Fig. 4

Sialylation of N-glycan chains at positions 6 and 15 of the β₂AR N-terminus. a Western blot analysis of whole-cell lysates from HEK-293 cells expressing wt or mutant HA-tagged β₂AR (description in Supplementary Fig. 7a). Intact cells were submitted to trypsin digestion (see Methods) to cleave the N-terminus of surface receptors. After the addition of Laemmli buffer directly to the mixture and PAGE separation, WB membranes were probed with the indicated antibodies. The 36-residue glycosylated N-termini produced by the tryptic digestion migrate faster than the corresponding un-cleaved receptors. The N-termini from N6A and N15A β₂AR, which contain only one Asn-branched glycan chain display a lower apparent MW than wt or (+11) β₂AR. b Supernatants of the same cells submitted to trypsin digestion were incubated with anti-HA-tag-coated beads to immunoprecipitate the released N-termini. c Immunoprecipitated N-termini were additionally digested with PNGase and chymotrypsin to generate glycan-free peptides for MS analysis. The shown chromatogram corresponds to the analysis of the wt β₂AR N-terminus. Spectrum annotation (b- and y-ion series) was done according to nomenclature of peptide fragmentation⁶¹. Indicated matched internal fragments (red bars) correspond to higher intensity peaks, mostly proline-starting fragments⁶² produced by higher energy collisional dissociation (HCD)-type high-resolution mass spectrometer. Indeed, the presence of proline residues causes increased peptide fragility at their N-terminal side during collisionnal-induced decay (CID)⁶³. The ^DEN residue corresponds to the change of an asparagine residue into an aspartic acid due to the enzymatic (PNGase) removal of N-linked glycans. d Receptor N-termini bound to HA-coated beads were heated in acidic conditions to release Neu5Ac from the glycan chains; liberated sialic acid was quantified by a colorimetric assay (see Methods). Left panel: amount of sialic acid released from the indicated N-termini. “WT control” corresponds to a sample of wt β₂AR bound to HA breads maintained in PBS buffer without acidic treatment. Values of Neu5Ac obtained with the assay were calibrated with the actual protein concentration of the N-terminal fragments determined by quantifying WB images (LI-COR software) (ANOVA: ****p < 0.0001, 9 samples for each condition, prepared from 3 independent batches of transfected cells). Middle panel: standard curve. Right panel: representative WB used to quantify the indicated N-terminal fragments. n.s., not significant