Competition between Core-2 GlcNAc-transferase and ST6GalNAc-transferase Regulates the Synthesis of the Leukocyte Selectin Ligand on Human P-selectin Glycoprotein Ligand-1

Cell Adhesion & Migration

Buffone

Neelamegham

2013

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The binding of selectins to carbohydrate epitopes expressed on leukocytes is the first step in a multi-step cell adhesion cascade that controls the rate of leukocyte recruitment at sites of inflammation. The glycans that function as selectin-ligands are post-translationally synthesized by the serial action of Golgi resident enzymes called glycosyltransferases (glycoTs). Whereas much of our current knowledge regarding the role of glycoTs in constructing selectin-ligands comes from reconstituted biochemical investigations or murine models, tools to assess the impact of these enzymes on the human ligands are relatively underdeveloped. This is significant since the selectin-ligands, particularly those that bind E-selectin, vary between different leukocyte cell populations and they are also different in humans compared with mice. To address this shortcoming, a recent study by Buffone et al. (2013) outlines a systematic strategy to knockdown upto three glycoTs simultaneously in human leukocytes. The results suggest that the fucosyltransferases (FUTs) regulating selectin-ligand synthesis may be species-specific. In particular, they demonstrate that FUT9 plays a significant role during human, but not mouse, leukocyte-endothelial interactions. Overall, this article discusses the relative roles of the FUTs during human L-, E-, and P-selectin-ligand biosynthesis, and the potential that the knockdown strategy outlined here may assess the role of other glycoTs in human leukocytes also.

Section: Role Of Carbohydrates In the Leukocyte Adhesion Cascadementioning

confidence: 99%

Section: Futs Regulating P- and L-selectin Binding Are Similar In Hummentioning

confidence: 99%

Distinct glycosyltransferases synthesize E-selectin ligands in human vs. mouse leukocytes

Cell Adhesion & Migration

Buffone

Neelamegham

2013

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“…The binding in these experiments was abrogated by an anti-E-selectin mAb HAE-1f ( Figure 3A-C). The antihuman (KPL-1, Figure 3A-B) and murine PSGL-1 mAbs (2PH1, Figure 3C) had a minor effect, 35,36 possibly due to the inhibition of secondary leukocyte-leukocyte capture in addition to primary leukocyte recruitment onto the substrate.…”

Section: Blood 22 January 2015 X Volume 125 Number 4 Regulation Of mentioning

confidence: 99%

“…Regulation of human myeloid cell binding to P-and L-selectin A core 2-based O-linked glycan bearing the sLe X epitope, located at the N terminus of PSGL-1, at Thr-57 in humans, 35,39 is the primary ligand for L-and P-selectin. Studies with knockout mice show that the biosynthesis of this O-glycan is initiated by the formation of the core 1 structure (Galb1,3GalNAca1-Thr) catalyzed by the core-1 b1,3galactosyltransferase/T-synthase.…”

Section: Application Of Crispr/cas9 For Studies Of Leukocyte Functionmentioning

confidence: 99%

ST3Gal-4 is the primary sialyltransferase regulating the synthesis of E-, P-, and L-selectin ligands on human myeloid leukocytes

Buffone

Stolfa

et al. 2015

Blood

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• A single a(2,3) sialyltransferase, ST3Gal-4, controls sLe X biosynthesis on N-and O-glycans in cells of human myeloid lineage.• Blocking this enzyme activity prevents human neutrophil adhesion to E-, P-, and L-selectin.The precise glycosyltransferase enzymes that mediate selectin-ligand biosynthesis in human leukocytes are unknown. This knowledge is important because selectin-mediated cell tethering and rolling is a critical component of both normal immune response and various vascular disorders. We evaluated the role of 3 a(2,3)sialyltransferases, ST3Gal-3, -4, and -6, which act on the type II N-Acetyllactosamine structure (Galb1,4GlcNAc) to create sialyl Lewis-X (sLe X ) and related sialofucosylated glycans on human leukocytes of myeloid lineage. These genes were either silenced using lentiviral short hairpin RNA (shRNA) or functionally ablated using the clustered regularly interspaced short palindromic repeat/ Cas9 technology. The results show that ST3Gal-4, but not ST3Gal-3 or -6, is the major sialyltransferase regulating the biosynthesis of E-, P-, and L-selectin ligands in humans. Reduction in ST3Gal-4 activity lowered cell-surface HECA-452 epitope expression by 75% to 95%. Glycomics profiling of knockouts demonstrate an almost complete loss of the sLe X epitope on both leukocyte N-and O-glycans. In cell-adhesion studies, ST3Gal-4 knockdown/knockout cells displayed 90% to 100% reduction in tethering and rolling density on all selectins. ST3Gal-4 silencing in neutrophils derived from human CD34 1 hematopoietic stem cells also resulted in 80% to 90% reduction in cell adhesion to all selectins. Overall, a single sialyltransferase regulates selectin-ligand biosynthesis in human leukocytes, unlike mice where multiple enzymes contribute to this function. (Blood. 2015;125(4):687-696)

“…Such studies have demonstrated that the sialyl Lewis-X (sLe X ) tetrasaccharide attached to a core-2 O-glycan at the N-terminus of P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a major ligand for P- and L-selectin in both humans and mice 7-9 . Whereas only 2% of the overall O-glycans of human PSGL-1 contain the sLe X core-2 O-glycan 10 , in myeloid cells, this structure constitutes ~20% of the carbohydrates at Thr-57 on the N-terminus of this glycoprotein 11 . With regard to E-selectin, the physiological carbohydrate ligands likely differ between mice and humans 1, 3 .…”

Section: Introductionmentioning

confidence: 99%

Glycosphingolipids on Human Myeloid Cells Stabilize E-Selectin–Dependent Rolling in the Multistep Leukocyte Adhesion Cascade

Stolfa

Antonopoulos

et al. 2016

ATVB

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Objective Recent studies suggest that the E-selectin ligands expressed on human leukocytes may differ from those in other species, particularly mice. To elaborate on this, we evaluated the impact of glycosphingolipids (GSLs) expressed on human myeloid cells in regulating E-selectin mediated cell adhesion. Approach and Results A series of modified human cell lines and primary neutrophils were created by targeting UDP-Glucose Ceramide Glucosyltransferase (UGCG) using either lentivirus delivered shRNA or CRISPR-Cas9 based genome editing. Enzymology and mass spectrometry confirm that the modified cells had reduced or abolished glucosylceramide biosynthesis. Glycomics profiling showed that UGCG disruption also increased prevalence of bisecting N-glycans and reduced overall sialoglycan expression on leukocyte N- and O-glycans. Microfluidics based flow chamber studies demonstrated that both the UGCG knockouts and knockdowns display ~60% reduction in leukocyte rolling and firm adhesion on E-selectin bearing stimulated endothelial cells (ECs), without altering cell adhesion to P-selectin. Consistent with the concept that the GSLs support slow rolling and the transition to firm arrest, inhibiting UGCG activity resulted in frequent leukocyte detachment events, skipping motion and reduced diapedesis across the endothelium. Cells bearing truncated O- and N-glycans also sustained cell rolling on E-selectin, although their ability to be recruited from free fluid flow was diminished. Conclusions GSLs likely contribute to human myeloid cell adhesion to E-selectin under fluid shear, particularly the transition of rolling cells to firm arrest.