| Receptor-mediated endocytosis plays an important role in cellular transport. Here weinvestigated the endocytosis of two proteins: Shiga toxin B subunit (STxB) and galectin-3(Gal-3), mediated by cell surface receptors glycolipids and glycoproteins, respectively. Gal-3,STxB and the recombinant proteins of STxB, were prepared by molecular cloning. Theirintracellular transport pathways were compared and the relationship between the primarystructure and transport was analyzed.STx is a cytotoxic factor. Its main receptor on membrane and transport pathways havebeen comfirmed: STxB can bind to its receptor globotriaosyl ceramide (Gb3), navigate theretrograde pathway from the plasma membrane to the endoplasmic reticulum. The research inthis article revealed that both the concentration of STxB and the serum in culture mediuminfluenced the transport of STxB，although the transport pathway did not changed. STxB hasno signal sequence guiding the transport from Golgi to endoplasmic reticulum. It is proposedthat STxB transport to endoplasmic reticulum (ER) by associating with detergent-resistantmembranes. We noticed that there were some differences in the transport of SBQ and GBQ,which had some changes only at the C-terminal of STxB, suggesting that the amino acidsequence at C-terminal might play a role in transport. So we prepared a series of recombinantproteins, and compared their intracellular transport. Our results showed that Myc sequencedirectly affected STxB transport from the Golgi to the endoplasmic reticulum. When the endamino acid Arg (R) was mutated to be Ser (S), the speed of B-fragment transported intoendoplasmic reticulum (ER) at37oC was slower. When acidic amino acid tail “Glu-Ser”(ES)was added behind the end amino acid “R”, B-fragment transport speed slowed down andtended to remain in Golgi apparatus. Further studies showed that the effects induced by themutations of amino acid tail were STxB-EEEES≥-EEES>-EES>-ES, suggesting that theretarded effect of the tail increased along with the length of acidic amino acid added. Theeffect was probably produced by acidic amino acid tail, not only by amino acid “E”. Thesignificant inhibitory effect on the speed of B-fragment retrograde transport was observedonly when the mutations of acidic amino acid tail were linked near to C-terminus. In addition,this phenomenon of slow down of STxB trafficking is more obvious in the cells cultured innewborn calf serum. When STxB was directly modified with Myc tag at its C-terminus, thetargeting of the recombinant protein changed. Myc tag protein was prone to be degradated,and STxB could thansport in the original path when the Myc tag was degradated.Gal-3is a member of a growing family of carbohydrate-binding proteins and is involvedin multiple biological processes through interaction with specific ligands, including cell growth, adhesion, differentiation, inflammation, apoptosis, and metastasis. However, therewere few studies on its endocytosis and transport. The research in this article revealed thatGal-3could specifically associate with HUVEC and quickly internalize into the cells. Thisprocess can be inhibited by lactose. The results also showed that there are at least two sortingpathway in the vesicle transport of Gal-3, a fraction of the internalized Gal-3were transportedto the late endosome/lysososme via early/recycling endosomes, and the other fraction wasexternalized directly from early/recycling endosomes. Similar results were also obtained inHMEC, HeLa and CHO cells, indicating that the transport pathways were not limited toHUVEC.Finally, we compared the endocytic transport pathways of Gal-3and STxB. We foundthat there were some co-localizations in the early stages of transport. Once through therecycling endosomes, the two proteins quickly separated into their own transport pathways. |
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