Deimination of Human Hornerin Enhances its Processing by Calpain-1 and its Cross-Linking by Transglutaminases

Abstract

Hornerin (HRNR) shares numerous features with filaggrin, a key contributor to the epidermal barrier functions. The two proteins display a related structural organization, are expressed by the granular keratinocytes as a large precursor processed by proteolysis, and are cross-linked to the cornified cell envelopes. Two main steps in the metabolism of filaggrin are its deimination and calpain-1 cleavage. Here, using ion-exchange chromatography and two-dimensional gel electrophoresis of human epidermis extracts, we determined that HRNR is deiminated in vivo. Accordingly, cornified envelopes, purified from plantar and abdominal human skin, were shown to contain deiminated proteins. A recombinant form of HRNR (HRNR) deiminated in vitro was shown to be a better substrate for transglutaminases 1 and 3 than the unmodified form. Our data also indicated that calpain-1 may be involved in the proteolytic processing of HRNR, because calpain-1 was co-located with HRNR in the cytoplasm of granular keratinocytes. Using Western blotting and mass spectrometry analysis, HRNR was shown to be cleaved by calpain-1 in vitro, its deimination enhancing its proteolysis. In HRNR full sequence, four calpain-1 cleavage sites were identified. Altogether, these data allowed a new role to be deciphered for deimination during cornification and provided further characterization of HRNR metabolism.

Peptidylarginine deiminase is involved in maintaining the cornified oral mucosa of rats

“…In our western blot analyses combined with 2D electrophoresis, the anti‐CCP antibody was capable of detecting FLG monomer, whereas the anti‐MC antibody likely detected proFLG, its processing intermediates, and the FLG monomer as well as all citrullinated proteins. The more intense FLG immunoreactivity in the 3 epithelial types and additional immunoreactive spots observed by 2D gel electrophoresis indicated the involvement of other citrullinated proteins such as K1, K10 and hornerin …”

Transient epidermal barrier deficiency and lowered allergic threshold in filaggrin‐hornerin (FlgHrnr−/−) double‐deficient mice

“…Hornerin is another component of the CE and shares several features with filaggrin including gene localization and organization, as well as protein structure, expression, and function. Overall, HRNR protein, complementary and similar to FLG, plays a role in keratinocyte cornification and contributes to the generation and maintenance of the epidermal barrier . Similar to FLG, HRNR protein is per se expressed in healthy epidermis, increased in skin barrier impaired epidermis, and reduced in the skin of patients with atopic dermatitis (AD) and hand eczema .…”

Peptidylarginine deiminases and deiminated proteins at the epidermal barrier

“…Hornerin probably strengthens the envelopes and contributes to their mechanical resistance. We have shown that hornerin is deiminated in vivo; in addition, its deimination improves in vitro its proteolysis by calpain 1 and its cross‐linking by transglutaminases 1 et 3; this suggests that deiminated fragments of hornerin are incorporated into cornified envelopes. In agreement with this hypothesis, cornified envelopes purified from plantar and abdominal human skin do contain deiminated proteins .…”

“…We have shown that hornerin is deiminated in vivo; in addition, its deimination improves in vitro its proteolysis by calpain 1 and its cross‐linking by transglutaminases 1 et 3; this suggests that deiminated fragments of hornerin are incorporated into cornified envelopes. In agreement with this hypothesis, cornified envelopes purified from plantar and abdominal human skin do contain deiminated proteins . Whether additional envelope components including filaggrin and keratins are deiminated and whether addition of deiminated components modifies the envelope properties remain to be demonstrated.…”

“…Four PAD targets have been definitively identified: keratins KRT1 and KRT10, filaggrin and hornerin. This was evident after two‐dimensional gel electrophoresis of epidermal extracts and immunoblotting with a rabbit antibody specific for deiminated proteins . This affinity purified antibody called AMC, for antimodified citrulline antibody, recognizes citrullinated residues of any proteins after their chemical modification in strong acidic conditions in the presence of diacetyl monoxime and antipyrine …”