α-Keratin and Associated Proteins
Immunocytochemistry on snake epidermis has shown that α-keratins react weakly with the AE3 antibody for mammalian basic keratins (Alibardi, 2002c). Apart from β-keratins, all the remaining immunoreactivities observed in gels are associated with α-keratin layers, suggesting that loricrin-, sciellin-, filaggrin/AE2-positive keratins and transglutaminase-like proteins are present in the epidermis of snakes. Specific preabsorbing controls using the specific antigen searched for (loricrin, sciellin, filaggrin, and transglutaminase) were unavailable in our immunoblotting study. Despite this shortage, our negative controls (omitting the primary antibody) clearly indicate that specific immunoreactive bands were extracted from snake epidermis.
Previous immunocytochemical studies (Alibardi, 2002c) have shown that, when present, loricrin-like immunoreactivity is associated with α-keratin bundles of lacunar and α-cells, but is not localized along the cornified cell envelope as in corneocytes of mammalian epidermis (Steven et al., 1990; Ishida-Yamamoto et al., 2000; Kalinin et al., 2002). Similarly, in the cornified cell envelope, transglutaminase substrates (loricrin, filaggrin, etc.) may have no antigens available for immunodetection, as is the case for mammalian epidermis (Ishida-Yamamoto et al., 2000; Kalinin et al., 2002). The immunolabeling pattern for loricrin, as observed in Western blots, suggests that proteins with some epitopes with mammalian loricrin are present in lacunar cells of snake epidermis (Alibardi, 2002c). A previous study on snake epidermis indicated that a weak protein band of low molecular weight (below 18 kDa) was present (Hohl et al., 1993). The present study instead suggests that the prevalent band has a molecular weight of 57–58 kDa. A cross-reactivity of the loricrin antibody with other proteins of the corneous cell envelope of snake corneocytes is also possible. For example, loricrin shares with involucrin and small proline-rich proteins (two other proteins of the cornified cell envelope of mammalian epidermis) numerous amino acidic sequences in the N- and C-terminal regions (Backendorf and Hohl, 1992), and it is possible that the employed antibody recognizes these common epitopes in different proteins of cornification in snake epidermis. Only a study after isolation of specific cornified envelope proteins from snake epidermis will further clarify the present preliminary data.
Also, the presence of sciellin-immunoreactive protein bands, especially at 62 kDa, suggests that similar protein may be present among keratin filaments of snake α-cells (Kvedar et al., 1992). The immunolocalization of this protein at the microscopic level has still to be done.
The weak filaggrin-like immunoreactivity observed after immunocytochemistry in α-cells and in the lacunar layer of the hinge region of snakes (Alibardi, 2002c) is probably due to a common epitope(s) associated with K1/K10 keratins recognized by the AE2-keratin antibody. The latter recognizes 67–68 and 56.5 kDa α-keratins in mammalian epidermis (Dale and Sun, 1983) and in lizard (Carver and Sawyer, 1987; Alibardi et al., 2000, 2001). The presence of soft α-keratins in the hinge regions of snakes allows the elasticity of these areas among scales, which are used for the movement of snakes. Among α-keratin and associated proteins, a large production of complex lipids and waxes forms the barrier against water loss (Roberts and Lillywhite, 1983; Tu et al., 2002).
Finally, the presence of some weak immunolabeling for transglutaminase in Western blots suggests that a small amount of isopeptide bonds (the product of transglutaminase reaction on proteins) is formed in α-cells, in the peripheral cytoplasm or even along the cornified cell envelope [the marginal layer of Landmann (1979)]. Immunocytochemistry on snake epidermis shows a very weak reaction for isopeptide bonds, especially in α-cells of hinge regions (data not shown).
All the above proteins appear absent in β-cells of the hard corneous layer of snakes. In the latter, β-keratin is the prevalent protein, which accumulates into a dense and compact corneous mass. The prevalent transglutaminase band at 57 kDa found in the present Western blot study is within the range of molecular weight reported for mammalian and chick transglutaminases (Polakowska and Goldsmith, 1991).
Further detailed studies on snake epidermis (also using immunogold cytochemistry) are needed to determine where these proteins enter in the formation of the corneous cell envelope or the internal corneous mass of α-keratinocytes. However, the presence of cross-reactive protein bands for the first time suggests that common molecular processes of cornification as those operating in mammalian epidermis are present in the epidermis of snakes.
β-keratin layers mechanically protect the softer α-layers underneath and therefore the integrity of the water barrier (Maderson et al., 1998; Tu et al., 2002). β-keratin is deposited during the transition from the last formed α-layer (the clear) of the outer epidermal generation to the first β-layer of the inner generation (the oberhautchen), with the result that a shedding complex is formed.
The electron-dense histidine-labeled fibrous material in clear cells and in dense β-keratin packets in oberhautchen cells during the transition between α-keratin to β-keratin synthesis indicates that histidine is mainly used for the production of new keratins (Landmann, 1979; Alibardi, 2002a, 2002b, 2002c; Alibardi and Thompson, 2003). A specific biochemical study on specific protein bands isolated from snake epidermis is required to show whether nonkeratin histidine-rich proteins are also produced, as is the case for oberhautchen cells of lizards and geckos (Alibardi, 2001, 2002b; Alibardi et al., 2003). Histidine-incorporating proteins rapidly decrease in β-cells and their β-packets. Taken together, these observations suggest that the dense β-keratin packets in oberhautchen cells, but not the pale β-keratin of β-cells, contain histidine-rich proteins.
β-keratin is deposited over or among α-keratin bundles, which rapidly disappear in maturing β-cells before the formation of the syncitial and mature outer β-layer. Most of β-keratin-labeled bands from whole epidermis in snakes have a lower molecular weight than α-keratins, as in the epidermis of the other reptiles (Wyld and Brush, 1979, 1983; Gillespie et al., 1982; Marshall and Gillespie, 1982; Carver and Sawyer, 1987; Sawyer et al., 2000). Bands of variable molecular weights (low sulfur fractions) have been reported for lizard (12–70 kDa) (Gillespie et al., 1982) or gecko (12–72 kDa) (Thorpe and Giddings, 1981) β-keratins, although the main fractions (high sulfur) are those at 13–16 and 20–22 kDa.
In the epidermis of snakes, the smaller β-keratins with a molecular weight of 13–16 kDa appear constantly formed. The evolutions of these small keratins in reptiles from α-keratins remain to be solved, but the abundance of glycine-glycine-Tyr or glycine-glycine-Cys sequences (Gillespie et al., 1982; Marshall and Gillespie, 1982) suggests that β-keratins may be derived from the evolution of the gly-gly-rich sequences at the N- or C-terminal of the external nonhelical sequences of α-keratins (Klinge et al., 1987). Future molecular biology studies will definitely disclose the trend followed during the evolution of these hard forms of keratins in reptiles.