Mannose-terminating N-linked glycosylation of high-affinity receptor V_H decreases antigen-affinity and antigen-induced BCR stimulation. (A) Flow-cytometric measurement of surface expression of TKO cells expressing the indicated B1-8 receptor variants in comparison with empty-vector–transduced cells. (B-C) Surface binding profile of ConA (B) and NIP₇-BSA (C) to B1-8 receptor and the indicated receptor variants compared with empty-vector–transduced TKO cells. Statistical analysis by nonparametric Mann-Whitney U test of NIP₇-BSA binding is shown (mean; n = 5: B1-8 and N variants; n = 3: Q variants; **P < .01, *P < .05). (D-E) Kinetics of Ca²⁺ mobilization upon addition of OHT and the indicated stimuli after 1-minute baseline measurement in TKO cells expressing ERT2-SLP65 and the indicated B1-8 receptor variants measured in 1 tube after distinct eFluor670 labeling. (F) Flow-cytometric measurement of surface expression of TKO cells expressing the indicated Hy10 receptor variants. (G-H) Surface binding profile of ConA (G) and sHEL (H) to the indicated receptor variants compared with empty-vector–transduced TKO cells. Statistical analysis by nonparametric Mann-Whitney U test of sHEL binding is shown (mean; n = 5). (I-J) Kinetics of Ca²⁺ mobilization upon addition of OHT and the indicated stimuli after 1-minute baseline measurement in TKO cells expressing ERT2-SLP65 and the indicated Hy10 receptor variants or empty-vector–transduced cells measured in 1 tube after distinct eFluor670 labeling. Data are representative of >3 independent experiments. EV, empty vector.