TLN-58, an Additional hCAP18 Processing Form, Found in the Lesion Vesicle of Palmoplantar Pustulosis in the Skin Masamoto Murakami, Kenji Kameda, Hiroki.

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TLN-58, an Additional hCAP18 Processing Form, Found in the Lesion Vesicle of Palmoplantar Pustulosis in the Skin Masamoto Murakami, Kenji Kameda, Hiroki Tsumoto, Teruko Tsuda, Kana Masuda, Ryo Utsunomiya, Hideki Mori, Yuri Miura, Koji Sayama Journal of Investigative Dermatology Volume 137, Issue 2, Pages 322-331 (February 2017) DOI: 10.1016/j.jid.2016.07.044 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Several fragments were processed from the GST–hCAP-18–His recombinant peptide in depleted PPP vesicles. (a) GST–hCAP-18–His was incubated in dep-PPP-VF. Several fragments were confirmed in lane P-hCAP18 by Western blotting. (b) ELA2 expression in both peripheral monocytes/macrophages and neutrophils sorted by flow cytometry was confirmed by Western blotting. (c) A schematic presentation of hCAP18 (upper column) with indication of its known fragments (lower column). aa, amino acid; Dep-PPP-VF, depleted palmoplantar pustulosis vesicular fluid; ELA2, elastase-2 recombinant peptide; GST–hCAP-18–His, glutathione S-transferase–human cathelicidin–histidine; LL-37, LL-37 synthetic peptide; PPP, palmoplantar pustulosis; TLN, TLN-58 synthetic peptide; 1, healthy volunteer #1; 2, healthy volunteer #2; 3, PPP patient #1; 4, PPP patient #2. Journal of Investigative Dermatology 2017 137, 322-331DOI: (10.1016/j.jid.2016.07.044) Copyright © 2016 The Authors Terms and Conditions

Figure 2 ELA2 is responsible for TLN-58 processing from the GST–hCAP-18–His recombinant peptide. GST–hCAP-18–His was incubated with ELA2, and the treated sample was examined by Western blotting. (a) Western blotting using a rabbit polyclonal anti-LL-37 antibody. (b) Western blotting using a mouse monoclonal anti-LL-37 antibody. (c) The proteinase activity was blocked with α-1 anti-trypsin and subsequently confirmed by Western blotting using a mouse monoclonal anti-LL-37 antibody. Crude, crude GST–hCAP-18–His recombinant peptide incubated without ELA2; e.b., GST–hCAP-18–His processed by elastase-2 in elution buffer; ELA2, GST–hCAP-18–His processed by elastase-2 in elution buffer; ELA+α1AT, GST–hCAP-18–His incubated with elastase-2 and alpha-1 anti-trypsin; GST–hCAP-18–His, glutathione S-transferase–human cathelicidin–histidine; LL-37, LL-37 synthetic peptide; TLN, TLN-58 synthetic peptide. Journal of Investigative Dermatology 2017 137, 322-331DOI: (10.1016/j.jid.2016.07.044) Copyright © 2016 The Authors Terms and Conditions

Figure 3 TLN-58 induced inflammatory cytokine mRNAs in both normal human keratinocytes and NCL-SG3 cells. (a–e) Normal human keratinocytes and (f–j) NCL-SG3 cells were stimulated with TLN-58 and LL-37 synthetic peptides. The relative mRNA expression levels (fold) compared with the control were evaluated by quantitative real-time reverse transcriptase–PCR. (a, f) IL-8 mRNA. (b, g) IL-17C mRNA. (c, h) IL-23 mRNA. (d, i) IL-1α mRNA. (e, j) IL-1β mRNA. ∗P < 0.05 versus control. M, mol/L. Journal of Investigative Dermatology 2017 137, 322-331DOI: (10.1016/j.jid.2016.07.044) Copyright © 2016 The Authors Terms and Conditions

Figure 4 TLN-58 induced inflammatory cytokines in normal human keratinocytes but not in NCL-SG3 cells. (a–e) Normal human keratinocytes and (f–j) NCL-SG3 cells were stimulated with TLN-58 and LL-37 synthetic peptides. The protein expression level was evaluated by ELISA and expressed in pg/ml. (a, f) IL-8. (b, g) IL-17C. (c, h) IL-23. (d, i) IL-1α. (e, j) IL-1β. ∗P < 0.05 versus control. M, mol/L. Journal of Investigative Dermatology 2017 137, 322-331DOI: (10.1016/j.jid.2016.07.044) Copyright © 2016 The Authors Terms and Conditions

Figure 5 TLN-58 showed antimicrobial activity against group A Streptococcus species, S. aureus, and S. epidermidis. Antimicrobial activity of TLN-58 was evaluated using a colony-forming unit assay in sweat butter and compared with that of LL-37. Values are percentages of living bacteria compared with the control (no synthetic peptide). (a) Group A Streptococcus NZ131, (b) S. aureus RN450 (MRSA), (c) S. aureus 209P (methicillin-sensitive SA), (d) S. aureus MCTC10443 (MRSA), (e) S. epidermidis Se11, (f) S. epidermidis U-31. M, mol/L; MRSA, methicillin-resistant Staphylococcus aureus; SA, Staphylococcus aureus. Journal of Investigative Dermatology 2017 137, 322-331DOI: (10.1016/j.jid.2016.07.044) Copyright © 2016 The Authors Terms and Conditions