A Genome-Wide CRISPR/Cas9-Based Screen Identifies Heparan Sulfate Proteoglycans as Ligands of Killer-Cell Immunoglobulin-Like Receptors

While human leukocyte antigen (HLA) and HLA-like proteins comprise an overwhelming majority of known ligands for NK-cell receptors, the interactions of NK-cell receptors with non-conventional ligands, particularly carbohydrate antigens, is less well described. We previously found through a bead-base...

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Autores principales:	Klara Klein, Angelique Hölzemer, Tim Wang, Tae-Eun Kim, Haley L. Dugan, Stephanie Jost, Marcus Altfeld, Wilfredo F. Garcia-Beltran
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	CRISPR screen KIR heparan sulfate NK cells Immunologic diseases. Allergy RC581-607
Acceso en línea:	https://doaj.org/article/817f14afd14f4171ab4d81ffe9b0d460
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spelling	oai:doaj.org-article:817f14afd14f4171ab4d81ffe9b0d4602021-12-01T15:33:35ZA Genome-Wide CRISPR/Cas9-Based Screen Identifies Heparan Sulfate Proteoglycans as Ligands of Killer-Cell Immunoglobulin-Like Receptors1664-322410.3389/fimmu.2021.798235https://doaj.org/article/817f14afd14f4171ab4d81ffe9b0d4602021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fimmu.2021.798235/fullhttps://doaj.org/toc/1664-3224While human leukocyte antigen (HLA) and HLA-like proteins comprise an overwhelming majority of known ligands for NK-cell receptors, the interactions of NK-cell receptors with non-conventional ligands, particularly carbohydrate antigens, is less well described. We previously found through a bead-based HLA screen that KIR3DS1, a formerly orphan member of the killer-cell immunoglobulin-like receptor (KIR) family, binds to HLA-F. In this study, we assessed the ligand binding profile of KIR3DS1 to cell lines using Fc fusion constructs, and discovered that KIR3DS1-Fc exhibited binding to several human cell lines including ones devoid of HLA. To identify these non-HLA ligands, we developed a magnetic enrichment-based genome-wide CRISPR/Cas9 knock-out screen approach, and identified enzymes involved in the biosynthesis of heparan sulfate as crucial for the binding of KIR3DS1-Fc to K562 cells. This interaction between KIR3DS1 and heparan sulfate was confirmed via surface plasmon resonance, and removal of heparan sulfate proteoglycans from cell surfaces abolished KIR3DS1-Fc binding. Testing of additional KIR-Fc constructs demonstrated that KIR family members containing a D0 domain (KIR3DS1, KIR3DL1, KIR3DL2, KIR2DL4, and KIR2DL5) bound to heparan sulfate, while those without a D0 domain (KIR2DL1, KIR2DL2, KIR2DL3, and KIR2DS4) did not. Overall, this study demonstrates the use of a genome-wide CRISPR/Cas9 knock-out strategy to unbiasedly identify unconventional ligands of NK-cell receptors. Furthermore, we uncover a previously underrecognized binding of various activating and inhibitory KIRs to heparan sulfate proteoglycans that may play a role in NK-cell receptor signaling and target-cell recognition.Klara KleinKlara KleinKlara KleinAngelique HölzemerAngelique HölzemerAngelique HölzemerTim WangTim WangTim WangTae-Eun KimHaley L. DuganHaley L. DuganStephanie JostStephanie JostMarcus AltfeldWilfredo F. Garcia-BeltranWilfredo F. Garcia-BeltranFrontiers Media S.A.articleCRISPRscreenKIRheparan sulfateNK cellsImmunologic diseases. AllergyRC581-607ENFrontiers in Immunology, Vol 12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR screen KIR heparan sulfate NK cells Immunologic diseases. Allergy RC581-607
spellingShingle	CRISPR screen KIR heparan sulfate NK cells Immunologic diseases. Allergy RC581-607 Klara Klein Klara Klein Klara Klein Angelique Hölzemer Angelique Hölzemer Angelique Hölzemer Tim Wang Tim Wang Tim Wang Tae-Eun Kim Haley L. Dugan Haley L. Dugan Stephanie Jost Stephanie Jost Marcus Altfeld Wilfredo F. Garcia-Beltran Wilfredo F. Garcia-Beltran A Genome-Wide CRISPR/Cas9-Based Screen Identifies Heparan Sulfate Proteoglycans as Ligands of Killer-Cell Immunoglobulin-Like Receptors
description	While human leukocyte antigen (HLA) and HLA-like proteins comprise an overwhelming majority of known ligands for NK-cell receptors, the interactions of NK-cell receptors with non-conventional ligands, particularly carbohydrate antigens, is less well described. We previously found through a bead-based HLA screen that KIR3DS1, a formerly orphan member of the killer-cell immunoglobulin-like receptor (KIR) family, binds to HLA-F. In this study, we assessed the ligand binding profile of KIR3DS1 to cell lines using Fc fusion constructs, and discovered that KIR3DS1-Fc exhibited binding to several human cell lines including ones devoid of HLA. To identify these non-HLA ligands, we developed a magnetic enrichment-based genome-wide CRISPR/Cas9 knock-out screen approach, and identified enzymes involved in the biosynthesis of heparan sulfate as crucial for the binding of KIR3DS1-Fc to K562 cells. This interaction between KIR3DS1 and heparan sulfate was confirmed via surface plasmon resonance, and removal of heparan sulfate proteoglycans from cell surfaces abolished KIR3DS1-Fc binding. Testing of additional KIR-Fc constructs demonstrated that KIR family members containing a D0 domain (KIR3DS1, KIR3DL1, KIR3DL2, KIR2DL4, and KIR2DL5) bound to heparan sulfate, while those without a D0 domain (KIR2DL1, KIR2DL2, KIR2DL3, and KIR2DS4) did not. Overall, this study demonstrates the use of a genome-wide CRISPR/Cas9 knock-out strategy to unbiasedly identify unconventional ligands of NK-cell receptors. Furthermore, we uncover a previously underrecognized binding of various activating and inhibitory KIRs to heparan sulfate proteoglycans that may play a role in NK-cell receptor signaling and target-cell recognition.
format	article
author	Klara Klein Klara Klein Klara Klein Angelique Hölzemer Angelique Hölzemer Angelique Hölzemer Tim Wang Tim Wang Tim Wang Tae-Eun Kim Haley L. Dugan Haley L. Dugan Stephanie Jost Stephanie Jost Marcus Altfeld Wilfredo F. Garcia-Beltran Wilfredo F. Garcia-Beltran
author_facet	Klara Klein Klara Klein Klara Klein Angelique Hölzemer Angelique Hölzemer Angelique Hölzemer Tim Wang Tim Wang Tim Wang Tae-Eun Kim Haley L. Dugan Haley L. Dugan Stephanie Jost Stephanie Jost Marcus Altfeld Wilfredo F. Garcia-Beltran Wilfredo F. Garcia-Beltran
author_sort	Klara Klein
title	A Genome-Wide CRISPR/Cas9-Based Screen Identifies Heparan Sulfate Proteoglycans as Ligands of Killer-Cell Immunoglobulin-Like Receptors
title_short	A Genome-Wide CRISPR/Cas9-Based Screen Identifies Heparan Sulfate Proteoglycans as Ligands of Killer-Cell Immunoglobulin-Like Receptors
title_full	A Genome-Wide CRISPR/Cas9-Based Screen Identifies Heparan Sulfate Proteoglycans as Ligands of Killer-Cell Immunoglobulin-Like Receptors
title_fullStr	A Genome-Wide CRISPR/Cas9-Based Screen Identifies Heparan Sulfate Proteoglycans as Ligands of Killer-Cell Immunoglobulin-Like Receptors
title_full_unstemmed	A Genome-Wide CRISPR/Cas9-Based Screen Identifies Heparan Sulfate Proteoglycans as Ligands of Killer-Cell Immunoglobulin-Like Receptors
title_sort	genome-wide crispr/cas9-based screen identifies heparan sulfate proteoglycans as ligands of killer-cell immunoglobulin-like receptors
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/817f14afd14f4171ab4d81ffe9b0d460
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A Genome-Wide CRISPR/Cas9-Based Screen Identifies Heparan Sulfate Proteoglycans as Ligands of Killer-Cell Immunoglobulin-Like Receptors

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