Neu5Gc and beyond: sialylation at the innate immune and thromboinflammatory interface of xenotransplantation

Abstract

Recent advances in genetic engineering have substantially reduced the major carbohydrate barriers to pig-to-human xenotransplantation. Deletion of GGTA1, CMAH, and B4GALNT2 decreases α Gal, Neu5Gc, and B4GALNT2-dependent Sda-like xenoantigens and thereby attenuates preformed antibody binding and complement-mediated injury. However, removal of these major xenoantigens does not necessarily generate a human-compatible cell-surface glycome. In particular, CMAH knockout eliminates Neu5Gc but leaves a Neu5Ac-dominant sialome whose linkage, chemical modification, glycoconjugate carrier, density, and spatial presentation are not necessarily human-like. This review examines sialylation and glycan-dependent recognition as an underappreciated regulatory layer linking innate immunity, blood-cell homeostasis, complement regulation, and thromboinflammation in xenotransplantation. Two conceptual distinctions are emphasized. First, the direction and evidence depth of glycan–receptor recognition must be explicitly considered. The best-established Siglec-1-dependent xenogeneic interaction is a graft-versus-host pathway, in which donor porcine Siglec-1-positive macrophages capture recipient human erythrocytes. Conversely, direct receptor-level binding of human Siglec-1 to CMAH-deficient gene-edited pig erythrocytes has recently been demonstrated, providing evidence for the reciprocal host-versus-graft direction; however, whether this interaction drives recipient macrophage phagocytosis or in vivo clearance remains unresolved. Donor ASGR1-dependent uptake of human platelets provides another direct example of glycan-dependent recipient blood-cell clearance. Second, α 2,3-linked Neu5Ac illustrates a fundamental functional trade-off in sialome engineering. Neu5Ac α 2,3Gal-containing glycans can serve as preferred ligands for Siglec-1-mediated capture, whereas α 2,3-linked sialoglycans also contribute to Factor H-dependent recognition of self-associated surfaces and alternative complement regulation. Conversely, increasing α 2,6-linked sialylation may reduce macrophage xenoreactivity and enhance inhibitory Siglec engagement but decrease α 2,3-dependent Factor H recognition; thus, ST6GAL1 engineering should not be considered uniformly protective or equivalent to glycan “humanization”. Other pathways, including CD24–Siglec-10 signaling, Siglec-9-mediated regulation of neutrophil activation and NETosis, and platelet Siglec signaling, provide additional mechanistic frameworks but remain incompletely validated in solid-organ xenotransplantation. We therefore propose that post-editing glycan compatibility should be evaluated according to receptor directionality, evidence depth, and functional trade-offs, rather than glycan abundance or donor genotype alone. Organ-, cell-, carrier-, and receptor-specific analyses integrating quantitative glycomics/glycoproteomics, human receptor and immune-cell assays, functional crossmatching, and ex vivo perfusion are needed to link donor genotype to post-editing glycan phenotype and guide precision optimization of xenograft compatibility.