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The workflow for constructing cell-based glycan arrays. a Distinct glycan epitopes are installed onto N-glycans of Lec2 CHO cells via sialyltransferase-mediated cell-surface in situ sialylation using the CMP-Neu5Ac donor (or its analogs) or fucosyltransferase-mediated cell-surface in situ fucosylation using the GDP-Fuc donor (or its analogs). b Cell-based glycan arrays displaying LeX or its derivatives. c N-glycans of Lec2 CHO cells. d Cell-based glycan arrays displaying α2-3- or α2-6-linked sialosides, or their derivatives. X represents unnatural substituents introduced at fucose C-5 or NeuNAc acetyl position
Source publication
Glycan microarrays provide a high-throughput means of profiling the interactions of glycan-binding proteins with their ligands. However, the construction of current glycan microarray platforms is time consuming and expensive. Here, we report a fast and cost-effective method for the assembly of cell-based glycan arrays to probe glycan-glycan-binding...
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Background
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Background:
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Methods:
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Citations
... Furthermore, we have experimentally verified, by X-ray crystallography (unpublished data), that the hMPV F protein is in a prefusion form consistent with the previously published study from Battles M. et al. (16). To study protein-glycan interactions, we used glycan array technology, a well-established tool (24), to obtain the glycan binding profile of HMPV F in the pre-fusion conformation. The glycan array library, immobilized on the glass slide, comprises a variety of oligosaccharide structures (368 structures) that broadly represent the human glycome. ...
Numerous viruses have been found to exploit glycoconjugates expressed on human cells as their initial attachment factor for viral entry and infection. The virus-cell glycointeractome, when characterized, may serve as a template for antiviral drug design. Heparan sulfate proteoglycans extensively decorate the human cell surface and were previously described as a primary receptor for human metapneumovirus (HMPV). After respiratory syncytial virus, HMPV is the second most prevalent respiratory pathogen causing respiratory tract infection in young children. To date, there is neither vaccine nor drug available to prevent or treat HMPV infection. Using a multidisciplinary approach, we report for the first time the glycointeractome of the HMPV fusion (F) protein, a viral surface glycoprotein that is essential for target-cell recognition, attachment, and entry. Our glycan microarray and surface plasmon resonance results suggest that Galβ1-3/4GlcNAc moieties that may be sialylated or fucosylated are readily recognized by HMPV F. The bound motifs are highly similar to the N-linked and O-linked glycans primarily expressed on the human lung epithelium. We demonstrate that the identified glycans have the potential to compete with the cellular receptors used for HMPV entry and consequently block HMPV infection. We found that lacto-N-neotetraose demonstrated the strongest HMPV binding inhibition in a cell infection assay. Our current findings offer an encouraging and novel avenue for the design of anti-HMPV drug candidates using oligosaccharide templates.
IMPORTANCE
All cells are decorated with a dense coat of sugars that makes a sugar code. Many respiratory viruses exploit this sugar code by binding to these sugars to cause infection. Human metapneumovirus is a leading cause for acute respiratory tract infections. Despite its medical importance, there is no vaccine or antiviral drug available to prevent or treat human metapneumovirus infection. This study investigates how human metapneumovirus binds to sugars in order to more efficiently infect the human host. We found that human metapneumovirus binds to a diverse range of sugars and demonstrated that these sugars can ultimately block viral infection. Understanding how viruses can take advantage of the sugar code on our cells could identify new intervention and treatment strategies to combat viral disease.
... A treatment with sialidase and the knockdown of ST6Gal-I revealed the involvement of alpha (2,6)-linked sialic acid in osteoclast differentiation [59]. Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) has been reported to regulate the development of osteoclasts [60][61][62][63][64][65]67,68]. Monocytes/macrophages derived from mice in which Siglec-15 was deleted showed an impaired ability for osteoclast differentiation [62]. ...
Glycosylation plays a crucial role in the maintenance of homeostasis in the body and at the onset of diseases such as inflammation, neurodegeneration, infection, diabetes, and cancer. It is also involved in bone metabolism. N- and O-glycans have been shown to regulate osteoblast and osteoclast differentiation. We recently demonstrated that ganglio-series and globo-series glycosphingolipids were essential for regulating the proliferation and differentiation of osteoblasts and osteoclasts in glycosyltransferase-knockout mice. Herein, we reviewed the importance of the regulation of bone metabolism by glycoconjugates, such as glycolipids and glycoproteins, including our recent results.
... In contrast to MGE, chemoenzymatic glycan labeling (CeGL) does not rely on intracellular biosynthetic pathways and can directly attach biotin-, fluorescent-, or even proteintagged sugars to specific substrate glycans via a one-step chemoenzymatic reaction. 51,52 In addition to specifically forming natural glycosidic linkages, CeGL also allows for labeling of higher-order glycan structures and glycan chain elongation. This is extremely important for researchers to identify glycan structures, elucidate the biological effects of specific glycan chains, and intervene in glycosylation. ...
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.
... Introduction of a new monosaccharide (e.g., Neu5Ac) on the cell surface is detected by increased binding of the cell to a lectin. However, direct detection of changes directly in cellular glycan composition by mass spectrometry is challenging 53 . The challenges present in state-of-the-art enzymatic glycosylation on-cells, onglass, and on-DNA prompted us to develop chemoenzymatic glycan synthesis "on-phage" with a complimentary analytical method to monitor reaction conversion. ...
Cellular glycosylation is characterized by chemical complexity and heterogeneity, which is challenging to reproduce synthetically. Here we show chemoenzymatic synthesis on phage to produce a genetically-encoded liquid glycan array (LiGA) of complex type N -glycans. Implementing the approach involved by ligating an azide-containing sialylglycosyl-asparagine to phage functionalized with 50–1000 copies of dibenzocyclooctyne. The resulting intermediate can be trimmed by glycosidases and extended by glycosyltransferases yielding a phage library with different N -glycans. Post-reaction analysis by MALDI-TOF MS allows rigorous characterization of N -glycan structure and mean density, which are both encoded in the phage DNA. Use of this LiGA with fifteen glycan-binding proteins, including CD22 or DC-SIGN on cells, reveals optimal structure/density combinations for recognition. Injection of the LiGA into mice identifies glycoconjugates with structures and avidity necessary for enrichment in specific organs. This work provides a quantitative evaluation of the interaction of complex N -glycans with GBPs in vitro and in vivo.
... CCR1-chemokines were produced by osteoclasts and played an essential role in inflammatory cell chemotaxis (71). In 2018, Jennie Briard et al. found SIGLEC15 positively regulated osteoclast differentiation, and loss of it could result in impaired osteoclast differentiation and osteopetrosis in SIGLEC15-deficient mice (72). ...
Background
Osteosarcoma is the most common primary malignant bone tumor. The existing treatment regimens remained essentially unchanged over the past 30 years; hence the prognosis has plateaued at a poor level. Precise and personalized therapy is yet to be exploited.
Methods
One discovery cohort (n=98) and two validation cohorts (n=53 & n=48) were collected from public data sources. We performed a non-negative matrix factorization (NMF) method on the discovery cohort to stratify osteosarcoma. Survival analysis and transcriptomic profiling characterized each subtype. Then, a drug target was screened based on subtypes’ features and hazard ratios. We also used specific siRNAs and added a cholesterol pathway inhibitor to osteosarcoma cell lines (U2OS and Saos-2) to verify the target. Moreover, PermFIT and ProMS, two support vector machine (SVM) tools, and the least absolute shrinkage and selection operator (LASSO) method, were employed to establish predictive models.
Results
We herein divided osteosarcoma patients into four subtypes (S-I ~ S-IV). Patients of S- I were found probable to live longer. S-II was characterized by the highest immune infiltration. Cancer cells proliferated most in S-III. Notably, S-IV held the most unfavorable outcome and active cholesterol metabolism. SQLE, a rate-limiting enzyme for cholesterol biosynthesis, was identified as a potential drug target for S-IV patients. This finding was further validated in two external independent osteosarcoma cohorts. The function of SQLE to promote proliferation and migration was confirmed by cell phenotypic assays after the specific gene knockdown or addition of terbinafine, an inhibitor of SQLE. We further employed two machine learning tools based on SVM algorithms to develop a subtype diagnostic model and used the LASSO method to establish a 4-gene model for predicting prognosis. These two models were also verified in a validation cohort.
Conclusion
The molecular classification enhanced our understanding of osteosarcoma; the novel predicting models served as robust prognostic biomarkers; the therapeutic target SQLE opened a new way for treatment. Our results served as valuable hints for future biological studies and clinical trials of osteosarcoma.
... This is typically done in batch reactions, however, we also showed that NAL can be immobilized on beads without loss of enzymatic activity and remained active in a continuous flow setup for at least a week. With this strategy several N-modified sialic acids were synthesized (47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59). 73 Besides modifications on the 5position, also small modifications at the 9-positions were tested, as well as shortened glycerol tails. ...
The biosynthesis of glycans is a highly conserved biological process and found in all domains of life. The expression of cell surface glycans is increasingly recognized as a target for therapeutic intervention given the role of glycans in major pathologies such as cancer and microbial infection. Herein, we summarize our contributions to the development of unnatural monosaccharide derivatives to infiltrate and alter the expression of both mammalian and bacterial glycans and their therapeutic application.
... However, direct detection of changes directly in cellular glycan composition by mass spectrometry is very challenging 54 . The challenges present in state-of-the-art enzymatic glycosylation on-cells, on-glass, and on-DNA prompted us to develop chemoenzymatic glycan synthesis "on-phage" with a complimentary analytical method to monitor reaction conversion. ...
A hallmark of cellular glycosylation is its chemical complexity and heterogeneity, which can be challenging to capture synthetically. Using chemoenzymatic synthesis on M13 phage, we produce a genetically-encoded liquid glycan array (LiGA) of biantennary complex type N-glycans. Ligation of azido-functionalized sialylglycosyl-asparagine derived from egg yolk to phage functionalized with 50–1000 copies of dibenzocyclooctyne produced divergent intermediate that can be trimmed by glycosidases and extended by glycosyltransferases to yield a library of phages with different N -glycans. Post-reaction analysis by MALDI-TOF MS provided a rigorous approach to confirm N -glycan structure and density, both of which were encoded in the bacteriophage DNA. The binding of this N -glycan LiGA by ten lectins, including CD22 or DC-SIGN expressed on live cells, uncovered an optimal structure/density combination for recognition. Injection of the LiGA into mice identified glycoconjugates with structures and avidity necessary for enrichment in specific organs. This work provides an unprecedented quantitative evaluation of the interaction of complex N -glycans with GBPs in vitro and in vivo .
... Introduction of a new monosaccharide (e.g., Neu5Ac) on the cell surface is detected by increased binding of the cell to a lectin. However, direct detection of changes directly in cellular glycan composition by mass spectrometry is very challenging 48 . The challenges present in state-of-the-art enzymatic glycosylation on-cells, on-glass, and on-DNA prompted us to develop chemoenzymatic glycan synthesis "on-phage" with a . ...
A hallmark of cellular glycosylation is its chemical complexity and heterogeneity, which can be challenging to capture synthetically. Using chemoenzymatic synthesis on M13 phage, we produce a genetically-encoded liquid glycan array (LiGA) of biantennary complex type N-glycans. Ligation of azido-functionalized sialylglycosyl-asparagine derived from egg yolk to phage functionalized with 50-1000 copies of dibenzocyclooctyne produced divergent intermediate that can be trimmed by glycosidases and extended by glycosyltransferases to yield a library of phages with different N-glycans. Post-reaction analysis by MALDI-TOF MS provided a rigorous approach to confirm N-glycan structure and density, both of which were encoded in the bacteriophage DNA. The binding of this N-glycan LiGA by ten lectins, including CD22 or DC-SIGN expressed on live cells, uncovered an optimal structure/density combination for recognition. Injection of the LiGA into mice identified glycoconjugates with structures and avidity necessary for enrichment in specific organs. This work provides an unprecedented quantitative evaluation of the interaction of complex N-glycans with GBPs in vitro and in vivo.
... Recent studies have shown that Siglec-15 plays an important role in regulating the tumor microenvironment and promoting tumor immunosuppression in two main ways. One is by preferentially binding to the structure of sialyl-Tn (Briard et al., 2018), which is commonly expressed in tumors and may induce TGF-b secretion via the intracellular DAP12-Syk signaling pathway of TAMs (Ishida-Kitagawa et al., 2012;Takamiya et al., 2013). Syk is mediated by a variety of intermediate kinases, such as AKT (Yi et al., 2014), PKC (Takada and Aggarwal, 2004;Aggarwal and Pittenger, 2005), ERK (Eliopoulos et al., 2006;Parsa et al., 2008), etc. ...
Siglec-15 is an important immunosuppressive molecule considered to be a key target in next generation tumor immunotherapy. In this study, we screened 22 high affinity antibodies that specifically recognize human siglec-15 by using a large human phage antibody library and 5 representative sequences were selected for further study. The results showed the binding activity of 5 antibodies to siglec-15 (EC50 ranged from 0.02368μg /mL to 0.07949 μg /mL) and in 2 siglec-15 overexpressed cell lines, 3 antibodies had the strongest binding activity, so the 2 clones were discarded for further study. Subsequently the affinity of 3 antibodies were measured by Bio-layer interferometry technology (5-9×10E-09M). As the reported ligands of siglec-15, the binding activity of siglec-15 and sialyl-Tn, CD44, MAG and LRRC4C can be blocked by 3 of the antibodies. Among these, 3F1 had a competitive advantage. Then, the antibody 3F1 showed an obvious ADCC effect (EC50 were 0.85 μg/mL). Further, antibody 3F1 can reverse the inhibitory effect of siglec-15 on lymphocyte proliferation (especially CD4+T, CD8+T) and cytokine release (IFN-γ). Given the above results, 3F1 was selected as a candidate for the in vivo pharmacodynamics study. In the tumor model of Balb/c Nude mice, 3F1 (10 mg/kg) showed certain anti-tumor effects (TGI was 31.5%) while the combination of 3F1 (5 mg/kg) and ERBITUX (5 mg/kg) showed significant antitumor effects (TGI was 48.7%) compared with the PBS group. In conclusion, novel human antibody 3F1 has anti-tumor activity and is expected to be an innovative candidate drug targeting siglec-15 for tumor immunotherapy Significance Statement Siglec-15 is considered as an important target in the next generation of tumor immunotherapy. 3F1 is expected to be the most promising potential candidate for targeting siglec-15 for cancer treatment, and could provide a reference for the development of anti-tumor drugs.
... Siglec-15 binds to α2-6-sialosides but also α2-3-linked sialosides. (Briard et al., 2018) Ligand 21 shown in the figure below, was identified as the best ligand of this series ( Figure 18). ...
