Human Gb3/CD77 synthase produces P1 glycotope-capped N-glycans, which mediate Shiga toxin 1 but not Shiga toxin 2 cell entry

Abstract

The human Gb3/CD77 synthase, encoded by the A4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Galα1→4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, P k ) and the P1 antigen. Gb3 is the major receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli . A single amino acid substitution (p.Q211E) ramps up the enzyme’s promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise to NOR1 and NOR2 GSLs. Human Gb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Galα1→4Galβ1→4GlcNAc-R) on a complex type N-glycan and a synthetic N-glycoprotein (saposin D). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminated N-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays show that Stx1 can use P1 N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonical GSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology.

Structural Diversities of Lectins Binding to the Glycosphingolipid Gb3

“…Stx1 interacts exclusively with the carbohydrate moiety of Gb3, while Stx2 needs additional interactions with the full glycolipid (Gallegos et al, 2012). This necessity has also been recently demonstrated in studies using the P1 glycotope, which was N-linked to the synthetic membrane protein Saposin D. This synthetic receptor mediated Stx1 entry into cells, but not the uptake of Stx2 (Szymczak-Kulus et al, 2021). Furthermore, Stx1 and Stx2 prefer to bind to Gb3 containing an α-hydroxyl fatty acyl chain, but not to a Gb3 analog without the hydroxyl group (Binnington et al, 2002).…”

“…In humans and other mammals, the αGal14Gal epitope was considered to be present only on glycolipids (Gb3 and P1 antigen), but it was recently demonstrated that the Gb3 synthase can also produce αGal14Gal-capped N-glycans in transfected CHO cells ( Szymczak-Kulus et al, 2021 ). This epitope is widely present on N-glycoproteins in birds with substantial similarity between pigeon α4GalT and human Gb3 synthase (72.5%) ( Suzuki et al, 2004 ).…”

Two Paralogous Gb3/CD77 Synthases in Birds Show Different Preferences for Their Glycoprotein and Glycosphingolipid Substrates

“…Recent reports proposed that human Gb3/CD77 synthase can also attach Gal residues to glycoprotein acceptors, contrary to the long-standing belief that it can only use glycosphingolipids [ 14 , 35 ]. If the human and avian enzymes indeed synthesize similar products, it would rule out the possibility that birds are refractory to Shiga toxins due to a lack of functional receptors.…”

“…The mechanism of Stx cytotoxicity is quite well understood, but some aspects of receptor recognition remain unclear. There is a general agreement that the main receptor for Stx is Gb3, while the hypothesis that glycoproteins containing Galα1→4Gal could be alternative receptors was long abandoned but recently revisited [ 12 , 13 , 14 ].…”

Chemical and Enzymatic Synthesis of Sialylated Glycoforms of Human Erythropoietin

“…Theu nexpectedly high reactivity of the bacterial 2,6sialyltransferase towards the folded EPO A glycoprotein provides rapid access to the sialylated glycoform EPO S in as ingle step.T his straight-forward transformation bypasses the need to synthesize the three sialoglycopeptide building blocks 33-35 and carry them through at ime-consuming process involving multiple ligations,p eptide modifications and protein refolding. Theo bserved high efficiency of the late-stage enzymatic sialylation of EPO A is very encouraging.M ost likely other homogeneous glycoproteins can also be modified analogously,h owever, the feasibility and efficiency of this approach need to be evaluated on ac ase-to-case basis.T he efficient enzymatic a-1,4-galactosylation [46] of afolded N-glycoprotein was recently shown using asynthetic glycoform of Saposin D containing ab iantennary N-glycan. [16] From our experiences well-behaved proteins are preferred substrates since they readily withstand the conditions of the enzymatic reactions and the following purifications leading to good overall yields.…”