N-Glycosylation of Band 3 is essential for the binding of Ganoderma lucidum immunomodulatory protein rLZ-8

Fungal immunomodulatory proteins (FIPs) are considered potential therapeutic candidates due to their immunomodulatory and anti-cancer activities. Meanwhile, they are capable of agglutinating mammalian red blood cells in a manner similar to lectins. Herein, GST-tagged rLZ-8, the first identified FIP in Ganoderma lucidum, was employed as bait to pull down its target proteins from mouse red blood cells. An important membrane protein, Band 3, was found to interact with rLZ-8. The result was confirmed by detecting the interactions of rLZ-8 with mBand 3 and hBand 3 heterologously expressed in HEK293 cells. Moreover, the interaction specifically required the sole N-glycosylation of Band 3 at its extracellular loop (between the seventh and eighth transmembrane helices), as evidenced by diminished binding both to the wild-type hBand 3 pretreated with the N-glycan trimming enzyme PNGaseF in vitro or with the mannosidase inhibitor (N-glycosylation processing enzyme) kifunensine (KIF) in vivo and to the N642D-hBand 3 (N660D-mBand 3) mutant. Consistently, both the binding of LZ-8 to Band 3 and its hemagglutinating property were inhibited by the highly glycosylated protein bovine mucin. These results suggest that the hemagglutinating ability of LZ-8 may be attributed to its binding to the N-glycosylated proteins, including Band 3, on the erythrocyte surface. Furthermore, supporting the clinical safety profile of FIPs, stopped-flow analysis performed on human red blood cell ghosts demonstrated that the Cl⁻/HCO₃⁻ exchange activity of Band 3 remained unaltered upon binding to LZ-8. KEY POINTS: This study first reported the interaction between Band 3 and FIPs. The sole N-glycosylation of Band 3 is critical for the binding of FIPs. The addition of LZ-8 does not alter the Cl⁻/HCO₃⁻ exchange activity of human red blood cell ghosts.