Download Evolution of hard proteins in the sauropsid integument in relation to

J. Anat. (2009) 214, pp560–586
doi: 10.1111/j.1469-7580.2009.01045.x
REVIEW
Blackwell Publishing Ltd
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 Nardi2 and Mattia Toni1
1
2
Dipartimento di Biologia evoluzionistica sperimentale, University of Bologna, Italy
Dipartimento di Biologia, University of Padova, Italy
Abstract
Hard skin appendages in amniotes comprise scales, feathers and hairs. The cell organization of these appendages
probably 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. The
evolution of these skin appendages was characterized by the production of specific coiled-coil keratins and
associated proteins in the inter-filament matrix. Unlike mammalian keratin-associated proteins, those of sauropsids
contain 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 of
these proteins into filaments indicated as beta-keratin filaments. The nucleotide and derived amino acid sequences
for these sauropsid keratin-associated proteins are presented in conjunction with a hypothesis about their evolution
in reptiles-birds compared to mammalian keratin-associated proteins. It is suggested that genes coding for ancestral
glycine-serine-rich sequences of alpha-keratins produced a new class of small matrix proteins. In sauropsids, matrix
proteins may have originated after mutation and enrichment in proline, probably in a central region of the ancestral
protein. This mutation gave rise to the core-box, and other regions of the original protein evolved differently in
the various reptilians orders. In lepidosaurians, two main groups, the high glycine proline and the high cysteine
proline proteins, were formed. In archosaurians and chelonians two main groups later diversified into the high
glycine proline tyrosine, non-feather proteins, and into the glycine-tyrosine-poor group of feather proteins, which
evolved in birds. The latter proteins were particularly suited for making the elongated barb/barbule cells of feathers. In
therapsids-mammals, mutations of the ancestral proteins formed the high glycine-tyrosine or the high cysteine
proteins but no core-box was produced in the matrix proteins of the hard corneous material of mammalian
derivatives.
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 different
species (Maderson, 1985; Landmann, 1986; Maderson et al.
1998; Alibardi, 2003, 2006c) (Fig. 1A–D). Details on the
morphology and cytology of reptilian integument have
Correspondence
Dr L. Alibardi, Dipartimento di Biologia evoluzionistica sperimentale,
University of Bologna, Italy. E: [email protected]
Accepted for publication 9 December 2008
been reported in the above references, and will not be
dealt with in the present review. The main goal of this
review is to report the latest data on the present state-ofthe-art in the study of the characteristics of keratinization
in reptilian epidermis. The present account represents a
further step forward from recently published reviews on
this rapidly growing topic (Alibardi & Toni, 2006a; Toni
et al. 2007b).
In general terms, from the basic anapsid cotylosaurs a
new form of corneous proteins, beta-keratins, was produced
from ancestral proteins. The new proteins mainly contributed to the formation of a hard and mechanically
resistant corneous layer. The production of these proteins
increased the strength of the epidermis but a thick and hard
beta-keratinized layer impeded skin elasticity, pliability,
and sensitivity in the derived reptiles.
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
Hard epidermal proteins in reptiles, L. Alibardi et al. 561
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.
Beta-keratins are produced in the epidermis of most
turtles and crocodilians, and in feathers, scales, claw and
beak of birds, whereas they tend to disappear in some
layers of the epidermis of lizards and snakes, where mainly
soft alpha-keratin remains. The evolution of modern
reptilian scales from primitive reptiles with an extensive
dermaskeleton took place by a specialization of the cornification process. Microscopy of scales in different species
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
562 Hard epidermal proteins in reptiles, L. Alibardi et al.
of reptiles is presented in Fig. 1(E–M), and gives a direct
view of their histological structure and layer stratification.
Recent immunological and molecular studies suggest that
the reduction in the production of these proteins may be
associated with the evolution in lepidosaurian reptiles of
a mechanism of shedding, which permits the cyclical loss of
the outer part of the corneous layer (Alibardi, 2006c;
Alibardi & Toni, 2006a). Lepidosaurians show a multilayered
epidermis whose external portion is cyclically shed (Maderson,
1985; Maderson et al. 1998).
Recent immunocytochemical, biochemical and molecular
biology studies have suggested that the cytological
differences among the six main layers of lepidosaurian
epidermis (oberhautchen, beta-, mesos-, alpha-, lacunar and
clear-layers; some layers are shown in Fig. 1E,F) are derived
from the genetic control of the production of their hard
proteins during the shedding cycle (Alibardi & Toni, 2006a–d;
Alibardi et al. 2007; Toni et al. 2007a,b). The molecular
control of the activation or repression of genes for the
synthesis of these proteins still remains to be discovered.
In the tuatara (Sphenodon punctatus), a primitive lepidosaurian, shedding of the outermost generation occurs
through the formation of an intermediate region where
cells possess mixed alpha and beta characteristics, a precursor
of the more specialized shedding layer of lizards and snakes
(Fig. 1G–H). The loss of the outer corneous layer occurs in
this 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 the
last alpha-layer of the previous epidermal generation, the
clear layer. These two layers form a specialized shedding
complex which separates along a shedding line (Fig. 1E).
The synchronization of cell differentiation of the epidermis
over discrete areas of the skin surface in lizards, or over the
entire surface of the skin in snakes, produces a patchy
molting in lizards and a single molt in snakes. After shedding,
the epidermis is covered by a mature beta-layer and a more
or less mature alpha-layer (Fig. 1F).
In crocodilians and turtles a shedding mechanism in the
epidermis is absent or limited, and epidermal histology
and the control on the production of hard-proteins appear
simpler than in lepidosaurians. Crocodilian epidermis
comprises a multilayered epithelium with a transitional
layer of flat cells that are incorporated into a variably thick
corneous layer (depending on the type of scales) (Fig. 1I).
In archosaurians (crocodilians and birds), the process of
shedding 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-rich
corneocytes, suggests that archosaurians may also use this
mechanism of a switch in cell differentiation to create a
scission layer. A different type of sloughing layer is also
formed in regenerating feathers and underneath the
sheath before it is lost to reveal the barb ramification.
In turtles, hard scutes are present in the plastron and softer
scutes or simple folds in the remaining body regions, with
a corneous layer made of soft-keratin (Fig. 1J). The cell
proliferation in the basal layer and the intense protein
synthesis of transitional cells near the hinge regions of the
growing scutes layer allow the growth of scutes (Fig. 1J–M).
In the scutes of the carapace and plastron of some species
of turtles (chelonians), the switch from thicker beta- into
thinner alpha-keratin cells determines the formation of a
shedding layer. The latter permits a slow intra-corneous
detachment of the more external layers with the sloughing
of the superficial part from the more internal part of the
corneous layer (Fig. 1K). A new beta-layer allows for the
next stratification of the stratum corneum; this process
occurs in shedding turtle but not in non-shedding turtle or
in the tortoises where the superficial part of the corneous
layer is gradually lost by natural wear.
Scale development and epidermal layers in reptiles
and birds
Scales in reptiles derive from the interaction between a likely
inductive mesenchyme and a large surface of the epidermis
(Fig. 2). Extensive reviews of studies on the embryology in
reptilian skin have been presented in previous accounts,
and the reader can find further information in these
publications (Maderson, 1985; Alibardi, 2003, 2004b, 2005).
It has been hypothesized that areas of dermal–epidermal
interactions (ADEIs) are relatively large in reptiles, including
pre-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).
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
Hard epidermal proteins in reptiles, L. Alibardi et al. 563
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
564 Hard epidermal proteins in reptiles, L. Alibardi et al.
directly or indirectly responsible for the activation of
several genes in the epidermis, including those coding for
the production of beta-keratins. Beneath an embryonic
epidermis made of two to six layers, depending on the
reptilian species, the accumulation of beta-keratins determines sloughing at hatching (Fig. 2A-A7). Among reptiles
the crocodilians are particularly interesting for their phylogenetic affinity with birds, and the analysis of their embryonic
epidermis reveals the highest sequence identity with that
of birds. In the latter, considered here to be a lineage of
archosaurian reptiles, a special morphogenetic process has
evolved for the production of feathers from embryonic
skin (Maderson, 1970; Brush, 1993; Sawyer et al. 2005b;
Alibardi et al. 2006b). The process has determined the
selection of small proteins the size of feather-keratins,
which have been utilized for the formation of barbs, as
shown in Fig. 2.
Recent studies on the embryonic epidermis of alligator
and birds have produced further indications that feathers
may derive from a generalized archosaurian epidermis
(Sawyer & Knapp, 2003; Sawyer et al. 2005b; Alibardi, 2006b;
Fig. 2A–F). During development, different layers with a
specific fate are produced in sequence in the epidermis.
These studies have suggested that specific cell populations
were selected from the generalized archosaurian epidermis
to produce specific epidermal structures such as scales, beaks
or claws, and for the formation of feathers. In particular,
some cell populations containing a specific type of featherlike keratin molecule were selected among others to
produce barb and barbule cells and eventually feathers.
In the alligator and in the avian embryonic epidermis,
outer and inner periderm layers are initially formed and
contain similar periderm granules (Fig. 2B–F). Over most of
the 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 inner
periderm cells are instead present, whereas this increases
to six to eight layers in the beak (Kingsbury et al. 1953;
Alibardi, 2002, and personal observations). Beneath the
inner periderm, one subperiderm layer is present which
contains feather-keratin immunoreactivity (Fig. 2C,E). It has
been suggested that the displacement of the subperiderm
layer into barbule plates and a ramus area eventually
generated feathers (Fig. 3D). The subperiderm layer in all
archosaurian epidermis contains a 24-amino acid featherkeratin epitope (AVGSTTSAAVGSILSEEGVPINSG) that
disappears at hatching except in feathers (Sawyer et al.
2005a,b). Therefore this epitope may be an ancient component of the epidermis of archosaurian reptiles.
Maderson & Alibardi, 2000), probably by a drastic alteration
of the morphogenetic pattern of dermo-epidermal interactions. Reptilian scales are either of the scute type, as in
extant crocodilians and chelonian shell, or appear as tuberculate or pebble-like scales, such as those described in
fossilized archosaurians, including dinosaurs (Martin &
Czerkas, 2000; Prum & Brush, 2002). It is believed that
during the evolution of the avian skin the rigid corneous
layer of archosaurian scales progressively became restricted
to smaller and smaller areas of ADEIs, while most of the
skin became non-specialized and softer, and was the origin
of the apteric skin (Alibardi, 2004b, 2005). The flat scale in
archosaurian ancestors became progressively narrower,
tuberculate and the epidermis was nourished from a
vascularized mesenchyme, a condition that resembles that
of feather filaments (Fig. 3A–C). The latter also exchanged
morphoregulative or inductive signals with the epidermis,
and this activity determined the formation of specialized
layers 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 or
filiform appendages are interpreted as both protofeathers
(summarized in Prum & Brush, 2002; Wu et al. 2004) or
collagenous structures (Lingham-Soliar et al. 2007). The
detailed cytological analysis of the process of feather
morphogenesis has, however, indicated that some fossilized
‘protofeathers’ may be genuine remnants of barbs with
different degrees of branching (Alibardi, 2006a,b,c).
According to the latter studies, the hairy-like elongation
was hollow, and when epithelial folds appeared in the
epidermis of these hypothesized filaments, the formation
of feathers began. The first stage was the formation of barb
ridges that could evolve into different morphogenetic
mechanisms to produce barbs with extensive ramification
(Fig. 3E-E3), short ramification (Fig. 3F-F3) or without any
ramification (Fig. 3G-G3) (Alibardi, 2006a,b). Similar
morphotypes of downfeathers have been found in fossilized
feathers or filamentous structures (summarized in Prum &
Brush, 2002; Wu et al. 2004).
The above hypothetical stages are supported by morphological and biochemical data. Reptilian and avian scales
and feathers are mainly composed of scale and featherkeratins, and feathers can develop from scales under some
natural or experimental circumstances (Dhouailly & Senegel,
1983; Chuong, 1993; Chuong & Widelitz, 1999; Sawyer et al.
2005b). The successive fusion of barb ridges into an axial
rachis gave rise to pennaceous feathers of different shapes
and sizes (Fig. 3H-H3).
Feathers as a skin specialization of archosaurian
epidermis
Hairs as a skin specialization of theropsid reptiles
It is still believed that feathers might be derived from
archosaurian scales (Spearman, 1966; Maderson, 1972;
It is believed that the evolution of hairs from basic reptiles
resulted from the reduction of a primordial, scaled skin
into a hairy and softer skin (Maderson et al. 1972; Alibardi,
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
Hard epidermal proteins in reptiles, L. Alibardi et al. 565
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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Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
566 Hard epidermal proteins in reptiles, L. Alibardi et al.
2003; Wu et al. 2004). Also in this case, it is hypothesized
that the initial expanded ADEIs of reptilian scales became
progressively confined to small areas, giving rise to
dermal 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 the
possible evolution of hairs, which cannot be considered
here. Unfortunately, while some feathers and corneous
filaments remain in fossils, no fossilized hairs have so
far been reported in the paleontological literature and
therefore we do not have any skin evidence about hair
evolution. Also the developmental stages of hair development provide few clues about the evolutive transition
from a scale to a hair as the two structures are very different
(Fig. 4B-B7).
Hairs develop from an invagination of an epidermal
placode in contact with a special mesenchymal condensation
to form a peg that grows downward into an elongated, clublike epidermal tongue (see stages of hair morphogenesis,
Philpot & Paus, 1999). From the neck of the elongated
peg some cells become organized in a cone-like layer that
grows upward and initiates the hair shaft that grows
toward the epidermal surface (Fig. 4B5–6). In the meantime,
the mesenchyme at the bottom of the club (the future hair
bulb) penetrates into a small cavity that is formed at the
base 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 open
tube, a study that, if confirmed, would provide some
evidence of a process of invagination (see references in
Alibardi, 2004a,b).
Other hard skin derivatives present in mammals are
represented by nails, quills, claws, horns, hooves, corneous
teeth in whales, etc., all considered to incorporate hard
keratins (Gillespie, 1991). Their embryological origin is
partially known but all of them appear to have an intimate
dermal–epidermal connection which directly or indirectly
induces 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 keratins
has been more precisely defined. Today, two main components are recognized in the cells (trichocytes) of mammalian hard corneous derivatives (hairs, nails, horns), a fibrous
component and a matrix or inter-filament component. The
fibrous component consists of a specific set of alpha-keratin
proteins, composed of the keratin intermediate filaments
(KIFs), with molecular weights of 40–70 kDa. These keratins
have been sequenced from their genes cloned from human
and mouse (Langbein et al. 1999, 2001; Langbein & Schweizer,
2005), and are classified into two types based on their
sequence homology, namely 11 type I (acidic) and nine
type II (basic). The spatial-temporal sequence of expression
of these hair keratins varies at the different layers and
level of developing hairs, suggesting different mechanical
roles in differentiating trichocytes (Fig. 4).
The matrix or inter-fibrillar component comprises ‘keratinassociated proteins’ (KAPs), generally with smaller molecular
weight (8–30 kDa) (Gillespie, 1991; Powell & Rogers, 1994;
Rogers et al. 2006). Over 100 KAPs have been identified and
form the matrix among KIFs that effectively results in the
hard and resistant corneous material of hairs, nails and
horns (Gillespie, 1991). The KAP proteins are not fibrous,
but more globular in conformation, but the molecular
organization of these proteins and their linkage with KIFs
is not well understood (Rogers, 2004; Rogers et al. 2006).
X-ray diffraction, electron microscopy and chemical
studies have demonstrated that hair KIFs contain about
70% of alpha-helical domains, the keratin intermediate
filaments are 7–10 nm thick (Filsie & Rogers, 1961; Fraser
et al. 1972), and disulfide links KIFs together and also links
KIFs 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.
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
Hard epidermal proteins in reptiles, L. Alibardi et al. 567
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
568 Hard epidermal proteins in reptiles, L. Alibardi et al.
When hair fibers or films of corneous material (produced
after chemical extraction or by direct application of X-rays
to thin pieces of the corneous material, see Fraser et al. 1972),
are stretched or heated, the alpha-type X-ray diffraction
pattern changes to what is termed the beta-pattern. The
alpha-helical conformation of the keratin chains in the
KIFs under stress elongates to form regions of beta-pleated
sheets. Early stages of stretching or heating can be reversible
(Fig. 5A). Reptilian/avian keratins, in which there are regions
containing beta-sheets, are referred to as beta-keratins and
are very different from the keratin proteins of intermediate
filaments.
Mammalian KAPs (mKAPs) are grouped into three types
with a molecular weight range of 6–36 kDa. They are
distinguished by their content of particular amino acids,
which include glycine-tyrosine-rich (HGTs), high sulfur
proteins (HSPs), and ultra high-sulfur proteins (UHSPs).
Each of these protein groups is a family of proteins of related
sequence. Their regulated expression in the differentiating
hair shaft in the follicle follows that of specific acidic/basic
trichocytic 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 synthesized
in the lower part of the differentiating zone (pre-cortex),
located above the matrix zone, HSP (KAPs 1–3) in the intermediate region, UHSPs (KAPs 4 and 12) in the maturing
cortex, 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). Some
examples of amino acid sequences of human KAPs are
shown in Fig. 5B, where four key amino acids (glycine,
cysteine, serine and proline) are colored for comparison
with the proteins present in sauropsids (reptiles including
birds). The number of amino acids varies from 57 to 90 in
HGT proteins, 98 to 298 in HS proteins, and 114 to 205 in
UHSs, with molecular weights variable from 5.4 to 36 kDa.
The prediction of the secondary structure in mammalian
KAPs (mKAPs) using the PSIPRED program (Alibardi et al.
2007; Toni and Alibardi, unpublished data) on 128 mKAPs
has shown that only few HSs present strand regions, and a
few (less of 5.5% in our analysis) show regions with a
core-box structure.
Figure 5D shows schematically that KAPs surround the
numerous alpha-keratin filaments and become organized
around them, forming the matrix of the corneous material.
The beta-keratins in sauropsid epidermis and its
derivatives
The keratin proteins of reptiles have remained poorly
known, compared with those of mammalian and avian
proteins, for over 30 years. They were considered to consist
of soft (alpha)-keratin and hard (beta or phi)-keratins
(Maderson, 1985; Landmann, 1986; Alibardi, 2003). The amino
acid sequences of keratins present in reptilian corneous
structures (scales, claws, ramphotheca, etc.) were unknown
until recently. The hard proteins of reptiles and birds are
proteins (beta-keratins) with a fibrous structure that gives
a beta-pattern by X-ray diffraction and a filament of 3 – 4 nm
in diameter, as first described by Filsie & Rogers (1961).
The amino acid composition and molecular weights of
beta-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 layers
had been sequenced, and only one small keratin from a
lizard claw was known in its primary amino acid structure
(Inglis et al. 1987). However, it was unclear whether this
protein was representative of reptilian scales or merely
reflected the composition of a specialized claw keratin.
Also, a 20-amino acid sequence localized in an inner
region of an alligator beta-keratin was known, and this
epitope shows high identity with that of avian betakeratins (Sawyer et al. 2000, 2005a).
It was known that different proportions of specific
monomers of beta-keratins, called phi-keratins, are present
in reptilian derivatives of different rigidity (Wyld &
Brush, 1979, 1983; Thorpe & Giddings, 1981; Gillespie et al.
1982; Marshall & Gillespie, 1982; Inglis et al. 1987). These
proteins are of small molecular weight, mainly in the
10–20 kDa range, but a component of higher molecular
weight was also found (30–44 kDa), although in minor
amounts. It was not clear whether they were true polymers
or aggregates derived from chemical extraction. Different
forms (monomers) are present in various skin derivatives
such as dorsal and ventral scales, shell scutes, claws, and
ramphotheca in turtles, lizards, snakes and crocodilians.
According to Wyld & Brush (1983), phi (beta)-keratins of
reptiles are believed to be phylogenetically distant from
beta-keratins of birds, or even unrelated to feather-keratins
(Brush, 1993). The latter study in particular pointed out
that feather-keratins and feathers are a completely new
innovation of avian skin.
Other studies on crocodilian and avian beta-keratins
have indicated that these proteins can be distinguished
into a few main types: scale, beak, claw, feather or featherlike 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 species
to species in their amino acidic sequences, and those in
archosaurian skin (crocodilians) are strictly correlated to
those of birds. Scale, claw, and beak keratins showed
a relatively higher molecular weight, 14–18 kDa, whereas
feather or feather-like keratins possessed a smaller molecular
weight, 10–12 kDa (Gregg & Rogers, 1986). Studies on
feather genes suggested that at least 30–40 genes alone
were present, and other 10–20 genes were found for scale,
claw and beak keratins.
Early knowledge on the process of cornification in
mammals vs. reptiles/birds indicated that some striking
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
Hard epidermal proteins in reptiles, L. Alibardi et al. 569
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.
570 Hard epidermal proteins in reptiles, L. Alibardi et al.
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.
differences were present. In the corneous skin appendages
of reptiles/birds, beta-keratin aggregates to form filaments
differently from what occurs with alpha-keratins (Gregg &
Rogers, 1986; Gillespie, 1991; Brush, 1993). This suggested
that a more central region of the molecule of beta-keratin
contributed to make the fibrous framework of the filamentous polymer, while the extremities formed the matrix.
This was a different mechanism from that present in
mammalian corneous appendages. It is not clear why such
different mechanisms of cornification originated in reptiles/
birds compared to mammals as they have a common
reptilian progenitor.
The resulting alpha-keratin pattern of mammals vs. the
beta-keratin pattern of sauropsids might be due not to a
difference in keratins but instead to a different composition
and organization between proteins or the fibrous component (alpha-keratins) vs. those of the matrix component
(keratin-associated proteins). This organization is different in
the corneous material of mammals compared with that in
reptiles/birds, but in both cases the corneous material
consists 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 studies
on reptilian epidermis have shown the presence of both
cytokeratin and beta-keratin immunoreactivity (Alibardi &
Sawyer, 2002; Alibardi, 2003, 2005, 2006c; Alibardi & Toni,
2006a–d; see examples in Fig. 6). Controls, omitting the
primary antibody, are unlabeled (data not shown). Alphakeratins in reptilian corneous material were revealed
using broad-spectrum cytokeratin antibodies such as AE3
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
Hard epidermal proteins in reptiles, L. Alibardi et al. 571
and Pan-cytokeratin antibodies, which recognized different acidic and basic cytokeratins (Alibardi & Toni, 2006a).
The latter indicated that beta-keratins in reptiles and
birds have broad cross-reactivity as shown by a chick general
antibody (beta-1) (Alibardi & Sawyer, 2002). Later analysis
indicated that most reptilian and avian beta-keratins also
contain a ‘universal’ antigen found in avian and alligator
beta-keratins with the sequence SRVVIQPSPVVVTLPGPILS
(Sawyer et al. 2000, 2005a). This sequence, or the homologous sequences, contained in a central region of all known
beta-keratins, were indicated as the core-box (Alibardi &
Toni, 2006a, 2007). The final confirmation of the precise
localization of beta-keratins in reptilian epidermis came
after the isolation of antibodies produced in rat and directed
to 14–17 kDa proteins isolated from reptilian epidermis
(Alibardi & Toni, 2006c,d).
Our proteomic studies on epidermal proteins in reptiles
have initially confirmed that beta-keratins generally fall
within the 12–20 kDa range (Alibardi & Toni, 2006b,c,d;
Toni & Alibardi, 2007a,b,c; Toni et al. 2007a,b). The use of
general and more specific antibodies for alpha- and betakeratins after two-dimensional electrophoretic separation
has identified mainly acidic and neutral alpha-keratins in
the 40–70-kDa range (Fig. 6G). The other large group of
proteins, beta-keratins of 10–20 kDa, are mainly basic and
neutral, and there is also a small fraction that appears to
be acidic (Fig. 6G). Therefore alpha- and beta-keratins may
be complementary and thereby associate into keratin
filaments, a unique condition of sauropsid keratins.
These immunological proteomic studies indicated that
beta-keratins seem to associate with alpha-keratins as
mammalian KAPs associate with keratin intermediate
filament keratin proteins (KIFs) in mammalian tissues,
suggesting that beta-keratins may actually function as
reptilian KAPs. Matrix proteins so far known in amniote
epidermis (filaggrin and trichohyaline) are basic and they
have charged interactions with neutral or negatively charged
groups present in keratin filaments (Mack et al. 1993). In
avian feathers two basic KAPs, indicated as CAP-1 and
CAP-2, and four basic histidine-rich proteins (HRPs) are
different from beta-keratins (Gregg & Rogers, 1986; Presland
et al. 1989a; Knapp et al. 1991,1993; Barnes & Sawyer, 1995).
In summary, all amniotes produce corneous material using
alpha-keratins as fibrous material and keratin-associated
proteins as a matrix. The peculiarity of reptilian and bird
KAPs is that the proteins form filaments that replace or
mask those of alpha-keratins. This derives from the special
amino acid composition of some key regions of reptilian
and avian KAPs, as it will be illustrated in the next section.
Beta-keratins in reptilian and avian keratinocytes of
scales and feathers form fibrous polymers and filaments
(Fraser et al. 1972; Brush, 1983) as compared to mammalian
hard corneous material of hairs, nail and horns, where
mKAPs are mainly amorphous. The beta-keratins are also
different from intermediate filament proteins in com-
position, chemical-physical properties, and mechanism
of polymerization. Therefore small proteins previously
indicated as beta-keratins (fibrous proteins with regions of
beta-pleated sheet) should be considered a fibrous type of
keratin-associated or matrix proteins (sauropsid KAPs)
(Alibardi et al. 2007; Toni et al. 2007a,b). The term betakeratins will be used in the present review as being equivalent
to sauropsid KAPs (sKAPs). However, it is generally agreed
that true keratins are defined as belonging to the keratin
intermediate filaments family (Steinert & Freedberg, 1991;
Fuchs & Weber, 1994; Coulombe & Omary, 2002).
The lack of knowledge about proteins and their genes
involved in the production of hard proteins in reptilian
scales has impeded a proper evaluation of the evolution of
these proteins from reptilian ancestors to birds. The essential,
missing information in reptiles was the protein sequence,
secondary and tertiary conformation, and the genes coding
for these hard proteins. The recent genomic and proteomic
studies on reptilian beta-keratins have rapidly clarified this
issue and the new information suggests a new interpretation
that 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-keratins
have been initially obtained by designing degenerate
primers from specific regions (core-box) of avian and a lizard
claw beta-keratins (Inglis et al. 1987). These primers were
then replaced by more specific primers selected on the
same reptilian regions (Dalla Valle et al. 2005, 2007a,b,
2008 and 2009). This work has aided the deduction of the
amino acid sequences of lepidosaurians (lizards, snakes),
chelonian, and crocodilian beta-keratins. The nucleotide
and protein sequences of different reptilian keratins are
now deposited in GenBank under specific accession
numbers.
This initial information has been now integrated with
the analysis of the lizard Anolis carolinensis and chicken
genomes (Dalla Valle et al. unpublished studies).
These data have allowed, for the first time, the complete
comparison of the nucleotide and amino acid sequences
and protein structure of different reptilian beta-keratins
with avian keratins. In fact, the nucleotide structure of betakeratin cDNAs so far sequenced from reptilian epidermis
(Dalla Valle et al. 2005, 2007a,b) show a general linear
organization that resembles the avian nucleotide structure
for 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, according
to the first data, also in turtle, show the same organization
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
572 Hard epidermal proteins in reptiles, L. Alibardi et al.
as previously found in avian beta-keratin genes: the coding
region is uninterrupted, whereas an intron of variable
length is present in the 5′-untranslated terminal region
(5′-UTR) (Fig. 7). The first reptilian gene coding for betakeratin containing an intron has been sequenced in the
snake Elaphe guttata (now called Panthoteris guttatus)
(Dalla Valle et al. 2007b). In the snake gene an intron of
226 bp is present after 29 bp of the 5′-UTR and it is followed
by a short non-translated region of 17 bp. A similar result has
been found in the crocodile Crocodylus niloticus, in which the
5′-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 cDNA
and the genomic sequences obtained by PCR using sense
primers located at the beginning of the 5′-UTR and antisense
primer covering the stop codon. However, this approach
gave a different result in the two species of geckos analysed:
a fragment of the same length was amplified with both
cDNA and genomic DNA. Sequencing showed that no intron
was present in the two genomic fragments obtained (Dalla
Valle 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 of
genes coding for beta-keratin proteins similar to those
found in other lizards. In total about 40 lizard genes are
present in this species; moreover, as all genes were found
in the same scaffold, these genes are probably localized in
one single chromosomal locus (Dalla Valle et al. work in
progress). The accessibility of A. carolinensis genome allowed
direct comparison of the cDNA with the genomic sequences.
A preliminary 5′-RACE analysis on some beta-keratin mRNAs
expressed in the epidermis of the green anole (Anolis
carolinensis), showed that they contain an intron in their
5′-UTR (Dalla Valle et al. unpublished results). At present it
is known that one intron of variable length is present in
the 5′-untranslated region of beta-keratins in all reptiles:
snake, crocodile and in the turtle. It is not known whether
the intron may be the regulative region for the transcription.
Although in gecko lizard genes so far analysed (from two
different species) no intron has been found, these observations suggest that other beta-keratin genes within the
lizard genome likely possess introns. The presence of one
intron in the 5′-UTR of reptilian beta-keratin gene is probably
general but not always constant. More work, such as the
5′-RACE analysis of beta-keratins of the green anole is
necessary to clarify this point.
Genomic Southern analysis using reptilian β-keratin cDNA
as a probe has indicated the presence of several related
genes in the genome of geckos, snake, crocodile and turtle
(Dalla Valle et al. 2007a,b, 2008 and 2009). These data
have confirmed that also in reptiles β-keratins belong to
a multigene family as demonstrated in the chick (Gregg
et al. 1983, 1984; Presland et al. 1989a,b; Whitbread et al.
1991). These results are also confirmed after some cloning
experiments leading to the sequencing of more then one
gene in each analysed reptilian species. When performed,
PCR analysis indicates a head-to-tail orientation of
these genes in their cluster. However, the analysis of
A. carolinensis and avian genomes reveals that, although
this is the common orientation, genes are also present that
are arranged with tail-to-tail or head-to-head orientation
(work in progress).
The in situ localization of messenger RNA for these proteins
reveals a strict epidermal expression, mainly localized in the
differentiating oberhautchen, in beta-layers of lepidosaurians, and in the pre-corneous layer of crocodilian
and turtle scales. (Fig. 8A,B,D–F). The ultrastructural in situ
hybridization analysis of mRNA gene expression has shown
that transcripts are present in the cytoplasm associated
with the initial sites of deposition of beta-keratins (betapackets; Fig. 8J). However, the periphery of larger bundles
of filaments often presents beads of gold particles that
identify antisense digoxigenin-labeled probes associated
with the filaments (Dalla Valle et al. 2005; Alibardi et al.
2006a). This finding suggests that the protein still in the
translation phase can be directly packed in the growing
bundle.
The genomic structure of avian beta-keratin genes is
organized on the reptilian model
Previous information on chick scale, claw, beak and featherkeratins indicated that their genes are clustered in the
same 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 betakeratin genes nor their location was indicated. Genes
appear to be arranged in multiple copies with the same
orientation in the genome. They contain one intron in the
5′-UTR, perhaps with a function involved in transcription
regulation. A previous summary of all known avian betakeratin genes indicated the presence of 18 genes for featherkeratins, 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.
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
Hard epidermal proteins in reptiles, L. Alibardi et al. 573
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Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
574 Hard epidermal proteins in reptiles, L. Alibardi et al.
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.
for claw keratins and nine genes for scale keratins. No
indication was available on the number of genes for beak
keratins (summary in Stevens, 1996). This was clearly a
large underestimation of avian beta-keratin genes, as
initial studies indicated the presence of 100–240 different
genes for feather-keratins alone (Kemp et al. 1974). The
latest analysis on the chick genome shows that probably
around 140 genes for feather, claw, scale and beak keratins
are present, not taking into account pseudogenes or
incomplete sequencing data (Dalla Valle et al. unpublished
results). Among beta-keratin genes, 56 are clustered in
chromosome 25, whereas the other 64 genes are present
in chromosome 27. A few beta-keratin genes (from a single
copy to seven copies) are present in five other chromosomes,
but they may be copies of some genes present in chromosomes 25 and 27.
The structure of the chick feather gene (gene C) (Gregg
et al. 1984; Gregg & Rogers, 1986) comprises a 5′-UTR of 93
bp, which is interrupted after 37 pb by an intron of 329 bp.
Like reptilian genes, the coding region of avian genes is
devoid 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 genome
analysis. The intron may be of some importance for the
regulation of the velocity of transcription of beta-keratin gene
(Koltunow et al. 1986). The possibility to increase transcription may explain the rapid production of feather-keratin
in 2–3 days, from 13–16 days of development in the chick
embryo (Hamburgher-Hamilton stages 37–39; Kemp et al.
1974; Gregg & Rogers, 1986). In a large part of the noncoding region of the beta-keratin gene cluster, is a highly
conserved nucleotide sequence, especially nucleotides 1–37,
indicating an essential physiological role in the transcription and translation process. The 3′-UTR part of the gene
is much less conserved than the other regions.
From the above description and the recent data on the
gene organization of crocodilian beta-keratins it is likely
that beta-keratins of birds are derived from an archosaurian
organization of this family of genes that pre-dated the origin
of birds. The latter inherited this basic organization and
evolved their own specific sequences for the formation of
scale and feather keratins (see description below).
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
Hard epidermal proteins in reptiles, L. Alibardi et al. 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).
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
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 sequenced
and 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; Hallahan
et 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 point
of some deduced proteins correspond to specific spots
obtained following two-dimensional separation of
epidermal proteins, which confirms molecular findings
(Toni et al. 2007a,b and unpublished results). Analysis of
the deduced amino acid sequences by specific software
can graphically predict secondary structure (Fig. 9). The
study of some representative beta-keratins in lizard, snake,
turtle, crocodile and birds has demonstrated the presence
of a central region where two strands (beta-folded
secondary conformation), made up of four to seven amino
acids each, are present in-tandem. The core-box represents
the region with the highest sequence identity and is
present within a 32-amino acid region that contains the
strand or beta-pleated sheets in beta-keratin monomeres
(Fraser et al. 1972; Brush, 1993; Fraser & Parry, 1996). A
recent study on feather and reptilian beta-keratins, based
on a different method of analysis of the secondary conformation, reported the presence of four beta-strands
in this region (Fraser & Parry, 2008). The predictions show
that short beta-sheets made of four to eight amino acids,
generally not organized in tandem, are present in other
regions of these small proteins (Fig. 9, and data not shown).
The role of the extra strands in these proteins remains
undetermined but awaits more information from the
prediction of their tertiary structures (ongoing analysis in
our laboratory).
The determination of the amino acid sequence for betakeratins from lizards, snake, turtle and crocodile, together
that for a lizard claw (Inglis et al. 1987), has indicated that
some regions of these proteins present high sequence
identity with avian keratins (Dalla Valle et al. 2005, 2007a,b).
These predictions show some of the structural homologies
of reptilian and bird proteins, especially in the core-box or
beta-pleated sheet region (Figs 9, 10).
Surprisingly, the predicted secondary structure of the
deduced proteins from turtle resembles those of crocodilians
Fig. 10 Comparative representation of some sKAPs from all different reptilian groups. Some key amino acids are specifically colored to reveal their preferred location within these proteins. Note in particular the conservation in the corebox region and the presence of cysteines mainly toward the N- and C-regions in all groups of reptiles and birds, where they probably intervene in cross-linking. Glycine-rich regions in HGPS proteins of lepidosaurians are present toward the
N- and C-regions. Cysteine residues in lepidosaurian HCPS proteins are also present toward the N- and C-terminals. Serines are mainly present outside the core-box where they may be involved in phosphorylation or in other post-translational
processes. In the chimeric-like chelonian/archosaurian proteins a cysteine-rich region is particularly evident, while the glycine-rich region is mainly present toward the C-terminus. Finally, the glycine-rich region disappears in the specialized,
smallest feather proteins (FK). Goanna-claw (Inglis et al. 1987); Anolis Ker 20, 22, 23, 24, 25; Ge-gprp-1 (CAJ44302); Ge-gprp-3 (CAK19321); Ge-gprp-4 (CAJ90467); Ge-gprp-5 (CAK19322); Li-gprp-1 (CAJ67601); Li-gprp-3 (CAJ90483);
Li-gprp-5 (CAJ90485); Sn-gprp-1 (CAL49457); Sn-gprp-5 (CA CAL51276); Sn-gcrp-1 (work in progress); Tu-gptrp-1 (CAO78677); Tu-gptrp-2 (CAO78678); Cr- gptrp-1 (CAO78748); Cr-gptrp-2 (CAO78749); Chick-scale (P04459), Chickbeak (AAO85139), Chick-claw (AAA62730), Chick-feather I–IV (P02450, P04458, P20307, P20308); Chick-keratinocyte (NP-001001310); Turkey-vulture-feather (Q98U06); Wood-stork-feather (Q98U05); Pigeon-feather (BAA33471); Duckfeather (PO8335).
576 Hard epidermal proteins in reptiles, L. Alibardi et al.
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
Hard epidermal proteins in reptiles, L. Alibardi et al. 577
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).
578 Hard epidermal proteins in reptiles, L. Alibardi et al.
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.
and birds, which can be included in a subgroup different
from those of lepidosaurians. This suggests that turtle and
archosaurians have a closer or common basic archosaur
progenitor.
While the concentration of proline distorts the polypeptide chains, the presence of numerous valines, isoleucine
and leucine in some cases, determines the formation of a
beta-sheet structure in this region (Fig. 11, top panel).
The beta-strands present in this region of the protein
are considered the site of polymerization of beta-keratin
monomers into the long polymer that forms the filaments
of beta-keratin 3–4 nm in diameter (Fraser et al. 1972;
Brush, 1983; Gregg & Rogers, 1986; Fraser & Parry, 1996). This
region probably has a three-dimensional conformation
acting as an anchoring point for the interaction with
similar proteins (monomers). The random coiled regions,
generally representing most of the protein sequence, are
regions with free rotation of R-groups of amino acids along
the fixed -CO-NH- peptide bond.
Our biochemical studies have shown that alpha-keratins
are present in hard corneous material of reptilian scales,
so that proteins of intermediate filaments are probably
masked by the deposition of beta-keratins (sKAPs). It
seems that in scales and claws of different reptiles and
birds, in the beak and feathers, the amount of sKAPs
capable of forming filaments is generally larger than that
of alpha-keratins.
The synthesized beta-keratins present in different appendages of sauropsid integument (scales, claws, ramphoteca
or beak, and feathers) can form bundles of corneous material
as shown in Figs 6, 12 and 13. Alpha-keratin is generally
replaced or degraded, whereas sKAPs accumulate and make
filaments through the polymerization of their core-boxes.
The abundance in proteins containing the beta-strands is
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
Hard epidermal proteins in reptiles, L. Alibardi et al. 579
probably responsible for the prevalent beta-keratin pattern
observed after X-ray diffraction on the corneous layer of
reptilian epidermis (Baden & Maderson, 1970; Fraser et al.
1972). It is believed that the stabilization of the filaments
of beta-keratin occurs by homophilic interactions in the
glycine-rich regions and by intra- and inter-molecular
disulfide bonds. The latter are favored by the prevalent
localization of cysteines toward the extremities of these
proteins, in proteins both with low and high amounts of
this 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 lepidosaurian proteins shows that, outside the core-box, most of
their molecule contains a random coil conformation and alphahelix regions are relatively scarce. According to the content
of some amino acids, lepidosaurian proteins can be generally
divided into high glycine proline serine (HGPS) and high
cysteine proline serine (HCPS) (Fig. 10). The latter presumably
form numerous disulfide bonds and may be prevalent in
the harder corneous material of reptilian scales or in claws.
Further analysis of the primary and secondary structure
of glycine-proline-rich proteins of crocodilian and turtle
epidermis shows that long regions of the proteins are
occupied by a random-coil conformation (Fig. 9).
In the crocodile, after the core-box, from amino acid 57
to 171, near the N-terminal, a region rich in glycine-X and
glycine-glycine-X sequences is found. In crocodile glycineproline-tyrosine-rich protein 1 (Cr-gptrp-1), 19 GGX repeats
are present. In particular, from amino acid 100 to 135 alternate
repeats of six GGY-GGL are found in Cr-gptrp-1 (Figs 9, 14,
Dalla Valle et al. submitted). Even more remarkable is the
23 GGX repeats found in Cr-gptrp-2 protein, the longest so
far isolated in any sauropsid protein. As in crocodilian betakeratins, chick scale (65–74% identity) and claw (66–68 %
identity) contain long glycine-rich sequences although they
are shorter than in crocodile. Feather-keratins are missing
such sequences (Gregg et al. 1984). This progressive reduction
of glycine-rich sequences from the C-terminus toward the
core-box of archosaurian scales vs. feather-keratins indicates
an evolution of the protein toward simplification. This
change was somehow favorable for the specialization of
feather-keratin to form long bundles for the elongation of
barb/barbule cells (Fig. 13). The small proteins form better
oriented filaments with fewer cross-links than larger proteins
with long glycine-rich regions (Fraser et al. 1972; Gregg &
Rogers, 1986). Feather-keratins tend to resist tension better
than scale keratins as they are more oriented. Scale proteins
present a less organized distribution, which increases
mechanical resistance (Brush, 1993).
Turtle proteins resemble those of archosaurian scales and
claws, both crocodilians and birds (Figs 9–11, Dalla Valle
et al. 2009). Among these characteristics are the richness
of tyrosine and the high sequence identity of the core-box
region and in surrounding amino acid regions. Therefore,
based on sKAPs in the epidermis, turtle, crocodile and birds
appear to share a common progenitor, and this reinforces
the recent evidence that turtle and archosaurians taxonomically are much closer to each other than to lepidosaurs
(Rest et al. 2003).
Some regions of these proteins show sequence identity
with mammalian KAPs (Alibardi et al. 2006a) that are present
in hard keratinized appendages (hairs, claws, horns, etc.,
see Gillespie, 1991; Marshall et al. 1991; Powell & Rogers,
1994). However, in comparison with mammalian high
glycine-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 and
archosaurian/chelonian proteins are also emphasized
by the further detection of different amino acid regions,
here provisionally indicated as pre- and post-core-boxes, that
characterize archosaurians and chelonian vs. lepidosaurian
beta-keratins (Fig. 11).
In summary, the central core-box of 20 amino acids
contains two beta-strands and has 75–90% identity within
archosaurian/chelonian KAPs, whereas a pre-core-box of
17 amino acids, indicated as archosaurian/chelonian-box,
has also high identity (Dalla Valle et al. 2007a,b, 2008 and
2009). These values fall to 60–75% when comparing the corebox of crocodilian/chelonians with those of lizards or snakes.
The latter contain a pre-core-box called the lepidosaurianbox (Fig. 11). The high sequence identity based on the corebox of their beta-keratins confirms that crocodilians and
birds are the closest living archosaurians. Another difference
between lepidosaurian and chelonian-archosaurian betakeratins is that in the former the core-box is localized from
halfway to the C-terminus of the protein, whereas in the
latter the core-box is moved toward the head to the middle
of the protein (Fig. 10). The specific role of these and of
other regions of reptilian KAPs remains to be discovered.
Evolution and phylogenetic relationships among
sauropsid KAPs
The recent knowledge on genes and protein sequences
has addressed for the first time the long-debated problem
of the evolution of these small proteins in reptiles and
birds (Wyld & Brush, 1979, 1983; Maderson, 1985). A general
phylogram obtained using the neighbor-joining method
(Saitou & Nei, 1987), showing some examples of sauropsid
KAPs, is presented in Fig. 15A. The phylogram shows the
initial divergence between lepidosaurian and chelonian/
archosaurian proteins, probably during the mid-upper
Permian (Pough et al. 2001).
The molecular evolution in lepidosaurians apparently
produced two main types of proteins, the HGPS and HCPS
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
580 Hard epidermal proteins in reptiles, L. Alibardi et al.
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 glycinerich 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.
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
Hard epidermal proteins in reptiles, L. Alibardi et al. 581
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 corebox, 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 featherspecific 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).
582 Hard epidermal proteins in reptiles, L. Alibardi et al.
proteins (lepidosaurian KAPs), which are poor in tyrosine.
These two subfamilies contain different members that will
be more properly classified when they have all been characterized. The variety of types, in amino acid composition and
molecular weight, are probably related to the specializations
of the integuments of these modern reptiles (such as
different types of scales over different body regions, soft
and hard with keels, frills, gastrosteges of snakes for body
movement, and claws). Some of the studies reported above
have indicated that HGP proteins are present in relatively
soft scales of lizards and snakes, while HCP proteins are
present in claws and climbing setae.
Proteins of archosaurians/chelonians so far identified differ
from those of lepidosaurians in that they are not divided
into HGPS and HCPS proteins. Their general structure
contains a tail region with HCPS characteristics and a tail
region with HGPS characteristics, resembling a chimeric
molecule (see Fig. 10). That typical diapsid (archosaurians)
and presumed anapsid (chelonians) reptiles possess more
similar proteins than the proteins of lizards and snakes
(diapsids) was unexpected. The study of a current representative of a basal lineage of diapsid lepidosaurians such
as Sphenodon would be very important to complete the
whole picture on the evolution of these keratins (Alibardi
& Toni, 2006b).
After this initial divergence, in archosaurians two main
groups later diversified, that of tyrosine-glycine-rich nonfeather proteins and the glycine-tyrosine-poor group of
feather proteins (see also Gregg et al. 1983). In chelonian/
archosaurian KAPs, the amount of tyrosine and glycine
increases somehow in relation to the irregular orientation
of the corneous material, which increases resistance
(Brush, 1993). These regions were either lost (or never
appeared) in the small and specialized proteins of feathers
(Figs 13, 14).
Interestingly, the initial archosaurian group includes the
proteins of turtle, suggesting that turtle proteins have
differentiated from common ancestral proteins with those
of archosaurs. Turtle proteins later diverged from crocodilian
and avian proteins. Turtles have maintained a quite uniform
typology of scutes in the shell, with marginals, coastal and
neural, and sternoplastron, hyoplastron, hypoplastron,
xyphiplastron, mainly changing their areas and thickness in
different species. More variety was generated in crocodilian
scutes, where tail keeled scutes, ventral softer scales, dorsal
tough scales, etc., are present. The scale proteins of dorsal
tough scales are the closest proteins to those of crocodilians.
This supports the evolution of scale proteins in birds from
an archosaurian protein (Figs 14, 15).
The molecular evolution of archosaurian proteins of
scales, claws and ramphoteca, seems to have affected the
C-terminus of their molecule, whereas the initial-mid part
has remained more similar to that of their possible common
ancestors. The molecular data indicate that archosaurian
proteins initially possessed extended glycine-rich regions,
as in the proteins of extant crocodilians (Fig. 15). Given the
ancient lineage of crocodilians, which were present with
similar features and adaptations 200 millions years ago,
we may speculate that similar long glycine-rich tails were
present in beta-keratins of the extinct Mesozoic archosaurians. Glycine-rich proteins may be suited for making
hard scales by giving high resistance and hydrophobicity
to these proteins. The glycine-rich tails in proteins present
in extant crocodiles suggest that similar molecules might
have been present in ancient archosaurians from which
feathers are believed to be derived (Spearman, 1966;
Maderson, 1972; Gregg et al. 1984). In this case, our data
support the previous view on the reduction of the glycine-tails
in beta-keratins suggesting an evolution from archosaurian
scales to feathers (Spearman, 1966; Maderson et al. 1972;
Molloy et al. 1982; Gregg et al. 1984; Fig. 14). However, our
cladogram in Fig. 15 (upper panel) also shows that featherkeratins separated early from the proteins of avian and
non-avian archosaurs, and the cladogram suggests that
feather-keratins are very ancient and underwent an
independent evolution from that of other skin appendages. This point is at present under detailed phylogenetic
analysis (Dalla Valle et al. 2008).
The comparison between feather- and scale keratin
showed a deletion of the glycine-rich region (52 amino
acids) in feathers from the ancestral sequence present in
scales (Gregg & Rogers, 1986). This observation supported
the morphological conclusions of the evolution of feathers
from scales (Gregg et al. 1984; Presland et al. 1989a,b).
Another change between scale-claws-beak and featherkeratins 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 proteins
may, however, also mean that scales, claws and beak
beta-keratins evolved differently from those of feathers.
The terminal part of this caudal region in feather proteins
also contains cysteine residues, probably involved in
intra-molecular and/or inter-molecular disulfide bonds
for making a strong corneous material. The latter produce
keratin bundles more regularly packed than in scales and
claws, permitting the axial elongation of barb/barbule
cells (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, one
evolutive lineage of amniotes leading to mammals (the
theropsids) produced mainly non-fibrous keratin-associated
proteins. The other lineage of amniotes, the sauropsids,
leading to modern reptiles and birds, instead produced
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
Hard epidermal proteins in reptiles, L. Alibardi et al. 583
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.
© 2009 The Authors
Journal compilation © 2009 Anatomical Society of Great Britain and Ireland
584 Hard epidermal proteins in reptiles, L. Alibardi et al.
fibrous keratin-associated proteins (the ‘beta-keratins’). In
particular in the lineage of archosaurian reptiles from
which birds originated, a small type of fibrous protein,
feather-keratin, was produced and its characteristics
permitted the formation of barbs and feathers (Fig. 15A).
As both sauropsids and theropsids probably possess
glycine-rich proteins it is hypothesized that some mutations
in genes of the ancestral short glycine-rich protein gave
rise to HGT proteins in theropsids and HGS in sauropsids.
This gene may be derived from that of variable regions of
cytokeratins, or by a divergent evolution from a similar
gene later incorporated into that of cytokeratins to form
their V-regions (Fig. 15B). In sauropsids, the mutation of
the first or the second base of coding triplets determined
the replacement of serine with proline, cysteine or tyrosine
in specific regions of the ancestral protein, giving rise to
the core-box and to cysteine-rich regions and tyrosine-rich
regions. In theropsids, similar mutation in other parts of the
ancestral protein might have produced the HGT and HS/UHS
proteins, as indicated in Fig. 15B. Also, in the evolutive
lineage from which mammals originated (synapsids and
therapsids) other mutations in the triplets of serine might have
introduced cysteine in the variable region of cytokeratins,
transforming this region in the E-regions of trichocytic
keratins.
The increasing knowledge of the nucleotide and amino
acid sequences of ‘beta-keratins’ in reptiles, will allow
understanding of the molecular evolution of these proteins
in archosaurians, chelonians, and lepidosaurians, from
common stem reptiles. After a core-box was formed in
stem reptiles, where a mechanically resistant and scaled
integument was likely present, two main lineages of
evolution 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 these
specialized beta-keratins for the appropriate skin appendages in different species of reptiles. Further studies are
needed to compare possible homologies between some
regions of sauropsid KAPs with mammalian KAPs to reveal
which proteins may have similar roles in hard derivatives
of sauropsids and mammalians. The latter goal will be
fully realized when reptilian genomes and proteomes are
sequenced in representatives of lizards, snakes, crocodilians
and turtles.
In conclusion, a broad generalization of the process of
cornification in the skin of all vertebrates can be made.
In all skin derivatives, the hard material results from the
interaction between two main protein components,
fibrous keratins and matrix or associated proteins. The
latter are now known in amniotes, where they are represented by sKAPs and mKAPs, whereas no molecular information is yet available for KAPs of fishes and amphibians.
The latter topic would be a fruitful field of investigation
for future studies.
Acknowledgements
Our studies have been financed with grants from the Universities
of 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 reptilian
beta-keratins, and for checking the language of the manuscript.
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