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Cell-surface N-glycan editing in a subtype-selective manner by a two-step strategy Left path: selective editing of core-fucosylated N-glycans by a ‘delete’ step with WT Endo-F3 and an ‘insert’ step with Endo-F3 mutant. Right path: selective editing of non-core-fucosylated N-glycans by a ‘delete’ step with WT Endo-M and an ‘insert’ step with Endo-M mutant.
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Cell surfaces are glycosylated in various ways with high heterogeneity, which usually leads to ambiguous conclusions about glycan-involved biological functions. Here, we describe a two-step chemoenzymatic approach for N-glycan-subtype-selective editing on the surface of living cells that consists of a first ‘delete’ step to remove heterogeneous N-g...
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... 24 Recently, chemoenzymatic labeling has been developed to profile core fucosylation for its good specificity and few side reactions. 22,23,25 There are two types of probes that have been widely used in chemoenzymatic labeling methods namely "two-step probes" and "one-step probes." 26 A "two-step probe" contains a biorthogonal reactive group, such as an alkynyl or azido group, and additional chemical reactions are required to introduce a reporter group. ...
Core fucosylation, a special type of N-linked glycosylation, is important in tumor proliferation, invasion, metastatic potential, and therapy resistance. However, the core-fucosylated glycoproteome has not been extensively profiled due to the low abundance and poor ionization efficiency of glycosylated peptides. Here, a “one-step” strategy has been described for protein core-fucosylation characterization in biological samples. Core-fucosylated peptides can be selectively labeled with a glycosylated probe, which is linked with a temperature-sensitive poly(N-isopropylacrylamide) (PNIPAM) polymer, by mutant endoglycosidase (EndoF3-D165A). The labeled probe can be further removed by wild-type endoglycosidase (EndoF3) in a traceless manner for mass spectrometry (MS) analysis. The feasibility and effectiveness of the “one-step” strategy are evaluated in bovine serum albumin (BSA) spiked with standard core-fucosylated peptides, H1299, and Jurkat cell lines. The “one-step” strategy is then employed to characterize core-fucosylated sites in human lung adenocarcinoma, resulting in the identification of 2494 core-fucosylated sites distributed on 1176 glycoproteins. Further data analysis reveals that 196 core-fucosylated sites are significantly upregulated in tumors, which may serve as potential drug development targets or diagnostic biomarkers. Together, this “one-step” strategy has great potential for use in global and in-depth analysis of the core-fucosylated glycoproteome to promote its mechanism research.
... Some of the most powerful approaches for saccharide editing in glycotechnology include skeletal editing via the acid-catalyzed transformation of furanosides to pyranosides 12 , Ferrier rearrangement for converting carbohydrates to cyclitols 13 , sugar-to-sugar transformations via oxidation followed by reduction 14 , uncontrolled editing of cell surface glycans by metabolic oligosaccharide engineering (MOE) 15 , and enzymatic addition of glycans (EAG) 16 . In MOE, cells are cultured with a monosaccharide containing a reporter moiety (e.g., alkyne/azide); hypothesizing that the conjugate of the reporter moiety and monosaccharide are incorporated into the cellular processes, the incorporated moiety can guide the investigation of biological pathways including cell imaging using bioorthogonal chemistry (Fig. 1A) 15 . ...
... This technique has been utilized for cell imaging and probing biosynthetic machinery. In contrast, EAG exploits the site-specific cleavage of glycosidic bonds using designer glycosidases, and the resulting hydrolyzed glycans are subjected to post-synthetic modifications using glycosyl transferases to attach new glycans/probes (Fig. 1B) 16 . To the best of our knowledge, no editing technique enables the insertion of a foreign glycan into the already synthesized glycans via chemical or enzymatic routes (Fig. 1C). ...
Post-synthetic surgical editing enables synthesizing diverse molecules from a common scaffold. Editing carbohydrates by inserting a foreign glycan is still a far-reaching goal for synthetic chemists. In this study, a one-pot-three-step chemical approach was employed to edit glycoconjugates. It is comprised of three steps: the first is a ‘cut’ step, cleaving one of the interglycosidic bonds and producing an intermediate that could be intercepted with 4-mercaptotoluene; second step activates the thiotolyl glycoside in the presence of an aglycon containing an orthogonally activatable ethynylcycloxyl carbonate moiety; and the third step involves ‘stitching’ by activating the carbonate donor. The cut-insert stitch-editing reaction (CIStER) is demonstrated by inserting branched and linear arabinans reminiscent of M. tuberculosis cell wall from the same designer trimannoside. Glycosylating an activated hydroxyacid (serinyl, steroidal, and lipid) after cutting the interglycosidic bond and stitching in the presence of base extendes the CIStER approach to the synthesis of glycohybrids.
... The copyright holder for this preprint this version posted https://doi.org/10.1101/2024.03.27.586941 doi: bioRxiv preprint specific "artificial targets" can be introduced to the surface of almost all types of tumor cells [62][63][64][65][66] . Therefore, it may be possible to incorporate bio-orthogonal chemical motifs onto CTCs via MGE to facilitate precise recognition, capturing, and subsequent release of CTCs. ...
Although strategies for circulating tumor cells (CTCs) enrichment have been proposed, the practical effects of clinical CTCs detection are far from satisfactory. Generally, the methodologies for CTCs detection aim at naturally occurring targets, but misdetection/interferences are prevalent due to the diverse phenotypes and subpopulations of CTCs with high heterogeneity. Herein, a CTCs isolation system based on the "labeling-capture-release" process is demonstrated for precise and high-efficient enrichment of CTCs from clinical blood samples. The mechanism which is based on abnormal glyco-metabolism of tumor cells including CTCs can be utilized for the surface decoration of CTCs with artificial azido groups. With the aid of bio-orthogonal plates designed with DBCO- and disulfide groups and exploiting the anti-fouling effects, the cells labeled with azido groups can be captured via a copper-free click reaction and released in a non-destructive manner during subsequent disulfide reduction. The technique is demonstrated to label multiple different types of tumor cells with the EpCAM+/- phenotypes and adherent/suspended status, and all the epithelial/interstitial/hybrid phenotypes of CTCs can be separated from clinical blood samples from 25 patients with 10 different cancer types. Moreover, our strategy is superior to the clinically approved CTCs detection system from the perspective of broad-spectrum and accurate recognition of heterogeneous CTCs. The capturing efficiency of this isolation system is over 80% and the release efficiency exceeds 90%. Most of the released CTCs survive with maintained glycolytic activity thus boding well for downstream applications such as drug susceptibility tests using viable CTCs.
... To achieve selective editing of N-glycans, Huang's group performed subtype-selective "delete" and "insert" operations on cell-surface glycans on the basis of the substrate selectivity of different endoglycosidases and their mutants. 190 Withers's group discovered that endo-O-glycan hydrolases can selectively cleave O-glycans at the cellular and protein level. 191 Proteinand cell-specific glycan editing helps us to better understand and intervene in the functions of glycans on glycoconjugates and cell conditions. ...
Besides proteins and nucleic acids, carbohydrates are also ubiquitous building blocks of living systems. Approximately 70% of mammalian proteins are glycosylated. Glycans not only provide structural support for living systems but also act as crucial regulators of cellular functions. As a result, they are considered essential pieces of the life science puzzle. However, research on glycans has lagged far behind that on proteins and nucleic acids. The main reason is that glycans are not direct products of gene coding, and their synthesis is nontemplated. In addition, the diversity of monosaccharide species and their linkage patterns contribute to the complexity of the glycan structures, which is the molecular basis for their diverse functions. Research in glycobiology is extremely challenging, especially for the in situ elucidation of glycan structures and functions. There is an urgent need to develop highly specific glycan labeling tools and imaging methods and devise glycan editing strategies. This Perspective focuses on the challenges of in situ analysis of glycans in living systems at three spatial levels (i.e., cell, tissue, and in vivo) and highlights recent advances and directions in glycan labeling, imaging, and editing tools. We believe that examining the current development landscape and the existing bottlenecks can drive the evolution of in situ glycan analysis and intervention strategies and provide glycan-based insights for clinical diagnosis and therapeutics.
... Enzymatic modification of glycoproteins outside of cells allows post-production manipulation of regiochemistry, anomeric configuration, and site-specificity that are not possible to control within cells [6,29]. This method benefits from the high specificity of modifications, resulting in single glycoforms. ...
Proteins continue to represent a large fraction of the therapeutics market, reaching over a hundred billion dollars in market size globally. One key feature of protein modification that can affect both structure and function is the addition of glycosylation following protein folding, leading to regulatory requirements for the accurate assessment of protein attributes, including glycan structures. The non-template-driven, innately heterogeneous N-glycosylation process thus requires accurate detection to robustly generate protein therapies. A challenge exists in the timely detection of protein glycosylation without labor-intensive manipulation. In this article, we discuss progress toward N-glycoprotein control, focusing on novel control strategies and the advancement of rapid, high-throughput analysis methods.
... Cell surface proteomics analyses of Lec1 and Lec4 cells, models of mammalian N-glycosylation deficiency, have unraveled a significant impact on the functioning of IGF-1 pathway. Lec1 and Lec4 cells have been probed previously for the glycan-processing potential of the Golgi apparatus [71]. ...
Cell surface proteins carrying N-glycans play important roles in inter- and intracellular processes including cell adhesion, development, and cellular recognition. Dysregulation of the glycosylation machinery has been implicated in various diseases, and investigation of global differential cell surface proteome effects due to the loss of N-glycosylation will provide comprehensive insights into their pathogenesis. Cell surface proteins isolated from Parent Pro–5 CHO cells (W5 cells), two CHO mutants with loss of N-glycosylation function derived from Pro–5 CHO (Lec1 and Lec4 cells), were subjected to proteome analysis via high-resolution LCMS. We identified 44 and 43 differentially expressed membrane proteins in Lec1 and Lec4 cells, respectively, as compared to W5 cells. The defective N-glycosylation mutants showed increased abundance of integrin subunits in Lec1 and Lec4 cells at the cell surface. We also found significantly reduced levels of IGF-1R (Insulin like growth factor-1 receptor); a receptor tyrosine kinase; and the GTPase activating protein IQGAP1 (IQ motif-containing GTPase activating protein), a highly conserved cytoplasmic scaffold protein) in Lec1 and Lec4 cells. In silico docking studies showed that the IQ domain of IQGAP1 interacts with the kinase domain of IGF-1R. The integrin signaling and insulin growth factor receptor signaling were also enriched according to GSEA analysis and pathway analysis of differentially expressed proteins. Significant reductions of phosphorylation of ERK1 and ERK2 in Lec1 and Lec4 cells were observed upon IGF-1R ligand (IGF-1 LR3) stimulation. IGF-1 LR3, known as Long arginine3-IGF-1, is a synthetic protein and lengthened analog of insulin-like growth factor 1. The work suggests a novel mechanism for the activation of IGF-1 dependent ERK signaling in CHO cells, wherein IQGAP1 plausibly functions as an IGF-1R-associated scaffold protein. Appropriate glycosylation by the enzymes MGAT1 and MGAT5 is thus essential for processing of cell surface receptor IGF-1R, a potential binding partner in IQGAP1 and ERK signaling, the integral components of the IGF pathway.
... [44] Recently, Huang and co-workers used several endoglycosidases (Endo-F3, M-Endo-F3, EndoM from Mucor hiemalis, and EndoM N175Q) and an azido biantennary-type N-glycan oxazoline (similar structure with probe 2 in scheme 1) to edit N-glycans on living cells. [45] The subsequent introduction of a biotin group via click chemistry reaction allowed the imaging analysis of core-fucosylated and non-core-fucosylated N-glycans, and the functional study of a cell-surface protein opioid receptor delta 1 (OPRD1). [45] We assumed that M-Endo-F3 could tolerate bulk tags to label cell-surface core fucose with probe 3 in a "one-step labeling" manner, resulting in a high labeling sensitivity (Scheme 1). ...
... [45] The subsequent introduction of a biotin group via click chemistry reaction allowed the imaging analysis of core-fucosylated and non-core-fucosylated N-glycans, and the functional study of a cell-surface protein opioid receptor delta 1 (OPRD1). [45] We assumed that M-Endo-F3 could tolerate bulk tags to label cell-surface core fucose with probe 3 in a "one-step labeling" manner, resulting in a high labeling sensitivity (Scheme 1). If the labeled probe can be broken by wild type Endo-F3 after enrichment by streptavidin resin, the on-bead traceless cleavage will allow the global mapping of cell-surface corefucosylated glycoproteins and glycosylation sites by MS (Scheme 1). ...
Core fucosylation, the attachment of α1,6‐fucose to the innermost N‐acetylglucosamine (GlcNAc) residue of N‐glycans, has a strong relationship with tumor growth, invasion, metastasis, prognosis, and immune evasion by regulating many membrane proteins. However, details about the functional mechanism are still largely unknown due to the lack of an effective analytical method to identify cell‐surface core‐fucosylated glycoproteins, and especially glycosylation sites. Here, we developed a sensitive and reversible labeling strategy for probing core fucosylation, by which core‐fucosylated glycoproteins that located on cell‐surface were selectively tagged by a biotinylated probe with high sensitivity. The labeled probe can be further broken enzymatically after the capture by affinity resin. The on‐bead traceless cleavage allowed the global mapping of core‐fucosylated glycoproteins and glycosylation sites by mass spectrometry (MS). The profile of core‐fucosylated glycoproteome provides an in‐depth understanding of the biological functions of core fucosylation.
... The complex glycans are composed of core fucosylated, terminal galactosylated, non-galactosylated and terminal sialylated structures. Based on the glycosylation site and kind of glycosylation, it can numerously influence the function, including the immunogenicity, vector binding, nAb binding, etc [79]. The biolayer interferometry and cryo-EM analysis prove that the N343 glycan, together with D405, R408 and D427, participate in a gating role in the facilitation of RBD opening [70]. ...
Glycan is an essential molecule that controls and drives life in a precise direction. The paucity of research in glycobiology may impede the significance of its role in the pandemic guidelines. The SARS-CoV-2 spike protein is heavily glycosylated, with 22 putative N-glycosylation sites and 17 potential O-glycosylation sites discovered thus far. It is the anchor point to the host cell ACE2 receptor, TMPRSS2, and many other host proteins that can be recognized by their immune system; hence, glycosylation is considered the primary target of vaccine development. Therefore, it is essential to know how this surface glycan plays a role in viral entry, infection, transmission, antigen, antibody responses, and disease progression. Although the vaccines are developed and applied against COVID-19, the proficiency of the immunizations is not accomplished with the current mutant variations. The role of glycosylation in SARS-CoV-2 and its receptor ACE2 with respect to other putative cell glycan receptors and the significance of glycan in host cell immunity in COVID-19 are discussed in this paper. Hence, the molecular signature of the glycan in the coronavirus infection can be incorporated into the mainstream therapeutic process.
... In this way, the authors generated an engineered glyco form of cetuximab with eightfold higher efficacy than the non engineered version. Similar approaches have also been used to edit glycans on cell surfaces 152 . Another approach towards selectivity is through introduction of N linked GlcNAc and (pseudo eukaryotic) Glc handles into proteins using bacterial glycosyltransferases [153][154][155] . ...
The 1013–1014 microorganisms present in the human gut (collectively known as the human gut microbiota) dedicate substantial percentages of their genomes to the degradation and uptake of carbohydrates, indicating the importance of this class of molecules. Carbohydrates function not only as a carbon source for these bacteria but also as a means of attachment to the host, and a barrier to infection of the host. In this Review, we focus on the diversity of carbohydrate-active enzymes (CAZymes), how gut microorganisms use them for carbohydrate degradation, the different chemical mechanisms of these CAZymes and the roles that these microorganisms and their CAZymes have in human health and disease. We also highlight examples of how enzymes from this treasure trove have been used in manipulation of the microbiota for improved health and treatment of disease, in remodelling the glycans on biopharmaceuticals and in the potential production of universal O-type donor blood. The human gut microbiota produces an extensive array of carbohydrate-active enzymes to degrade carbohydrates derived from the diet, host and other microorganisms. Withers and colleagues discuss the vast diversity and activities of these enzymes and their potential applications.
... A second engineering technique is to remove subclasses of N-glycans on mammalian cells by trimming them with the appropriate glycosidases and insert whole N-glycans with a chemical handle, an oxazoline (Tang et al. 2020). This technique could potentially homogenize the cell surface. ...
All bacteria display surface-exposed glycans that can play an important role in their interaction with the host and in select cases mimic the glycans found on host cells, an event called molecular or glycan mimicry. In this review, we highlight the key bacteria that display human glycan mimicry and provide an overview of the involved glycan structures. We also discuss the general trends and outstanding questions associated with human glycan mimicry by bacteria. Finally, we provide an overview of several techniques that have emerged from the discipline of chemical glycobiology, which can aid in the study of the composition, variability, interaction and functional role of these mimicking glycans.
