Display of the human mucinome with defined O-glycans by gene engineered cells

Nason, Rebecca; Büll, Christian; Konstantinidi, Andriana; Sun, Lingbo; Ye, Zilu; Halim, Adnan; Du, Wenjuan; Sørensen, Daniel Madriz; Durbesson, Fabien; Furukawa, Shoei; Mandel, Ulla; Joshi, Hiren J.; Dworkin, Leo Alexander; Hansen, Lars; David, Leonor; Iverson, T.M.; Bensing, Barbara A.; Sullam, Paul M.; Varki, Ajit; Vries, Erik de; Haan, Cornelis A. M. de; Vincentelli, Renaud; Henrissat, Bernard; Vakhrushev, Sergey Y.; Clausen, Henrik; Narimatsu, Yoshiki

doi:10.1038/s41467-021-24366-4

Cited by 88 publications

(122 citation statements)

References 95 publications

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“…However, proteins such as mucins also exist in invertebrates and seem to carry different O -linked glycans than those in vertebrates ( Staudacher, 2015 ), suggesting the need for more research in this area. In vertebrates, O -linked glycans are important for the immune system and for the synthesis of mucus ( Giovannone et al, 2018 ; Nason et al, 2021 ), a substance modulating pathogen adherence in animals ( Gonzalez-Morelo et al, 2020 ). As a consequence, disruption of this type of glycosylation is associated with many human diseases ( Hansson, 2019 ).…”

Section: Discussionmentioning

confidence: 99%

The Role of Fucose-Containing Glycan Motifs Across Taxonomic Kingdoms

Thomès

Bojar

2021

Front. Mol. Biosci.

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The extraordinary diversity of glycans leads to large differences in the glycomes of different kingdoms of life. Yet, while most monosaccharides are solely found in certain taxonomic groups, there is a small set of monosaccharides with widespread distribution across nearly all domains of life. These general monosaccharides are particularly relevant for glycan motifs, as they can readily be used by commensals and pathogens to mimic host glycans or hijack existing glycan recognition systems. Among these, the monosaccharide fucose is especially interesting, as it frequently presents itself as a terminal monosaccharide, primed for interaction with proteins. Here, we analyze fucose-containing glycan motifs across all taxonomic kingdoms. Using a hereby presented large species-specific glycan dataset and a plethora of methods for glycan-focused bioinformatics and machine learning, we identify characteristic as well as shared fucose-containing glycan motifs for various taxonomic groups, demonstrating clear differences in fucose usage. Even within domains, fucose is used differentially based on an organism’s physiology and habitat. We particularly highlight differences in fucose-containing motifs between vertebrates and invertebrates. With the example of pathogenic and non-pathogenic Escherichia coli strains, we also demonstrate the importance of fucose-containing motifs in molecular mimicry and thereby pathogenic potential. We envision that this study will shed light on an important class of glycan motifs, with potential new insights into the role of fucosylated glycans in symbiosis, pathogenicity, and immunity.

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Section: Discussionmentioning

confidence: 99%

The Role of Fucose-Containing Glycan Motifs Across Taxonomic Kingdoms

Thomès

Bojar

2021

Front. Mol. Biosci.

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“…To further evaluate sulfation capacities on O-glycosylation, we expressed previously designed human-secreted mucin TR O-glycan reporters in the engineered cells ( Figs. 1 B and S5 ) ( 38 , 40 ). We first analyzed the purified mucin1 reporter by SDS-PAGE and Western blot analysis and observed clear shifts in mobility when expressed in cells with different O-glycosylation capacities ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Genetic engineering of the glycosylation capacities in a cell line can be used to dissect biosynthesis and genetic regulation of specific glycosylation features, and with more comprehensive and systematic engineering to develop a cell-based glycan array that displays the glycome on cells for biological interrogation ( 38 , 39 ). We obtained engineered HEK293 cells that display and produce 3- O -sulfated core1 and core2 O-glycans by installation of the GAL3ST2 and GAL3ST4 sulfotransferases and used these to express small GPF-tagged reporters containing tandem repeat (TR) sequences derived from human mucins ( 40 ). We used the platform to dissect the binding specificity of galectin-4 (GAL-4) for 3- O -sulfo-core1 O-glycans.…”

mentioning

confidence: 99%

Installation of O-glycan sulfation capacities in human HEK293 cells for display of sulfated mucins

Sun

Konstantinidi

et al. 2022

Journal of Biological Chemistry

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The human genome contains at least 35 genes that encode Golgi sulfotransferases that function in the secretory pathway, where they are involved in decorating glycosaminoglycans, glycolipids, and glycoproteins with sulfate groups. Although a number of important interactions by proteins such as selectins, galectins, and sialic acid–binding immunoglobulin-like lectins are thought to mainly rely on sulfated O-glycans, our insight into the sulfotransferases that modify these glycoproteins, and in particular GalNAc-type O-glycoproteins, is limited. Moreover, sulfated mucins appear to accumulate in respiratory diseases, arthritis, and cancer. To explore further the genetic and biosynthetic regulation of sulfated O-glycans, here we expanded a cell-based glycan array in the human embryonic kidney 293 (HEK293) cell line with sulfation capacities. We stably engineered O-glycan sulfation capacities in HEK293 cells by site-directed knockin of sulfotransferase genes in combination with knockout of genes to eliminate endogenous O-glycan branching (core2 synthase gene GCNT1 ) and/or sialylation capacities in order to provide simplified substrates (core1 Galβ1–3GalNAcα1–O-Ser/Thr) for the introduced sulfotransferases. Expression of the galactose 3- O -sulfotransferase 2 in HEK293 cells resulted in sulfation of core1 and core2 O-glycans, whereas expression of galactose 3- O -sulfotransferase 4 resulted in sulfation of core1 only. We used the engineered cell library to dissect the binding specificity of galectin-4 and confirmed binding to the 3- O -sulfo-core1 O-glycan. This is a first step toward expanding the emerging cell-based glycan arrays with the important sulfation modification for display and production of glycoconjugates with sulfated O-glycans.

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“…Multivalent Siglec ligands can be formed at the cell surface through clustering of individual sialoglycoproteins or lipids or are formed by densely glycosylated protein such as the mucins ( 151 – 155 ). Mucins are a family of large secreted and membrane-bound glycoproteins consisting for up to 60% of tandem repeat domains formed by repeating serine, threonine and proline sequences ( 156 , 157 ).…”

Section: Siglec Clustering and Multivalent Ligandsmentioning

confidence: 99%

Siglec Signaling in the Tumor Microenvironment

et al. 2021

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Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of receptors that recognize sialoglycans – sialic acid containing glycans that are abundantly present on cell membranes. Siglecs are expressed on most immune cells and can modulate their activity and function. The majority of Siglecs contains immune inhibitory motifs comparable to the immune checkpoint receptor PD-1. In the tumor microenvironment (TME), signaling through the Siglec-sialoglycan axis appears to be enhanced through multiple mechanisms favoring tumor immune evasion similar to the PD-1/PD-L1 signaling pathway. Siglec expression on tumor-infiltrating immune cells appears increased in the immune suppressive microenvironment. At the same time, enhanced Siglec ligand expression has been reported for several tumor types as a result of aberrant glycosylation, glycan modifications, and the increased expression of sialoglycans on proteins and lipids. Siglec signaling has been identified as important regulator of anti-tumor immunity in the TME, but the key factors contributing to Siglec activation by tumor-associated sialoglycans are diverse and poorly defined. Among others, Siglec activation and signaling are co-determined by their expression levels, cell surface distribution, and their binding preferences for cis- and trans-ligands in the TME. Siglec binding preference are co-determined by the nature of the proteins/lipids to which the sialoglycans are attached and the multivalency of the interaction. Here, we review the current understanding and emerging conditions and factors involved in Siglec signaling in the TME and identify current knowledge gaps that exist in the field.

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Display of the human mucinome with defined O-glycans by gene engineered cells

Cited by 88 publications

References 95 publications

The Role of Fucose-Containing Glycan Motifs Across Taxonomic Kingdoms

The Role of Fucose-Containing Glycan Motifs Across Taxonomic Kingdoms

Installation of O-glycan sulfation capacities in human HEK293 cells for display of sulfated mucins

Siglec Signaling in the Tumor Microenvironment

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