The cross-linked (cornified) envelope is a characteristic product of terminal differentiation in the keratinocyte of the epidermis and related epithelia. This envelope contains many proteins of which involucrin was the first to be discovered and shown to become cross-linked by a cellular transglutaminase. Involucrin has evolved greatly in placental mammals, but retains the glutamine repeats that make it a good substrate for the transglutaminase. Until recently, it has been impossible to detect involucrin outside the placental mammals, but analysis of the GenBank and Ensembl databases that have become available since 2006 reveals the existence of involucrin in marsupials and birds. We describe here the properties of these involucrins and the ancient history of their evolution.aves ͉ marsupial ͉ evolution ͉ glutamine repeats T he outer surface of the skin of humans and other mammals consists of dead cells, each of which contains a chemically resistant envelope. The nature of this envelope has been extensively reviewed (1, 2). Envelopes formed in cultures of epidermal cells are insoluble in ionic detergents at 100°C, but are dissolved by proteolytic enzymes (3). The envelopes are Ϸ120 Å in thickness (4) and composed of proteins heavily cross-linked by -(␥ glutamyl) lysine (isopeptide) bonds introduced by the action of transglutaminase (5). A protein ultimately incorporated into the cross-linked envelope was discovered as a soluble precursor before the activation of the cross-linking (6, 7). This precursor was named involucrin (from the Latin for envelope: involucrum) and was present only in enlarging cells undergoing terminal differentiation (8,9). Because involucrin is a substrate of transglutaminase, it is not surprising that it contains numerous glutamines capable of participating in the cross-linking reaction. Human involucrin contains 38-42 repeats of a 10 amino acid sequence, each repeat containing 3 glutamine residues (10-13).Other protein precursors of the cross-linked envelope were soon discovered (14). One had a molecular mass of 210 kDa and was later named envoplakin (15). Another had a molecular mass of 195 kDa and was later named periplakin (16). Still other precursors were discovered, including filaggrin (17), small proline-rich repeat proteins or SPRRs (18), and loricrin (19). The genes encoding most envelope precursors (but not periplakin or envoplakin) are located in the region of human chromosome 1q21, the so-called EDC or epidermal differentiation complex (20). This has been shown for involucrin (13), filaggrin (21), loricrin (22), cornifin (23), the SPRRs (24), and others (25). Other proteins encoded in the EDC, such as cornulin (26), appear late in terminal differentiation, but are not envelope precursors. Still others are precursors that are incorporated after the envelope is formed, particularly the ''late envelope proteins'' (27). It has been proposed that because involucrin, loricrin, and SPRR proteins have similar amino acid sequences in their N-terminal and C-terminal domains, they probab...