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Restricted processing of CD16a/Fc ? receptor IIIa N-glycans from primary human NK cells impacts structure and function.

ABSTRACT: CD16a/Fc ? receptor IIIa is the most abundant antibody Fc receptor expressed on human natural killer (NK) cells and activates a protective cytotoxic response following engagement with antibody clustered on the surface of a pathogen or diseased tissue. Therapeutic monoclonal antibodies (mAbs) with greater Fc-mediated affinity for CD16a show superior therapeutic outcome; however, one significant factor that promotes antibody-CD16a interactions, the asparagine-linked carbohydrates (N-glycans), remains undefined. Here, we purified CD16a from the primary NK cells of three donors and identified a large proportion of hybrid (22%) and oligomannose N-glycans (23%). These proportions indicated restricted N-glycan processing and were unlike those of the recombinant CD16a forms, which have predominantly complex-type N-glycans (82%). Tethering recombinant CD16a to the membrane by including the transmembrane and intracellular domains and via coexpression with the Fc ? receptor ?-chain in HEK293F cells was expected to produce N-glycoforms similar to NK cell-derived CD16a but yielded N-glycoforms different from NK cell-derived CD16a and recombinant soluble CD16a. Of note, these differences in CD16a N-glycan composition affected antibody binding: CD16a with oligomannose N-glycans bound IgG1 Fc with 12-fold greater affinity than did CD16a having primarily complex-type and highly branched N-glycans. The changes in binding activity mirrored changes in NMR spectra of the two CD16a glycoforms, indicating that CD16a glycan composition also affects the glycoprotein's structure. These results indicated that CD16a from primary human NK cells is compositionally, and likely also functionally, distinct from commonly used recombinant forms. Furthermore, our study provides critical evidence that cell lineage determines CD16a N-glycan composition and antibody-binding affinity.

SUBMITTER: Patel KR

PROVIDER: S-EPMC5846152 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Restricted processing of CD16a/Fc γ receptor IIIa <i>N</i>-glycans from primary human NK cells impacts structure and function.

Patel Kashyap R KR Roberts Jacob T JT Subedi Ganesh P GP Barb Adam W AW

The Journal of biological chemistry 20180112 10

CD16a/Fc γ receptor IIIa is the most abundant antibody Fc receptor expressed on human natural killer (NK) cells and activates a protective cytotoxic response following engagement with antibody clustered on the surface of a pathogen or diseased tissue. Therapeutic monoclonal antibodies (mAbs) with greater Fc-mediated affinity for CD16a show superior therapeutic outcome; however, one significant factor that promotes antibody-CD16a interactions, the asparagine-linked carbohydrates (<i>N</i>-glycans ...[more]

PMID: 29330305

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Project description:Accumulating evidence suggests an important role for Natural Killer (NK) cells in the control of HIV-1 infection. Recently, it was shown that NK cell-mediated immune pressure can result in the selection of HIV-1 escape mutations. A potential mechanism for this NK cell escape is the selection of HLA class I-presented HIV-1 epitopes that allow for the engagement of inhibitory killer cell immunoglobulin-like receptors (KIRs), notably KIR2DL2. We therefore investigated the consequences of sequence variations within HLA-Cw*0102-restricted epitopes on the interaction of HLA-Cw*0102 with KIR2DL2 using a large panel of overlapping HIV-1 p24 Gag peptides. 217 decameric peptides spanning the HIV-1 p24 Gag consensus sequence were screened for HLA-Cw*0102 stabilization by co-incubation with Cw*0102?/TAP-deficient T2 cells using a flow cytometry-based assay. KIR2DL2 binding was assessed using a KIR2DL2-IgG fusion construct. Function of KIR2DL2? NK cells was flow cytometrically analyzed by measuring degranulation of primary NK cells after co-incubation with peptide-pulsed T2 cells. We identified 11 peptides stabilizing HLA-Cw*0102 on the surface of T2 cells. However, only one peptide (p24 Gag??????? AAEWDRLHPV) allowed for binding of KIR2DL2. Notably, functional analysis showed a significant inhibition of KIR2DL2? NK cells in the presence of p24 Gag???????-pulsed T2 cells, while degranulation of KIR2DL2? NK cells was not affected. Moreover, we demonstrated that sequence variations in position 7 of this epitope observed frequently in naturally occurring HIV-1 sequences can modulate binding to KIR2DL2. Our results show that the majority of HIV-1 p24 Gag peptides stabilizing HLA-Cw*0102 do not allow for binding of KIR2DL2, but identified one HLA-Cw*0102-presented peptide (p24 Gag???????) that was recognized by the inhibitory NK cell receptor KIR2DL2 leading to functional inhibition of KIR2DL2-expressing NK cells. Engagement of KIR2DL2 might protect virus-infected cells from NK cell-mediated lysis and selections of sequence polymorphisms that increase avidity to KIR2DL2 might provide a mechanism for HIV-1 to escape NK cell-mediated immune pressure.

Dataset Information

Restricted processing of CD16a/Fc ? receptor IIIa N-glycans from primary human NK cells impacts structure and function.

Publications

Restricted processing of CD16a/Fc γ receptor IIIa <i>N</i>-glycans from primary human NK cells impacts structure and function.

Similar Datasets

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