Allotype-specific processing of the CD16a N45-glycan from primary human natural killer cells and monocytes

Abstract

Fc γ receptor IIIa/CD16a is an activating cell surface receptor with a well-defined role in natural killer (NK) cell and monocyte effector function. The extracellular domain is decorated with five asparagine (N)-linked glycans; N-glycans at N162 and N45 directly contribute to high-affinity antibody binding and protein stability. N-glycan structures at N162 showed significant donor-dependent variation in a recent study of CD16a isolated from primary human NK cells, but structures at N45 were relatively homogeneous. In this study, we identified variations in N45 glycan structures associated with a polymorphism coding for histidine instead of leucine at position 48 of CD16a from two heterozygous donors. It is known that H48 homozygous individuals suffer from immunodeficiency and recurrent viral infections. A mass spectrometry analysis of protein isolated from the primary natural killer cells of individuals expressing both CD16a L48 and H48 variants demonstrated clear processing differences at N45. CD16a H48 displayed a greater proportion of complex-type N45 glycans compared to the more common L48 allotype with predominantly hybrid N45-glycoforms. Structures at the four other N-glycosylation sites showed minimal differences from data collected on donors expressing only the predominant L48 variant. CD16a H48 purified from a pool of monocytes similarly displayed increased processing at N45. Here, we provide evidence that CD16a processing is affected by the H48 residue in primary NK cells and monocytes from healthy human donors.

The oligomannose N-glycans 3D architecture and its response to the FcγRIIIa structural landscape

“…The stabilization of this glycoform by the FcγRIIIa surface landscape, renders the (1-3) branch on the (1-6) arm virtually inaccessible for further functionalization. This result agrees with recent work highlighting the unique prevalence of hybrid and oligomannose type N-glycans at N45 32, 39 . The N162 position determines very little steric hindrance to the dynamics of Man5, which retains most of the degrees of freedom characterized for the glycan free in solution.…”

“…Indeed, the two FcγRIIIa glycosylation sites studied in this work present different sets of constraints to different glycoforms and accordingly shift each conformational equilibrium specifically. This determines a diverse degree of accessibility of the arms for further functionalization by glycotransferases and glycohydrolases at N45 32, 39 , which has been found to have an unusually high degree of hybrid N-glycoforms, and ultimately exposure of the arms at N162 for contact with the IgG1 Fc N-glycans, which is implicated in modulating ADCC 19, 31, 40, 41 . Work in this direction is currently ongoing in our lab.…”

“…To understand how the protein affects the presentation of the glycans to potential receptors we have looked at the human FcγRIIIa (CD16a). Human FcγRIIIa has two N-glycosylation sites, namely N45 and N162, where the type of glycosylation affects the receptor's binding affinity to IgG1s 31,32,39 . The surface landscape around these two sites is quite different, with N162 exposed to the solvent, while N45 located in the core of one of the two structural domains, see highlighting the unique prevalence of hybrid and oligomannose type N-glycans at N45 32,39 .…”

Fc γ receptor compositional heterogeneity: Considerations for immunotherapy development

“…Homozygosity of the H48-encoding allele is believed to be responsible for an NK cell–related immunodeficiency ( 45 ). N45 from NK cell CD16a H48 in a heterozygous donor revealed greater processing, unlike the comparable processing at the four other N-glycosylation sites ( 46 ). It is possible that these N45-glycan differences are attributable to H48 disrupting CD2 binding and exposing the N45 site for further glycan processing ( 45 ).…”

Fc γ receptor IIIa/CD16a processing correlates with the expression of glycan-related genes in human natural killer cells

“…CD16a also exhibits substantial structural and functional variability. Two predominant CD16a polymorphic variants provide either greater (V158) or weaker (F158) antibody binding affinity ( 12 , 13 , 14 ). Other mechanisms to increase affinity exist, including altering posttranslation modifications that may be more widely tolerated than engineered polypeptides.…”