Blood group P1 antigen–bearing glycoproteins are functional but less efficient receptors of Shiga toxin than conventional glycolipid-based receptors

Abstract

Shiga toxin (STx) is a virulence factor produced by enterohemorrhagic Escherichia coli. STx is taken up by mammalian host cells by binding to the glycosphingolipid (GSL) globotriaosylceramide (Gb3; Galα1-4Galβ1-4Glc-ceramide) and causes cell death after its retrograde membrane transport. However, the contribution of the hydrophobic portion of Gb3 (ceramide) to STx transport remains unclear. In pigeons, blood group P1 glycan antigens (Galα1-4Galβ1-4GlcNAc-) are expressed on glycoproteins that are synthesized by α1,4-galactosyltransferase 2 (pA4GalT2). To examine whether these glycoproteins can also function as STx receptors, here we constructed glycan-remodeled HeLa cell variants lacking Gb3 expression but instead expressing pA4GalT2-synthesized P1 glycan antigens on glycoproteins. We compared STx binding and sensitivity of these variants with those of the parental, Gb3-expressing HeLa cells. The glycan-remodeled cells bound STx1 via N-glycans of glycoproteins and were sensitive to STx1 even without Gb3 expression, indicating that P1-containing glycoproteins also function as STx receptors. However, these variants were significantly less sensitive to STx than the parent cells. Fluorescence microscopy and correlative light EM revealed that the STx1 B subunit accumulates to lower levels in the Golgi apparatus after glycoprotein-mediated than after Gb3-mediated uptake but instead accumulates in vacuole-like structures probably derived from early endosomes. Furthermore, coexpression of Galα1-4Gal on both glycoproteins and GSLs reduced the sensitivity of cells to STx1 compared with those expressing Galα1-4Gal only on GSLs, probably because of competition for STx binding or internalization. We conclude that lipid-based receptors are much more effective in STx retrograde transport and mediate greater STx cytotoxicity than protein-based receptors. Shiga toxin (STx) is a virulence factor produced by enterohemorrhagic Escherichia coli. STx is taken up by mammalian host cells by binding to the glycosphingolipid (GSL) globotriaosylceramide (Gb3; Galα1-4Galβ1-4Glc-ceramide) and causes cell death after its retrograde membrane transport. However, the contribution of the hydrophobic portion of Gb3 (ceramide) to STx transport remains unclear. In pigeons, blood group P1 glycan antigens (Galα1-4Galβ1-4GlcNAc-) are expressed on glycoproteins that are synthesized by α1,4-galactosyltransferase 2 (pA4GalT2). To examine whether these glycoproteins can also function as STx receptors, here we constructed glycan-remodeled HeLa cell variants lacking Gb3 expression but instead expressing pA4GalT2-synthesized P1 glycan antigens on glycoproteins. We compared STx binding and sensitivity of these variants with those of the parental, Gb3-expressing HeLa cells. The glycan-remodeled cells bound STx1 via N-glycans of glycoproteins and were sensitive to STx1 even without Gb3 expression, indicating that P1-containing glycoproteins also function as STx receptors. However, these variants were significantly less sensitive to STx than the parent cells. Fluorescence microscopy and correlative light EM revealed that the STx1 B subunit accumulates to lower levels in the Golgi apparatus after glycoprotein-mediated than after Gb3-mediated uptake but instead accumulates in vacuole-like structures probably derived from early endosomes. Furthermore, coexpression of Galα1-4Gal on both glycoproteins and GSLs reduced the sensitivity of cells to STx1 compared with those expressing Galα1-4Gal only on GSLs, probably because of competition for STx binding or internalization. We conclude that lipid-based receptors are much more effective in STx retrograde transport and mediate greater STx cytotoxicity than protein-based receptors. Shiga toxin (STx) is a well-studied bacterial exotoxin and is produced by enterohemorrhagic Escherichia coli and Shigella dysenteriae. The toxin causes watery diarrhea, hemorrhagic colitis, and life-threatening hemolytic-uremic syndrome. STx consists of one A subunit (STxA) and five B fragments that constitute the homopentameric B subunit (STxB) (1Fraser M.E. Chernaia M.M. Kozlov Y.V. James M.N. Crystal structure of the holotoxin from Shigella dysenteriae at 2.5 Å resolution.Nat. Struct. Biol. 1994; 1 (7656009): 59-6410.1038/nsb0194-59Crossref PubMed Scopus (252) Google Scholar, 2Stein P.E. Boodhoo A. Tyrrell G.J. Brunton J.L. Read R.J. Crystal structure of the cell-binding B oligomer of verotoxin-1 from E. coli.Nature. 1992; 355 (1741063): 748-75010.1038/355748a0Crossref PubMed Scopus (256) Google Scholar). Each B fragment possesses receptor-binding sites that bind to specific glycan structures on host cells (3Shimizu H. Field R.A. Homans S.W. Donohue-Rolfe A. 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The binding of in cells than in cells. the of to P1 antigens in cells To whether P1 antigens were in N-glycans or in is for and and 1 is for of were by of RNA in cells. that of reduced the binding of specific of indicating that in cells of the In of reduced the binding of structures in indicating that in cells of the these only reduced STx indicating that P1 were on To the of P1 on the structures were in cells as the following cell HeLa parent cells, cells, cells, cells, and using N-glycans from cells were by using into Each with using a in several in 1 and were by the and by both and in a and The of N-glycans from HeLa parent cells, cells, and cells were the that of cells different and P1 were by and to The in and that more than of N-glycans more than one Galα1-4Gal P1 at the in cells The P1 antigens were in and and and of P1 antigens glycan with the that cells including Gb3 we in cells that expressed P1 glycan antigens only on the glycoproteins. that of N-glycans with the Galα1-4Gal were on the from and cells with the that the P1 on glycoproteins of cells were using that only a activity of P1 on and P1 antigens were synthesized only STx were compared the cells HeLa parent cells were sensitive to STx at the and cells were to the toxin even at the expressed in cells and the cells sensitive to of indicating that glycoproteins can receptors of However, cells were more than less sensitive to STx than the parent cells in the parent cells to a in the of STx bound to the cell than that on the parent cells that the activity of P1 antigens on glycoproteins as STx receptors lower than that of A that is for the of STx with in the endoplasmic reticulum for a T. Johannes L. The of toxin with is by and is in the endoplasmic reticulum for a Biol. PubMed Scopus Google Scholar). However, the of GSLs the for the sensitivity of because cells, expressed Gb3 but expressed as as a of STx receptors on also lower sensitivity to STx than the parent cells the P1 antigens on glycoproteins were less as STx receptors than we that only a of STx the cytosol from the cell the P1 antigens on glycoproteins were as the STx receptors. To examine transport of from the cell compared between cells and parent cells. internalized into the parent cells and the Golgi In cells, on the cell even after and Golgi up to at The structures of at in cells were with the early and only with the To in the membrane structures where the correlative light and EM were to the of with the of structures by in vacuole-like derived from endosomes, because these structures were However, less in that STx in early P1 antigens on glycoproteins were as the of cells. on for and then to 37 for microscopy and are The that P1 antigens on glycoproteins were only as STx compared with Gb3 to the of P1 antigens on glycoproteins on Gb3-mediated STx cytotoxicity on the cell To cells that both P1 antigens on glycoproteins and the into the parent cells. of bound to cells and the expression of Gb3 significantly different between the cell However, STx sensitivity in cells lower than that in the parent cells as as in cells that P1 antigens on glycoproteins as and Gb3-mediated STx cytotoxicity the P1 antigens were with Gb3 is the STx in mammalian cells, and of Gb3 to However, it to the moiety of Gb3 to retrograde transport of STx to the for a activity and whether membrane In we and HeLa cells expressed the Galα1-4Gal only on and that P1 antigens on glycoproteins were a STx However, the STx sensitivity in cells lower than that in the parent cells, and P1 antigens on glycoproteins and Gb3 were the P1 antigens on glycoproteins as a to Gb3-mediated cell death in that the moiety of Gb3 is for STx to its cytotoxicity and only a of STx1 internalized by glycoproteins can the retrograde transport pathway leading to cell P1 antigens on glycoproteins are to bind and with P1-containing to STx M. 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