Initiation of assembly of the cell envelope barrier structure of stratified squamous epithelia

The cell envelope (CE) is a specialized structure that is important for bar rier function in terminally differentiated stratified squamous epithelia. T he CE is formed inside the plasma membrane and becomes insoluble as a resul t of cross-linking of constituent proteins by isopeptide bonds formed by tr ansglutaminases. To investigate the earliest:stages of assembly of the CE, we have studied human epidermal keratinocytes induced to terminally differe ntiate in submerged liquid culture as a model system for epithelia in gener al. CEs were harvested from 2-, 3-, 5-, or 7-d cultured cells and examined by 1) immunogold electron microscopy-using antibodies to known CE or other junctional proteins and 2) amino acid sequencing of cross-linked peptides d erived by proteolysis of CEs. Our data document that CE assembly is initiat ed: along the plasma membrane between desmosomes by head-to-tail and head-t o-head cross-linking of involucrin to itself and to envoplakin and perhaps periplakin. Essentially only one lysine and two glutamine residues of invol ucrin and two glutamines of envoplakin were used initially. In CEs of 3-d c ultured cells, involucrin, envoplakin, and small proline-rich proteins were physically located at desmosomes and had become cross-linked to desmoplaki n, and in 5-d CEs, these. three proteins had formed a continuous layer exte nding uniformly along the cell periphery. By this time >15 residues of invo lucrin were used for cross-linking. The CEs of 7-d cells contain significan t amounts of the protein loricrin, typically expressed at a later stage of CE assembly. Together, these data stress the importance of juxtaposition of membranes, transglutaminases, and involucrin and envoplakin in the, initia tion of CE assembly of stratified squamous epithelia.