Using CRISPR-Cas9 to quantify the contributions of O-glycans, N-glycans and Glycosphingolipids to human leukocyte-endothelium adhesion.

Gino Stolfa,Alexander Buffone,Sriram Neelamegham,Yuqi Zhu,Nandini Mondal,Xinheng Yu

doi:10.1038/srep30392

Gino Stolfa, Alexander Buffone + Show 4 more

Open Access

https://doi.org/10.1038/srep30392

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Abstract

There is often interest in dissecting the relative contributions of the N-glycans, O-glycans and glycosphingolipids (GSLs) in regulating complex biological traits like cell signaling, adhesion, development and metastasis. To address this, we developed a CRISPR-Cas9 toolkit to selectively truncate each of these commonly expressed glycan-types. Here, O-glycan biosynthesis was truncated by knocking-out Core 1 β3Gal-T Specific Molecular Chaperone (COSMC), N-glycans by targeting the β1,2 GlcNAc-transferase (MGAT1) and GSLs by deleting UDP-glucose ceramide glucosyltransferase (UGCG). These reagents were applied to reveal the glycoconjugates regulating human myeloid cell adhesion to selectins under physiological shear-flow observed during inflammation. These functional studies show that leukocyte rolling on P- and L-selectin is ablated in cells lacking O-glycans, with N-glycan truncation also increasing cell rolling velocity on L-selectin. All three glycan families contributed to E-selectin dependent cell adhesion with N-glycans contributing to all aspects of the leukocyte adhesion cascade, O-glycans only being important during initial recruitment, and GSLs stabilizing slow cell rolling and the transition to firm arrest. Overall, the genome editing tools developed here may be broadly applied in studies of cellular glycosylation.

Highlights

N-linked glycans, O-glycans and glycosphingolipids (GSLs) represent three common forms of glycoconjugates on mammalian cell surfaces
The current manuscript describes the use of the CRISPR-Cas[9] method to dissect, for the first time, the relative contributions of O-glycans, N-glycans and GSLs during shear-dependent selectin mediated human cell adhesion. We study this molecular interaction due to the broad importance of carbohydrate ligands that function as selectin-ligands during hematopoietic stem and progenitor cell (HSPC) homing to the bone marrow vascular niche[13], cancer cell metastasis[3], and leukocyte trafficking during immunity and inflammation[4,14]
O-linked glycans expressed on peripheral node addressins (PNAd) were considered to exclusively facilitate L-selectin mediated lymphocyte homing, until Mitoma et al.[15] demonstrated a critical role for N-glycans as well using mice lacking O-glycan biosynthesis pathways

Summary

Introduction

N-linked glycans, O-glycans and glycosphingolipids (GSLs) represent three common forms of glycoconjugates on mammalian cell surfaces. The current manuscript describes the use of the CRISPR-Cas[9] method to dissect, for the first time, the relative contributions of O-glycans, N-glycans and GSLs during shear-dependent selectin mediated human cell adhesion We study this molecular interaction due to the broad importance of carbohydrate ligands that function as selectin-ligands during hematopoietic stem and progenitor cell (HSPC) homing to the bone marrow vascular niche[13], cancer cell metastasis[3], and leukocyte trafficking during immunity and inflammation[4,14]. We select HL-60 s since these cells bear close resemblance to human neutrophils in terms of glycosyltransferase expression as well as selectin binding phenotype[20] Such studies are currently not possible in short-lived primary human neutrophils since CRISPR-Cas and related technologies still need to be developed to efficiently knock-out multiple genes in the hard-to-transfect primary cells, and track these knockouts in functional assays

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