Selective N-glycan editing on living cell surfaces to probe glycoconjugate function.

Cell surfaces are glycosylated in various ways with high heterogeneity, which usually leads to ambiguous conclusions about glycan-involved biological functions. Here, we describe a two-step chemoenzymatic approach for N-glycan-subtype-selective editing on the surface of living cells that consists of a first 'delete' step to remove heterogeneous N-glycoforms of a certain subclass and a second 'insert' step to assemble a well-defined N-glycan back onto the pretreated glyco-sites. Such glyco-edited cells, carrying more homogeneous oligosaccharide structures, could enable precise understanding of carbohydrate-mediated functions. In particular, N-glycan-subtype-selective remodeling and imaging with different monosaccharide motifs at the non-reducing end were successfully achieved. Using a combination of the expression system of the Lec4 CHO cell line and this two-step glycan-editing approach, opioid receptor delta 1 (OPRD1) was investigated to correlate its glycostructures with the biological functions of receptor dimerization, agonist-induced signaling and internalization.

Dynamic States of the Ligand-Free Class A G Protein-Coupled Receptor Extracellular Side.

G protein-coupled receptors (GPCRs) make up the largest family of drug targets. The second extracellular loop (ECL2) and extracellular end of the third transmembrane helix (TM3) are basic structural elements of the GPCR ligand binding site. Currently, the disulfide bond between the two conserved cysteines in the ECL2 and TM3 is considered to be a basic GPCR structural feature. This disulfide bond has a significant effect on receptor dynamics and ligand binding. Here, molecular dynamics simulations and experimental results show that the two cysteines are distant from one another in the highest-population conformational state of ligand-free class A GPCRs and do not form a disulfide bond, indicating that the dynamics of the GPCR extracellular side are different from our conventional understanding. These surprising dynamics should have important effects on the drug binding process. On the basis of the two distinct ligand-free states, we suggest two kinetic processes for binding of ligands to GPCRs. These results challenge our commonly held beliefs regarding both GPCR structural features and ligand binding.

N-glycosylation of the ß2 adrenergic receptor regulates receptor function by modulating dimerization.

N-glycosylation is a common post-translational modification of G-protein-coupled receptors (GPCRs). However, it remains unknown how N-glycosylation affects GPCR signaling. ß2 adrenergic receptor (ß2 AR) has three N-glycosylation sites: Asn6, Asn15 at the N-terminus, and Asn187 at the second extracellular loop (ECL2). Here, we show that deletion of the N-glycan did not affect receptor expression and ligand binding. Deletion of the N-glycan at the N-terminus rather than Asn187 showed decreased effects on isoproterenol-promoted G-protein-dependent signaling, ß-arrestin2 recruitment, and receptor internalization. Both N6Q and N15Q showed decreased receptor dimerization, while N187Q did not influence receptor dimerization. As decreased ß2 AR homodimer accompanied with reduced efficiency for receptor function, we proposed that the N-glycosylation of ß2 AR regulated receptor function by influencing receptor dimerization. To verify this hypothesis, we further paid attention to the residues at the dimerization interface. Studies of Lys60 and Glu338, two residues at the receptor dimerization interface, exhibited that the K60A/E338A showed decreased ß2 AR dimerization and its effects on receptor signaling were similar to N6Q and N15Q, which further supported the importance of receptor dimerization for receptor function. This work provides new insights into the relationship among glycosylation, dimerization, and function of GPCRs. ENZYMES: Peptide-N-glycosidase F (PNGase F, EC 3.2.2.11); endo-ß-N-acetylglucosaminidase A (Endo-A, EC 3.2.1.96).

Glycoengineering and glycosite-specific labeling of serum IgGs from various species.

Chemoenzymatic glycoengineering of immunoglobulin G (IgG) catalyzed by Endo-S is a powerful approach to remodel the heterogeneous N-glycoforms of Fc domain with a homogeneous synthetic glycan structure for enhanced Fc receptor-mediated effector functions. The previous researches on the method development mainly focused on human or humanized IgGs with therapeutic potentials. Here, for the first time we report the extended application of this method on glycan-remodeling of serum IgGs from other species including rabbit, mouse, and goat. Harnessing an azido-tagged non-natural N-glycan substrate and successive click reaction, glycosite-specific fluorescent labeling of IgGs was enabled. This study provided a new avenue for glycoengineering and Fc-specific labeling of IgGs with minimized influence on antigen-binding domains, and this method was adaptive to thousands of commercial antibody reagents from various species with great application potentials.