(174al) Pleiotropic Effect of Glycan Perturbation on Leukocyte Immunological Response

Glycans decorate a majority of mammalian secreted and cell surface proteins. They regulate multiple biological processes includingdevelopment, inflammation, thrombosis, tumorigenesis and cancer metastasis. While glycosylation primarily affects immunological response like molecular recognition and cell adhesion, we report that glycan perturbations also alter seemingly unrelated pathways related to intracellular signaling. Here we characterized a panel of CRISPR-Cas9 human leukocytic HL-60 cell lines that lack the necessary enzymes for O-glycan ([O]‾), N-glycan ([N]‾) and glycosphingolipid/GSL ([G]‾) biosynthesis. Differentiating these cells to terminal neutrophils resulted in leukocytes that efficiently rolled and adhered on stimulated vascular endothelial cells. RNA-Seq differential expression analysis revealed that ~2-3% of the transcripts are differentially expressed in the knock-out cell lines compared to wild-type controls. While the specific perturbed transcripts in the different knockouts were distinct, they all affected common pathways related to innate immune response, cell stress response and cell communication. Ontology and detailed biochemical pathway analysis also suggest potential crosstalk among biosynthesis pathways that result in the formation of O-glycans, N-glycans, glycolipids and glycosaminoglycans. Consistent with this RNA-Seq analysis, we observed that the ablation of glycolipids resulted in an increase in bisecting N-glycan structures, while knocking out O-glycans reduced such glycans. Additionally, whereas the differentiated wild-type neutrophils exhibited a robust calcium flux response upon chemokine IL-8 stimulation, diminished flux was observed in the differentiated [O]‾ and [G]‾ cells and was absent in [N]‾. The effect was specific to inflammatory chemokines, since the bacterial mimetic formyl peptide did not alter cell calcium flux or phagocytotic capacity. Consistent with the altered response to chemokine signaling, all glycan knockouts also displayed diminished migration capacity compared to wild-type cells. Mechanistic analysis revealed that the depressed cell function may be related to reduction in the expression of the CXCR1 and CXCR2 chemokine receptors on leukocytes. Overall, the study demonstrates for the first time that in addition to controlling molecular recognition, glycan perturbations may also control cell signaling processes via their action on defined gene regulatory mechanisms.