77Se-Enriched Selenoglycoside Enables Significant Enhancement in NMR Spectroscopic Monitoring of Glycan–Protein Interactions

Detailed investigation of ligand–protein interactions is essential for better understanding of biological processes at the molecular level. Among these binding interactions, the recognition of glycans by lectins is of particular importance in several diseases, such as cancer; therefore, inhibition of glycan-lectin/galectin interactions represents a promising perspective towards developing therapeutics controlling cancer development. The recent introduction of 77Se NMR spectroscopy for monitoring the binding of a selenoglycoside to galectins prompted interest to optimize the sensitivity by increasing the 77Se content from the natural 7.63% abundance to 99%. Here, we report a convenient synthesis of 77Se-enriched selenodigalactoside (SeDG), which is a potent ligand of the medically relevant human galectin-3 protein, and proof of the expected sensitivity gain in 2D 1H, 77Se correlation NMR experiments. Our work opens perspectives for adding isotopically enriched selenoglycans for rapid monitoring of lectin-binding of selenated as well as non-selenated ligands and for ligand screening in competition experiments.

Investigating Constraints Along the Plant Secretory Pathway to Improve Production of a SARS-CoV-2 Spike Vaccine Candidate

Given the complex maturation requirements of viral glycoproteins and the challenge they often pose for expression in plants, the identification of host constraints precluding their efficient production is a priority for the molecular farming of vaccines. Building on previous work to improve viral glycoprotein production in plants, we investigated the production of a soluble SARS-CoV-2 spike comprising the ectopic portion of the glycoprotein. This was successfully transiently expressed in N. benthamiana by co-expressing the human lectin-binding chaperone calreticulin, which substantially increased the accumulation of the glycoprotein. The spike was mostly unprocessed unless the protease furin was co-expressed which resulted in highly efficient processing of the glycoprotein. Co-expression of several broad-spectrum protease inhibitors did not improve accumulation of the protein any further. The protein was successfully purified by affinity chromatography and gel filtration, although the purified product was heterogenous and the yields were low. Immunogenicity of the antigen was tested in BALB/c mice, and cellular and antibody responses were elicited after low dose inoculation with the adjuvanted protein. This work constitutes an important proof-of-concept for host plant engineering in the context of rapid vaccine development for SARS-CoV-2 and other emerging viruses.

Novel antifungal activity of Q-Griffithsin, a broad-spectrum antiviral lectin

Background There is a rising global trend in candida strains with high resistance to fluconazole and other antifungal drugs, hence the need for novel agents. Here, we investigated the anti-Candida activity of Q-Griffithsin (Q-GRFT), a lectin naturally produced by the red-sea algae, Griffithsia spp. Methods To assess in vitro growth inhibitory activity, C. albicans was incubated with Q-GRFT on agar plates and in broth media. We investigated GFP-bound Q-GRFT’s ability to adhere to C. albicans using fluorescence microscopy and fluorescence intensity assessments. To demonstrate in vivogrowth inhibitory activity, CBA/J mice were treated per vaginam with Q-GRFT followed by challenge with C. albicans, and fungal burden determined following vaginal lavage. Results Wild type fluorescently labeled Q-GRFT displayed higher fluorescence than the lectin-binding site deficient variant following incubation with C. albicans. Q-GRFT localized around the fungal cells and bound to α-mannan in the cell wall. Q-GRFT significantly inhibited C. albicans growth in broth and on agar plates, disrupted the integrity of the cell wall, and induced ROS formation. The lectin significantly inhibited the growth of C. glabrata, C. parapsilosis and C. krusei, with modest activity against C. auris CDC388 and C. auris CDC389 strains in vitro. Topical treatment resulted in a lower fungal burden compared to the vehicle control group in vaginal candidiasis. Conclusion Q-GRFT binds to and inhibits C. albicans growth both in vitro and in vivo. Further studies are needed to establish the mechanism of growth inhibition.

Oxygen-Dependent Changes in the N-Glycome of Murine Pulmonary Endothelial Cells

Supplemental oxygen is frequently used together with mechanical ventilation to achieve sufficient blood oxygenation. Despite the undoubted benefits, it is vigorously debated whether too much oxygen can also have unpredicted side-effects. Uncertainty is also due to the fact that the molecular mechanisms are still insufficiently understood. The lung endothelium is covered with an exceptionally broad glycocalyx, carrying N- and O-glycans, proteoglycans, glycolipids and glycosaminoglycans. Glycan structures are not genetically determined but depend on the metabolic state and the expression level and activity of biosynthetic and glycan remodeling enzymes, which can be influenced by oxygen and the redox status of the cell. Altered glycan structures can affect cell interactions and signaling. In this study, we investigated the effect of different oxygen conditions on aspects of the glycobiology of the pulmonary endothelium with an emphasis on N-glycans and terminal sialylation using an in vitro cell culture system. We combined a proteomic approach with N-glycan structure analysis by LC-MS, qRT-PCR, sialic acid analysis and lectin binding to show that constant and intermittent hyperoxia induced time dependent changes in global and surface glycosylation. An siRNA approach identified St6gal1 as being primarily responsible for the early transient increase of α2-6 sialylated structures in response to hyperoxia.

Functional analysis of N-acetylglucosaminyltransferase-I knockdown in 2D and 3D neuroblastoma cell cultures

Tumor development can be promoted/suppressed by certain N-glycans attached to proteins at the cell surface. Here we examined aberrant neuronal properties in 2D and 3D rat neuroblastoma (NB) cell cultures with different N-glycan populations. Lectin binding studies revealed that the engineered N-glycosylation mutant cell line, NB_1(-Mgat1), expressed solely oligomannose N-glycans, and verified that the parental cell line, NB_1, and a previous engineered N-glycosylation mutant, NB_1(-Mgat2), expressed significant levels of higher order N-glycans, complex and hybrid N-glycans, respectively. NB_1 grew faster than mutant cell lines in monolayer and spheroid cell cultures. A 2-fold difference in growth between NB_1 and mutants occurred much sooner in 2D cultures relative to that observed in 3D cultures. Neurites and spheroid cell sizes were reduced in mutant NB cells of 2D and 3D cultures, respectively. Cell invasiveness was highest in 2D cultures of NB_1 cells compared to that of NB_1(-Mgat1). In contrast, NB_1 spheroid cells were much less invasive relative to NB_1(-Mgat1) spheroid cells while they were more invasive than NB_1(-Mgat2). Gelatinase activities supported the ranking of cell invasiveness in various cell lines. Both palladin and HK2 were more abundant in 3D than 2D cultures. Levels of palladin, vimentin and EGFR were modified in a different manner under 2D and 3D cultures. Thus, our results support variations in the N-glycosylation pathway and in cell culturing to more resemble in vivo tumor environments can impact the aberrant cellular properties, particularly cell invasiveness, of NB.

Whole-Genome CRISPR Screening Identifies N-Glycosylation As an Essential Pathway and a Potential Novel Therapeutic Target in CALR-Mutant MPN

Somatic mutations in the ER chaperone calreticulin (CALR) are frequent and disease-initiating in myeloproliferative neoplasms (MPN). Although the mechanism of mutant CALR-induced MPN is known to involve pathogenic binding between mutant CALR and MPL, this insight has not yet been exploited therapeutically. Consequently, a major deficiency is the lack of clonally selective therapeutic agents with curative potential. Hence, we set out to discover and validate unique genetic dependencies for mutant CALR-driven oncogenesis. We first performed a whole-genome CRISPR knockout screen in CALR Δ52 MPL-expressing hematopoietic cells to identify genes that were differentially required for the growth of cytokine-independent, transformed CALR Δ52 cells as compared to control cells. Using gene-set enrichment analyses, we identified the N-glycan biosynthesis, unfolded protein response, and the protein secretion pathways to be amongst the most significantly differentially depleted pathways (FDR q values <0.001, 0.014, and 0.025, respectively) in CALR Δ52 cells. We performed a secondary CRISPR pooled screen focused on significant pathways from the primary screen and confirmed these findings. Strikingly, seven of the top ten hits in both screens were linked to protein N-glycosylation. Four of those genes encode proteins involved in the enzymatic activity of dolichol-phosphate mannose synthase (DPM1, DPM2, DPM3, and MPDU1). This enzyme synthesizes dolichol D-mannosyl phosphate, an essential substrate for protein N-glycosylation. Importantly, these findings from an unbiased whole-genome screen align with prior mechanistic studies demonstrating that both the N-glycosylation sites on MPL and the lectin-binding sites on CALR Δ52 are required for mutant CALR-driven oncogenesis. We next performed single gene CRISPR Cas9 validation studies and found that DPM2 is required for CALR Δ52-mediated transformation, as demonstrated by increased cell death, reduced p-STAT5 and decreased MPL cell-surface levels, when Dpm2 is knocked out. Importantly, cells cultured in cytokine-rich medium were unaffected by DPM2 loss. Upon cytokine withdrawal, a sub-clone of non-edited Dpm2WT CALR Δ52 cells grew out, further demonstrating requirement for DPM2 for the survival of CALR Δ52 cells. Additionally, we observed a >50% reduction in ex vivo myeloid colony formation of murine CalrΔ52 Dpm2 ko bone marrow (BM) compared with CRISPR-Cas9 non-targeting controls, with non-significant effects on CalrWT BM cells. To enable clinical translation, we performed a pharmacological screen targeting pathways significantly depleted in our CRISPR screens. Screening 70 drugs, we found that the N-glycosylation pathway was the only pathway in which all tested compounds preferentially killed CALR Δ52 transformed cells. We then treated primary Calr Δ52/+ mice with a clinical grade N-glycosylation (N-Gi) inhibitor and found platelet counts (Sysmex) to be significantly reduced (vehicle 3x10 6/mL, N-Gi 1x10 6/mL after 18 days, p<.0001). Concordantly, the proportion of megakaryocyte erythrocyte progenitors (MEPs) was significantly reduced in CalrΔ52 BM (p=0.03). We next performed competitive BM transplantation assays using CD45.2 UBC-GFP MxCre CalrΔ52 knockin and CD45.1 mice. We found that mice treated with N-Gi had significantly reduced platelet counts (vehicle 1440x10 6/mL, N-Gi 845x10 6/mL, p=0.005) as well as significantly reduced platelet chimerism (vehicle 55%, N-Gi 27%, p<0.001), indicating a distinct vulnerability of CalrΔ52 over WT cells. Finally, we interrogated RNA-sequencing data from primary human MPN platelets. We found N-glycosylation-related pathways to be significantly upregulated in CALR-mutated platelets (n = 13) compared to healthy control platelets (n = 21), highlighting the relevance of our findings to human MPN. In summary, using unbiased genetic and focused pharmacological screens, we identified the N-glycan biosynthesis pathway as essential for mutant CALR-driven oncogenesis. Using a pre-clinical MPN model, we found that in vivo inhibition of N-glycosylation normalizes key features of MPN and preferentially targets CalrΔ52 over WT cells. These findings have therapeutic implications through inhibiting N-glycosylation alone or in combination with other agents to advance the development of clonally selective therapeutic approaches in CALR-mutant MPN. AEM and JSJ contributed equally. Figure 1 Figure 1. Disclosures Mullally: Janssen, PharmaEssentia, Constellation and Relay Therapeutics: Consultancy.

Comprehensive analysis of lectin-glycan interactions reveals determinants of lectin specificity

Lectin-glycan interactions facilitate inter- and intracellular communication in many processes including protein trafficking, host-pathogen recognition, and tumorigenesis promotion. Specific recognition of glycans by lectins is also the basis for a wide range of applications in areas including glycobiology research, cancer screening, and antiviral therapeutics. To provide a better understanding of the determinants of lectin-glycan interaction specificity and support such applications, this study comprehensively investigates specificity-conferring features of all available lectin-glycan complex structures. Systematic characterization, comparison, and predictive modeling of a set of 221 complementary physicochemical and geometric features representing these interactions highlighted specificity-conferring features with potential mechanistic insight. Univariable comparative analyses with weighted Wilcoxon-Mann-Whitney tests revealed strong statistical associations between binding site features and specificity that are conserved across unrelated lectin binding sites. Multivariable modeling with random forests demonstrated the utility of these features for predicting the identity of bound glycans based on generalized patterns learned from non-homologous lectins. These analyses revealed global determinants of lectin specificity, such as sialic acid glycan recognition in deep, concave binding sites enriched for positively charged residues, in contrast to high mannose glycan recognition in fairly shallow but well-defined pockets enriched for non-polar residues. Focused fine specificity analysis of hemagglutinin interactions with human-like and avian-like glycans uncovered features representing both known and novel mutations related to shifts in influenza tropism from avian to human tissues. As the approach presented here relies on co-crystallized lectin-glycan pairs for studying specificity, it is limited in its inferences by the quantity, quality, and diversity of the structural data available. Regardless, the systematic characterization of lectin binding sites presented here provides a novel approach to studying lectin specificity and is a step towards confidently predicting new lectin-glycan interactions.

Assembly of tetraspanins, galectin-3, and distinct N-glycans defines the solubilization signature of seminal prostasomes from normozoospermic and oligozoospermic men

Background: Prostasomes, extracellular vesicles (EVs) abundantly present in seminal plasma, express distinct tetraspanins (TS) and galectin-3 (gal-3), which are supposed to shape their surface by an assembly of different molecular complexes. In this study, detergent-sensitivity patterns of membrane-associated prostasomal proteins were determined aiming at the solubilization signature as an intrinsic multimolecular marker and a new parameter suitable as a reference for the comparison of EVs populations in health and disease. Methods: Prostasomes were disrupted by Triton X-100 and analyzed by gel filtration under conditions that maintained complete solubilization. Redistribution of TS (CD63, CD9, and CD81), gal-3, gamma-glutamyltransferase (GGT), and distinct N-glycans was monitored using solid-phase lectin-binding assays, transmission electron microscopy, electrophoresis, and lectin blot. Results: Comparative data on prostasomes under normal physiology and conditions of low sperm count revealed similarity regarding the redistribution of distinct N-glycans and GGT, all presumed to be mainly part of the vesicle coat. In contrast to this, a greater difference was found in the redistribution of integral membrane proteins, exemplified by TS and gal-3. Accordingly, they were grouped into two molecular patterns mainly consisting of overlapped CD9/gal-3/wheat germ agglutinin-reactive glycoproteins and CD63/GGT/concanavalin A-reactive glycoproteins. Conclusions: Solubilization signature can be considered as an all-inclusive distinction factor regarding the surface properties of a particular vesicle since it reflects the status of the parent cell and the extracellular environment, both of which contribute to the composition of spatial membrane arrangements.

A Useful Guide to Lectin Binding: Machine-Learning Directed Annotation of 57 Unique Lectin Specificities

ABSTRACTGlycans are critical to every facet of biology and medicine, from viral infections to embryogenesis. Tools to study glycans are rapidly evolving, however the majority of our knowledge is deeply dependent on binding by glycan binding proteins (e.g., lectins). The specificities of lectins, which are often naturally isolated proteins, have not been well- defined, making it difficult to leverage their full potential for glycan analysis. Herein, we use glycan microarray analysis of 116 commercially available lectins, including different preparations of the same lectin, to extract the specific glycan features required for lectin binding. Data was obtained using the Consortium for Functional Glycomics microarray (CFG v5.0) containing 611 glycans. We use a combination of machine learning algorithms to define lectin specificity, mapping inputs (glycan sequences) to outputs (lectin-glycan binding) for a large-scale evaluation of lectin-glycan binding behaviours. Our motif analysis was performed by integrating 68 manually defined glycan features with systematic probing of computational rules for significant binding motifs using mono- and disaccharides- and linkages. Using a combination of machine learning and manual annotation of the data, we created a detailed interpretation of glycan-binding specificity for 57 unique lectins, categorized by their major binding motifs: mannose, complex-type N-glycan, O-glycan, fucose, sialic acid and sulfate, GlcNAc and chitin, Gal and LacNAc, and GalNAc. Our work provides fresh insights into the complex binding features of commercially available lectins in current use, providing a critical guide to these important reagents.