Citation

  • Authors: Verhallen L. et al.
  • Year: 2023
  • Journal: Cell Mol Life Sci 80 55
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: CHO
    Description: Chinese hamster ovary cells

Method

Transient transfection of CHO cells The transfection mix was made consisting of 200 µl serum-free medium (Opti-SFM, Gibco) with 0.2 µg receptor and 0.8 µg CAMYEL sensor (cAMP sensor using YFP-Epac-RLuc) for BRET experiments or 1 µg receptor for western blotting for each transfection of 2 × 106 cells. At the end, 4 µl of FectoPro (Polyplus, Illkirch, France) was added to each transfection and the mixture was kept at room temperature for 15 min. Cells were resuspended in 500 µl serum-free medium (Opti-SFM, Gibco) with 1% GlutaMAX (Gibco) per 2 × 106 cells. Next, they were transferred to a 6-well plate at 2 × 106 cells/well and incubated at 37 °C. Afterwards, 200 µl of the mixture was added and the cells were incubated for 3 h at 37 °C and 10% CO2. After incubation, 2 µl of FectoBooster (Polyplus) was added together with 2.5 ml of CHO media without penicillin and streptomycin to each cell. For inhibition of tyrosine sulfation, 100 mM sodium chlorate (NaClO3) (Sigma-Aldrich) was added to the CHO media without penicillin and streptomycin. The cells were incubated overnight at 37 °C and 10% CO2.

Abstract

Chemokine ligands and receptors regulate the directional migration of leukocytes. Post-translational modifications of chemokine receptors including O-glycosylation and tyrosine sulfation have been reported to regulate ligand binding and resulting signaling. Through in silico analyses, we determined potential conserved O-glycosylation and sulfation sites on human and murine CC chemokine receptors. Glyco-engineered CHO cell lines were used to measure the impact of O-glycosylation on CC chemokine receptor CCR5, while mutation of tyrosine residues and treatment with sodium chlorate were performed to determine the effect of tyrosine sulfation. Changing the glycosylation or tyrosine sulfation on CCR5 reduced the receptor signaling by the more positively charged CCL5 and CCL8 more profoundly compared to the less charged CCL3. The loss of negatively charged sialic acids resulted only in a minor effect on CCL3-induced signal transduction. The enzymes GalNAc-T1 and GalNAc-T11 were shown to be involved in the process of chemokine receptor O-glycosylation. These results indicate that O-glycosylation and tyrosine sulfation are involved in the fine-tuning and recognition of chemokine interactions with CCR5 and the resulting signaling.

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