Dataset Information

An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells

ABSTRACT: The structural diversity of glycans on cells – the glycome – is vast and complex to decipher. Glycan arrays have played a pivotal role in exploring the informational content of glycans. Current glycan arrays display oligosaccharides generated by chemical and chemoenzymatic synthesis or isolated from biological sources and are used to report glycan hapten binding epitopes. Glycan arrays are limited resources, produced through considerable efforts, and they display individual saccharides without the natural context of other glycans and glycoconjugates at the cell surface. We usedmaps of glycosylation pathways to generate a library of isogenic HEK293 cells with combinatorially engineered glycosylation capacities designed to display and dissect the genetic, biosynthetic and structural basis for glycan binding epitopes in a natural context. This expandable and self-renewable cell-based glycan array reports glycosyltransferase genes required (or blocking) for interactions through logic sequential biosynthetic steps, which not only predicts essential structural features of involved glycans including the glycoconjugate context, but importantly provides instructions for enzymatic synthesis, recombinant production, and genetic strategies to dissect biological functions. The broad utility of this cell-based glycan array and its comprehensive read-out is demonstrated by dissecting glycan-binding specificities of microbial adhesins, and the discovery power is demonstrated by uncovering higher order binding of microbial adhesins to clustered patches of O-glycans organized by their presentation on proteins.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Sergey Vakhrushev

LAB HEAD: Henrik Clausen

PROVIDER: PXD013676 | Pride | 2019-06-24

REPOSITORIES: Pride

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Publications

An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells.

Narimatsu Yoshiki Y Joshi Hiren J HJ Nason Rebecca R Van Coillie Julie J Karlsson Richard R Sun Lingbo L Ye Zilu Z Chen Yen-Hsi YH Schjoldager Katrine T KT Steentoft Catharina C Furukawa Sanae S Bensing Barbara A BA Sullam Paul M PM Thompson Andrew J AJ Paulson James C JC Büll Christian C Adema Gosse J GJ Mandel Ulla U Hansen Lars L Bennett Eric Paul EP Varki Ajit A Vakhrushev Sergey Y SY Yang Zhang Z Clausen Henrik H

Molecular cell 20190618 2

The structural diversity of glycans on cells-the glycome-is vast and complex to decipher. Glycan arrays display oligosaccharides and are used to report glycan hapten binding epitopes. Glycan arrays are limited resources and present saccharides without the context of other glycans and glycoconjugates. We used maps of glycosylation pathways to generate a library of isogenic HEK293 cells with combinatorially engineered glycosylation capacities designed to display and dissect the genetic, biosynthet ...[more]

PMID: 31227230

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Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus utilizes an extensively glycosylated spike protein that protrudes from the viral envelope to bind to angiotensin-converting enzyme-related carboxypeptidase (ACE2) as its primary receptor to mediate host-cell entry. Currently, the predominant recombinant spike protein production hosts are Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. In this study, recombinant full-size spike protein was produced transiently in CHO and HEK cell suspensions. To further evaluate the sialic acid linkages presenting on spike glycans, a two-step amidation process, employing dimethylamine and ammonium hydroxide reactions in a solid support system, was developed to differentially modify the sialic acid linkages on glycans and glycopeptides from spike protein. We determined global and site-specific N-linked glycosylation patterns in soluble SARS-CoV-2 spike using MALDI-TOF and LC-MS/MS with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation. We identified the glycan compositions at 21 and 19 out of the 22 predicted N-glycosylation sites of the SARS-CoV-2 spike proteins produced in CHO and HEK, respectively. The N-glycan site at 1158 position (N1158) and the N-glycan sites at 122 ,282 and 1158 positions (N122, N282 and N1158) were found to be unoccupied on spike secreted from CHO and HEK cells, respectively. The structural mapping of glycans of recombinant human spike proteins revealed that CHO-Spike presented more complex and higher sialylation (α2,3-linked) content while HEK-Spike displayed more high-mannose and minor content of α2,3- and α2,6-linked sialic acids. The N74 site represents the most heavily N-glycosylated site on both spike proteins while some high-mannose abundant sites (N17, N234, N343, N616, N709, N717, N801 and N1134) on HEK-Spike may differentially shield the virus compared to CHO-spike and provide a different host immune system interaction strategy. Collectively, these data underscore the importance of characterizing site-specific glycosylation on recombinant human spike protein from HEK and CHO cells in order to better understand the impact of the production host on this complex and important protein used in diagnostics and vaccines.

Dataset Information

An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells

Publications

An Atlas of Human Glycosylation Pathways Enables Display of the Human Glycome by Gene Engineered Cells.

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