The sialyltransferase ST6GAL1 protects against radiation-induced gastrointestinal damage.

High-dose irradiation poses extreme risk of mortality from acute damage to the hematopoietic compartment and gastrointestinal tract. While bone marrow transplantation can reestablish the hematopoietic compartment, a more imminent risk of death is posed by gastrointestinal acute radiation syndrome (GI-ARS), for which there are no FDA-approved medical countermeasures. Although the mechanisms dictating the severity of GI-ARS remain incompletely understood, sialylation by ST6GAL1 has been shown to protect against radiation-induced apoptosis in vitro. Here, we used a C57BL/6 St6gal1-KO mouse model to investigate the contribution of ST6GAL1 to susceptibility to total body irradiation in vivo. Twelve gray total body ionizing γ-irradiation (TBI) followed by bone marrow transplant is not lethal to wild-type mice, but St6gal1-KO counterparts succumbed within 7 d. Both St6gal1-KO and wild-type animals exhibited damage to the GI epithelium, diarrhea and weight loss, but these symptoms became progressively more severe in the St6gal1-KO animals while wild-type counterparts showed signs of recovery by 120 h after TBI. Increased apoptosis in the GI tracts of St6gal1-KO mice and the absence of regenerative crypts were also observed. Together, these observations highlight an important role for ST6GAL1 in protection and recovery from GI-ARS in vivo.

Extrinsic sialylation is dynamically regulated by systemic triggers in vivo.

Recent reports have documented that extracellular sialyltransferases can remodel both cell-surface and secreted glycans by a process other than the canonical cell-autonomous glycosylation that occurs within the intracellular secretory apparatus. Despite association of the abundance of these extracellular sialyltransferases, particularly ST6Gal-1, with disease states such as cancer and a variety of inflammatory conditions, the prevalence of this extrinsic glycosylation pathway in vivo remains unknown. Here we observed no significant extrinsic sialylation in resting mice, suggesting that extrinsic sialylation is not a constitutive process. However, extrinsic sialylation in the periphery could be triggered by inflammatory challenges, such as exposure to ionizing radiation or to bacterial lipopolysaccharides. Sialic acids from circulating platelets were used in vivo to remodel target cell surfaces. Platelet activation was minimally sufficient to elicit extrinsic sialylation, as demonstrated with the FeCl3 model of mesenteric artery thrombosis. Although extracellular ST6Gal-1 supports extrinsic sialylation, other sialyltransferases are present in systemic circulation. We also observed in vivo extrinsic sialylation in animals deficient in ST6Gal-1, demonstrating that extrinsic sialylation is not mediated exclusively by ST6Gal-1. Together, these observations form an emerging picture of glycans biosynthesized by the canonical cell-autonomous glycosylation pathway, but subjected to remodeling by extracellular glycan-modifying enzymes.

Platelets support extracellular sialylation by supplying the sugar donor substrate.

Sizable pools of freely circulating glycosyltransferases are in blood, but understanding their physiologic contributions has been hampered because functional sources of sugar donor substrates needed to drive extracellular glycosylation have not been identified. The blood-borne ST6Gal-1 produced and secreted by the liver is the most noted among the circulatory glycosyltransferases, and decorates marrow hematopoietic progenitor cells with α2,6-linked sialic acids and restricts blood cell production. Platelets, upon activation, secrete a plethora of bioactive molecules including pro- and anti-inflammatory mediators. Cargos of sugar donor substrates for glycosyltransferase activity have also been reported in platelets. Here, we implemented a cell-based system to interrogate platelets for their ability to deliver effectively the sugar donor substrate for extracellular ST6Gal-1 to function. We report that thrombin-activated platelets, at physiologic concentration and pH, can efficiently and effectively substitute for CMP-sialic acid in extracellular ST6Gal-1-mediated sialylation of target cell surfaces. Activated platelets can also supply the sialic acid donor to sialylate the synthetic acceptor, Gal(ß1,4)GlcNAcα-o-benzyl, with the product Sia(α2,6)Gal(ß1,4)GlcNAcα-o-benzyl structurally confirmed by LC/MS. Platelet-secreted donor substrate was recovered in the 100,000 × g sediment, strongly suggesting the association of this otherwise soluble substrate, putatively CMP-sialic acid, within platelet microparticles. Sequestration within microparticles may facilitate delivery of glycosylation substrate at effective dosages to sites of extracellular glycosylation while minimizing excessive dilution.

Leukocyte-borne α(1,3)-fucose is a negative regulator of ß2-integrin-dependent recruitment in lung inflammation.

Leukocyte recruitment in inflammation is a multistep, sequential cascade where the initial step is the selectin-dependent tethering, followed by the formation of firmer integrin-mediated adhesive forces leading to extravasation. The α(1,3)-fucose-containing sialyl-Lewis X (sLeX) is the archetypical ligand on leukocyte surfaces mediating selectin interactions. Canonically, disruption of α(1,3)-fucose formation ablates selectin-mediated adhesion, dramatically reducing trafficking. We report a paradoxical response to α(1,3)-fucose deficiency in which the loss exacerbated rather than attenuated leukocyte recruitment in a murine model of acute airway inflammation. The architecture of the capillary-dominated vasculature in the lung minimized the importance of the selectin dependent step, and we observed that α(1,3)-fucose deficiency augmented CXCR2-mediated Rap1-GTP signaling to enhance the ß2-integrin-ICAM-1-binding axis. The data disclose a previously unknown function for α(1,3)-fucose, in which this structure negatively regulates the integrin activation step in leukocyte recruitment.