Genome wide effects of glycogene knockouts on cellular pathways

Glycans regulate multiple biological processes including development, inflammation, thrombosis, tumorigenesis and cancer metastasis. While glycosylation primarily affects molecular recognition and protein folding, glycan perturbations may also alter unrelated pathways related to intracellular signaling. Here we characterized a panel of CRISPR-Cas9 human leukocytic HL60 cell lines that lack the necessary enzymes for O-glycan , N-glycan and glycosphingolipid/GSL 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. Perturbed transcripts in different knockouts affected common pathways related to innate immune response, cell stress response and cell communication. Ontology and detailed biochemical pathway analysis also suggestedpotential crosstalk among different glycosylation pathways. Additionally, whereas the differentiated wild-type neutrophils exhibited a robust calcium flux response upon chemokine IL-8 stimulation, diminished flux was observed in the GSL knockout cells and was absent in O-linked and N-linked knockouts. 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 cellular cell signaling processes via their action on defined gene regulatory mechanisms.