Disulfide-bond-mediated cross-linking of corneous beta-proteins in lepidosaurian epidermis

•Corneous layers of lizards and snakes mainly contain intermediate filament (IF) keratins and corneous beta proteins (CBPs).•The interactions and chemical bonds in the differentiating and mature corneous beta- and alpha-layers are not known.•Protein extraction from fresh epidermis or molts shows bands in the IF-keratin and beta-range.•Electrophoresis under reductive or oxidative conditions indicates that CBPs bond to IF-keratins.•This occurs by the formation of disulfide bonds between IF-keratins and CBPs in the beta-layer. Corneous beta-proteins (CBPs), formerly referred to as beta-keratins, are major protein components of the epidermis in lepidosaurian reptiles and are largely responsible for their material properties. These proteins have been suggested to form filaments of 3.4nm in thickness and to interact with themselves or with other proteins, including intermediate filament (IF) keratins. Here, we performed immunocytochemical labeling of CBPs in the epidermis of different lizards and snakes and investigated by immunoblotting analysis whether the reduction of disulfide bonds or protein oxidation affects the solubility and mobility of these CBPs. Immunogold labeling suggested that CBPs are partly co-localized with IF-keratins in differentiating and mature beta-cells. The chemical reduction of epidermal proteins from lizard and snake epidermis increased the abundance of CBP-immunoreactive bands in the size range of CBP monomers on Western blots. Conversely, in vitro oxidation of epidermal proteins reduced the abundance of putative CBP monomers. Some modifications in the IF-keratin range were also noted. These results strongly indicate that CBPs associate with IF-keratins and other proteins via disulfide bonds in the epidermis of lizards and snakes, which likely contributes to the resilience of the cornified beta- and alpha-layers of the lepidosaurian epidermis in live animals and after shedding.