CD16a with oligomannose-type N-glycans is the only “low-affinity” Fc γ receptor that binds the IgG crystallizable fragment with high affinity in vitro

Fc receptors (FcRs) bind circulating IgG (IgG1) at the surface of leukocytes. Antibodies clustered at the surface of a targeted particle trigger a protective immune response through activating FcRs. Three recent reports indicate that the composition of the asparagine-linked carbohydrate chains (N-glycans) of FcRIIIa/CD16a impacted IgG1-binding affinity. Here we determined how N-glycan composition affected the affinity of the low-affinity FcRs for six homogeneous IgG1 Fc N-glycoforms (G0, G0F, G2, G2F, A2G2, and A2G2F). Surprisingly, CD16a with oligomannose N-glycans bound to IgG1 Fc (A2G2) with a K-D = 1.0 +/- 0.1 nm. This affinity represents a 51-fold increase over the affinity measured for CD16a with complex-type N-glycans (51 +/- 8 nm) and is comparable with the affinity of FcRI/CD64, the sole high-affinity FcR. CD16a N-glycan composition accounted for increases in binding affinity for the other IgG1 Fc glycoforms tested (10-50-fold). This remarkable sensitivity could only be eliminated by preventing glycosylation at Asn(162) with an Asn-to-Gln mutation; mutations at the four other N-glycosylation sites preserved tighter binding in the Man5 glycoform. None of the other low-affinity FcRs showed more than a 3.1-fold increase upon modifying the receptor N-glycan composition, including CD16b, which differs from CD16a by only four amino acid residues. This result indicates that CD16a is unique among the low-affinity FcRs, and modifying only the glycan composition of both the IgG1 Fc ligand and receptor provides a 400-fold range in affinities.