophoresis for comparative purpose. Tissues were immediately fixed or frozen in liquid nitrogen and stored at -80 °C for biochemical analysis or extraction of the RNA (see later). Few samples of skin were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4, rinsed in buffer for about 20 min, postfixed for 1 h with 2% OsO4 in phosphate buffer, dehydrated with ethanol, infiltrated with propylene oxide for 30-40 min, and embedded in Durcupan Resin (Fluka-Aldrich, Germany). The latter tissues were used for immunocytochemistry. Other pieces were fixed in freshly made 4% paraformaldehyde solution in 0.1 M phosphate buffer, pH 7.4. The tissues were dehydrated in ethanol and embedded in Bioacryl Resin at 0-4 °C under UV light as indicated in the method by Scala et al.25 Tissues embedded in resin were sectioned with an ultramicrotome to collect 1-3 µm thick sections for light microscopy and immunocytochemistry. Other 40-90 nm thin sections were collected with nickel grids for ultrastructiural examination and immunocytochemistry.

research articles Immunocytochemistry. Nonspecific antigenic sites were blocked by incubating the sections for 20 min with 5% normal goat serum in 0.05 M Tris-buffered saline at pH 7.6 containing 2% bovine serum albumin. The sections were incubated overnight at 4 °C in the primary antibody, while the negative controls were incubated with preimmune sera or buffer only. Four different antibodies against β-keratins were used. The Beta-1 keratin antibody, which was the most used in our study, was produced in rabbit against a chick scale β-keratin,21 and was diluted 1:200. Another antibody (A68b), produced in rat and directed against a lizard β-keratin of 15-16 kDa was used at a dilution of 1:50.26 The specific epitopes targeted by the above two antibodies are not known. Another antibody, produced in rabbit against a 20 amino acid epitope common among β-keratins (SRVVIQPSPVVVTLPGPILS) called β-universal,21,27 was also used at a dilution of 1:200. Finally FBK, a feather-specific antibody developed against the epitope indicated as AVGSTTSAAVGSILSEEGVPINSG,21,27 was tested in some cases for cross reactivity. After incubation with the primary antibody, sections were rinsed and then incubated for 1 h at room temperature with a goat anti-rabbit or anti-rat FITC-conjugated secondary antibody (Fuorescein-Iso-ThioCyanate, Sigma) diluted 1:50 in Tris-buffered saline (TBS). After rinsing, sections were mounted in Fluoromount (EM Sciences) and observed with a Zeiss epifluorescence microscope. Thin sections collected on nickel grids were incubated at room temperature for about 20 min in TBS containing 0.1% TritonX100 and 1% cold-water fish gelatin (to block nonspecific antigenic sites). Sections were then incubated overnight in the same buffer at 4 °C with different β-keratin antibodies indicated above (controls were incubated with the buffer only or using pre-sera). After rinsing in buffer, grids were incubated for 1 h at room temperature with the secondary anti-rabbit or antirat antibodies conjugated to 10 nm gold particles (Sigma or Chemicon) at 1:40 dilution in the same buffer. After a rinsing step in the buffer, grids were washed in distilled water, dried, and observed unstained or lightly stained (5 min with 2% uranyl acetate) under the electron microscope. Protein Extraction. The remaining epidermis from the above specimens, including that containing the digit pads, was collected over the whole body and used for the electrophoretic analysis. Epidermal proteins were extracted by the method of Sybert et al.28 as previously reported. Briefly, the skin was incubated in 5 mM EDTA in phosphate-buffered saline (PBS) for 3-5 min at 50 °C and 2-4 min in cold PBS. The epidermis was separated from the dermis by dissection using a stereomicroscope. The epidermis was homogenized in 8 M urea/50 mM Tris-HCl (pH 7.6)/0.1 M 2-mercaptoethanol/1 mM dithiothreithol/1 mM phenylmethylsulphonyl fluoride, and the particulate matter was removed by centrifugation at 10 000g for 10 min. Protein concentration was assayed by the Lowry method. Electrophoresis and Immunoblotting. For monodimensional electrophoresis analysis, proteins were denatured by boiling in the Sample Buffer for 5 min. Then, 50 µg of proteins was loaded in each lane and separated in 15% SDS-polyacrylamide gels (SDS-PAGE) according to Laemmli.29 For BD electrophoresis analysis, the Ettan IPGphor III IEF System (Amersham, U.K.) was used for the isoelectrofocusing (IEF). A 150 µg protein sample (containing 2% CHAPS (Sigma) and 1% carrier ampholyte mixture, pH 3,5-10 (Amersham, U.K.) was loaded onto a 7 cm (pH 3-10) or a 13 cm (pH 6-11) strip (Amersham, U.K.), depending on the experiment. Application of the strips and the running procedure were carried out as Journal of Proteome Research • Vol. 6, No. 5, 2007 1793

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Figure 1. (A) T. mauritanica with regenerating tail (arrowhead). The location of the pads on the digits is indicated by arrows. Bar, 0.5 mm. (B) H. turcicus with arrowed digits indicating the pads. Bar, 0.5 mm. (C) Detail of limb scale with the thin corneous layer (arrow). Toluidine-blue stain. Bar, 20 µm. (D) Detail of pad lamella (modified scale of digital pads) with the long setae above the thin epidermis (arrow). Toluidine blue stain. Bar, 20 µm. Legends: d, dermis; h, hinge region; i, inner (ventral) scale surface; o, outer (dorsal) scale surface; s, spinulae/setae.

described by the manufacturer. The following protocol was used. Rehydratation was performed for 12 h at room temperature and was followed by the IEF, step by step, from 0.5 h 500 V, 0.5 h 1000 V, 0.5 h 5000 or 8000 V gradually, and for 1 h at 5000 or 8000 V (5000 and 8000 V were applied to 7 and 13 cm strip, respectively). Strips were kept at 50 V until loaded on the second dimension. Before starting the second dimension, the strips were equilibrated for 10 min in 6 M urea, 30% glycerol, 50 mM Tris, pH 6.8, and 2% DTT. Afterward, strips were briefly rinsed with double distilled water and equilibrated in 6 M urea, 30% glycerol, 50 mM Tris, pH 6.8, and 2.5% 1794

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iodoacetamide for an additional 10 min. The second dimension was carried out in a MiniProtean III electrophoresis apparatus (Bio-Rad) for 7 cm strips and in a 16 × 18 cm gel electrophoresis apparatus for 13 cm strips. Ten or 15% SDS-polyacrylammide gels were used, depending on the experiment. Successively, gels were immunoblotted on nitrocellulose paper (Hybond C+ Extra, Amersham, U.K.). In electrophoresis experiments, Wide Range (MW 6500-20 500) molecular weight marker (Sigma) were used. After Western blot, membranes were stained with Ponceau red to verify the protein transfer and incubated with primary antibodies. Two general antibodies

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Epidermis of Scales in Gecko Lizards Contains β-Keratins

Figure 2. (A) Beta-1 immunofluorescent staining of setae of T. mauritanica near the tip of a pad lamella (dashes underline the epidermis). Bar, 10 µm. (B) Ultrastructural detail of mature oberhautchen-β-layer with spinulae of H. turcicus diffusely immunolabeled with the Beta-universal antibody. Bar, 250 nm. (C) Intense Beta-1 immunolabeling of mature oberhautchen-β-layer at the base of a seta in H. turcicus. Bar, 250 nm. Legends: ob, oberhautchen merged with β-layer; s, spinulae/setae.

against R-keratins were tested for R-keratin detection in gecko molts. One, termed A1, was generously supplied by Drs. L. W. Knapp and R. H. Sawyer, University of South Carolina. It is a concentrated goat antiserum directed toward a range of chick R-keratins.30 It was used at high dilution in buffer since it was very sensitive (1:50000-120 000). The other R-keratin antibody (Pan-cytokeratin-ab, Sigma, diluted 1:2000-5000 dilution in Buffer) is a broad-spectrum antibody against mammalian cytokeratins. For β-keratin detection, the following antibodies were used: Beta-1 (diluted 1:5000), Beta-universal (diluted 1:2000), the A68b lizard β-keratin antiserum (diluted 1:50), FBK

Figure 3. (A) Partial view of pad lamella of H. turcicus featuring the mature (outer) setae and, underneath, the forming (inner) setae. The asterisk indicates an artefactual tissue detachment. Toluidine blue stain. Bar 20 µm. (B) Close-up to forming setae of the inner generation from oberhautchen cells in H. turcicus contacting clear cells of the outer generation. Toluidine blue stain. Bar, 10 µm. (C) Ultrastructural detail of Beta-1 immunogoldlabeled base of an inner seta (arrows) of H. turcicus. Bar, 250 nm. Legends: a, R-layer; ba, basal layer of the epidermis; cl, clear layer; de, dermis; e, epidermis; I, inner scale surface; ib, inner β-cells; iob, inner differentiating oberhautchen cells; is, inner setae generation; ob, oberhautchen merged with β-layer; os, outer scale surface; s, setae; t, scale tip.

(diluted 1:1000) (see above in “Immunocytochemistry” section). The secondary antibodies HRP-conjugated (Sigma) wad diluted 1:1000 in TBS-Tween + 5% non-fat milk powder. Detection was done using the enhanced chemiluminescence procedure developed by Amersham (ECL, Amersham, U.K.).

Results Histology and Cellular Structure of Pad Lamellae. Both species (T. mauritanica and H. turcicus, Figure 1A,B) are skillful and fast moving crawlers on both vertical walls and ceilings (Figure 1A,B). Most of the scales are covered by a corneous layer (Figure 1C) made of a compact β-layer with microornaJournal of Proteome Research • Vol. 6, No. 5, 2007 1795

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Figure 4. Schematic representation of the shedding cycle in gecko epidermis from early renewal (A), to mid-renewal phase (B) and late renewal phase (C), and finally to shedding (C2). A indicates digit pad lamellae shown in longitudinal view (square and A1) and in detailed cytological view with all the different epidermal layers (square and A2). Panel B shows the coexistence of outer and inner generations with their cytological details (square and B1). The granulated clear layer (yellow) sends cytoplasmic projection that surround growing inner setae of the oberhautchen (pale green). The tip of outer setae (enlarging circle) is branched and terminates in the adhesive spatulae. Panel C shows the completion of the renewal phase with the separation of the outer from the inner setae. The latter reach the length of outer setae (cytological details in C1) after which shedding occurs (C2). The molt is lost, and the epidermis enters a post-shedding (resting) phase. 1796

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Figure 5. BD-gels of epidermal proteins separated in 10% polyacrylammide from fresh epidermis of T. mauritanica immunostained for R-keratins (see text for details). (A) Ponceau red stain of body scales and (B) the relative pan-cytokeratin immunostain; (C) ponceau red stain of pad lamellae (with setae) and (D) the relative immunostaining; (E) A1-immunostaining; (F) Pan-cytokeratin immunostaining. Numbers in abscissa indicate pH; those in ordinals indicate molecular weight.

mentation, which is underlain by a less compact R-layer. In the digit pads, the scales are modified into more expanded lamellae where setae are present. Setae vary in length, from a few micrometers at the base of the lamella to long setae (over 20 µm) by the tip of the lamella (Figure 1D). After immunostaining with specific β-keratin antibodies (Beta-1, Beta-universal, or lizard β-keratin) only the β-layer and the oberhautchen with spinulae or setae are immunoreactive (Figure 2). The β-keratin layer of both normal and regenerated scales shows a specific immunolabeling with all three types of antibodies against β-keratins (data not shown). Under the electron microscope, the structure of the epidermis in resting phase of the outer scale surface shows the short spinulae of the oberhautchen layer that are merged with the pale β-layer (Figure 2B,C). The structure of the epidermis in renewal phase shows the presence of the outer and inner setae generations (Figure 3A,B). The forming (inner) setae in pad lamellae are immunoreactive for β-keratin antibodies which specifically labeled their long keratin bundles, oriented along the main axis of setae (Figure 3C). Figure 4 schematically illustrates the cellular events that shape setae during the resting and renewal phases of the epidermis in geckos (the shedding cycle). At the beginning of the renewal phase, an outer generation of setae is present, followed underneath by β-, mesos- and R-layers. The lowermost

part of the R-layer, termed clear layer, often contains granules and interfaces with the small but growing spinules of early differentiating oberhautchen cells, belonging to the inner generation (Figure 4A-A2). At this stage, beneath the oberhautchen, differentiating layers of β-cells of the inner generation are present. In a following stage, spinulae have grown into longer setae that are surrounded by the cytoplasm of the clear layer, and the two epidermal generations are evident (Figures 3A,B and 4B-B1). In a later stage, the two epidermal generations become even more evident, and the setae of the inner generation have further elongated. Finally, toward the final stage of the shedding cycle, the inner setae have grown to almost the same length of those of the outer generation, and they begin the process of detachment from the clear layer of the outer epidermal generation (Figure 4C-C1). This occurs by a process of junctional degradation between the two layers that continues until shedding (Figure 4C2). After the detachment of the molt (cornified outer epidermal generation), the inner setae have the same length than those lost with the molt, and the epidermis enters in resting phase. Beneath the β-layer of the inner generation, an intermediate thin mesos-region made of 2-4 stratified thin cells is present, and is followed by differentiating R-cells (Figure 4). In the still differentiating R-cells present above suprabasal layers, tonofilaJournal of Proteome Research • Vol. 6, No. 5, 2007 1797

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Figure 6. Monodimensional-gels (A-C) and BD-gels at broad pH range (D-H) of epidermal proteins of H. turcicus immunostained with β-keratin antibodies. (A) Ponceau red (Pnc-R) and relative Beta-1 immunostaining (B), and Beta-universal immunostaining (C). BD-gels immunostained with the different indicated antibodies (Beta-1, Beta-universal, and A68b, see text for detail) are shown (D-H). The arrow and arrowheads indicates the more likely protein spots close to the cloned proteins in this species shown in Figure 12 (GE-GPRP4 and GE-GPRP5). Numbers in abscissa indicate pH; those in ordinals indicate molecular weight.

Figure 7. Monodimensional gels (A-D) and BD-gels at broad range (pH 3-10, E-G) of molts of body scales in T. mauritanica. Panel A shows the protein pattern stained by Ponceau Red (Pnc-R). Panels B, E, and F show the pattern using Beta-1 antibody, panels C and G show the immunostaining using Beta-universal antibody, and panel D shows the pattern using FBK antibody.

ments aggregate but they pack into a dense corneous mass only in mature R-cells. 1798

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Immunoblotting at Wide Range of pH for r-Keratins. In specimens of H. turcicus, due to the small size and number of

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Figure 8. BD-gels at broad pH range of fresh epidermis from body scales (A-D, I-J) and pad lamellae (with setae; E-H) of T. mauritanica. Panels A, C, E, and G show the protein pattern stained by Ponceau Red; panels B, D, F, and H show the pattern using the Beta-1 antibody. Panels I and J are panels showing proteins from the same animals immunostained with different antibodies against β-keratins (Beta-universal or A68b, see text for more details). The arrowheads indicate the protein spots closer to the cloned proteins in this species shown in Figure 11 (GE-GPRP 1 and 2 have different polynucleotide sequences but the same protein, see ref 24 and GE-GPRP 3). Numbers in abscissa indicate pH; those in ordinals indicate molecular weight.

available specimens, the electrophoretic study was limited to molts of body scales and pad lamellae pooled together. In the larger specimens, and more numerous samples of T. mauritanica, the analysis was more detailed. This comprises both molts from body scales, molts from pad lamellae (containing

mainly setae), and from fresh epidermis (comprising living and corneous layers) of body scales and from separated pad lamellae. In the epidermis of T. maturitanica, after protein migration in BD-gels and blotting, the Pan-Cytok antibody (Pan-K) or the Journal of Proteome Research • Vol. 6, No. 5, 2007 1799

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Figure 9. BD-gels and Beta-1 immunoblots at restricted pH of molts (A-D), fresh epidermis (E-H) of body scales (A, B, E, and G), and pad lamellae (with setae; C, D, F, and H) in T. mauritanica. Protein patterns stained by Ponceau Red are presented in panels A and C and immunostained by the Beta-1 antibody are presented in panels B, D-H. The arrowheads indicate the protein spots closer to the sequenced proteins in this species (GE-GPRP 1-3; see text for more details). Numbers in abscissa indicate pH; those in ordinals indicate molecular weight.

chick R-keratin antibody (A1) show labeled proteins in the range of R-keratins. In fresh epidermis (including living and corneous layers), most of the protein spots in the R-keratin range react with the Pan-K antibody (Figure 5A,B). Spots at 5356 kDa present pI at 4.2-4.4 or at 5.0-5.2. Another spot of 5960 kDa has pI at 4.3, and another spot at 66-67 kDa has pI at 4.9-5.2. A similar pattern is observed in fresh epidermis of pad lamellae comprising setae (Figure 5C,D). The A1 antibody used on molt proteins T. mauritanica shows unresolved spots at 3840 and 50-62 kDa, with pI at 4.5-5.5 (Figure 5E). The PanCytok antibody on proteins extracted from molts shows similar spots as those observed in fresh epidermis, with three main spots at 45-47, 55-57, and 60 kDa, with pI at 4.8-5.2 (Figure 5F). Cytokeratins, therefore, appear all acidic. Immunoblotting at Wide Range of pH for β-Keratins. In monodimensional gel separation of proteins extracted from body molts of H. turcicus, weak bands are seen in the β-keratin range (Figure 6A). Immunolabeled bands at 13, 14, 16-20, and 26 kDa are observed after immunostaining with Beta-1 antibody (Figure 6B). Bands at 14 and 17 are seen with the Betauniversal antibody (Figure 6C). In BD-gels of molt proteins at broad pH range (3-10), a weak but large area is observed at 55-65 kDa and pI at 4.8-5.5 using Ponceau Red (Figure 6D). The Beta-1 antibody produces spots at 15 kDa with pI 8 (Figure 6E). Other spots at 16-17 kDa show pI at 6.3 and 6.7. Two more spots at 18-19 kDa show pI at 7.6 and 8.6. Using the Beta-universal antibody, spots at 16 kDa with pI at 8.3 (Figure 6G,H) and 6.6 (Figure 6H) are seen. 1800

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Finally, the A68 lizard β-keratin antibody shows specifically labeled spots (not present in the pre-serum, data not shown) at 18-19 kDa with pI at 5-6, at 22-23 kDa with pI at 5, and at 33 kDa with pI at 6.7 and 7.2 (Figure 6F). Among the immunoreactive spots, arrowheads in Figure 6 indicate those spots that correspond to identified protein sequences deduced from molecular biology analysis (see later). In T. mauritanica, after extraction of epidermal proteins from molts of body scales and monodimensional migration (Figure 7A), labeled bands at 16, 20, and 29-30 kDa are seen using the Beta-1 antibody (Figure 7B). Bands at 14, 16-18, 20, 27, 36, and 45 kDa are seen using the Beta-universal antibody (Figure 7C). No bands are seen with the FBK antibody against feather keratin (Figure 7D). In BD-gels at broad pH range (3-10), most proteins of molts are localized at 45-66 kDa with pI 4.8-5.2 and at 16-18 kDa with pI ranging from 6.2 to 9.0 (Figures 7 and 8). The Beta-1 antibody shows mainly spots at 16-18 kDa with pI varying from 9.4 to 5.8 (Figure 7E,F). Other weaker spots are seen at 10-14 kDa with acidic pI (3.5, 4.0, 4.3, and 5.3), and in some samples at 25 kDa with pI at 8.0, and 30 kDa with pI at 6.2-6.8 (Figure 7E). The Beta-universal antibody stains spots at 16-17 kDa and pI at 8.4, 7.5, 6.5, 4.8, and 4.0 (Figure 7G). Also in this species, among immunolabeled protein spots, the arrowheads in Figure 7E,F indicate the spots that correspond to the identified protein sequences deduced from molecular biology analysis (see later). In fresh epidermis of body scales (Figure 8A-D,I,J), and pad lamellae (Figure 8E-H), the BD-protein pattern at broad range

Epidermis of Scales in Gecko Lizards Contains β-Keratins

Figure 10. Schematic drawing summarizing BD-gels patterns with more constant spots found in both molts and fresh body epidermis of H. turcicus (A) and body epidermis (B) and pad lamellae (with setae; C) in T. mauritanica. More common spots are represented, and those indicated by arrows in the basic range are those that should correspond to the deduced sequences derived from molecular biology techniques (GE-GPRP 1-5).

(pH 3-10), stained by Ponceau red, also shows most protein spots in the R-keratin range (40-57 kDa) and in the β-keratin range (16-20 kDa, Figure 8A,C,E,G,I). In body scales, the Beta-1 antibody stains protein spot at 14-17 kDa with pI at 3.9, 4.4, 4.7, 6.4, 6.9, 7.3, and around 8.0 (Figure 8 B). In another sample of body scales (Figure 8 D), a similar Beta-1 immunolabeling pattern is seen, with strong spots at 18 kDa with pI at 7.2 and at 23 kDa with pI at 6.5. Another small protein spots at 21-22 kDa shows pI at 7.8 and 8.6, and a spot at 35 kDa has pI at 6.2 and 6.6. A very weak spot is detected at 18 kDa with a pI around 9 (see Figure 8D). From the general protein pattern obtained in pad lamellae (mainly made of setae) (Figure 8E), immunopositive spots at 15-22 kDa are seen with pI at 5.3, 6.0, 6.8, 7.4, 8.0, and around 9.0 (Figure 8F). Another sample from pad lamellae shows mainly proteins in the R-keratin range (36-68 kDa, Figure 8G), but the Beta-1 antibody stains spots at 14-18 kDa with pI at 3.5, 4.3, 5.0, 6.0, 7.0, 9.2 (Figure 8H). The Beta-universal antibody produces immunolabeled spots at 16-17 kDa, with pI 4.2, 5.5, 6.5, 7.5, and around 9.0 (Figure 8I). Finally, the A68b lizard β-keratin antibody immunostains spots at 13-14 kDa with pI at 4.0, 7.2, and 8.0, and a spot at 22-25 kDa, and pI at 6.0 (Figure 8J). Among the various spots

research articles obtained, arrowheads in Figure 8D,F,H,I indicate the specific protein spots immunolabeled by the latter antibody that correspond to the identified protein sequences deduced from molecular biology analysis (see later). Immunoblotting for β-Keratins at Narrow Range of pH. Other bidimensional-gel analysis are conducted at a more restricted molecular (up to 30 kDa) and pH range (6-11). This allows improved resolution of protein spots in the β-keratin range (10-30 kDa) and eliminated the nonspecific capture of antibodies sometimes observed in the region where R-keratins are abundant (40-70 kDa, pI 4.5-6.0). Proteins extracted from molts of body scales (Figure 9A) produce immunolabeled spots at 13-14 kDa and pI at 9.6 and 10, 16-17 kDa, and pI at 6.8, 7.3, 7.8, and 10, and main spots at 18-20 kDa with pI at 8.8 and 9.2-9.6 using the Beta-1 antibody (Figure 9B). After protein extraction in molts of pad lamellae (mainly constituted by setae; Figure 9C) mainly spots at 18 and 20 kDa with pI at 8.2, 8.8, and 9.0-9.2 are immunolabeled (Figure 9D). In fresh epidermis of body scales (Figure 9E-G) and of pad lamellae (Figure 9F-H) a different Beta-1 immunolabeled pattern from that seen in molts is obtained. Although the number of protein spots changes in different samples, most of the spots represented basic proteins in the 17-20 kDa range and with pI of 8.5-10.5. Among different spots, the arrowheads in Figure 9 indicate the spots that correspond to the identified protein sequences deduced from molecular biology analysis (see later). Finally, the general Beta-1 immunostaining patterns observed in most samples have been schematically summarized in Figure 10. Correlation between Protein Spots and Deduced Proteins from Isolated cDNAs. The deduced proteins presented in Figures 11 and 12 derive from previous data after mRNA extraction, RT-PCR, and RACE analysis of gecko β-keratins (GEGPRP 1-5).24 The predicted secondary structure of the five gecko β-keratins so far sequenced is presented (Figures 11 and 12). This prediction has been obtained using the “PSIPRED Protein Structure Prediction Server” at http://www2.ebi.ac.uk/ clustalw/.31 The latter gives a visual representation of the prediction of the position and extension of R helix regions, random coil regions, and beta-strand regions within the protein. From the epidermis of T. mauritanica, three transcripts were translated into proteins of slightly different length (two of 179 and one of 177 amino acids, GE-GPRP 1-3; Figure 11). From the epidermis of H. turcicus, two proteins were deduced, one made of 191 and another of 169 amino acids (GE-GPRP 4-5, Figure 12). These are proteins of 16-18 kDa with predicted pI from 8.8 to 9.5 as indicated in Figures 11 and 12. The analysis of these proteins shows the general homology of various parts of the protein and in particular the presence of a core region with two strand regions (arrows in the boxes in Figures 11 and 12). The proteins present a chiefly random coil conformation (82-88%), some strand regions (8.0-10.5%), and limited areas of R helix (2.8-8.4%). A core region of 20 amino acids containing five prolines (28% of the prolines in the whole protein), presents two strand β-folds, and the specific analysis of this region has shown a high degree of homology in the corebox (average of 55%) among reptilian β-keratins.23,24 The most representative amino acids of β-keratins from reptilian scales so far sequenced in comparison with an avian scale and feather keratins are presented in Table 1. All reptilian β-keratins are rich in glycine (19-29%) and serine (7-17%), Journal of Proteome Research • Vol. 6, No. 5, 2007 1801

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Figure 11. The molecular weight, pI, and the predicted secondary structure using the PSIPRED-program of three sequenced proteins in the gecko T. mauritanica24 are shown. The square indicated the common motif (core box) where at least two strand regions are present. The percentages of secondary conformation regions (helix, strand, and random coil) are indicated.

expecially in gecko β-keratins. Proline varies from 8 to above 10%, while cysteine is low (2-6%) in β-keratins of reptiles so far sequenced. Cysteine is higher in small proteins (131 amino acids) of the claw in two lizard species (Varanus sp, 13-16%). Also avian scale and feather keratins are glycine-proline-serinerich proteins, although feather keratin contains the lowermost percentage of glycine (11-16% in different types here not reported).

amount of β-keratins increases, as they constitute most of the long setae present in these specialized scales. The abundance of proteins in the R-keratin range is in part responsible for the nonspecific capture of the antibodies in this range. The presence of epitopes rich in glycine-sequences in the variable regions of R-keratins may also cross-react with antibodies directed against β-keratins. Finally, another possibility is the presence of some β-keratin polymers in the range of 40-70 kDa that are still associated with R-keratins.

Discussion and Conclusion

R-Keratins of gecko epidermis, as in the epidermis of other reptiles, appear mainly at 55-66 kDa and are of acidic type (pI in the 4.5-6.0 range). The amino acidic sequence of R-keratins and the modality of their association with β-keratins remain to be analyzed.

Proteins of Scales and Setae. The protein patterns found in the present study show protein spots immunoreactive for β-keratins that correspond to those cloned24 but also other protein spots recognized as β-keratins of unknown nature (mainly, neutral or acidic). This indicates that aside from basic β-keratins of known amino acid sequences, other types of β-keratin may compose the epidermis and setae of geckos. The present study shows that some proteins extracted from gecko epidermis are recognized by the three antibodies for β-keratins (Beta-1, Beta-universal, and A68b), but not by the feather-keratin antibody. Generally, most extracted proteins from molts and fresh epidermis are localized within the molecular weight of R-keratins (40-70 kDa) and β-keratins (1025 kDa). Molts from scales in geckos are composed by a thin β-layer and a thicker R-layer. R-Keratins are present in both layers, although they decrease or are masked by β-keratins in mature β-layers.15,16,32 In the specialized pad lamellae, the 1802

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The present study has shown that proteins within the β-keratin range comprise spots at acidic, neutral, and basic range. The latter are those with molecular weight and pI closer to that of the two sequenced glycine-proline-rich proteins of both T. mauritanica and H. turcicus (compare the arrowed spots in bidimensional gels in Figures 6-10 with the values of molecular weight and pI of the deduced sequences in Figures 11 and 12). A specific immunoreactivity is observed, especially using the Beta-1 antibody, in the low range corresponding to that for β-keratins.15,21,27,32 The study supports previous electrophoretic analysis on β-keratins from different reptiles (turtles, tortoises, crocodilians, sphenodontids, snakes, and other lizards), which

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Epidermis of Scales in Gecko Lizards Contains β-Keratins

Figure 12. The molecular weight, pI, and the predicted secondary structure using the PSIPRED-program of three sequenced proteins in the gecko H. turcicus24 are shown. The square indicated the common motive (core box) where strand regions are present. The percentages of secondary conformation regions (helix, strand, and random coil) are indicated.

suggested that basic β-keratins may combine with acidic R-keratins to form dense β-keratin filaments.15,32 β-Keratins of the corneous layer of gecko scales or pad lamellae have molecular weight ranging between 14 and 19 kDa, and some of them are basic proteins. This further suggests that neutral or basic “β-keratins” have a role as matrix proteins and associate with cytokeratins to form the hard (β) corneous layer of reptilian epidermis. β and oberhautchen layers, and the setae of digit pads, are made of weakly elastic but resistant proteins. The present results suggest that some of the proteins of 10-16 kDa form long, axially oriented filaments of β-keratin for the growth of setae, as feather keratins are needed for the elongation of barb/ barbules in feathers.19,20,33 These microfibrils of β-keratins support cell elongation and possess a high resistance coupled with the pliability and deformation necessary to sustain climbing, gliding or flying. The present study has also confirmed that different R- and β-keratins compose the corneous layers (represented by the molts) of scales and pad lamellae of geckos. Immunocytochemistry, however, indicates that the oberhautchen and β-layer contain essentially β-keratins or that R-keratins are absent, degraded, or masked by the massive deposition of β-keratin.15,16 Previous biochemical studies showed the pres-

Table 1. Percentage of Five Characteristic Amino Acids in Known Sequences of Reptilian β-Keratins (from Scales Except the Protein from the Claw in Varanus gouldii) and from Avian β-Keratins % amino acids species

cysteine tyrosine serine proline glycine references

Podarcis sicula (wall lizard, scale) Tarentola mauritanica (mediterranean gecko, scale) Hemidactylus turcicus (small geko, scale) Varanus gouldii (goanna claw) Varanus varanus (goanna claw) Elaphe guttata (corn snake, scale) Gallus gallus (scutate scale) Gallus gallus (feather)

4.3

4.9

7.4

8.0

29.4

22

2.2

2.2

17.3

10.6

20.7

24

2.6

2.6

16.2

8.4

23.0

24

16.0

2.1

9.0

8.0

29.0

35

13.1

5.6

6.3

10.2

27.6

41

5.8

1.5

10.2

12.4

19.0

5.2

12.3

16.1

9.7

23.2

Dalla Valle et al., (unpublished) 42

9.2

1.0

15.3

11.2

11.3

42

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Table 2. Predicted Phosphorylation Sites of Gecko Cloned Sequence24 Performed by NetPhos 2.0 Server40 at http://www.cbs.dtu.dk/services/NetPhos/a predicted phosphorylation sites serine gecko β-keratin

GE-GPRP 1-2 GE-GP 3 GE-GP 4 GE-GP 5

threonine

tyrosine

sites average sites average sites average number score number score number score

4 5 7 7

0.886 0.839 0.904 0.836

1 2 1 1

0.963 0.916 0.559 0.963

2 1 1 0

0.891 0.890 0.759 0.000

a Number of predicted phosphorylation sites and average score are reported for serine, threonine, and tyrosine. The score indicates the probability of phosphorilation.40

Table 3. Predicted Molecular Weight and Isoelectric Point Variation of GE-GPRP1-2 Regarding the Number of Putative Phosphorylation of the Protein Performed by Using “Scansite Molecular Weight & Isoelectric Point Calculator” at http://scansite.mit.edu/cgi-bin/calcpi no. of putative phosphates

molecular weight (Da)

isoelectric point

0 1 2 3 4 5 6 7 8

16793 16871 16949 17027 17105 17183 17261 17339 17417

8.86 7.86 6.82 5.65 4.53 3.86 3.23 2.81 2.56

ence of high sulfur and low sulfur components in gecko scales.14 The high sulfur components showed molecular weight at 16, 18, and 20 kDa, but represents a different protein components of setae from the sequenced glycine-proline-serine-rich proteins, as the latter contain low level of cysteine (Table 1). Conversely, the sequenced proteins may correspond to some of the 15-20 kDa protein bands within the low-sulfur fraction found in the above studies. The identified basic β-keratins are the only proteins of reptilian epidermis so far detected which correspond to the proteins which have been sequenced by RACE-analysis.22,24 As for avian β-keratins,34 it is possible that neutral or acidic β-keratins with the same molecular weight are processed or degraded, or may derive from post-translational phosphorylation modifications (phosphorylation lowers the pI) of initially basic proteins. The gecko proteins sequenced show the presence of several predicted phosphorylation sites (Table 2) that could explain the shift of the Beta-1 immunolabeled spot pI toward neutral and basic range (Table 3). A phosphorylation process occurs for some keratins of avian epidermis.21,34 The presence of β-keratins rich in sulfur and with acidic pI, like that of a lizard claw (13.1 kDa and calculated pI at 5.6),35 might also explain some of the spots with neutral or acidic pI. The latter point remains to be further analyzed, but so far, no neutral or acidic scale β-keratin has been sequenced. Structural and Comparative Analysis of Gecko β-Keratins. The deduced amino acid sequence of the proteins in T. mauritanica (Figure 11) and H. turcicus (Figure 12) shows that they are slightly longer (169-191 amino acids) than the scale (β-) keratins recently isolated and sequenced in the lizard Podarcis sicula (163 amino acids).22,24 The presence of more 1804

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copies of genes for β-keratins allow the rapid production of high amounts of different varieties of glycine-proline-rich proteins in scales and in pad lamellae of geckos during the renewal phase of the epidermis. Despite the number of amino acids, glycine represents above 19% of the amino acids in all β-keratins so far sequenced, while proline and serine are both present over 7-8% (Table 1). These proteins present three main regions: two glycine-rich regions, respectively, toward the N- and C-termini, and a proline-rich central region. The predicted secondary structure has shown that most of the glycine-rich regions possess a random coil conformation and show little homology with mammalian “glycine-tyrosine-rich keratin associated proteins.”23,35 The central region (boxed in Figures 11 and 12) has high homology (60-80%) with the core region of avian β-keratins, and this detail indicates that a common precursor for β-keratins was probably present in stem reptiles from which reptiles and birds derived. From the ancestral proteins, a diversification probably occurred in the evolutionary lineages of snakes, lizards, turtles, crocodilians, and birds. The core-box present in β-keratins is probably responsible for the polymerization of β-keratin monomers to form long and resistant β-keratin filaments.20,33 The core-box also allows the folding of the molecules and may be responsible for the appearing of a β-(feather-like) keratin pattern in reptilian corneous layer as indicated by X-ray analysis.20,36,37 Two to three potential strand regions are predicted for these proteins, and in the longer protein of H. turcicus, a fourth strand region might allow a further folding of the protein (Figure 12). Chemical-physical studies are required to understand the three-dimensional structure and aggregation of these glycine-proline-serine-rich proteins to form β-keratin filaments inside the setae. While the core region contains proline and is hydrophobic, serine and arginine (hydrophilic and polar amino acids) are mainly present in lateral regions of β-keratins and can interact with ions and possibly water of hydration. The state of hydration of β-keratins or of other proteins present in setae is unknown, but water may influence the stiffness and resistance of β-keratins, and can affect the adhesive properties of setae.9,10 The latter studies have indicated an increase in adhesion at moderate degree of humidity in the environment. It is known that water decreases the stiffness and stress resistance of keratin material in feathers.11,12 Apparently, small, hydrophobic, and resistant glycine-serineproline-rich proteins have been selected in scales of both archosaurians (crocodiles and birds) and lepidosaurians (lizards and snakes) to build branching microornamentation that allow flying (birds) or climbing (geckos). It appears that reptilian β-keratins have some of the properties of keratin-associated proteins in mammalians, and functionally, they may represent the reptilian counterpart of glycine-tyrosine-rich proteins present in mammalian cornified derivatives such as hairs, nails, horns, and so forth.38,39

Acknowledgment. The A1, Beta-1, and Beta-universal antibodies were generously supplied by Dr. R. H. Sawyer, Biological Science Department, University of South Carolina, Columbia, SC. Study mainly supported by a University of Bologna Grant (60%) and by self-support. Thanks to Prof. V. Tomasi (Department of Biology, University of Bologna) for making available the Ettan IPGphor 3 IEF System (GE Healthcare, Sweden) for bidimensional electrophoresis.

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Epidermis of Scales in Gecko Lizards Contains β-Keratins

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(23) Alibardi, L.; Dalla Valle, L.; Toffolo, V.; Toni, M. Scale keratin in lizard epidermis reveals amino acid regions homologous with avian and mammalian epidermal proteins. Anat. Rec., Part A 2006, 288, 734-52. (24) Dalla Valle, L.; Nardi, A.; Toffolo, V.; Niero, C.; Toni, M.; Alibardi, L. Cloning and characterization of scale β-keratins in the differentiating epidermis of geckos show they are glycine-prolineserine-rich proteins with a central motif homologous to avian beta-keratins. Dev. Dyn. 2007, 236, 374-88. (25) Scala, C.; Cenacchi, G.; Ferrari, C.; Pasquinelli, G.; Preda, P.; Manara, G. C. A new acrylic resin formulation: a useful tool for histological, ultrastructural, and immunocytochemical investigations. J. Histochem. Cytochem. 1992, 40, 1799-804. (26) Alibardi, L.; Toni, M. Immunological characterization and fine localization of a lizard beta-keratin. J. Exp. Zool., Part B 2006, 306, 528-38. (27) Sawyer, R. H.; Glenn, T. C.; French, J. O.; Knapp, L. W. Developing antibodies to synthetic peptides based on comparative DNA sequencing of multigene families. Methods Enzymol. 2005, 395, 636-52. (28) Sybert, V. P.; Dale, B. A.; Holbrook, K. A. Ichthyosis vulgaris: identification of a defect in synthesis of filaggrin correlated with an absence of keratohyaline granules. J. Invest. Dermatol. 1985, 84, 191-4. (29) Laemmli, U. K. Cleavage of structural proteins during the assemblage of the head of the bacteriophage T4. Nature 1970, 227, 680-85. (30) O’Guin, W. M.; Sawyer, R. H. Avian scale development. VII. Relationships between morphogenetic and biosynthetic differentiation. Dev. Biol. 1982, 89, 485-92. (31) McGuffin, L. The PSIPRED protein structure prediction server. Bioinformatics 2000, 16, 404-5. (32) Alibardi, L.; Toni, M. Cytochemical, biochemical and molecular aspects of the process of keratinization in the epidermis of reptilian scales. Prog. Histochem. Cytochem. 2006, 40, 73-134. (33) Brush, A. H. The origin of feather: a novel approach. In Avian Biology; Farner, D., Kling, J., Parker, K., Eds.; Academic Press: New York, 1993; pp 121-62. (34) Knapp, L. W.; Shames, R. B.; Barnes, G. L.; Sawyer, R. H. Regionspecific patterns of beta keratin expression during avian skin development. Dev. Dyn. 1993, 196, 283-90. (35) Inglis, A.; Gillespie, J. M.; Roxburgh, C.; Whittaker, L.; Casagranda, F. Sequence of a glycine-rich protein from lizard claw: unusual dilute acid and heptafluorobutyric acid cleavage. In Protein, Structure and Function; L’Italien, Ed.; Plenum Press: New YorkLondon, 1987; pp 757-64. (36) Baden, H. P.; Maderson, P. F. Morphological and biophysical identification of fibrous proteins in the amniote epidermis. J. Exp. Zool. 1970, 174, 225-32. (37) Baden, H.; Sviokla, S.; Roth, I. The structural protein of reptilian scales. J. Exp. Zool. 1974, 187, 287-94. (38) Gillespie, J. M. The structural proteins of hair: isolation, characterization and regulation of biosynthesis. In Physiology, Biochemistry and Molecular Biology of the Skin; Goldsmith, L., Ed.; Oxford University Press: Oxford, U.K., 1991; pp 625-59. (39) Powell, B.; Rogers, G. Differentiation in hard keratin tissues: hair and related structure. In The Keratinocyte Handbook; Leigh, I., Lane, B., Watt, F., Eds.; Cambridge University Press, Cambridge, U.K., 1994; pp 401-36. (40) Blom, N.; Gammeltoft, S.; Brunak, S. Sequence and structurebased prediction of eukaryotic protein phosphorylation sites. J. Mol. Biol. 1999, 294, 1351-62. (41) Frenkel, M. J.; Gillespie, J. M. The proteins of the keratin component of bird’s beaks. Aust. J. Biol. Sci. 1976, 29, 467-79. (42) Gregg, K.; Wilton, S.; Parry, D. A.; Rogers, G. A comparison of genomic coding sequences for feather and scale keratins: structural and evolutionary implications. EMBO J. 1984, 3, 175-78.

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