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Functional genomics identifies extension of complex N-glycans as a mechanism to evade lysis by natural killer cells

ABSTRACT: Somatic mutations can lead to the transformation of healthy cells into malignant cells and allow their evasion from immune surveillance. To uncover genes that play a role in the detection and lysis of tumor cells by natural killer (NK) cells, a B lymphoblastoid cell line was subjected to a genome-wide CRISPR screen. Deletion of genes for death receptor and ligands for NK activation receptors was found in cells that survived incubation with primary NK cells. Among the top hits that facilitated NK evasion was SPPL3, a gene for an endoprotease that cleaves transmembrane glycosyl transferases. SPPL3-deficient cells accumulated glycosyl transferases, such as MGAT5, and displayed increased N-glycosylation. Binding of NK receptors NKG2D and CD2 to their corresponding ligands MICB and CD58, and binding of rituximab to CD20, was disrupted. Inhibition of N-glycan maturation restored receptor binding and sensitivity to NK cells. To investigate the cause of this resistant phenotype, a secondary CRISPR screen using a glycosylation-focused library was performed in SPPL3-deficient cells. This screen identified transferases that promote the formation of highly branched N-glycans and N-acetyl-lactosamine (LacNAc) extensions as key regulators that prevent killing. A significant enrichment of poly-LacNAc-containing tetra-antennary species was confirmed by glycoproteomic analysis. These findings provide a basis for understanding why SPPL3 deletions have been linked to cancer.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): B Cell

DISEASE(S): Lymphoma

SUBMITTER: Yanlong Ji

LAB HEAD: Henning Urlaub

PROVIDER: PXD040769 | Pride | 2024-03-19

REPOSITORIES: Pride

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