Effect of N-glycosylation on constitutive signal transduction by mutated cytokine receptor-like factor 2

• Published in: Biochimica et biophysica acta. General subjects Vol. 1867; no. 11; p. 130465
• Main Authors: Yamamoto, Rio, Segawa, Ryosuke, Liu, Jianwei, Isaji, Tomoya, Gu, Jianguo, Hiratsuka, Masahiro, Hirasawa, Noriyasu
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.11.2023
• Subjects: B-cell acute lymphoblastic leukemia; Cytokine receptor-like factor 2; cytokines; flow cytometry; Glu-Ile-Met; glutamine; Glycosylation; Golgi apparatus; lymphocytic leukemia; polysaccharides; signal transduction; somatic mutation; Thymic stromal lymphopoietin; transactivators; PBS; B-ALL; SDS; TSLP; KO; Glu-Ile-Met; EIM; PNGase F; Glycosylation; EndoH; Cytokine receptor-like factor 2; STAT; HEK293; GnT-I; Fut8; Thymic stromal lymphopoietin; IL-7Rα; JAKs; GAPDH; WT; B-cell acute lymphoblastic leukemia; CRLF2
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2023.130465

Cytokine receptor-like factor 2 (CRLF2) is a subunit of the receptor for thymic stromal lymphopoietin (TSLP). A somatic mutation (insEIM) in the transmembrane domains of CRLF2 has been identified in acute lymphocytic leukemia (ALL), and Glu-Ile-Met (EIM) CRLF2 induces constitutive activation of signals. However, the signaling mechanism remains unclear. HEK293 cells were transfected with expression vectors encoding wild-type (WT), insEIM CRLF2, or their mutants which N-glycosylation site was replaced with a glutamine. Cell surface expression of CRLF2 was assessed by flow cytometry. Total CRLF2 and phosphorylated signal transducer and activator of transcription 5 (STAT5) were detected by western blotting. Three major species of CRLF2 (53-, 57- and 58-kDa) were identified. Deglycosylation analysis revealed that they were modified with complex-type and oligomannose-type glycans. The expression of both WT and EIM CRLF2 decreased in N-acetylglucosaminyltransferase (GnT)-I (MGAT1) knockout (KO) cells and slightly decreased in α1,6-fucosyltransferase (Fut8) KO cells compared to that in the control cells. In GnT-I or Fut8 KO cells, WT CRLF2 did not induce ligand-independent activation. Both WT and EIM CRLF2 contained four N-glycosylation sites. N55 of CRLF2 was required for the cell surface expression and activation by EIM CRLF2. We found that N-glycosylation of CRLF2 plays crucial roles for its cell surface expression and signaling. However, N-glycan processing in the Golgi apparatus does not seem to be essential for ligand-independent activation of EIM CRLF2. Our studies provide a crucial role of glycosylation in the cell surface expression of receptors. •CRLF2 existed in three molecular species, two with complex-type sugar chains and the other with oligomannose-type N-glycans.•Glycans of EIM CRLF2 were rich in immature form.•N-glycan processing in the Golgi apparatus was not essential for the ligand-independent activation in the EIM CRLF2.•N-glycosylation on CRLF2 plays crucial roles in its expression on the cell surface and cellular signaling.