LEKTI domains D6, D7 and D8+9 serve as substrates for transglutaminase 1: implications for targeted therapy of Netherton syndrome

Abstract

Background Transglutaminase (TG)1 plays a key role in the formation of the cornified envelope and thus in the maintenance of the epidermal barrier. Patients with Netherton syndrome (LEKTI deficiency) have increased activity of both TG1 and serin proteases. Objectives To determine whether there is a functional biochemical link between TG1 and LEKTI and whether LEKTI domains could possibly serve as substrates for TG1. Methods We analysed the protein sequence of LEKTI for possible TG1 recognition sites using bioinformatics. Synthetic peptides and recombinant LEKTI domains D6, D7 and D8+9 were examined in vitro and in situ for possible substrate specificity. The recombinant LEKTI domains were studied for inhibitory activity in a kallikrein (KLK)5 activity test. Results We identified possible TG1 consensus sequences in LEKTI domains D6, D7 and D8+9, pointing to a novel biological link between these two proteins. Indeed, synthesized short peptides from these consensus sequences were incorporated into the TG1 activity zone of the epidermis. In vitro the entire recombinant domains of LEKTI showed substrate specificity for TG1, which was again confirmed in situ. The inhibitory activity of the recombinant LEKTI domains was confirmed by a KLK5 inhibition test. The strongest inhibition was observed for domains D8+9. Conclusions There are specific domains of LEKTI that are recognized and processed by TG1. LEKTI domains D6, D7 and D8+9 contribute to the formation and protection of the cornified envelope. These results impact the development of protein replacement therapy approaches for Netherton syndrome.What's already known about this topic?• LEKTI and transglutaminase (TG)1 are key proteins involved in the terminal differentiation of the epidermis.• Lack of LEKTI causes Netherton syndrome; TG1 deficiency causes lamellar ichthyosis.• The serine protease inhibitor LEKTI is processed into different functional units. • Among different target proteases, kallikrein (KLK)5 appears to be a key player in disease pathology.• It has been demonstrated that LEKTI domain 6 inhibits KLK5 and KLK7; LEKTI domains 8-11 also inhibit KLK14.

Knockdown of SDR9C7 Impairs Epidermal Barrier Function

“…Other crucial genes for epidermal barrier function are TGM1 and SPINK5. It has been found that LEKTI serves as TGase-1 substrate and patients with Netherton syndrome have increased activity of both TGase-1 and serine proteases (Wiegmann et al, 2019). Similarly, we observed elevated expression of TGase-1 in SPINK5-siRNA treated 3D skin constructs.…”

Molecular Mechanism of Epidermal Barrier Dysfunction as Primary Abnormalities

“…Under certain conditions, more than one abnormal finding is present. Examples include abnormal lipid composition of the SC associated with impaired epidermal calcium gradients in KID syndrome [14,15], altered calcium gradients accompanied by increased skin pH and CE rearrangement in aging skin [16], loss-of-function mutations in filaggrin in atopic dermatitis [28], and increased activity of TG1 and serine proteases in Netherton syndrome [61]. These accompanying findings could provide a direction for future research to identify more organized mechanism involved in barrier dysfunction.…”

“…These findings emphasize that corneodesmosome degradation depends on the sum of protease and inhibitor activities. In addition to increased activity of serine proteases, TGase1 activity has been reported to increase in Netherton syndrome, which contributes to the novel functional link between LEKTI and TGase1 [61]. Considering that impaired corneodesmosome degradation can lead to hyperkeratosis as a clinical symptom, hyperkeratosis is not considered as a cause of barrier dysfunction related to desquamation.…”

The matriptase‐prostasin proteolytic cascade in dermatologic diseases

“…LEKTI, the protein product of the SPINK5 gene, is cleaved by furin into specific domains, with some of these domains undergoing further activation by the epidermal enzyme transglutaminase 1. [ 49 ] These activated subunits of LEKTI then inhibit the activation of KLK5, KLK6, KLK7 and KLK14, thus inhibiting corneodesmosome breakdown and desquamation and inhibiting the pro‐inflammatory effects of KLK6. [ 20,50 ] This is consistent with absence or splitting of the cornified layer sometimes seen in NS, [ 26 ] in which dysfunction of LEKTI leads to uninhibited breakdown of corneodesmosomes.…”