Probing the binding specificities of human Siglecs by cell-based glycan arrays

Abstract

Siglecs are a family of sialic acid–binding receptors expressed by cells of the immune system and a few other cell types capable of modulating immune cell functions upon recognition of sialoglycan ligands. While human Siglecs primarily bind to sialic acid residues on diverse types of glycoproteins and glycolipids that constitute the sialome, their fine binding specificities for elaborated complex glycan structures and the contribution of the glycoconjugate and protein context for recognition of sialoglycans at the cell surface are not fully elucidated. Here, we generated a library of isogenic human HEK293 cells with combinatorial loss/gain of individual sialyltransferase genes and the introduction of sulfotransferases for display of the human sialome and to dissect Siglec interactions in the natural context of glycoconjugates at the cell surface. We found that Siglec-4/7/15 all have distinct binding preferences for sialylated GalNAc-type O-glycans but exhibit selectivity for patterns of O-glycans as presented on distinct protein sequences. We discovered that the sulfotransferase CHST1 drives sialoglycan binding of Siglec-3/8/7/15 and that sulfation can impact the preferences for binding to O-glycan patterns. In particular, the branched Neu5Acα2–3(6-O-sulfo)Galβ1–4GlcNAc (6′-Su-SLacNAc) epitope was discovered as the binding epitope for Siglec-3 (CD33) implicated in late-onset Alzheimer’s disease. The cell-based display of the human sialome provides a versatile discovery platform that enables dissection of the genetic and biosynthetic basis for the Siglec glycan interactome and other sialic acid–binding proteins.

Carbohydrate sulfation as a mechanism for fine-tuning Siglec ligands

“…51 Kifunensine and benzyl-α-GalNAc had only minor effects in blocking Siglec binding in HEK293 cells (Figure 4k). Previous studies overexpressing CHSTs in HEK293 using genetic knockouts of subclasses of glycans made conclusions about where sulfated glycans are presented, 35 which differ to some degree with the results presented here b-h) binding of CD22, CD33, Siglec-7, 8, 9, 14, and 15 to the WT, CHST1-, and CHST2-overexpressing U937 cells with inhibition of each glycosylation pathway. (i-l) K562, Jurkat, HEK293, and A549 overexpressing CHST1 or EV were probed for ligands of CD33, Siglec-5/14 and Siglec-15 in cells treated with kifunensine or benzylα-GalNAc.…”

“…Similar trends were observed for many of the key CHST-upregulated Siglec binding interactions in U937 cells (Figure 3d-g). Interestingly, enhanced binding of Siglec-5/14 to the CHST1-overexpressing HEK293 cells was not observed previously, 35 but was readily observed in our studies, which we speculate may be attributed to non-functional commercial Siglec-5/14-Fc. In contrast with the other cells, CD33, Siglec-5/14 and Siglec-15 showed either minimal or no increase of binding to the CHST1overexpressing Jurkat cells.…”

“…Results for Siglec-8 were anticipated based on previous work, 24,32,33 while results with CD33, Siglec-7, and Siglec-15 are consistent with a recent study that overexpressed CHST1 in HEK293 cells. 35 However, the ability of CHST1 overexpression to upregulate ligands for Siglec-5 and -14, which share an identical amino acid sequence in their first three domains 41 (described from now on as Siglec-5/14), have not been described previously. A modest enhancement in binding of CD22 to the CHST1-overexpressing U937 cells was also not anticipated, but this may be due to enhanced expression of α2-6 linked sialosides, as evidenced by increased staining of SNA (Figure 1c,d).…”

“…25 Very recently, probing Siglec ligands on HEK293 cells transfected with a series of carbohydrate sulfotransferases (CHSTs) genes demonstrated that CHST1, which catalyzes the installation of the sulfate onto the 6-hydroxyl of Gal, 34 enhanced the binding of Siglec-3, -7, -8, and -15. 35 Although it is increasingly clear that sulfation plays a role in Siglec biology, numerous unanswered questions remain about the ability of CHST1 to enhance Siglec ligands, including context dependence, which was not studied in earlier work. Here, we overexpressed eight CHSTs in a variety of cell lines and discovered both enhanced and diminished binding of human and mouse Siglec-Fc proteins.…”

The Crossroads of Glycoscience, Infection, and Immunology

“…By eliminating catalytic activity and enhancing affinity using directed evolution informed by computational predictions of known molecular interactions, enhanced GBPs are generated. In theory, this innovative approach could convert any glycoactive enzyme to a binding reagent that is far more specific than traditional lectins, providing valuable reagents to further our understanding of glycobiology ( Angel et al, 2021 ; Büll et al, 2021 ).…”