Transglutaminase Cross-linking Properties of the Small Proline-rich 1 Family of Cornified Cell Envelope Proteins

Abstract

Small proline-rich 1 (SPR1) proteins are important for barrier function in stratified squamous epithelia. To explore their properties, we expressed in bacteria a recombinant human SPR1 protein and isolated native SPR1 proteins from cultured mouse keratinocytes. By circular dichroism, they possess no ␣ or ␤ structure but have some organized structure associated with their central peptide repeat domain. The transglutaminase (TGase) 1 and 3 enzymes use the SPR1 proteins as complete substrates in vitro but in different ways: head domain A sequences at the amino terminus were used preferentially for cross-linking by TGase 3, whereas those in head domain B sequences were used for cross-linking by TGase 1. The TGase 2 enzyme cross-linked SPR1 proteins poorly. Together with our data base of 141 examples of in vivo cross-links between SPRs and loricrin, this means that both TGase 1 and 3 are required for cross-linking SPR1 proteins in epithelia in vivo. Double in vitro cross-linking experiments suggest that oligomerization of SPR1 into large polymers can occur only by further TGase 1 cross-linking of an initial TGase 3 reaction. Accordingly, we propose that TGase 3 first cross-links loricrin and SPRs together to form small interchain oligomers, which are then permanently affixed to the developing CE by further cross-linking by the TGase 1 enzyme. This is consistent with the known consequences of diminished barrier function in TGase 1 deficiency models.A large body of recent amino acid sequencing data has demonstrated that members of the three known classes of small proline-rich (SPR) 1 proteins serve as constituents of the cornified cell envelope (CE) of stratified squamous epithelia (1-5), which is a specialized structure essential for barrier function (6 -9). The SPR1 (two members), SPR2 (8 -11 members) and SPR3 (one member) are assembled from a common plan (10 -14). Their amino (head) and carboxyl (tail) domains are enriched in Gln and Lys residues and consist of sequences that have been conserved between each member of an SPR class but differ between classes. These flank a central domain consisting of a series of Pro-rich peptide repeats of sequences that likewise have been conserved between members of a class but vary between classes. The SPRs become cross-linked to themselves and other CE structural protein constituents by both disulfide bonds and N ⑀ -(␥-glutamyl)lysine or N 1 ,N 8 -bis(␥-glutamyl)-spermidine isopeptide bonds formed by transglutaminases (TGases), resulting in an insoluble macromolecular protein complex ideal for barrier function (6 -9). Moreover, for reasons that are not yet understood, individual SPR family members are differentially expressed in highly variable ways in many different types of epithelia (10 -31).Examination of the way in which the SPRs were cross-linked to protein partners in human and mouse CE preparations revealed several novel features of their properties and functions (1-5). First, only head and tail domain Gln and Lys residues were used for cross-linking. Indeed, multiple ad...

Bricks and mortar of the epidermal barrier

“…Interestingly different residues on the amino termini are used by the two enzymes, indicating that both enzymes are also required for the appropriate assimilation of SPRs into the CE in vivo. In addition, double cross-linking experiments have shown that the TGase 3 enzyme first cross-links the SPRs into short oligomers, which are later affixed to the CE by the TGase 1 enzyme (Candi et al, 1999). We have noted a correlation between the amount of SPRs present in CEs and the presumed requirements of epithelia for resistance to physical trauma.…”

“…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).…”

“…The repeats are flanked by short glutamine-, lysine-and proline-rich amino and carboxy terminal domains showing distant homology to the head and tail regions in involucrin and loricrin (Gibbs et al, 1993). Recombinant human SPR2 (Candi et al, 1999) and SPR1 (Tarcsa et al, 1998) proteins have been studied in vitro. Circular dichroism measurements indicate a random coil secondary structure for the termini, but a limited protein turn conformation for the repeat motifs.…”

A model for the reaction mechanism of the transglutaminase 3 enzyme

“…Typically, TGases recognize the generic sequence motif Gln-Gln*-Val for the first step of the reaction (where Gln* represents the targeted residue), although different isoforms display specificity as to which substrates bearing the motif may approach the enzyme . Anecdotal data suggest less specificity for Lys substrates (Tarcsa et al, 1998;Candi et al, 1999;.…”

“…Typically, TGases recognize the generic sequence motif Gln-Gln*-Val for the first step of the reaction (where Gln* represents the targeted residue), although different isoforms display specificity as to which substrates bearing the motif may approach the enzyme . Anecdotal data suggest less specificity for Lys substrates (Tarcsa et al, 1998;Candi et al, 1999;.The three dimensional structures of four TGases have now been reported, i.e. human factor XIIIa (hfXIIIa) ), TGase 2 (Liu et al, 2002, TGase 3 enzymes (Ahvazi et al, 2002;2003), and a fish enzyme (fTG, equivalent to mammalian TGase 2, Noguchi et al, 2001).…”