Abstract
Post-translational modification confers diverse functional properties to immune system proteins. The composition of serum proteins such as immunoglobulin G (IgG) strongly associates with disease including forms lacking a fucose modification of the crystallizable fragment (Fc) asparagine(N)-linked glycan that show increased effector function, however, virtually nothing is known about the composition of cell surface receptors or their bound ligands in situ because of low abundance in the circulating blood. We isolated primary NK cells from apheresis filters following plasma or platelet donation to characterize the compositional variability of Fc γ receptor IIIa/CD16a and its bound ligand, IgG1. CD16a N162-glycans showed the largest differences between donors; one donor displayed only oligomannose-type N-glycans at N162 that correlate with high affinity IgG1 Fc binding whereas the other donors displayed a high degree of compositional variability at this site. Hybrid-type N-glycans with intermediate processing dominated at N45 and highly modified, complex-type N-glycans decorated N38 and N74 from all donors. Analysis of the IgG1 ligand bound to NK cell CD16a revealed a sharp decrease in antibody fucosylation (43.2 ± 11.0%) versus serum from the same donors (89.7 ± 3.9%). Thus, NK cells express CD16a with unique modification patterns and preferentially bind IgG1 without the Fc fucose modification at the cell surface.
Highlights
Primary Human Natural Killer Cells Retain Proinflammatory IgG1 at the Cell Surface and Express CD16a Glycoforms with Donordependent Variability*□S
Fc ␥ receptor IIIa (Fc␥RIIIa/ CD16a) is a protein expressed on natural killer (NK)1 cells that binds immunoglobulin G (IgG), triggering a protective immune response (Fig. 1A)
Donor allotype was confirmed for four of the five donors by sequencing CD16a cDNA; we observed peptides corresponding to both alleles from the NK09 donor but only found cDNA corresponding to F158 that may be explained by the greater abundance of the F158-containing transcripts in the NK09 NK cells biasing the cDNA sequencing results
Summary
Experimental Design—The healthy human donors were de-identified and not directly enrolled for the study but rather randomly selected based on scheduled donations at the blood centers. Mass Spectrometry—Samples were resuspended in 5 l of 5% ACN, 0.1% TFA acid in water separated on a 75 m ϫ 20 cm fused silica capillary (Agilent Technologies, Santa Clara, CA) column/ emitter attached to an EASY nLC-1200 LC system with a Nanospray FlexIon source (Thermo) The tip of this column was fabricated using a laser puller (Sutter P-200, Novato, CA) and was packed with 5 m Agilent Zorbax C18 medium using a pressure cell ( Advance, Troy, NY). Once retention times for each glycopeptide were identified, we generated a comprehensive mass list for all possible CD16a glycopeptides based on species identified by manual assignment in one NK cell data set (NK13) and N-glycans identified in a previous NK cell CD16a glycomics study as well as any potential N-glycan variations [6]. The segments were cloned into pGEM-T Easy Vector (Promega) and sequenced
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