title: Discovery of the cell-penetrating function of A2 domain derived from LTA subunit of Escherichia coli heat-labile enterotoxin.
abstract: Heat-labile enterotoxin (LT) is a protein toxin produced by enterotoxigenic Escherichia coli (ETEC). As a bacterial toxin, LT holotoxin can enter intestinal epithelial cells and cause diarrhea. In addition, LT is also a powerful mucosal adjuvant capable of enhancing the strong immune responses to co-administered antigens. However, the LT immunological mechanism is still not clear in some aspects, especially with the respect to how the LTA subunit functions alone. Here, we discovered that the A2 domain of LTA could carry a fluorescent protein into cells, whose function is similar to a cell-penetrating peptide. The transmembrane-transporting ability of the A2 domain is non-specific in its cell-penetrating function, which was shown through testing with different cell types. Moreover, the LTA2 fusion protein penetrated a fluorescently labeled cell membrane that identified LTA2 internalization through membrane transport pathways, and showed it finally localized in the endoplasmic reticulum. Furthermore, low-temperature stress and pharmacological agent treatments showed that the LTA2 internalization route is a temperature-dependent process involving the clathrin-mediated endocytosis and the macropinocytosis pathways. These results could explain the internalization of the LTA subunit alone without the LTB pentamer, contributing to a better understanding of LTA working as a mucosal adjuvant; they also suggest that the A2 domain could be used as a novel transport vehicle for research and treatment of disease.
authors.affiliation: School of Biotechnology and State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Di
authors.initials: D
authors.lastname: Liu
authors.affiliation: School of Biotechnology and State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Guo
authors.affiliation: School of Pharmacy, Shanghai Key Laboratory of New Drug Design, East China University of Science and Technology, Shanghai, China.
authors.forename: Wenyun
authors.initials: W
authors.lastname: Zheng
authors.affiliation: School of Biotechnology and State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Na
authors.initials: N
authors.lastname: Zhang
authors.affiliation: School of Biotechnology and State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Tianwen
authors.initials: T
authors.lastname: Wang
authors.affiliation: School of Biotechnology and State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Wang
authors.affiliation: School of Biotechnology and State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China. xymy@ecust.edu.cn.
authors.forename: Xingyuan
authors.initials: X
authors.lastname: Ma
mesh_terms: D000072283:A549 Cells; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D045744:Cell Line, Tumor; D057846:Cell-Penetrating Peptides / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D004705:Endocytosis; D054307:Enterotoxigenic Escherichia coli / Q000235:genetics / Q000378:metabolism*; D004768:Enterotoxins / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D045325:HCT116 Cells; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D007422:Intestines / Q000166:cytology; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000235:genetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013682:Teichoic Acids / Q000737:chemistry* / Q000235:genetics; D000097573:Red Fluorescent Protein
chemical_list: D001427:Bacterial Toxins; D057846:Cell-Penetrating Peptides; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D013682:Teichoic Acids; D000097573:Red Fluorescent Protein; C009900:lipoteichoic acid; C027538:heat-labile enterotoxin, E coli
keywords: A2 domain; Cell-penetrating; Drugs delivery; Heat-labile enterotoxin (LT); Multi-endocytosis pathway