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

Abstract

AbstractSomatic 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. Among the top hits that facilitated NK evasion wasSPPL3, which encodes an intramembrane protease that cleaves transmembrane glycosyltransferases in the Golgi apparatus.SPPL3-deficient cells accumulated glycosyltransferases, such as acetylglucosaminyltransferase 5 (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 bySPPL3-deletion. Inhibition of N-glycan maturation restored receptor binding and sensitivity to NK cells. To elucidate the mechanism of this resistant phenotype, a secondary CRISPR screen was performed inSPPL3-deficient cells. This screen identified glycosyltransferases that catalyze 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 mechanistic insight into howSPPL3deletions have been linked to cancer.