Suprabasal change and subsequent formation of disulfide-stabilized homo- and hetero-dimers of keratins during esophageal epithelial differentiation

Abstract

Rabbit esophageal epithelium, a parakeratinized stratified epithelium, synthesizes as one of its major differentiation products a keratin pair consisting of a basic K4 (59 kDa) and an acidic K13 (41 kDa) keratin. Although immunohistochemical staining data suggest that in esophageal epithelia of some other species these two keratins are suprabasally located, antigenic masking of the epitopes in the basal cells has not been ruled out. Using several well-characterized monoclonal antibodies including AE8, which specifically recognizes K13, coupled with biochemical analysis of keratins of basal and suprabasal cells isolated from confluent rabbit esophageal epithelial culture, we have obtained direct evidence that K4 and K13 keratins are largely absent in the undifferentiated basal cells, but are present in large amounts in suprabasal cells. We also show that in the cornified cell layers that are formed during the terminal stage of esophageal epithelial differentiation, K4 and K13 keratins become disulfide-crosslinked to form three different dimers. Two of them (110 kDa and 100 kDa) are heterodimers and consist of equimolar amounts of K4 and K13; they presumably represent isomers crosslinked via different cysteine residues. The third dimer (90 kDa) was found to be a homodimer of the acidic K13 keratin. Trypsinization experiment established that at least some of the disulfide crosslinks in the K4/K13 heterodimer must involve cysteine residues residing in the trypsin-resistant rod domains of keratins. Air-oxidation of in vitro reconstituted filaments reproduced the two heterodimers, which most likely involve the crosslinking between type I and type II keratins of different coiled coils. The formation of these disulfide-crosslinked keratin dimers, instead of higher molecular mass oligomers or polymers as occurring in the epidermis and hair, may contribute to the formation of cornified cells with a physical stability and rigidity that are optimal for esophageal function. Our data also suggest that interactions involved in the formation of homodimers, thought to be metastable and unimportant during the initial step of filament assembly (i.e. tetramer formation), may actually play an important role in stabilizing a higher order structure in mature keratin filaments.