Figure 6. Nicotine exposure induces the modification of α4β2R N-linked glycans to complex forms.

(A) Endo H and PNGase F cleavage of surface α4 subunits from untreated or nicotine-treated α4β2R cells. Cells were untreated or nicotine-treated for 17 hr. Proteins on the cell surface were then biotinylated, solubilized, precipitated with streptavidin agarose and glycosidase-treated with Endo H or PNGase F enzymes on the agarose. Afterward, eluted proteins were analyzed on sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) and immunoblotted using anti-α4 antibody. (B) Swainsonine treatment blocks nicotine-induced glycan modification of surface α4 subunits. α4β2R cells were treated with the α-mannosidase inhibitor, swainsonine, for 2 hr and then swainsonine and nicotine for 17 hr. Afterward, samples were prepared as in A. (C) Neuraminidase (NMdase) cleavage of surface α4 subunits from untreated (-nicotine) or nicotine-treated (+nicotine) α4β2R cells. α4β2R cells were prepared as in A except with NMdase, which cleaves sialic acid, replacing the other glycosidase enzymes. (D) Sambucus Nigra (SNA) lectin recognizes α4 subunits after nicotine treatment. Samples were prepared as in A, and after solubilization, proteins were precipitated with the agarose-conjugated sialic acid-recognizing, lectin SNA and then analyzed by immunoblotting with anti-α4 antibody (left) and compared to samples precipitated with with streptavidin agarose as in A–C (right). (E) Ammonium chloride treatment blocks nicotine-induced glycan modification of surface α4 subunits. α4β2 cells were treated with or without ammonium chloride (NH₄Cl; 10 mM) to inhibit activity of sialyltransferases that require an acidic environment to function. Samples were processed as in B, with NH₄Cl replacing swainsonine. (F) Time course of surface α4 subunit glycan modification. α4β2R cells were nicotine-treated (+Nic) or untreated (-Nic) for displayed times, after which the cells were surface biotinylated and surface α4 subunits processed as in the previous panels. (G) Quantification of the time course of surface α4 subunit glycan modification. Densitometry of α4 subunit bands from three separate experiments, as in F, was preformed and the ratio of complex-trimmed (upper) band to the high-mannose (lower) bands are plotted as the mean ± SEM.