Extrinsic sialylation is dynamically regulated by systemic triggers in vivo

Abstract

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.

Keywords: CMAH; Neu5Gc; ST6Gal-1; inflammation; platelet; platelets; sialic acid; sialylation; sialyltransferase; thrombosis.

Figure 1.

Irradiation triggers extrinsic sialylation in vivo. St6gal1-null marrow cells (CD45.2⁺) were transplanted into irradiated C57BL/6 (CD45.1⁺) recipients as described previously (12). Donor-derived CD45.2⁺ cells in the blood were assessed for SNA reactivity to determine cell-surface α2,6-sialylation. A, D0 represents St6gal1-null blood cells harvested from the native St6gal1-null mouse as baseline negative α2,6-sialylation profile. D7, D15, and D50 represent CD45.2⁺ cells recovered from recipients at 7, 15, and 50 days after transplantation, respectively. D50+IR represents samples from animals that received 12 grays of irradiation 24 h prior to blood collection at D50. B, comparison of geometric mean of SNA signal in CD45.2⁺ cells collected at D50 and D50+IR. Shown are total marrow nucleated cells (Total Marrow), marrow GR-1⁺ cells (Marrow Gr-1⁺), or total circulating cells (Total Blood). Dashed lines signify the geometric mean of equivalent cell fractions from the native St6gal1-null animal (D0) as baseline negative α2,6-sialylation. n of 3 animals were used for each data point. Statistical significance for the difference is indicated by * (p < = 0.05) or ** (p < = 0.005).

Figure 2.

Inflammation and platelet activation triggers extrinsic sialylation. A, wild-type platelets from C57/BL6 animals, which contain the Neu5Gc form of sialic acid, were transfused into CMAH-null animals natively lacking Neu5Gc and expressing only Neu5Ac (A1), circulating white cells were probed for Neu5Gc either not subjected to or 3 h after being subjected to intratracheal inoculation of LPS to elicit a localized bout of acute airway inflammation (A2 and A3, respectively), as described under “Experimental Procedures.” B, Neu5Gc staining of frozen lung sections. B1, uniform staining of a C57BL/6 lung. B2 and B3, respectively, Neu5Gc staining of CMAH-null recipients transfused with C57BL/6 platelets either not subjected to or 3 h after being subjected to intratracheal inoculation of LPS. C, mesentery arteries stained for Neu5Gc. C1, C57BL/6 at baseline. C2 and C3, respectively, CMAH-null recipients transfused with C57BL/6 platelets either without or with a 10-min application of FeCl3 to induce localized thrombosis as described under “Experimental Procedures.” I and L denote intima and lumen of the artery, respectively. D, comparison of CMAH-null and CMAH-null/St6gal1-null recipients receiving Neu5Gc⁺ wild-type platelets and 3 h after being subjected to intratracheal inoculations of LPS (D1 and D2, respectively), demonstrating the ability to extrinsically transfer platelet Neu5Gc despite the absence of systemic ST6Gal-1, which still occurs using platelets from ST6Gal-1–deficient animals (D3). D4 shows the absence of SNA reactivity in the CMAH-null/St6gal1-null recipients despite receiving SNA⁺ wild-type platelets. For all panels, CD41-phycoerythrin (CD41-PE) (platelet marker) is visualized in red.