Characterization of the ganglioside recognition profile of Escherichia coli heat-labile enterotoxin LT-IIc.

The heat-labile enterotoxins of Escherichia coli and cholera toxin of Vibrio cholerae are related in structure and function. Each of these oligomeric toxins is comprised of one A polypeptide and five B polypeptides. The B-subunits bind to gangliosides, which are followed by uptake into the intoxicated cell and activation of the host's adenylate cyclase by the A-subunits. There are two antigenically distinct groups of these toxins. Group I includes cholera toxin and type I heat-labile enterotoxin of E. coli; group II contains the type II heat-labile enterotoxins of E. coli. Three variants of type II toxins, designated LT-IIa, LT-IIb and LT-IIc have been described. Earlier studies revealed the crystalline structure of LT-IIb. Herein the carbohydrate binding specificity of LT-IIc B-subunits was investigated by glycosphingolipid binding studies on thin-layer chromatograms and in microtiter wells. Binding studies using a large variety of glycosphingolipids showed that LT-IIc binds with high affinity to gangliosides with a terminal Neu5Acα3Gal or Neu5Gcα3Gal, e.g. the gangliosides GM3, GD1a and Neu5Acα3-/Neu5Gcα3--neolactotetraosylceramide and Neu5Acα3-/Neu5Gcα3-neolactohexaosylceramide. The crystal structure of LT-IIc B-subunits alone and with bound LSTd/sialyl-lacto-N-neotetraose d pentasaccharide uncovered the molecular basis of the ganglioside recognition. These studies revealed common and unique functional structures of the type II family of heat-labile enterotoxins.

Preparation and Characterization of the Self-releasing Intracellular Transporter LCA2 Domain / 中国生物化学与分子生物学报

Protein drugs play an extremely important role in the prevention and treatment of diseases. But the properties of macromolecules hinder their effects on intracellular targets. Among the existing delivery strategies, penetrating peptides are more suitable for clinical research and treatment, and have gradually become the most important tool to deliver protein drugs. Therefore, the development of safe and effective penetrating peptide delivery vehicles is of great significance to the basic research and clinical application of biomedicine. In this paper, a self-releasing intracellular transporter LCA2 based on the enterotoxin A2 domain is designed. This carrier is composed of three parts: a linker, self-releasing enzyme sensitive sites (Cs), and the transmembrane domain LTA2. The fluorescent protein mCherry was used as the model protein to detect the properties of LCA2. The results of electrophoresis showed that the high-purity mCherryLCA2 fusion protein was obtained from the engineered bacteria containing pET24a(+)-ma2 recombinant plasmids, and mCherry could be effectively separated from LCA2 by low concentration trypsin. It was observed under a fluorescence microscope that LCA2 could transport mCherry into different types of cells. Flow cytometry has detected that the transport capacity of LCA2 has certain cellular differences. Confocal microscope fluorescence analysis and Western blotting results showed that the mCherry was transported to the endoplasmic reticulum by the LCA2 carrier, separated from LCA2 by cleavage of enzyme sensitive sites and released into the cell. The CCK-8 results showed that there was no significant change in cell viability within the dose range of 5-40 μg/ mL. These results demonstrate that LCA2 is a safe and effective self-releasing delivery vehicle, which can transport and release active proteins or protein drugs into cells.

Discovery of the cell-penetrating function of A2 domain derived from LTA subunit of Escherichia coli heat-labile enterotoxin.

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.

A Novel Method for Efficient Preparation of Mucosal Adjuvant Escherichia coli Heat-Labile Enterotoxin Mutant (LTm) by Artificially Assisted Self-Assembly In Vitro.

As well-known powerful mucosal adjuvant proteins, Escherichia coli heat-labile enterotoxin (LT) and its non-toxic or low-toxic mutants (LTm) are capable of promoting strong mucosal immune responses to co-administered antigens in various types of vaccines. However, due to the complex composition and special structure, the yield of LTm directly from the recombinant genetic engineering strains is quite low. Here, we put forward a novel method to prepare LTm protein which designed, expressed, and purified three kinds of component subunits respectively and assembled them into a hexamer structure in vitro by two combination modes. In addition, by simulated in vivo environment of polymer protein assembly, the factors of the protein solution system which include environment temperature, pH, ionic strength of the solution, and ratio between each subunit were taken into consideration. Finally, we confirmed the optimal conditions of two assembly strategies and prepared the hexamer holotoxin in vitro. These results are not only an important significance in promoting large-scale preparation of the mucosal adjuvant LTm but also an enlightening to produce other multi-subunit proteins.