Chunk #25 — Results — The Mr of G118/D40-hMOPR was lower than that of A118/N40-hMOPR, due to different N-glycosylation, when both were stably expressed in cultured cells
CHO cells stably expressing A118/N40-hMOPR or G118/D40-hMOPR were solubilized with Triton X-100, and hMOPRs enriched using WGL Sepharose 6MB affinity purification. Immunoblotting with anti-HA revealed A118/N40-hMOPR and G118/D40-hMOPR as two diffuse bands with median Mr's of 81 and 76 kDa, respectively (Fig. 5, lanes 1 and 2). Following removal of N-glycans by treatment with PNGase F, the difference in the Mr's of A118/N40-hMOPR and G118/D40-hMOPR disappeared, and either hMOPR was recognized as a diffuse band with identical median Mr of 48 kDa (Fig. 5, lanes 3 and 4). These results indicate that the differences in the Mr's of the two MOPR variants are due to varying degrees of N-linked glycosylation.
