REVIEW Evolution of hard proteins in the sauropsid ... · arrowhead) and the darker neck epidermis...

J. Anat.

(2009)

214

, pp560–586 doi: 10.1111/j.1469-7580.2009.01045.x

© 2009 The AuthorsJournal compilation © 2009 Anatomical Society of Great Britain and Ireland

Blackwell Publishing Ltd

REVIEW

Evolution of hard proteins in the sauropsid integument in relation to the cornification of skin derivatives in amniotes

Lorenzo Alibardi,

1

Luisa Dalla Valle,

2

Alessia Nardi

2

and Mattia Toni

1

1

Dipartimento di Biologia evoluzionistica sperimentale, University of Bologna, Italy

2

Dipartimento di Biologia, University of Padova, Italy

Abstract

Hard skin appendages in amniotes comprise scales, feathers and hairs. The cell organization of these appendagesprobably derived from the localization of specialized areas of dermal–epidermal interaction in the integument.The horny scales and the other derivatives were formed from large areas of dermal–epidermal interaction. Theevolution of these skin appendages was characterized by the production of specific coiled-coil keratins andassociated proteins in the inter-filament matrix. Unlike mammalian keratin-associated proteins, those of sauropsidscontain a double beta-folded sequence of about 20 amino acids, known as the core-box. The core-box shows 60%–95% sequence identity with known reptilian and avian proteins. The core-box determines the polymerization ofthese proteins into filaments indicated as beta-keratin filaments. The nucleotide and derived amino acid sequencesfor these sauropsid keratin-associated proteins are presented in conjunction with a hypothesis about their evolutionin reptiles-birds compared to mammalian keratin-associated proteins. It is suggested that genes coding for ancestralglycine-serine-rich sequences of alpha-keratins produced a new class of small matrix proteins. In sauropsids, matrixproteins may have originated after mutation and enrichment in proline, probably in a central region of the ancestralprotein. This mutation gave rise to the core-box, and other regions of the original protein evolved differently inthe various reptilians orders. In lepidosaurians, two main groups, the high glycine proline and the high cysteineproline proteins, were formed. In archosaurians and chelonians two main groups later diversified into the highglycine proline tyrosine, non-feather proteins, and into the glycine-tyrosine-poor group of feather proteins, whichevolved in birds. The latter proteins were particularly suited for making the elongated barb/barbule cells of feathers. Intherapsids-mammals, mutations of the ancestral proteins formed the high glycine-tyrosine or the high cysteineproteins but no core-box was produced in the matrix proteins of the hard corneous material of mammalianderivatives.

Key words

corneous proteins; epidermis; evolution; genes; reptiles; sequencing.

Scales of extant reptiles in comparison with feathers and hairs

Scales in extant reptiles are specialized structures

Most living reptiles are covered by scales of different size,thickness, variety and color, and these characterize differentspecies (Maderson, 1985; Landmann, 1986; Maderson et al.1998; Alibardi, 2003, 2006c) (Fig. 1A–D). Details on themorphology and cytology of reptilian integument have

been reported in the above references, and will not bedealt with in the present review. The main goal of thisreview is to report the latest data on the present state-of-the-art in the study of the characteristics of keratinizationin reptilian epidermis. The present account represents afurther step forward from recently published reviews onthis rapidly growing topic (Alibardi & Toni, 2006a; Toniet al. 2007b).

In general terms, from the basic anapsid cotylosaurs anew form of corneous proteins, beta-keratins, was producedfrom ancestral proteins. The new proteins mainly con-tributed to the formation of a hard and mechanicallyresistant corneous layer. The production of these proteinsincreased the strength of the epidermis but a thick and hardbeta-keratinized layer impeded skin elasticity, pliability,and sensitivity in the derived reptiles.

Correspondence

Dr L. Alibardi, Dipartimento di Biologia evoluzionistica sperimentale, University of Bologna, Italy. E: [email protected]

Accepted for publication

9 December 2008

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Hard epidermal proteins in reptiles, L. Alibardi et al.

© 2009 The Authors Journal compilation © 2009 Anatomical Society of Great Britain and Ireland

561

Beta-keratins are produced in the epidermis of mostturtles and crocodilians, and in feathers, scales, claw andbeak of birds, whereas they tend to disappear in somelayers of the epidermis of lizards and snakes, where mainly

soft alpha-keratin remains. The evolution of modernreptilian scales from primitive reptiles with an extensivedermaskeleton took place by a specialization of the cornifi-cation process. Microscopy of scales in different species

Fig. 1 Macroscopic aspect of reptilian scales (A–D) and histology of the epidermis in scales of different reptiles (E–M). (A) Overlapped trunk scale of snake (Natrix natrix). Bar, 0.5 mm. (B) Little overlapping scale of ventral region of midtrunk region of the tuatara (Sphenodon punctatus). Bar, 0.5 mm. (C) Large dorsal scales (arrow) and lateral small scales (arrowhead) of the alligator (Alligator mississippiensis). Bar, 1 mm. (D) Plastron scale (gular, arrowhead) and the darker neck epidermis (arrow) of the turtle Emydura macquarii. The latter shows a rough surface with a scale-like pattern, represented by a rough skin. Bar, 1 mm. (E) Epidermis of the snake N. natrix showing differentiating fusiform beta-cells beneath the shedding line (arrow). Bar, 15 μm. (F) Normal epidermis (resting) of the lizard Podarcis sicula showing the outer beta-layer and the complete alpha-layer. Bar, 15 μm. (G) Epidermis of S. punctatus in resting phase showing the outer beta-layer and inner alpha layer (arrows). Bar, 15 μm. (H) Stratified wound epidermis of S. punctatus showing a thick corneous alpha-layer. Bar, 10 μm. (I) Epidermis of ventral scale in saltwater crocodile (Crocodylus porosus) showing flat cells in the transitional layer before the pale, lower part of the corneous layer. Bar, 15 μm. (J) Tail scale of the turtle Chrysemys picta showing thymidine-labeled nuclei (arrows) 1 day after injection and autoradiography. The arrowhead indicates the transitional layer of the epidermis in the outer scale surface. Bar, 20 μm. (K) Tip of a plastron scute of C. picta showing the shedding line (arrows) along which the outer part of the corneous layer will detach. Bar, 20 μm. (L) Tip of plastron scute of C. picta 3 days post-injection of tritiated histidine. Most autoradiographic labeling (arrows) is present in cells of the intermediate and transitional layer beneath the thick corneous layer. Bar, 20 μm. (M) Autoradiographic detail of the intense silver grains localized in the transitional layer of the carapace 1 day after injection of tritiated histidine in C. picta. Bar, 10 μm. a, alpha-layer; ba, basal layer; be, beta-layer; c, corneous layer; ci, inner corneous layer; co, outer corneous layer; de, dermis; e, epidermis; h, hinge region; in, intermediate layer beneath the wound epidermis; pc, pre-corneous or transitional layer; sb, suprabasal layer; t, tip of scute; tr, transitional layer; w, wound (regenerating) epidermis. Dashes underline the basal layer of the epidermis.



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of reptiles is presented in Fig. 1(E–M), and gives a directview of their histological structure and layer stratification.Recent immunological and molecular studies suggest thatthe reduction in the production of these proteins may beassociated with the evolution in lepidosaurian reptiles ofa mechanism of shedding, which permits the cyclical loss ofthe outer part of the corneous layer (Alibardi, 2006c;Alibardi & Toni, 2006a). Lepidosaurians show a multilayeredepidermis whose external portion is cyclically shed (Maderson,1985; Maderson et al. 1998).

Recent immunocytochemical, biochemical and molecularbiology studies have suggested that the cytologicaldifferences among the six main layers of lepidosaurianepidermis (oberhautchen, beta-, mesos-, alpha-, lacunar andclear-layers; some layers are shown in Fig. 1E,F) are derivedfrom the genetic control of the production of their hardproteins during the shedding cycle (Alibardi & Toni, 2006a–d;Alibardi et al. 2007; Toni et al. 2007a,b). The molecularcontrol of the activation or repression of genes for thesynthesis of these proteins still remains to be discovered.

In the tuatara (

Sphenodon punctatus

), a primitive lepi-dosaurian, shedding of the outermost generation occursthrough the formation of an intermediate region wherecells possess mixed alpha and beta characteristics, a precursorof the more specialized shedding layer of lizards and snakes(Fig. 1G–H). The loss of the outer corneous layer occurs inthis area, under which a new beta-layer is formed. Finally,in squamates the shedding mechanism reaches perfection:a single layer, the oberhautchen layer, is adjacent to thelast alpha-layer of the previous epidermal generation, theclear layer. These two layers form a specialized sheddingcomplex which separates along a shedding line (Fig. 1E).The synchronization of cell differentiation of the epidermisover discrete areas of the skin surface in lizards, or over theentire surface of the skin in snakes, produces a patchymolting in lizards and a single molt in snakes. After shedding,the epidermis is covered by a mature beta-layer and a moreor less mature alpha-layer (Fig. 1F).

In crocodilians and turtles a shedding mechanism in theepidermis is absent or limited, and epidermal histologyand the control on the production of hard-proteins appearsimpler than in lepidosaurians. Crocodilian epidermis

comprises a multilayered epithelium with a transitionallayer of flat cells that are incorporated into a variably thickcorneous layer (depending on the type of scales) (Fig. 1I).In archosaurians (crocodilians and birds), the process ofshedding of epidermal layers is not specifically known.However, the presence of a scission layer in avian scales(Cane & Spearman, 1967), made by thinner and lipid-richcorneocytes, suggests that archosaurians may also use thismechanism of a switch in cell differentiation to create ascission layer. A different type of sloughing layer is alsoformed in regenerating feathers and underneath thesheath before it is lost to reveal the barb ramification.

In turtles, hard scutes are present in the plastron and softerscutes or simple folds in the remaining body regions, witha corneous layer made of soft-keratin (Fig. 1J). The cellproliferation in the basal layer and the intense proteinsynthesis of transitional cells near the hinge regions of thegrowing scutes layer allow the growth of scutes (Fig. 1J–M).

In the scutes of the carapace and plastron of some speciesof turtles (chelonians), the switch from thicker beta- intothinner alpha-keratin cells determines the formation of ashedding layer. The latter permits a slow intra-corneousdetachment of the more external layers with the sloughingof the superficial part from the more internal part of thecorneous layer (Fig. 1K). A new beta-layer allows for thenext stratification of the stratum corneum; this processoccurs in shedding turtle but not in non-shedding turtle orin the tortoises where the superficial part of the corneouslayer is gradually lost by natural wear.

Scale development and epidermal layers in reptiles and birds

Scales in reptiles derive from the interaction between a likelyinductive mesenchyme and a large surface of the epidermis(Fig. 2). Extensive reviews of studies on the embryology inreptilian skin have been presented in previous accounts,and the reader can find further information in thesepublications (Maderson, 1985; Alibardi, 2003, 2004b, 2005).It has been hypothesized that areas of dermal–epidermalinteractions (ADEIs) are relatively large in reptiles, includingpre-avian archosaurians (Fig. 2). These interactions are

Fig. 2 Drawing illustrating the embryonic layers formed on the scale of embryonic crocodilians (A) in comparison with those of bird embryos (B). The arrowed square illustrates the sequence of layers in crocodilian scale (A1) vs. avian beak (C), downfeather (D), claw (E), and scutate scales (F). The same colors indicate homologous layers, including those of downfeathers after the cell displacement within the barb ridge (see details in the text). The arrows pointing down in downfeathers indicate the direction of formation of barb ridges. Whereas feather-keratin form oriented and parallel bundles (arrow to D6), the other beta-keratins form an irregular orientation (arrows to A7, E3, F3. a, alpha (intermediate) layer; ADEI, areas of dermal–epidermal interaction; (B) Definitive (post-hatching and adult) beta-keratin layer containing different beta-keratins in scales, claws, beak and feather (colored with different colors); BA, barb (ramus); BL, barbule cells (equivalent to subperiderm cells); BR, barb ridges; BBK, adult beak beta-keratin with specific epitopes in black; CBK, adult claw beta-keratin with specific epitopes in black; CCBK, crocodilian claw beta-keratin; CSBK, crocodilian scale beta-keratin; CY, cylindrical cells; FBK, feather beta-keratin; FF, forming follicle; G, germinal layer; GFG, growing feather germ; HIS, tritiated histidine autoradiography; IP, inner periderm; OP, outer periderm; RB/PG, reticulate or periderm granules of the periderm; S, sheath (supportive cells corresponding to external layers derived from inner periderm cells); SP, subperiderm (containing the feather-keratin epitope, FBK); SBK, adult scale beta-keratin with specific epitopes in black; T, transitional layer along which the embryonic epidermis is shed around hatching (A3). THY, tritiated thymidine autoradiography; V, barb vane ridge cells of the axial plate of barb ridges (supportive cells equivalent to inner layers derived from inner periderm cells).



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directly or indirectly responsible for the activation ofseveral genes in the epidermis, including those coding forthe production of beta-keratins. Beneath an embryonicepidermis made of two to six layers, depending on thereptilian species, the accumulation of beta-keratins deter-mines sloughing at hatching (Fig. 2A-A7). Among reptilesthe crocodilians are particularly interesting for their phylo-genetic affinity with birds, and the analysis of their embryonicepidermis reveals the highest sequence identity with thatof birds. In the latter, considered here to be a lineage ofarchosaurian reptiles, a special morphogenetic process hasevolved for the production of feathers from embryonicskin (Maderson, 1970; Brush, 1993; Sawyer et al. 2005b;Alibardi et al. 2006b). The process has determined theselection of small proteins the size of feather-keratins,which have been utilized for the formation of barbs, asshown in Fig. 2.

Recent studies on the embryonic epidermis of alligatorand birds have produced further indications that feathersmay derive from a generalized archosaurian epidermis(Sawyer & Knapp, 2003; Sawyer et al. 2005b; Alibardi, 2006b;Fig. 2A–F). During development, different layers with aspecific fate are produced in sequence in the epidermis.These studies have suggested that specific cell populationswere selected from the generalized archosaurian epidermisto produce specific epidermal structures such as scales, beaksor claws, and for the formation of feathers. In particular,some cell populations containing a specific type of feather-like keratin molecule were selected among others toproduce barb and barbule cells and eventually feathers.

In the alligator and in the avian embryonic epidermis,outer and inner periderm layers are initially formed andcontain similar periderm granules (Fig. 2B–F). Over most ofthe body, especially in inter-follicular, apteric and scale skin,only one layer of inner periderm cells is present (Sawyer &Knapp, 2003) (Fig. 2F). In the claw, two to four layers of innerperiderm cells are instead present, whereas this increasesto six to eight layers in the beak (Kingsbury et al. 1953;Alibardi, 2002, and personal observations). Beneath theinner periderm, one subperiderm layer is present whichcontains feather-keratin immunoreactivity (Fig. 2C,E). It hasbeen suggested that the displacement of the subperidermlayer into barbule plates and a ramus area eventuallygenerated feathers (Fig. 3D). The subperiderm layer in allarchosaurian epidermis contains a 24-amino acid feather-keratin epitope (AVGSTTSAAVGSILSEEGVPINSG) thatdisappears at hatching except in feathers (Sawyer et al.2005a,b). Therefore this epitope may be an ancient com-ponent of the epidermis of archosaurian reptiles.

Feathers as a skin specialization of archosaurian epidermis

It is still believed that feathers might be derived fromarchosaurian scales (Spearman, 1966; Maderson, 1972;

Maderson & Alibardi, 2000), probably by a drastic alterationof the morphogenetic pattern of dermo-epidermal inter-actions. Reptilian scales are either of the scute type, as inextant crocodilians and chelonian shell, or appear as tuber-culate or pebble-like scales, such as those described infossilized archosaurians, including dinosaurs (Martin &Czerkas, 2000; Prum & Brush, 2002). It is believed thatduring the evolution of the avian skin the rigid corneouslayer of archosaurian scales progressively became restrictedto smaller and smaller areas of ADEIs, while most of theskin became non-specialized and softer, and was the originof the apteric skin (Alibardi, 2004b, 2005). The flat scale inarchosaurian ancestors became progressively narrower,tuberculate and the epidermis was nourished from avascularized mesenchyme, a condition that resembles thatof feather filaments (Fig. 3A–C). The latter also exchangedmorphoregulative or inductive signals with the epidermis,and this activity determined the formation of specializedlayers of cells in both scales and feathers.

The fossil record of feathers is relatively abundant,already showing the typical contour feather morphology(Prum & Brush, 2002; Wu et al. 2004). Simpler forms orfiliform appendages are interpreted as both protofeathers(summarized in Prum & Brush, 2002; Wu et al. 2004) orcollagenous structures (Lingham-Soliar et al. 2007). Thedetailed cytological analysis of the process of feathermorphogenesis has, however, indicated that some fossilized‘protofeathers’ may be genuine remnants of barbs withdifferent degrees of branching (Alibardi, 2006a,b,c).

According to the latter studies, the hairy-like elongationwas hollow, and when epithelial folds appeared in theepidermis of these hypothesized filaments, the formationof feathers began. The first stage was the formation of barbridges that could evolve into different morphogeneticmechanisms to produce barbs with extensive ramification(Fig. 3E-E3), short ramification (Fig. 3F-F3) or without anyramification (Fig. 3G-G3) (Alibardi, 2006a,b). Similarmorphotypes of downfeathers have been found in fossilizedfeathers or filamentous structures (summarized in Prum &Brush, 2002; Wu et al. 2004).

The above hypothetical stages are supported by mor-phological and biochemical data. Reptilian and avian scalesand feathers are mainly composed of scale and feather-keratins, and feathers can develop from scales under somenatural or experimental circumstances (Dhouailly & Senegel,1983; Chuong, 1993; Chuong & Widelitz, 1999; Sawyer et al.2005b). The successive fusion of barb ridges into an axialrachis gave rise to pennaceous feathers of different shapesand sizes (Fig. 3H-H3).

Hairs as a skin specialization of theropsid reptiles

It is believed that the evolution of hairs from basic reptilesresulted from the reduction of a primordial, scaled skininto a hairy and softer skin (Maderson et al. 1972; Alibardi,



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Fig. 3 Hypothetical evolution of feathers and their keratins from reptilian scales (A), through coniform scales (B) and a long coniform proto-feather (C). The latter contained a specific feather-keratin box (FK-box) together with the ancestral core-box. The glycine-rich box (GG-box) was lost from B to C. The formation of barb ridges (D) shows the inside view of the base of a feather filament and the different cell displacement (E–G) gives rise to three different branching phenotypes (see text for explanation). Finally, the fusion of barb ridges with the rachidial ridge gives rise to pennaceous feathers (H1, contour feathers; H2, bristles; H3, filoplume). AX, axial plate; BA, barbula; BL, barbule; BR, barb ridge; CO, collar; NA, no axial plate; RA, ramus; RM, ramogenic zone; RR, rachidial ridge; SA, short axial plate.



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2003; Wu et al. 2004). Also in this case, it is hypothesizedthat the initial expanded ADEIs of reptilian scales becameprogressively confined to small areas, giving rise todermal papillae and the formation of hairs among scales,as in the tails of rodents (Alibardi, 2004a,b,c; Fig. 4A-A3).The above references give more information on thepossible evolution of hairs, which cannot be consideredhere. Unfortunately, while some feathers and corneousfilaments remain in fossils, no fossilized hairs have sofar been reported in the paleontological literature andtherefore we do not have any skin evidence about hairevolution. Also the developmental stages of hair develop-ment provide few clues about the evolutive transitionfrom a scale to a hair as the two structures are very different(Fig. 4B-B7).

Hairs develop from an invagination of an epidermalplacode in contact with a special mesenchymal condensationto form a peg that grows downward into an elongated, club-like epidermal tongue (see stages of hair morphogenesis,Philpot & Paus, 1999). From the neck of the elongatedpeg some cells become organized in a cone-like layer thatgrows upward and initiates the hair shaft that growstoward the epidermal surface (Fig. 4B5–6). In the meantime,the mesenchyme at the bottom of the club (the future hairbulb) penetrates into a small cavity that is formed at thebase of the club, and forms the dermal papilla (Fig. 4B5–B7).A classic study on developing hairs in monotremes (Poulton,1894) showed that their hair canal may develop as an opentube, a study that, if confirmed, would provide someevidence of a process of invagination (see references inAlibardi, 2004a,b).

Other hard skin derivatives present in mammals arerepresented by nails, quills, claws, horns, hooves, corneousteeth in whales, etc., all considered to incorporate hardkeratins (Gillespie, 1991). Their embryological origin ispartially known but all of them appear to have an intimatedermal–epidermal connection which directly or indirectlyinduces epidermal cells to produce ‘hard keratins’ (Bragulla& Hirschberg, 2003).

Corneous proteins of hairs, scales and feathers: alpha-keratin vs. keratin-associated proteins of mammals, birds and reptiles

General features of corneous proteins in mammalian epidermis and its derivatives

In the last 25 years the concept of mammalian hard keratinshas been more precisely defined. Today, two main com-ponents are recognized in the cells (trichocytes) of mam-malian hard corneous derivatives (hairs, nails, horns), a fibrouscomponent and a matrix or inter-filament component. Thefibrous component consists of a specific set of alpha-keratinproteins, composed of the keratin intermediate filaments(KIFs), with molecular weights of 40–70 kDa. These keratinshave been sequenced from their genes cloned from humanand mouse (Langbein et al. 1999, 2001; Langbein & Schweizer,2005), and are classified into two types based on theirsequence homology, namely 11 type I (acidic) and ninetype II (basic). The spatial-temporal sequence of expressionof these hair keratins varies at the different layers andlevel of developing hairs, suggesting different mechanicalroles in differentiating trichocytes (Fig. 4).

The matrix or inter-fibrillar component comprises ‘keratin-associated proteins’ (KAPs), generally with smaller molecularweight (8–30 kDa) (Gillespie, 1991; Powell & Rogers, 1994;Rogers et al. 2006). Over 100 KAPs have been identified andform the matrix among KIFs that effectively results in thehard and resistant corneous material of hairs, nails andhorns (Gillespie, 1991). The KAP proteins are not fibrous,but more globular in conformation, but the molecularorganization of these proteins and their linkage with KIFsis not well understood (Rogers, 2004; Rogers et al. 2006).X-ray diffraction, electron microscopy and chemicalstudies have demonstrated that hair KIFs contain about70% of alpha-helical domains, the keratin intermediatefilaments are 7–10 nm thick (Filsie & Rogers, 1961; Fraseret al. 1972), and disulfide links KIFs together and also linksKIFs with KAPs (Rogers, 2004; Rogers et al. 2006).

Fig. 4 (A) Schematic representation of the hypothesis of hair evolution following the reduction of areas of dermal–epidermal interactions (ADEI) (A 1–3). (A1) In a large synapsid scale the area of contact (red layer) between the active dermis (green beads) and epidermis was extended over most of the outer scale surface, forming expanded ADEIs. (A2) ADEIs become smaller and smaller in later mammal-like reptiles (therapsids), producing dermal condensation near the hinge region while scales became reduced or disappeared. The concentration of active dermal cells, a precursor of the dermal papilla, induced the proliferation of a rod of corneous material out of the hinge region, a proto-hair. (A3) In advanced therapsids or true mammals the formation of a (condensed) dermal papilla stimulated the production of longer rods of corneous cells, the hairs. (B) Schematic representation of hair morphogenesis (1–6) to the mature hair (7). From the linear epidermis (1) a hair placode is formed (2) that grows downward into a hair germ (3). The latter forms a hair peg (4 and 4′) that grows into a bulbous peg in which the active dermis starts to penetrate into its cup-like basal epithelium to form a dermal papilla (5–6). From the matrix cell precursor of the hair initially only trichocytic keratins synthesize, and later KAPs are formed at different levels of the differentiating cortical and cuticle cells. Also cells of the inner root sheath express specific keratins and later trichohyalin (7). AD, active dermis (competent to send inductive signals to the epidermis); ADEIs, areas of dermal–epidermal interactions; BU, bulge (stem cell repository); CL, companion layer; CO, cortical cells (containing long bundles of corneous proteins); CU, hair cuticle; DC, dermal condensation; DP, dermal papilla; EP, epidermis; EIRS, elongating inner root sheath; FDP, forming dermal papilla; FIRS, forming inner root sheath; GE, germinal epidermis (matrix); HCU, cuticle of the hair; HE, Henle’s layer; HGT, high glycine tyrosine proteins; HL, hair-like primordia; HR, hair; HS, high sulfur proteins; S, high-sulfur proteins; HUX, Huxley layer; ICU, cuticle of the IRS (serrations correspond to those of the IRS); IK, intermediate filament keratins; IRS, inner root sheath; KAPs, keratin-associated proteins; MD, medulla cells; P, periderm; SG, sebaceous gland; SZ, sloughing zone; TH, trichohyalin; TK, trichocyte keratins; UD, undifferentiated dermis (does not produce signaling molecules); UHS, ultra-high sulfur proteins; VE, vesicles.

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When hair fibers or films of corneous material (producedafter chemical extraction or by direct application of X-raysto thin pieces of the corneous material, see Fraser et al. 1972),are stretched or heated, the alpha-type X-ray diffractionpattern changes to what is termed the beta-pattern. Thealpha-helical conformation of the keratin chains in theKIFs under stress elongates to form regions of beta-pleatedsheets. Early stages of stretching or heating can be reversible(Fig. 5A). Reptilian/avian keratins, in which there are regionscontaining beta-sheets, are referred to as beta-keratins andare very different from the keratin proteins of intermediatefilaments.

Mammalian KAPs (mKAPs) are grouped into three typeswith a molecular weight range of 6–36 kDa. They aredistinguished by their content of particular amino acids,which include glycine-tyrosine-rich (HGTs), high sulfurproteins (HSPs), and ultra high-sulfur proteins (UHSPs).Each of these protein groups is a family of proteins of relatedsequence. Their regulated expression in the differentiatinghair shaft in the follicle follows that of specific acidic/basictrichocytic keratins to which they are associated (Rogers,2004; Langbein & Schweizer, 2005; Rogers et al. 2006;Plowman et al. 2007). HGT (KAPs 6–8) are mainly synthesizedin the lower part of the differentiating zone (pre-cortex),located above the matrix zone, HSP (KAPs 1–3) in the inter-mediate region, UHSPs (KAPs 4 and 12) in the maturingcortex, and other UHSPs (KAPs 5, 9, 10) in the upper,maturing cells of the hair shaft cuticle (Powell & Rogers,1994; Rogers, 2004; Rogers et al. 2006) (Fig. 4). Someexamples of amino acid sequences of human KAPs areshown in Fig. 5B, where four key amino acids (glycine,cysteine, serine and proline) are colored for comparisonwith the proteins present in sauropsids (reptiles includingbirds). The number of amino acids varies from 57 to 90 inHGT proteins, 98 to 298 in HS proteins, and 114 to 205 inUHSs, with molecular weights variable from 5.4 to 36 kDa.The prediction of the secondary structure in mammalianKAPs (mKAPs) using the

PSIPRED

program (Alibardi et al.2007; Toni and Alibardi, unpublished data) on 128 mKAPshas shown that only few HSs present strand regions, and afew (less of 5.5% in our analysis) show regions with acore-box structure.

Figure 5D shows schematically that KAPs surround thenumerous alpha-keratin filaments and become organizedaround them, forming the matrix of the corneous material.

The beta-keratins in sauropsid epidermis and its derivatives

The keratin proteins of reptiles have remained poorlyknown, compared with those of mammalian and avianproteins, for over 30 years. They were considered to consistof soft (alpha)-keratin and hard (beta or phi)-keratins(Maderson, 1985; Landmann, 1986; Alibardi, 2003). The aminoacid sequences of keratins present in reptilian corneous

structures (scales, claws, ramphotheca, etc.) were unknownuntil recently. The hard proteins of reptiles and birds areproteins (beta-keratins) with a fibrous structure that givesa beta-pattern by X-ray diffraction and a filament of 3–4 nmin diameter, as first described by Filsie & Rogers (1961).

The amino acid composition and molecular weights ofbeta-keratins were available for some reptilian species(Baden & Maderson, 1970; Baden et al. 1974; Wyld &Brush, 1979, 1983; Sawyer et al. 2000; Homer et al. 2001).No reptilian alpha-keratins of the softer and harder layershad been sequenced, and only one small keratin from alizard claw was known in its primary amino acid structure(Inglis et al. 1987). However, it was unclear whether thisprotein was representative of reptilian scales or merelyreflected the composition of a specialized claw keratin.Also, a 20-amino acid sequence localized in an innerregion of an alligator beta-keratin was known, and thisepitope shows high identity with that of avian beta-keratins (Sawyer et al. 2000, 2005a).

It was known that different proportions of specificmonomers of beta-keratins, called phi-keratins, are presentin reptilian derivatives of different rigidity (Wyld &Brush, 1979, 1983; Thorpe & Giddings, 1981; Gillespie et al.1982; Marshall & Gillespie, 1982; Inglis et al. 1987). Theseproteins are of small molecular weight, mainly in the10–20 kDa range, but a component of higher molecularweight was also found (30–44 kDa), although in minoramounts. It was not clear whether they were true polymersor aggregates derived from chemical extraction. Differentforms (monomers) are present in various skin derivativessuch as dorsal and ventral scales, shell scutes, claws, andramphotheca in turtles, lizards, snakes and crocodilians.According to Wyld & Brush (1983), phi (beta)-keratins ofreptiles are believed to be phylogenetically distant frombeta-keratins of birds, or even unrelated to feather-keratins(Brush, 1993). The latter study in particular pointed outthat feather-keratins and feathers are a completely newinnovation of avian skin.

Other studies on crocodilian and avian beta-keratinshave indicated that these proteins can be distinguishedinto a few main types: scale, beak, claw, feather or feather-like keratins (Gregg et al. 1983; Gregg & Rogers, 1986;Presland et al. 1989b; Sawyer et al. 2000, 2005a; Sawyer &Knapp, 2003). Beta-keratins probably vary from speciesto species in their amino acidic sequences, and those inarchosaurian skin (crocodilians) are strictly correlated tothose of birds. Scale, claw, and beak keratins showeda relatively higher molecular weight, 14–18 kDa, whereasfeather or feather-like keratins possessed a smaller molecularweight, 10–12 kDa (Gregg & Rogers, 1986). Studies onfeather genes suggested that at least 30–40 genes alonewere present, and other 10–20 genes were found for scale,claw and beak keratins.

Early knowledge on the process of cornification inmammals vs. reptiles/birds indicated that some striking

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Fig. 5 Mammalian proteins involved in cornification. (A) Alpha keratins that normally produce an alpha-pattern can be deformed under some conditions (stretching or steaming the corneous material above 70–80 °C). These conditions induce a change of secondary conformation, from alpha-helix to random coil and even beta-sheets, which produce a beta-keratin. (B) Some examples of mammalian KAPs are shown. The key amino acids are colored with different colors to illustrate the three main groups – HGT, HS and UHS matrix proteins. (C) Examples of the main mammalian skin derivatives containing trichocytic keratins and KAPs to form very hard corneous layers. (D) Schematic representation of the progressive phases (1–3) of association between coiled alpha-keratins (trichocytic) and KAPs. In phase 3, KAPs are probably regularly ordered around alpha-keratins and the resulting pattern at X-ray is indicated as alpha pattern.



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differences were present. In the corneous skin appendagesof reptiles/birds, beta-keratin aggregates to form filamentsdifferently from what occurs with alpha-keratins (Gregg &Rogers, 1986; Gillespie, 1991; Brush, 1993). This suggestedthat a more central region of the molecule of beta-keratincontributed to make the fibrous framework of the fila-mentous polymer, while the extremities formed the matrix.This was a different mechanism from that present inmammalian corneous appendages. It is not clear why suchdifferent mechanisms of cornification originated in reptiles/birds compared to mammals as they have a commonreptilian progenitor.

The resulting alpha-keratin pattern of mammals vs. thebeta-keratin pattern of sauropsids might be due not to adifference in keratins but instead to a different compositionand organization between proteins or the fibrous com-ponent (alpha-keratins) vs. those of the matrix component

(keratin-associated proteins). This organization is different inthe corneous material of mammals compared with that inreptiles/birds, but in both cases the corneous materialconsists of a fibrous and a matrix component.

Immunocytochemical and biochemical studies suggest that beta-keratins function as keratin-associated proteins in sauropsids

Numerous ultrastructural and immunocytochemical studieson reptilian epidermis have shown the presence of bothcytokeratin and beta-keratin immunoreactivity (Alibardi &Sawyer, 2002; Alibardi, 2003, 2005, 2006c; Alibardi & Toni,2006a–d; see examples in Fig. 6). Controls, omitting theprimary antibody, are unlabeled (data not shown). Alpha-keratins in reptilian corneous material were revealedusing broad-spectrum cytokeratin antibodies such as AE3

Fig. 6 Localization of alpha-keratins (A–B) and beta-keratins (C–F) in reptilian corneous layers. (A) AE3-immunogold labeling (arrows) in lizard (Podarcis sicula) beta cells among pale beta-keratin bundles. Bar, 150 nm. (B) Detail of AE3-labeling over filaments (arrow) surrounding the pale beta-keratin bundle in a beta-cell of lizard (P. sicula). Bar, 100 nm. (C) Beta-1 immunolabeled, thick corneous layer of carapace scute in turtle (Chrysemys picta). Dashes underline the basal epidermis. Bar 10 μm. (D) Beta-1 gold immunolabeled corneous layer of scute of C. picta that disappears along the border with the underlying living keratinocyte (arrows). Bar, 250 nm. (E) Beta-lizard (A68B-antibody) immunolabeled keratin bundles in a beta-cell of P. sicula. Bar, 250 nm. (F) Beta-universal (β-U) labeling over keratin bundles of a differentiating beta-cell in snake (Natrix natrix). Bar, 250 nm. (G) Schematic general pattern of epidermal proteins from the epidermis of different species of reptiles (lizard, gecko lizard, turtle, alligator, crocodile and snake) showing the relative amount of alpha- vs. beta-keratins. AE3, immunolabeling positive for alpha-keratin AE3; be, beta-keratin bundles; β1, immunolabeling for beta-keratins using the chick scale β1 antibody; c, corneous layer.



571

and Pan-cytokeratin antibodies, which recognized differ-ent acidic and basic cytokeratins (Alibardi & Toni, 2006a).

The latter indicated that beta-keratins in reptiles andbirds have broad cross-reactivity as shown by a chick generalantibody (beta-1) (Alibardi & Sawyer, 2002). Later analysisindicated that most reptilian and avian beta-keratins alsocontain a ‘universal’ antigen found in avian and alligatorbeta-keratins with the sequence SRVVIQPSPVVVTLPGPILS(Sawyer et al. 2000, 2005a). This sequence, or the homo-logous sequences, contained in a central region of all knownbeta-keratins, were indicated as the core-box (Alibardi &Toni, 2006a, 2007). The final confirmation of the preciselocalization of beta-keratins in reptilian epidermis cameafter the isolation of antibodies produced in rat and directedto 14–17 kDa proteins isolated from reptilian epidermis(Alibardi & Toni, 2006c,d).

Our proteomic studies on epidermal proteins in reptileshave initially confirmed that beta-keratins generally fallwithin the 12–20 kDa range (Alibardi & Toni, 2006b,c,d;Toni & Alibardi, 2007a,b,c; Toni et al. 2007a,b). The use ofgeneral and more specific antibodies for alpha- and beta-keratins after two-dimensional electrophoretic separationhas identified mainly acidic and neutral alpha-keratins inthe 40–70-kDa range (Fig. 6G). The other large group ofproteins, beta-keratins of 10–20 kDa, are mainly basic andneutral, and there is also a small fraction that appears tobe acidic (Fig. 6G). Therefore alpha- and beta-keratins maybe complementary and thereby associate into keratinfilaments, a unique condition of sauropsid keratins.

These immunological proteomic studies indicated thatbeta-keratins seem to associate with alpha-keratins asmammalian KAPs associate with keratin intermediatefilament keratin proteins (KIFs) in mammalian tissues,suggesting that beta-keratins may actually function asreptilian KAPs. Matrix proteins so far known in amnioteepidermis (filaggrin and trichohyaline) are basic and theyhave charged interactions with neutral or negatively chargedgroups present in keratin filaments (Mack et al. 1993). Inavian feathers two basic KAPs, indicated as CAP-1 andCAP-2, and four basic histidine-rich proteins (HRPs) aredifferent from beta-keratins (Gregg & Rogers, 1986; Preslandet al. 1989a; Knapp et al. 1991,1993; Barnes & Sawyer, 1995).In summary, all amniotes produce corneous material usingalpha-keratins as fibrous material and keratin-associatedproteins as a matrix. The peculiarity of reptilian and birdKAPs is that the proteins form filaments that replace ormask those of alpha-keratins. This derives from the specialamino acid composition of some key regions of reptilianand avian KAPs, as it will be illustrated in the next section.

Beta-keratins in reptilian and avian keratinocytes ofscales and feathers form fibrous polymers and filaments(Fraser et al. 1972; Brush, 1983) as compared to mammalianhard corneous material of hairs, nail and horns, wheremKAPs are mainly amorphous. The beta-keratins are alsodifferent from intermediate filament proteins in com-

position, chemical-physical properties, and mechanismof polymerization. Therefore small proteins previouslyindicated as beta-keratins (fibrous proteins with regions ofbeta-pleated sheet) should be considered a fibrous type ofkeratin-associated or matrix proteins (sauropsid KAPs)(Alibardi et al. 2007; Toni et al. 2007a,b). The term beta-keratins will be used in the present review as being equivalentto sauropsid KAPs (sKAPs). However, it is generally agreedthat true keratins are defined as belonging to the keratinintermediate filaments family (Steinert & Freedberg, 1991;Fuchs & Weber, 1994; Coulombe & Omary, 2002).

The lack of knowledge about proteins and their genesinvolved in the production of hard proteins in reptilianscales has impeded a proper evaluation of the evolution ofthese proteins from reptilian ancestors to birds. The essential,missing information in reptiles was the protein sequence,secondary and tertiary conformation, and the genes codingfor these hard proteins. The recent genomic and proteomicstudies on reptilian beta-keratins have rapidly clarified thisissue and the new information suggests a new interpretationthat unifies the mechanism of cornification in all amniotes.

Genomic structure and expression of sauropsid keratin-associated proteins: nucleotide sequences, genes and chromosomal localization

Genes encoding reptilian beta-keratins

The recent molecular biology results on reptilian beta-keratinshave been initially obtained by designing degenerateprimers from specific regions (core-box) of avian and a lizardclaw beta-keratins (Inglis et al. 1987). These primers werethen replaced by more specific primers selected on thesame reptilian regions (Dalla Valle et al. 2005, 2007a,b,2008 and 2009). This work has aided the deduction of theamino acid sequences of lepidosaurians (lizards, snakes),chelonian, and crocodilian beta-keratins. The nucleotideand protein sequences of different reptilian keratins arenow deposited in GenBank under specific accessionnumbers.

This initial information has been now integrated withthe analysis of the lizard

Anolis carolinensis

and chickengenomes (Dalla Valle et al. unpublished studies).

These data have allowed, for the first time, the completecomparison of the nucleotide and amino acid sequencesand protein structure of different reptilian beta-keratinswith avian keratins. In fact, the nucleotide structure of beta-keratin cDNAs so far sequenced from reptilian epidermis(Dalla Valle et al. 2005, 2007a,b) show a general linearorganization that resembles the avian nucleotide structurefor their scale, claw and feather genes (Gregg et al. 1983,1984; Presland et al. 1989a,b; Whitbread et al. 1991).

The gene structure in snake, crocodiles and, accordingto the first data, also in turtle, show the same organization

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572

as previously found in avian beta-keratin genes: the codingregion is uninterrupted, whereas an intron of variablelength is present in the 5

′

-untranslated terminal region(5

′

-UTR) (Fig. 7). The first reptilian gene coding for beta-keratin containing an intron has been sequenced in thesnake

Elaphe guttata

(now called

Panthoteris guttatus

)(Dalla Valle et al. 2007b). In the snake gene an intron of226 bp is present after 29 bp of the 5

′

-UTR and it is followedby a short non-translated region of 17 bp. A similar result hasbeen found in the crocodile

Crocodylus niloticus

, in which the5

′

-UTR contains an intron of 621 nt (Dalla Valle et al. 2008).The presence of intron and the exon-intron borders was

identified based on the direct comparison of the cDNAand the genomic sequences obtained by PCR using senseprimers located at the beginning of the 5

′

-UTR and antisenseprimer covering the stop codon. However, this approachgave a different result in the two species of geckos analysed:a fragment of the same length was amplified with bothcDNA and genomic DNA. Sequencing showed that no intronwas present in the two genomic fragments obtained (DallaValle unpublished data).

The available genome of the American chameleon lizard

Anolis carolinensis

(at http://genome.ucsc.edu/cgibin/hgGateway?hgsid = 94953969&clade = vertebrate&org =Lizard&db = 0) has allowed analysis of the presence ofgenes coding for beta-keratin proteins similar to thosefound in other lizards. In total about 40 lizard genes arepresent in this species; moreover, as all genes were foundin the same scaffold, these genes are probably localized inone single chromosomal locus (Dalla Valle et al. work inprogress). The accessibility of

A. carolinensis

genome alloweddirect comparison of the cDNA with the genomic sequences.A preliminary 5

′

-RACE analysis on some beta-keratin mRNAsexpressed in the epidermis of the green anole (

Anoliscarolinensis

), showed that they contain an intron in their5

′

-UTR (Dalla Valle et al. unpublished results). At present itis known that one intron of variable length is present inthe 5

′

-untranslated region of beta-keratins in all reptiles:snake, crocodile and in the turtle. It is not known whetherthe intron may be the regulative region for the transcription.Although in gecko lizard genes so far analysed (from twodifferent species) no intron has been found, these obser-vations suggest that other beta-keratin genes within thelizard genome likely possess introns. The presence of oneintron in the 5

′

-UTR of reptilian beta-keratin gene is probablygeneral but not always constant. More work, such as the5

′

-RACE analysis of beta-keratins of the green anole isnecessary to clarify this point.

Genomic Southern analysis using reptilian

β

-keratin cDNAas a probe has indicated the presence of several relatedgenes in the genome of geckos, snake, crocodile and turtle(Dalla Valle et al. 2007a,b, 2008 and 2009). These datahave confirmed that also in reptiles

β

-keratins belong toa multigene family as demonstrated in the chick (Gregget al. 1983, 1984; Presland et al. 1989a,b; Whitbread et al.1991). These results are also confirmed after some cloningexperiments leading to the sequencing of more then onegene in each analysed reptilian species. When performed,PCR analysis indicates a head-to-tail orientation ofthese genes in their cluster. However, the analysis of

A. carolinensis

and avian genomes reveals that, althoughthis is the common orientation, genes are also present thatare arranged with tail-to-tail or head-to-head orientation(work in progress).

The

in situ

localization of messenger RNA for these proteinsreveals a strict epidermal expression, mainly localized in thedifferentiating oberhautchen, in beta-layers of lepido-saurians, and in the pre-corneous layer of crocodilianand turtle scales. (Fig. 8A,B,D–F). The ultrastructural

in situ

hybridization analysis of mRNA gene expression has shownthat transcripts are present in the cytoplasm associatedwith the initial sites of deposition of beta-keratins (beta-packets; Fig. 8J). However, the periphery of larger bundlesof filaments often presents beads of gold particles thatidentify antisense digoxigenin-labeled probes associatedwith the filaments (Dalla Valle et al. 2005; Alibardi et al.2006a). This finding suggests that the protein still in thetranslation phase can be directly packed in the growingbundle.

The genomic structure of avian beta-keratin genes is organized on the reptilian model

Previous information on chick scale, claw, beak and feather-keratins indicated that their genes are clustered in thesame locus on chromosomes (Molloy et al. 1982; Gregg et al.1983, 1984; Gregg & Rogers, 1986; Presland et al. 1989a,b;Whitbread et al. 1991; Brush, 1993; see Fig. 7). However,neither the number of chromosomes containing beta-keratin genes nor their location was indicated. Genesappear to be arranged in multiple copies with the sameorientation in the genome. They contain one intron in the5

′

-UTR, perhaps with a function involved in transcriptionregulation. A previous summary of all known avian beta-keratin genes indicated the presence of 18 genes for feather-keratins, three genes for feather-like keratins, four genes

Fig. 7 Genomic organization (A–F) and examples of nucleotide structure (G–I) of avian and reptilian beta-keratin genes. A cluster of genes coding for related proteins is localized in a chromosome locus (A) and they are linearly arranged in multiple copies (B). In the genes of chick (chicken feather-keratin gene, AC J00847, and snake (Elaphe guttata, AC AM404188), a variably-long intron is present in the 5′-non coding region (C,F,G,H). In contrast, in the gecko lizard species studied so far (here exemplified by one sequence of Tarentola mauritanica, AC AM162665), no intron has been found (E,I). ATG (boxed) initiation codon; TAA or TAG (boxed), termination codon; AATAAA (boxed), polyadenylation signal. Uppercase letters, coding region; lowercase letters, 5′ and 3′ untranslated terminal regions; bold lowercase letters, intron sequence. The splice signals of introns are boxed and shaded.

http://genome.ucsc.edu/cgibin/hgGateway?hgsid = 94953969&clade = vertebrate&org = Lizard&db = 0





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for claw keratins and nine genes for scale keratins. Noindication was available on the number of genes for beakkeratins (summary in Stevens, 1996). This was clearly alarge underestimation of avian beta-keratin genes, asinitial studies indicated the presence of 100–240 differentgenes for feather-keratins alone (Kemp et al. 1974). Thelatest analysis on the chick genome shows that probablyaround 140 genes for feather, claw, scale and beak keratinsare present, not taking into account pseudogenes orincomplete sequencing data (Dalla Valle et al. unpublishedresults). Among beta-keratin genes, 56 are clustered inchromosome 25, whereas the other 64 genes are presentin chromosome 27. A few beta-keratin genes (from a singlecopy to seven copies) are present in five other chromosomes,but they may be copies of some genes present in chromo-somes 25 and 27.

The structure of the chick feather gene (gene C) (Gregget al. 1984; Gregg & Rogers, 1986) comprises a 5

′

-UTR of 93bp, which is interrupted after 37 pb by an intron of 329 bp.Like reptilian genes, the coding region of avian genes isdevoid of introns (Fig. 7G). The intron position is conserved

in other avian beta-keratin genes sequenced (Gregg &Rogers, 1986) and this is also confirmed by the avian genomeanalysis. The intron may be of some importance for theregulation of the velocity of transcription of beta-keratin gene(Koltunow et al. 1986). The possibility to increase transcrip-tion may explain the rapid production of feather-keratinin 2–3 days, from 13–16 days of development in the chickembryo (Hamburgher-Hamilton stages 37–39; Kemp et al.1974; Gregg & Rogers, 1986). In a large part of the non-coding region of the beta-keratin gene cluster, is a highlyconserved nucleotide sequence, especially nucleotides 1–37,indicating an essential physiological role in the transcrip-tion and translation process. The 3

′

-UTR part of the geneis much less conserved than the other regions.

From the above description and the recent data on thegene organization of crocodilian beta-keratins it is likelythat beta-keratins of birds are derived from an archosaurianorganization of this family of genes that pre-dated the originof birds. The latter inherited this basic organization andevolved their own specific sequences for the formation ofscale and feather keratins (see description below).

Fig. 8 Examples of in situ hybridization localization using antisense probes for specific messengers coding for beta-keratins, and visualized by immunofluorescence (A–D,I), alkaline-phosphatase (E–H), or ultrastructural localization (M). (A) Regenerating scale of gecko (Tarentola mauritanica) with expression (arrows) in cells of the forming beta-layer (arrows). Bar, 10 μm. (B) Beta layer (arrow) of snake (Elaphe guttata). Bar, 10 μm. (C) transitional (arrow) and lower corneous layer of turtle (Pseudemys nelsonii ). Bar, 10 μm. (D) transitional layer (arrow) of crocodile (Crocodylus niloticus). Bar, 10 μm. (E) beta-cell layers (arrows) of regenerating scales in lizard (Podarcis sicula). Bar, 20 μm. (F) beta-layer cells (arrow) in scales in renewal stage of snake (E. guttata). Bar, 10 μm. G, fusiform cells of the transitional layer (arrow) of scutes in Chrysemys picta. Bar, 10 μm. H, forming beta-layer (arrow) of scale in the tuatara (Sphenodon punctatus). Bar, 10 μm. I, detail on the spindle-shaped cells of the transitional layer of crocodile (C. niloticus). Bar, 10 μm. J, ultrastructural detail of the cytoplasm of differentiating beta-cell in lizard (P. sicula). Clusters of 5-nm diameter gold particles indicate the position of mRNAs among small keratin material (arrows). The arrowhead points to gold particles associated with a larger keratin bundle. Bar, 50 nm. a, alpha-layer; b, beta-layer; c, corneous layer; d, dermis; e, epidermis.



575

Fig. 9 Examples of protein prediction using the PSIPRED Protein Structure Prediction Server at http://bioinf.cs.ucl.ac.uk/psipred/ for representative proteins of lizard, snake, crocodile, and turtle. The molecular weight and isoelectric point (pI) of these proteins are indicated. They all present inner amino acid regions with two close strands (beta-folded sheats) indicated as core-box (boxed). Note the variable extension of the alpha-helical region (green rods) in relation to the random-coiled regions (indicated with black lines).

http://bioinf.cs.ucl.ac.uk/psipred/



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Proteome analysis of sauropsid KAPs: types of proteins in different groups of reptiles and their comparison with those of birds

Comparative analysis of sauropsid KAPs shows that they possess a high homologous beta-pleated region: the core-box

To date about 100 proteins have been completely sequencedand analysed in five species of lizards (Podarcis sicula,Tarentola mauritanica, Hemidactylus turcicus, Gekko gecko,Anolis carolinensis), one snake (Elaphe guttata), a turtle(Pseudemys nelsonii), and the Nile crocodile (C. niloticus)(Dalla Valle et al. 2005, 2007a,b, 2008 and 2009; Hallahanet al. 2008). Partial sequences are known for the alligator(Alligator mississippiensis; (Sawyer et al. 2000), the tuatara(S. punctatus) and the soft-shelled turtle (Trionix spiniferus)(Dalla Valle et al. unpublished results).

The predicted molecular weight and isoelectric pointof some deduced proteins correspond to specific spotsobtained following two-dimensional separation ofepidermal proteins, which confirms molecular findings(Toni et al. 2007a,b and unpublished results). Analysis ofthe deduced amino acid sequences by specific softwarecan graphically predict secondary structure (Fig. 9). Thestudy of some representative beta-keratins in lizard, snake,turtle, crocodile and birds has demonstrated the presenceof a central region where two strands (beta-foldedsecondary conformation), made up of four to seven aminoacids each, are present in-tandem. The core-box representsthe region with the highest sequence identity and ispresent within a 32-amino acid region that contains thestrand or beta-pleated sheets in beta-keratin monomeres(Fraser et al. 1972; Brush, 1993; Fraser & Parry, 1996). Arecent study on feather and reptilian beta-keratins, basedon a different method of analysis of the secondary con-formation, reported the presence of four beta-strandsin this region (Fraser & Parry, 2008). The predictions showthat short beta-sheets made of four to eight amino acids,generally not organized in tandem, are present in otherregions of these small proteins (Fig. 9, and data not shown).The role of the extra strands in these proteins remainsundetermined but awaits more information from theprediction of their tertiary structures (ongoing analysis inour laboratory).

The determination of the amino acid sequence for beta-keratins from lizards, snake, turtle and crocodile, togetherthat for a lizard claw (Inglis et al. 1987), has indicated thatsome regions of these proteins present high sequenceidentity with avian keratins (Dalla Valle et al. 2005, 2007a,b).These predictions show some of the structural homologiesof reptilian and bird proteins, especially in the core-box orbeta-pleated sheet region (Figs 9, 10).

Surprisingly, the predicted secondary structure of thededuced proteins from turtle resembles those of crocodilians Fi

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Fig. 11 Examples of the specific amino acid sequences that characterize each group of reptiles. (Top panel) Comparative representation of some examples of proteins in which the position of three key amino acids such as tyrosine, valine and isoleucine is indicated. Tyrosine is mainly lateral (hydrophilic interactions?), whereas valine and isoleucine are more concentrated in the core-box and nearby regions, where they contribute to the formation of strand regions. (Other panels) Examples of group-specific (lepidosaurians vs. chelonians/arcosaurians) amino acid sequences found so far in pre- and post-core-boxes of sauropsids (see text).



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and birds, which can be included in a subgroup differentfrom those of lepidosaurians. This suggests that turtle andarchosaurians have a closer or common basic archosaurprogenitor.

While the concentration of proline distorts the poly-peptide chains, the presence of numerous valines, isoleucineand leucine in some cases, determines the formation of abeta-sheet structure in this region (Fig. 11, top panel).

The beta-strands present in this region of the proteinare considered the site of polymerization of beta-keratinmonomers into the long polymer that forms the filamentsof beta-keratin 3–4 nm in diameter (Fraser et al. 1972;Brush, 1983; Gregg & Rogers, 1986; Fraser & Parry, 1996). Thisregion probably has a three-dimensional conformationacting as an anchoring point for the interaction withsimilar proteins (monomers). The random coiled regions,generally representing most of the protein sequence, are

regions with free rotation of R-groups of amino acids alongthe fixed -CO-NH- peptide bond.

Our biochemical studies have shown that alpha-keratinsare present in hard corneous material of reptilian scales,so that proteins of intermediate filaments are probablymasked by the deposition of beta-keratins (sKAPs). Itseems that in scales and claws of different reptiles andbirds, in the beak and feathers, the amount of sKAPscapable of forming filaments is generally larger than thatof alpha-keratins.

The synthesized beta-keratins present in different append-ages of sauropsid integument (scales, claws, ramphotecaor beak, and feathers) can form bundles of corneous materialas shown in Figs 6, 12 and 13. Alpha-keratin is generallyreplaced or degraded, whereas sKAPs accumulate and makefilaments through the polymerization of their core-boxes.The abundance in proteins containing the beta-strands is

Fig. 12 Schematic drawing illustrating the synthesis of corneous material in beta-keratin cells of various skin derivatives in sauropsids. Beta-cells (B) of claw (A), scale (A1) and beak (A2) accumulate proteins with glycine-rich regions which produce irregularly organized filaments (B2) for high mechanical resistance. Only feather-keratins (A3) do not possess glycine-rich sequences. Beta-keratin filaments (in black in B3 and B4) increase among the decreasing alpha-keratin filaments. The molecular organization of keratin-associated proteins (KAPs) and histidine rich proteins (HRPs) among fibrous proteins is not known. The detail in B5 schematically shows the antiparallel overlapping of keratin monomers (see orientation of arrows inside the core-box) to form the framework of the protein polymer. Numerous inter- and intra-molecular disulfide bonds strengthen the stability of the filament/s.



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probably responsible for the prevalent beta-keratin patternobserved after X-ray diffraction on the corneous layer ofreptilian epidermis (Baden & Maderson, 1970; Fraser et al.1972). It is believed that the stabilization of the filamentsof beta-keratin occurs by homophilic interactions in theglycine-rich regions and by intra- and inter-moleculardisulfide bonds. The latter are favored by the prevalentlocalization of cysteines toward the extremities of theseproteins, in proteins both with low and high amounts ofthis amino acid, as indicated in the next section (Fig. 10).

Specific amino acid regions define different sKAPs: lepidosaurians vs. chelonian and archosaurian proteins

The analysis of the predicted secondary structures in lepido-saurian proteins shows that, outside the core-box, most oftheir molecule contains a random coil conformation and alpha-helix regions are relatively scarce. According to the contentof some amino acids, lepidosaurian proteins can be generallydivided into high glycine proline serine (HGPS) and highcysteine proline serine (HCPS) (Fig. 10). The latter presumablyform numerous disulfide bonds and may be prevalent inthe harder corneous material of reptilian scales or in claws.

Further analysis of the primary and secondary structureof glycine-proline-rich proteins of crocodilian and turtleepidermis shows that long regions of the proteins areoccupied by a random-coil conformation (Fig. 9).

In the crocodile, after the core-box, from amino acid 57to 171, near the N-terminal, a region rich in glycine-X andglycine-glycine-X sequences is found. In crocodile glycine-proline-tyrosine-rich protein 1 (Cr-gptrp-1), 19 GGX repeatsare present. In particular, from amino acid 100 to 135 alternaterepeats of six GGY-GGL are found in Cr-gptrp-1 (Figs 9, 14,Dalla Valle et al. submitted). Even more remarkable is the23 GGX repeats found in Cr-gptrp-2 protein, the longest sofar isolated in any sauropsid protein. As in crocodilian beta-keratins, chick scale (65–74% identity) and claw (66–68 %identity) contain long glycine-rich sequences although theyare shorter than in crocodile. Feather-keratins are missingsuch sequences (Gregg et al. 1984). This progressive reductionof glycine-rich sequences from the C-terminus toward thecore-box of archosaurian scales vs. feather-keratins indicatesan evolution of the protein toward simplification. Thischange was somehow favorable for the specialization offeather-keratin to form long bundles for the elongation ofbarb/barbule cells (Fig. 13). The small proteins form betteroriented filaments with fewer cross-links than larger proteinswith long glycine-rich regions (Fraser et al. 1972; Gregg &Rogers, 1986). Feather-keratins tend to resist tension betterthan scale keratins as they are more oriented. Scale proteinspresent a less organized distribution, which increasesmechanical resistance (Brush, 1993).

Turtle proteins resemble those of archosaurian scales andclaws, both crocodilians and birds (Figs 9–11, Dalla Valle

et al. 2009). Among these characteristics are the richnessof tyrosine and the high sequence identity of the core-boxregion and in surrounding amino acid regions. Therefore,based on sKAPs in the epidermis, turtle, crocodile and birdsappear to share a common progenitor, and this reinforcesthe recent evidence that turtle and archosaurians taxonomi-cally are much closer to each other than to lepidosaurs(Rest et al. 2003).

Some regions of these proteins show sequence identitywith mammalian KAPs (Alibardi et al. 2006a) that are presentin hard keratinized appendages (hairs, claws, horns, etc.,see Gillespie, 1991; Marshall et al. 1991; Powell & Rogers,1994). However, in comparison with mammalian highglycine-tyrosine proteins (7–13 kDa, see Gillespie, 1991),high glycine-proline-tyrosine proteins of turtle (HGPTs)are larger proteins (12–17 kDa).

The above differences between lepidosaurian andarchosaurian/chelonian proteins are also emphasizedby the further detection of different amino acid regions,here provisionally indicated as pre- and post-core-boxes, thatcharacterize archosaurians and chelonian vs. lepidosaurianbeta-keratins (Fig. 11).

In summary, the central core-box of 20 amino acidscontains two beta-strands and has 75–90% identity withinarchosaurian/chelonian KAPs, whereas a pre-core-box of17 amino acids, indicated as archosaurian/chelonian-box,has also high identity (Dalla Valle et al. 2007a,b, 2008 and2009). These values fall to 60–75% when comparing the core-box of crocodilian/chelonians with those of lizards or snakes.The latter contain a pre-core-box called the lepidosaurian-box (Fig. 11). The high sequence identity based on the core-box of their beta-keratins confirms that crocodilians andbirds are the closest living archosaurians. Another differencebetween lepidosaurian and chelonian-archosaurian beta-keratins is that in the former the core-box is localized fromhalfway to the C-terminus of the protein, whereas in thelatter the core-box is moved toward the head to the middleof the protein (Fig. 10). The specific role of these and ofother regions of reptilian KAPs remains to be discovered.

Evolution and phylogenetic relationships among sauropsid KAPs

The recent knowledge on genes and protein sequenceshas addressed for the first time the long-debated problemof the evolution of these small proteins in reptiles andbirds (Wyld & Brush, 1979, 1983; Maderson, 1985). A generalphylogram obtained using the neighbor-joining method(Saitou & Nei, 1987), showing some examples of sauropsidKAPs, is presented in Fig. 15A. The phylogram shows theinitial divergence between lepidosaurian and chelonian/archosaurian proteins, probably during the mid-upperPermian (Pough et al. 2001).

The molecular evolution in lepidosaurians apparentlyproduced two main types of proteins, the HGPS and HCPS

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Fig. 13 Protein predictions using the PSIPRED Protein Structure Prediction Server at http://bioinf.cs.ucl.ac.uk/psipred/ of avian scale and claw keratins (both HTPS proteins) in comparison with the shorter feather protein (HCPS protein). The molecular weight, isoelectric point and type of proteins are indicated. The core-box is present in all these proteins despite the specific amino acid length and composition in other regions. The lack of a glycine-rich region in the feather protein allows its polymerization into long filaments/bundles tending to be parallel, as indicated in the lower right panel (compare Figs 13 and 12). The accumulation of corneous material mainly with a parallel orientation in feather cells during progressive stages of morphogenesis is indicated by the drawing in the bottom right panel. The small amount of alpha-keratin (represented by short coils) is rapidly replaced by the small feather protein (BFK). The detail in the drawing schematically shows the antiparallel overlapping of keratin monomers (see orientation of arrows in the core-box) to form the framework of feather-keratin polymer. Numerous inter- and intra-molecular disulfide bonds strengthen the stability of the filament(s). BL, barbule cells; BCC, barb cortical cells; BMC, barb medullary cells (become vacuolated); HCPS, high cysteine proline serine proteins; HRP, histidine-rich proteins; HTPS, high tyrosine proline serine proteins; HCPS, high cysteine proline serine protein. KAP, keratin-associated proteins; KB, keratin bundles; PF, parallel filaments.

http://bioinf.cs.ucl.ac.uk/psipred/

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Fig. 14 Skin derivatives of sauropsids (A), and schematic mechanism of formation of their hard corneous material (B). This occurs as in mammals (compare with panel D in Fig. 5) but, because of the core-box, sauropsid KAPs form themselves into filaments that replace most intermediate filaments. This process determines the prevalence of a beta-keratin pattern over the initial alpha-pattern. The embryo of a crocodilian (C) and its scale (C1) synthesize a protein with a long glycine-rich region that forms irregularly oriented corneous bundles (C2). (D) A bird embryo, with scale keratin (D1) and claw keratin (D2) with a shorter glycine-rich sequence. These keratins form irregular corneous bundles (D3) as for beak keratin (D4). (D5) The short feather-keratin where the glycine-rich regions have disappeared but corneous bundles parallel (D6). One possibility is that an ancestral, crocodilian-like protein gave rise (short arrow on the left) to proteins present in avian scales, claws and beak and, eventually, feathers. The other possibility (long arrow on the right) is that feather-keratin was directly derived from a large deletion of the glycine-rich region from an ancestral protein, and that a specific evolution gave rise to the feather-specific sequence. According to immunological studies (see Fig. 2) the feather-epitope may be very ancient in arcosaurian (embryonic) epidermis but is later eliminated in scale, claw or beak proteins. Color codes: Glycine (red), Serine (yellow), Cysteine (green), Proline (blue).



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proteins (lepidosaurian KAPs), which are poor in tyrosine.These two subfamilies contain different members that willbe more properly classified when they have all been charac-terized. The variety of types, in amino acid composition andmolecular weight, are probably related to the specializationsof the integuments of these modern reptiles (such asdifferent types of scales over different body regions, softand hard with keels, frills, gastrosteges of snakes for bodymovement, and claws). Some of the studies reported abovehave indicated that HGP proteins are present in relativelysoft scales of lizards and snakes, while HCP proteins arepresent in claws and climbing setae.

Proteins of archosaurians/chelonians so far identified differfrom those of lepidosaurians in that they are not dividedinto HGPS and HCPS proteins. Their general structurecontains a tail region with HCPS characteristics and a tailregion with HGPS characteristics, resembling a chimericmolecule (see Fig. 10). That typical diapsid (archosaurians)and presumed anapsid (chelonians) reptiles possess moresimilar proteins than the proteins of lizards and snakes(diapsids) was unexpected. The study of a current repre-sentative of a basal lineage of diapsid lepidosaurians suchas Sphenodon would be very important to complete thewhole picture on the evolution of these keratins (Alibardi& Toni, 2006b).

After this initial divergence, in archosaurians two maingroups later diversified, that of tyrosine-glycine-rich non-feather proteins and the glycine-tyrosine-poor group offeather proteins (see also Gregg et al. 1983). In chelonian/archosaurian KAPs, the amount of tyrosine and glycineincreases somehow in relation to the irregular orientationof the corneous material, which increases resistance(Brush, 1993). These regions were either lost (or neverappeared) in the small and specialized proteins of feathers(Figs 13, 14).

Interestingly, the initial archosaurian group includes theproteins of turtle, suggesting that turtle proteins havedifferentiated from common ancestral proteins with thoseof archosaurs. Turtle proteins later diverged from crocodilianand avian proteins. Turtles have maintained a quite uniformtypology of scutes in the shell, with marginals, coastal andneural, and sternoplastron, hyoplastron, hypoplastron,xyphiplastron, mainly changing their areas and thickness indifferent species. More variety was generated in crocodilianscutes, where tail keeled scutes, ventral softer scales, dorsaltough scales, etc., are present. The scale proteins of dorsaltough scales are the closest proteins to those of crocodilians.This supports the evolution of scale proteins in birds froman archosaurian protein (Figs 14, 15).

The molecular evolution of archosaurian proteins ofscales, claws and ramphoteca, seems to have affected theC-terminus of their molecule, whereas the initial-mid parthas remained more similar to that of their possible commonancestors. The molecular data indicate that archosaurianproteins initially possessed extended glycine-rich regions,

as in the proteins of extant crocodilians (Fig. 15). Given theancient lineage of crocodilians, which were present withsimilar features and adaptations 200 millions years ago,we may speculate that similar long glycine-rich tails werepresent in beta-keratins of the extinct Mesozoic archo-saurians. Glycine-rich proteins may be suited for makinghard scales by giving high resistance and hydrophobicityto these proteins. The glycine-rich tails in proteins presentin extant crocodiles suggest that similar molecules mighthave been present in ancient archosaurians from whichfeathers are believed to be derived (Spearman, 1966;Maderson, 1972; Gregg et al. 1984). In this case, our datasupport the previous view on the reduction of the glycine-tailsin beta-keratins suggesting an evolution from archosaurianscales to feathers (Spearman, 1966; Maderson et al. 1972;Molloy et al. 1982; Gregg et al. 1984; Fig. 14). However, ourcladogram in Fig. 15 (upper panel) also shows that feather-keratins separated early from the proteins of avian andnon-avian archosaurs, and the cladogram suggests thatfeather-keratins are very ancient and underwent anindependent evolution from that of other skin append-ages. This point is at present under detailed phylogeneticanalysis (Dalla Valle et al. 2008).

The comparison between feather- and scale keratinshowed a deletion of the glycine-rich region (52 aminoacids) in feathers from the ancestral sequence present inscales (Gregg & Rogers, 1986). This observation supportedthe morphological conclusions of the evolution of feathersfrom scales (Gregg et al. 1984; Presland et al. 1989a,b).Another change between scale-claws-beak and feather-keratins was the appearance of a feather-specific epitope(TVVGSSTSAAVGSILSCEGVPINS, or similar, see Sawyer et al.2005a). The role of this feather-specific amino acid sequence(underlined in Fig. 14) remains unknown.

The lack of the glycine-tail sequence in feather proteinsmay, however, also mean that scales, claws and beakbeta-keratins evolved differently from those of feathers.The terminal part of this caudal region in feather proteinsalso contains cysteine residues, probably involved inintra-molecular and/or inter-molecular disulfide bondsfor making a strong corneous material. The latter producekeratin bundles more regularly packed than in scales andclaws, permitting the axial elongation of barb/barbulecells (Alibardi, 2002, 2006b).

Concluding remarks and future directions: comparison between the process of cornification in sauropsids and that in mammals

During evolution from reptile-like stem amniotes, oneevolutive lineage of amniotes leading to mammals (thetheropsids) produced mainly non-fibrous keratin-associatedproteins. The other lineage of amniotes, the sauropsids,leading to modern reptiles and birds, instead produced

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Fig. 15 (A) Summarizing cladogram showing the affinities of some representative of sKAPs in all groups (see text for further details). Note in particular the initial separation between lepidosaurian and chelonian/archosaurian proteins, and the following separation of HGPS from HCPS in lepidosaurian. In chelonian/archosaurians note the early separation between feather vs. non-feather proteins. The scale bar indicates the percentage of distance. (B) Hypothetical scheme of the point mutations in ancestral genes responsible for the transformation of a small glycine-serine-rich protein into mammalian or sauropsid KAPs, or histidine-rich proteins in arcosaurians (see text for a more detailed explanation). Whereas in sauropsids similar mutations invested a region that originated the core-box (green) in the lineage of synapsid-mammals, the mutations invested another region (blue) from which no core-box was (generally) formed. Also note that the mutated gene might or not belong to that coding for the variable regions of cytokeratins, assumed to be phylogenetically more ancient than KAPs. Finally, in the mammalian line, the enrichment of cysteine in the V1 and V2 regions of cytokeratins may also have contributed to the formation of the E-regions of trichocytic keratins.



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fibrous keratin-associated proteins (the ‘beta-keratins’). Inparticular in the lineage of archosaurian reptiles fromwhich birds originated, a small type of fibrous protein,feather-keratin, was produced and its characteristicspermitted the formation of barbs and feathers (Fig. 15A).

As both sauropsids and theropsids probably possessglycine-rich proteins it is hypothesized that some mutationsin genes of the ancestral short glycine-rich protein gaverise to HGT proteins in theropsids and HGS in sauropsids.This gene may be derived from that of variable regions ofcytokeratins, or by a divergent evolution from a similargene later incorporated into that of cytokeratins to formtheir V-regions (Fig. 15B). In sauropsids, the mutation ofthe first or the second base of coding triplets determinedthe replacement of serine with proline, cysteine or tyrosinein specific regions of the ancestral protein, giving rise tothe core-box and to cysteine-rich regions and tyrosine-richregions. In theropsids, similar mutation in other parts of theancestral protein might have produced the HGT and HS/UHSproteins, as indicated in Fig. 15B. Also, in the evolutivelineage from which mammals originated (synapsids andtherapsids) other mutations in the triplets of serine might haveintroduced cysteine in the variable region of cytokeratins,transforming this region in the E-regions of trichocytickeratins.

The increasing knowledge of the nucleotide and aminoacid sequences of ‘beta-keratins’ in reptiles, will allowunderstanding of the molecular evolution of these proteinsin archosaurians, chelonians, and lepidosaurians, fromcommon stem reptiles. After a core-box was formed instem reptiles, where a mechanically resistant and scaledintegument was likely present, two main lineages ofevolution of beta-keratins began, the one of arcosaurian/chelonians and the other of lepidosaurians (Fig. 15A).

The regional differences in scale keratins vs. claw, beak,and feather-keratins will clarify the specific role of thesespecialized beta-keratins for the appropriate skin append-ages in different species of reptiles. Further studies areneeded to compare possible homologies between someregions of sauropsid KAPs with mammalian KAPs to revealwhich proteins may have similar roles in hard derivativesof sauropsids and mammalians. The latter goal will befully realized when reptilian genomes and proteomes aresequenced in representatives of lizards, snakes, crocodiliansand turtles.

In conclusion, a broad generalization of the process ofcornification in the skin of all vertebrates can be made.In all skin derivatives, the hard material results from theinteraction between two main protein components,fibrous keratins and matrix or associated proteins. Thelatter are now known in amniotes, where they are repre-sented by sKAPs and mKAPs, whereas no molecular infor-mation is yet available for KAPs of fishes and amphibians.The latter topic would be a fruitful field of investigationfor future studies.

Acknowledgements

Our studies have been financed with grants from the Universitiesof Bologna and Padova, and largely by self-support (L. Alibardi).Dr. Vania Toffolo helped in the initial study on lizard beta-keratins.This paper is dedicated to Prof. G. E. Rogers (University of Adelaide,Australia) for his fundamental studies on the sauropsid keratins,for his influence on the start-up of our molecular studies on reptilianbeta-keratins, and for checking the language of the manuscript.

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REVIEW Evolution of hard proteins in the sauropsid ... · arrowhead) and the darker neck epidermis...

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