Correlation Between The Ligand Structure And Their Intracellular Transport

The glycosphingolipid globotriaosylceramide (Gb3), which is enriched in the plasma membrane of mammalian cells, plays an important role in cell growth, differentiation, recognition and signaling transduction. Gb3 is the cellular receptor of Shiga toxin (STx), which is produced by Shigella dysenteriae. Shiga toxin is composed of two moieties, one of which (A subunit, STxA) carries a deadenylase activity that inhibits protein biosynthesis by acting on the ribosomal 28S rRNA, whereas the other one (B subunit, STxB) allows the binding of the toxin to the receptor Gb3. In mammalian cells, it has been shown that Shiga toxin or Shiga toxin B subunit (STxB) follows the retrograde transport route from the plasma membrane to the endoplasmic reticulum (ER), via the early endosome/recyling endosome (EE/RE) and the Golgi apparatus, circumventing the degrading environment of the late endocytic pathway. And other studies reveal that fluorescently labeled Gb3 added into cells can also target to the Golgi apparatus. However, available evidence has suggested that there might be two different endocytic routes employed by Gb3 and its ligand STxB, the endogenous Gb3 route and the ligand route induced by STxB.The aim of this study was to investigate the correlation between the ligand structure and their intracellular transport in Hela cells. First, three STxB mutants bearing different binding strengths were constructed with molecular cloning and then their endocytic pathways were studied in contrast to the wild type. In the second part, endocytic pathways of different ligands (the natural ligand STxB and a synthetic ligand Gb3 antibody 38-13) were compared to elucidate whether the cellular trafficking route of STxB was induced by itself or not.According to literatures, we decided to construct three mutants: W34A, D17E and F30A. W34A shows the strongest binding strength, while D17E second and F30A weakest. The cDNA sequences were amplified with overlap extension PCR. They were digested by Sph I and Not I restriction enzymes and then ligated via T4 DNA ligase with pSU108 vector. The recombinant plasmids were transformed into E.coli DH5αand positive colonies were identified by gene sequencing.Recombinant and wide-type proteins were expressed by temperature induction under 42℃. Periplasmic extracts were prepared by osmosis shock. SDS-PAGE revealed that there were positive protein bands between 10 kDa and 15 kDa. To purify proteins, periplasmic extracts were loaded on Q-Sepharose FF column, and then on MonoQ HR 5/5 column, eluted by a linear NaCl gradient in 20 mM Tris·HCl, pH 7.5. The resulting proteins, esitimated to be 95% pure by SDS-PAGE, were further confirmed by Western Blot. Pure proteins were quantified by Bradford assay. Here using HeLa cells as the material, trafficking of both wild-type and mutant B subunits were detected by immunofluorescence analysis. Hela cells were first incubated with proteins at 18℃for 2 h or 37℃for 3 h, and subsequently visualized by immunofluorescence microcopy. Morphological studies and colocalization analysis showed that they were all delivered from the plasma membrane via early endosome/recyling endosome to the Golgi apparatus. However, part of STxB had showed characteristics of the endoplasmic reticulum staining, suggesting weaker binding strength had influenced the transport rate from early endosome/recyling endosome to the Golgi apparatus. After 4 h at 37℃, the wild type displayed a typical endoplasmic reticulum staining while mutant ligands were in the Golgi apparatus, indicating that they transported more slowly. And it seemd as if F30A whose binding strength weakest was the slowest. After 16 h at 37℃, the endoplasmic reticulum staining of the wild type were especially typical while mutant ligands were still localized in the Golgi apparatus, implying that they might not target to the endoplasmic reticulum. Further studies indicated that they were degraded from the Golgi apparatus owing to weaker binding strength and the intracellular transport of STxB was depended on the stable interaction of STxB-Gb3 complex.Then endocytic pathways of different ligands(STxB and the monoclonal anti-Gb3 antibody 38-13)were compared. STxB is the natural ligand of Gb3 while the monoclonal anti-Gb3 antibody 38-13 can be seen as some kind of synthetic ligand. They all bind the oligosaccharide part of Gb3. Results showed that the monoclonal anti-Gb3 antibody 38-13 was able to bind to the cell surface but could not enter into cells, suggesting the intracellular transport route of STxB was induced by itself, i.e. STxB had not employed the endogenous transport pathway of Gb3.