Deimination of Human Hornerin Enhances its Processing by Calpain-1 and its Cross- Linking by Transglutaminases  Chiung-Yueh Hsu, Géraldine Gasc, Anne-Aurélie Raymond, Odile Burlet-Schiltz, Hidenari Takahara, Guy Serre, Marie-Claire Méchin, Michel Simon  Journal of Investigative Dermatology  Volume 137, Issue 2, Pages 422-429 (February 2017) DOI: 10.1016/j.jid.2016.09.030 Copyright © 2016 The Authors Terms and Conditions

Figure 1 In the epidermis hornerin is deiminated. (a–d) A 6-mol/L urea extract of human normal epidermis was separated by ion-exchange chromatography. (a) The optical density and the NaCl concentration of the elution gradient were recorded. Proteins of the indicated collected fractions were separated by SDS-gel electrophoresis and (b) stained with Coomassie blue or (c) immunodetected with anti-hornerin (HRNR) and (d) anti-deiminated proteins (AMC) antibodies. Basic filaggrin, arrow; deiminated filaggrin, arrowhead. (e) Proteins of the urea extract, separated by two-dimensional gel electrophoresis, were immunodetected with the same antibodies. The boxes indicate protein areas recognized by both antibodies. The position of molecular mass standards is indicated in kDa on the right of the gels. AMC, anti-modified citrulline; HRNR, hornerin; IEF, isoelectric focusing; Ker, keratin; pI, isoelectric point. Journal of Investigative Dermatology 2017 137, 422-429DOI: (10.1016/j.jid.2016.09.030) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Deimination of HRNRHis enhances its crosslinking by TGases. Casein as a control, recombinant HRNRHis, and its deiminated form (HRNRHis*) were incubated with either TGase-1 or TGase-3 for 2, 60, and 240 minutes in the presence of CaCl2 and monodansylcadaverine 1–3, respectively. Samples were separated by SDS-gel electrophoresis, proteins were stained using Coomassie blue, and incorporation of monodansylcadaverine was shown by UV illumination (fluorescence). The • symbol indicates high-molecular-weight cross-linked complexes, asterisk indicates appearing band at 70–100 kDa, arrows indicate unmodified or deiminated HRNRHis. HRNR, hornerin; TGase, transglutaminase. Journal of Investigative Dermatology 2017 137, 422-429DOI: (10.1016/j.jid.2016.09.030) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Purified cornified cell envelopes contain deiminated proteins. CEs were purified from abdominal and plantar human skin. (a) They were observed in DF and immunodetected with the anti-hornerin (HRNR) and anti-deiminated proteins (AMC) antibodies. Note the difference in the morphology of CEs, depending on the anatomical origin, as observed in DF. Scale bar = 50 μm. (b) They were analyzed by immunoblotting with the same antibodies after incubations without (V8–) or with (V8+) V8 protease for 0–72 hours, as indicated. AMC, anti-modified citrulline; CE, cornified envelope; DF, dark field; HRNR, hornerin. Journal of Investigative Dermatology 2017 137, 422-429DOI: (10.1016/j.jid.2016.09.030) Copyright © 2016 The Authors Terms and Conditions

Figure 4 HRNRHis is a substrate of calpain-1. (a) Recombinant filaggrin (FLGHis) and HRNRHis were incubated with active calpain-1 for 3 and 16 hours, respectively. As a control, incubation was performed in the presence of the inactive protease. At the indicated times, aliquots were removed and analyzed by immunoblotting with AHF3, a monoclonal antibody directed against filaggrin, or the anti-HRNR antibody. (b) HRNRHis was separated by SDS-gel electrophoresis and stained with Coomassie blue before and after digestion with calpain-1, as indicated. The three fragments (H1–H3) produced by calpain-1 cleavage were selected for Edman degradation sequencing. From the amino-terminal sequences of the fragments (in bold letters), one calpain-1 cleavage site (indicated by a vertical arrow) was identified on the sequence of HRNRHis. The sequences recognized by the anti-HRNR antibody are underlined. h, hour; HRNR, hornerin; t, time. Journal of Investigative Dermatology 2017 137, 422-429DOI: (10.1016/j.jid.2016.09.030) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Indirect immunofluorescence localization of calpain-1 and HRNR in human epidermis. Normal human skin cryosections were analyzed by confocal microscopy using anti-calpain-1 and anti-hornerin (HRNR) antibodies. The merged images show calpain-1 (red) and HRNR (green) co-localization in granular keratinocytes and lowest corneocytes (yellow, arrows). An enlargement of the boxed area is shown at lower right. The dotted lines indicate the dermal-epidermal junction. Scale bar = 10 μm. On control sections incubated in the absence of primary antibody, no significant immunoreactivity was observed (data not shown). HRNR, hornerin. Journal of Investigative Dermatology 2017 137, 422-429DOI: (10.1016/j.jid.2016.09.030) Copyright © 2016 The Authors Terms and Conditions

Figure 6 Cleavage of deiminated HRNRHis by calpain-1. HRNRHis was deiminated by PAD1, PAD2, or PAD3 as indicated. The unmodified and deiminated (HRNRHis∗) proteins were then incubated for 0, 1, 10, and 30 minutes at 30 °C with calpain-1 at an enzyme:substrate molar ratio of 1:250 (PAD1) or 1:50 (PAD2 and PAD3). The extent of cleavage was determined by immunoblotting with the anti-His antibody (left) followed by a densitometric analysis (right). The lower PAD1 ratio used explains the apparent resistance of HRNRHis to this isoform. HRNR, hornerin; PAD, peptidylarginine deiminase; T, time. Journal of Investigative Dermatology 2017 137, 422-429DOI: (10.1016/j.jid.2016.09.030) Copyright © 2016 The Authors Terms and Conditions