Biochemical, Structural, and Transglutaminase Substrate Properties Of Human Loricrin, the Major Epidermal Cornified Cell Envelope Protein

Abstract

Loricrin is the major protein of the cornified cell envelope of terminally differentiated epidermal keratinocytes which functions as a physical barrier. In order to understand its properties and role in cornified cell envelope, we have expressed human loricrin from a fulllength cDNA clone in bacteria and purified it to homogeneity. We have also isolated loricrin from newborn mouse epidermis. By circular dichroism and fluorescence spectroscopy, the in vivo mouse and bacterially expressed human loricrins possess no ␣ or ␤ structure but have some organized structure in solution associated with their multiple tyrosines and can be reversibly denatured by either guanidine hydrochloride or temperature. The transglutaminase (TGase) 1, 2, and 3 enzymes expressed during epidermal differentiation utilized loricrin in vitro as a complete substrate, but the types of cross-linking were different. The TGase 3 reaction favored certain lysines and glutamines by forming mostly intrachain cross-links, whereas TGase 1 formed mostly large oligomeric complexes by interchain crosslinks involving different lysines and glutamines. Together, the glutamines and lysines used in vitro are almost identical to those seen in vivo. The data support a hypothesis for the essential and complementary roles of both TGase 1 and TGase 3 in cross-linking of loricrin in vivo. Failure to cross-link loricrin by TGase 1 may explain the phenotype of lamellar ichthyosis, a disease caused by mutations in the TGase 1 gene.Terminal differentiation in the epidermis involves the expression of a number of specific proteins that ultimately fulfill different structural roles in the cornified, dead stratum corneum cell. One set of proteins is the keratin intermediate filaments and the interfilamentous matrix protein filaggrin (1-3). A second set of proteins is used to construct the cornified cell envelope (CE), 1 a 15-nm-thick layer of protein deposited on the inner surface of the cell periphery, which serves as a physical barrier for the epidermis (4, 5). The CE proteins are rendered insoluble by cross-linking by both disulfide bonds and the N ⑀ -(␥-glutamyl)lysine isopeptide bond formed by the action of one or more of the three known epidermal transglutaminases (TGases) (4 -6). Several proteins have now been documented as CE constituents by direct sequencing analyses of cross-linked peptides (7), including loricrin, small proline-rich proteins 1 and 2 (SPR1 and SPR2), elafin, keratins, filaggrin, and desmoplakin. The proteins involucrin and cystatin ␣ are also likely constituents, but direct sequencing of cross-linked peptides involving these proteins has not yet been reported (reviewed in Ref. 8).In particular, a variety of data have suggested that loricrin comprises about 75% of the total CE protein mass (reviewed in Ref. 9), or 85-95% of the cytoplasmic two-thirds of the CE. In fact, amino acid sequencing of many peptides recovered by the proteolysis has now provided rigorous support for this idea (7). About 90% of the molar mass of peptides from the cytoplasmic...

Transglutaminase crosslinking and structural studies of the human small proline rich 3 protein

“…The data show that there are no significant differences in the kinetic efficiencies between the three TGases. This observation is in marked contrast to previous data for recombinant SPR1, 16 trichohyalin 15 and loricrin 14 (Table 1), where the TGase 3 enzyme was the most efficient. On the other hand, the kinetic efficiency values for the three enzymes with SPR3 are two-to eightfold higher than The bar marks the position of the 67 kDa form of the enzymes; the asterisk marks the full-length enzyme.…”

“…A similar observation has been made previously in the in vitro cross-linking of loricrin. 14 The most likely explanation for this is that the enzymes inserted extensive intrachain cross-links. However, this observation makes little biological sense, as the available data shows that the SPR3 protein forms extensive interchain linkages instead 10 in its proposed cross-bridging role in CE structures.…”

Bricks and mortar of the epidermal barrier

“…Seven members of the TGase family have been identified in the human genome so far, which are listed in Table 1. Four of these, TGases 1, 2, 3 and ✕ are commonly expressed in epithelia such as the epidermis (Kim et al , 1991;Aeschlimann et al, 1998), although to date only TGases 1 and 3 have proven importance in CE assembly (Candi et al, 1995;Tarcsa et al, 1997;Tarcsa et al, 1998Candi et al, 1999. It has also been proposed that the cross-linking by these enzymes coordinates mechanically the association between the CE and the underlying intracellular keratin intermediate filaments , and perhaps also in the bundling of keratin filaments (Clement et al, 1998).…”

“…Loricrin contains three glycine rich domains which are thought to form uniquely flexible glycine loops (Steinert et al, 1991), interspersed by glutamine-rich motifs and flanked by lysine-and glutamine-rich amino and carboxy terminal domains (Mehrel et al, 1990;Hohl et al, 1991a). In vitro cross-linking experiments using recombinant human loricrin have demonstrated that the TGase 1 and 3 enzymes utilize different glutamine and lysine residues, implying that both enzymes have distinctly complementary and essential functions in the utilization of loricrin for CE assembly in vivo (Candi et al, 1995).…”

Loricrin and involucrin expression is down-regulated by Th2 cytokines through STAT-6

“…Loricrin (LOR) and involucrin (IVL) are important proteins that facilitate terminal differentiation of the epidermis and formation of the skin barrier [7][8][9][10][11][12]. Human LOR is an insoluble protein initially expressed in the granular layer of the epidermis during cornification, and comprises 80% of the total protein mass of the cornified envelope (CE) [7,[13][14][15][16].…”