Shotgun label-free proteomic analysis for identification of proteins in HaCaT human skin keratinocytes regulated by the administration of collagen from soft-shelled turtle

Abstract

Soft-shelled turtles (Pelodiscus sinensis) are widely distributed in some Asian countries, and we previously reported that soft-shelled turtle tissue could be a useful material for collagen. In the present study, we performed shotgun liquid chromatography (LC)/mass spectrometry (MS)-based global proteomic analysis of collagen-administered human keratinocytes to examine the functional effects of collagen from soft-shelled turtle on human skin. Using a semiquantitative method based on spectral counting, we were able to successfully identify 187 proteins with expression levels that were changed more than twofold by the administration of collagen from soft-shelled turtle. Based on Gene Ontology analysis, the functions of these proteins closely correlated with cell-cell adhesion. In addition, epithelial-mesenchymal transition was induced by the administration of collagen from soft-shelled turtle through the down-regulation of E-cadherin expression. Moreover, collagen-administered keratinocytes significantly facilitated wound healing compared with nontreated cells in an in vitro scratch wound healing assay. These findings suggest that collagen from soft-shelled turtle provides significant benefits for skin wound healing and may be a useful material for pharmaceuticals and medical care products. © 2017 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2403-2413, 2018.

Keywords: E-cadherin; collagen; epithelial-mesenchymal transition; soft-shelled turtle; wound healing.

Figure 1

Cytotoxic effect of collagen administration in HaCaT cells. Suitable concentrations of collagen that are not cytotoxic to HaCaT cells were determined. No effect was observed on cell proliferation of HaCaT cells with collagen administration.

Figure 2

Semiquantitative comparison of identified proteins in collagen‐administered and nontreated HaCaT cells. R _sc and normalized spectral abundance factor (NSAF) values calculated for identified proteins are on the X‐axis. Protein expression is compared for collagen versus control. Proteins highly expressed in either collagen‐administered cells or nontreated cells are near the right or left side of the X‐axis. Housekeeping proteins are located around the center of the X‐axis.

Figure 3

Gene ontology (GO) analysis for identified proteins. (A) Proteins assigned to biological process, (B) cellular component, and (C) molecular function GO term categories. Only significant categories (p < 0.05) are shown.

Figure 4

Expression levels of E‐cadherin and EMT markers in HaCaT cells. E‐cadherin expression was decreased with the administration of collagen, whereas the expression levels of vimentin and snail were increased by the administration of collagen compared with nontreated cells.

Figure 5

Wound healing assay. (A) Microscopic images of wound healing over 8 h. (B) The percentage of wounded area in collagen‐administered HaCaT cells was significantly larger than in nontreated cells. *p < 0.01.