Transglutaminase 5 Cross-links Loricrin, Involucrin, and Small Proline-rich Proteins in Vitro

Abstract

Transglutaminases (TGases) are seven enzymes, cross-linking proteins by ␥-glutamil-⑀-lysine bonds, four of which are expressed in the skin. A new member of the TGase family, TGase 5, has been identified recently, and in the present study we evaluated its role in keratinocyte differentiation in vitro. In addition to the previously described isoforms, full-length TGase 5 and ⌬3 (deletion of exon 3), we identified two new splicing variants, ⌬11 and ⌬3⌬11 (deletion of exons 11 or 3, 11). We expressed full-length TGase 5, ⌬3, ⌬11, and ⌬3⌬11 isoforms in the keratinocyte and baculovirus systems. The results indicate that both full-length TGase 5 and ⌬11 are active, whereas ⌬3 and ⌬3⌬11 have very low activity. Expression studies show that full-length TGase 5 is induced during the early stages of keratinocyte differentiation and is differently regulated in comparison with the other epidermal TGases. Kinetic and in vitro crosslinking experiments indicate that full-length TGase 5 is very efficient in using specific epidermal substrates (loricrin, involucrin, and SPR3). In keratinocyte expression system, TGase 5 isoforms are retained in an intermediate filament-enriched fraction, suggesting its association with insoluble proteins. Indeed, TGase 5 co-localize with vimentin and it is able to cross-link vimentin in vitro.

Transglutaminase 5 is regulated by guanine–adenine nucleotides

“…The probe, comprising a C-terminal fragment of the cDNA encoding human TGase5, was hybridized to a commercially available poly(A + ) (polyadenylated) RNA dot-blot ( Figures 1A and 1B). To establish the specificity of the TGase5 probe, we performed Northern-blot analysis using mRNAs extracted from NHEKs (normal human epidermal keratinocytes) induced to differentiate with calcium, which express TGase1, TGase3 and TGase5 [18], and mRNA extracted from normal human fibroblasts, which express mainly TGase2 [7]. As already reported in our previous studies [18], we detected an accumulation of TGase5 mRNA within only 1 day of treatment of NHEK with Ca 2+ ( Figure 1C).…”

“…Originally, TGase5 cDNA was identified in human foreskin keratinocytes and further studies confirmed its involvement in keratinocyte differentiation in vitro and in vivo [17,18]. In the present study, we report for the first time that TGase5 is negatively regulated by adenine-guanine nucleotides, and is capable of hydrolysing GTP.…”

“…Recombinant purified TGase5 enzyme was obtained as described previously [18]. Human full-length TGase3 was expressed in the baculovirus system and purified as described previously [15].…”

“…Human full-length TGase3 was expressed in the baculovirus system and purified as described previously [15]. The TGase3 proenzyme was activated using dispase as described previously [18]. Guinea-pig liver TGase2 was purchased from Sigma and purified in-house by expression in Escherichia coli.…”

“…TGase5 is expressed in cultured keratinocytes induced to differentiate with calcium [17,18], but has also been detected in vitro in other cell lines [16]. To obtain a more detailed picture of its expression pattern, we performed a dot-blot Northern-blot analysis.…”