J Biol Chem. 2024 Nov 16;300(12):107997.  doi: 10.1016/j.jbc.2024.107997

题目：

O-glycosylation is essential for cell surface expression of the transcobalamin receptor CD320.

作者：

Chunyu Du^1,2,4, Wenjun Guo^1,2,4, Mengting Wang^1,2, Zibin Zhou³, Tiantian Zhou², Meng Liu², Ningzheng Dong^1,2,*, and Qingyu Wu^2,*

单位：

¹NHC Key Laboratory of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, Soochow University Suzhou Medical College, Suzhou 215006, China;

²Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology, State Key Laboratory of Radiation Medicine and Prevention, Soochow University, Suzhou 215123, China;

³Department of Orthopedics, the Second Affiliated Hospital of Soochow University, Suzhou 215004, China

⁴These authors contributed equally to this work

摘要：

CD320 is a cell surface receptor that mediates vitamin B12 uptake in most tissues. To date, the mechanisms that regulate CD320 expression on the cell surface are not fully understood. In this work, we studied CD320 expression in transfected human embryonic kidney (HEK) 293 and hepatoma HepG2 cells. By glycosidase and trypsin digestion, monensin and brefeldin treatment, western blotting, flow cytometry, and lectin biding, we found that CD320 underwent N- and O- glycosylation and sialylation, resulting in a ~70-kDa band that formed a high-molecular weight complex on the cell surface. Site-directed mutagenesis altering Asn126, Asn195 and Asn213 residues, individually or together, abolished N-glycosylation in CD320 but did not block its intracellular trafficking and expression on the cell surface in HEK293 and HepG2 cells. In contrast, treatment of the cells with Ben-gal, a structural analog of GalNAc-a-1-O-Ser/Thr, inhibited O- glycosylation and cell surface expression of CD320, and decreased vitamin B12 uptake. Analysis of CD320 deletion mutants indicated that O-glycosylation sites in a Ser/Thr-rich region near the transmembrane domain were important for CD320 expression on the cell surface. These results reveal an important role of O-glycans, but not N-glycans, in the intracellular trafficking and cell surface expression of CD320, providing new insights into the cellular mechanisms in regulating CD320 function and vitamin B12 metabolism.