Biognos (Sweden)

SummaryEbola virus glycoprotein is one of the most heavily O-glycosylated viral envelope glycoproteins. Using glycoengineered cell lines we demonstrate that O-linked glycan truncation and perturbed initiation retarded the production of viral particles and decreased infectivity of progeny virus. Next, using TMT isobaric labelling, we performed quantitative differential O-glycoproteomics on proteins produced in wild type HEK293 cells and cell lines ablated for three key GalNAc-transferases, GalNAc-T1, -T2, and -T3, as well as compared it to patterns on wild type virus-like particles. We demonstrate selective initiation of a subset of O-glycosites by each enzyme, with GalNAc-T1 having the largest contribution. This work represents a comprehensive site-specific analysis of EBOV GP, with 47 O-glycosites identified, and sheds light on differential regulation of EBOV GP glycosylation initiated by host GalNAc-Ts. Together with the effect on viral propagation it opens prospective avenues for tailored intervention approaches and means for modulating immunogen O-glycan density.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a public health crisis, and the vaccines that can induce highly potent neutralizing antibodies are essential for ending the pandemic. The spike (S) protein on the viral envelope mediates human angiotensin-converting enzyme 2 (ACE2) binding and thus is the target of a variety of neutralizing antibodies. In this work, we built various S trimer-antibody complex structures on the basis of the fully glycosylated S protein models described in our previous work, and performed all-atom molecular dynamics simulations to get insight into the structural dynamics and interactions between S protein and antibodies. Investigation of the residues critical for S-antibody binding allows us to predict the potential influence of mutations in SARS-CoV-2 variants. Comparison of the glycan conformations between S-only and S-antibody systems reveals the roles of glycans in S-antibody binding. In addition, we explored the antibody binding modes, and the influences of antibody on the motion of S protein receptor binding domains. Overall, our analyses provide a better understanding of S-antibody interactions, and the simulation-based S-antibody interaction maps could be used to predict the influences of S mutation on S-antibody interactions, which will be useful for the development of vaccine and antibody-based therapy.

A central tenet inthe design of vaccines is the display of native-likeantigens in the elicitation of protective immunity. The abundanceof N-linked glycans across the SARS-CoV-2 spike protein is a potentialsource of heterogeneity among the many different vaccine candidatesunder investigation. Here, we investigate the glycosylation of recombinantSARS-CoV-2 spike proteins from five different laboratories and comparethem against S protein from infectious virus, cultured in Vero cells.We find patterns that are conserved across all samples, and this canbe associated with site-specific stalling of glycan maturation thatacts as a highly sensitive reporter of protein structure. Moleculardynamics simulations of a fully glycosylated spike support a modelof steric restrictions that shape enzymatic processing of the glycans.These results suggest that recombinant spike-based SARS-CoV-2 immunogenglycosylation reproducibly recapitulates signatures of viral glycosylation.

Tartrate-resistant acid phosphatase (TRAP/ACP5) occurs as two isoforms-TRAP 5a with low enzymatic activity due to a loop interacting with the active site and the more active TRAP isoform 5b generated upon proteolytic cleavage of this loop. TRAP has been implicated in several diseases, including cancer. Thus, this study set out to identify small-molecule inhibitors of TRAP activity. A microplate-based enzymatic assay for TRAP 5b was applied in a screen of 30,315 compounds, resulting in the identification of 90 primary hits. After removal of promiscuous compounds, unwanted groups, and false positives by orthogonal assays and three-concentration validation, the properties of 52 compounds were further investigated to better understand their mechanism of action. Full-concentration-response curves for these compounds were established under different enzyme concentrations and (pre)incubation times to remove compounds with inconsistent results and low potencies. Full-concentration-response curves were also performed for both isoforms, to examine isoform prevalence. Filtering led to six prioritized compounds, representing different clusters. One of these, CBK289001 or (6S)-6-[3-(2H-1,3-benzodioxol-5-yl)-1,2,4-oxadiazol-5-yl]-N-(propan-2-yl)-1H,4H,5H,6H,7H-imidazo[4,5-c]pyridine-5-carboxamide, demonstrated efficacy in a migration assay and IC50 values from 4 to 125μm. Molecular docking studies and analog testing were performed around CBK289001 to provide openings for further improvement toward more potent blockers of TRAP activity.

Oligosaccharide structures derived from the lipooligosaccharide of M. catarrhalis show that the highly branched glucose-rich inner core of the oligosaccharide has an altered conformation compared to the most truncated tetra-glucose-Kdo lgt1/4Δ oligosaccharide structure. Addition of one residue each to the (1-4) and (1-6) chains to give the lgt2Δ oligosaccharide is the minimum requirement for this conformational change to occur. Extensive molecular modeling and NMR investigations have shown that the (1-3), (1-4), and (1-6) glycosidic linkages from the central α-d-Glcp have significantly altered conformational preferences between the two structures. For the lgt1/4Δ oligosaccharide the (1-3) and (1-4) linkage populates predominantly the syn minimum on the conformational free energy map and for the (1-6) linkage conformational flexibility is observed, which is supported by 1H-NMR T1 measurements. For the lgt2Δ oligosaccharide the unusual “(1-4)anti-ψ(1-6)gg” conformation, which could be confirmed by long-range NOE signals, is a dominant conformation in which the oligosaccharide is very compact with the terminal α-d-GlcNAc residue folding back towards the center of the molecule leading to an extensive intra-molecular hydrophobic interaction between the terminal residues. Comparing effective H-H distances, which were calculated for conformational sub-ensembles, with the NOE distances revealed that typically multiple conformations could be present without significantly violating the measured NOE restraints. For lgt2Δ the presence of more than one conformation is supported by the NOE data.

Docking methods are a valuable tool for the prediction of carbohydrate binding sites and the design of carbohydrate-based drugs. However, there are also significant limitations and care needs to be taken when evaluating the docking results. In this chapter the challenges, limitations, and possible pitfalls in docking of carbohydrates are described. Practical examples explain the use of docking methods for the rational design of carbohydrate-based inhibitors as well as the prediction of carbohydrate binding sites.

Viruses within a family often vary in their cellular tropism and pathogenicity. In many cases, these variations are due to viruses switching their specificity from one cell surface receptor to another. The structural requirements that underlie such receptor switching are not well understood especially for carbohydrate-binding viruses, as methods capable of structure-specificity studies are only relatively recently being developed for carbohydrates. We have characterized the receptor specificity, structure and infectivity of the human polyomavirus BKPyV, the causative agent of polyomavirus-associated nephropathy, and uncover a molecular switch for binding different carbohydrate receptors. We show that the b-series gangliosides GD3, GD2, GD1b and GT1b all can serve as receptors for BKPyV. The crystal structure of the BKPyV capsid protein VP1 in complex with GD3 reveals contacts with two sialic acid moieties in the receptor, providing a basis for the observed specificity. Comparison with the structure of simian virus 40 (SV40) VP1 bound to ganglioside GM1 identifies the amino acid at position 68 as a determinant of specificity. Mutation of this residue from lysine in BKPyV to serine in SV40 switches the receptor specificity of BKPyV from GD3 to GM1 both in vitro and in cell culture. Our findings highlight the plasticity of viral receptor binding sites and form a template to retarget viruses to different receptors and cell types.

Riproximin is a cytotoxic type II ribosome-inactivating protein showing high selectivity for tumor cell lines. Its binding to cell surface glycans is crucial for subsequent internalization and cytotoxicity. In this paper, we describe a unique mechanism of interaction and discuss its implications for the cellular targeting and cytotoxicity of riproximin. On a carbohydrate microarray, riproximin specifically bound to two types of asialo-glycans, namely to bi- and triantennary complex N-glycan structures (NA2/NA3) and to repetitive N-acetyl-D-galactosamine (GalNAc), the so-called clustered Tn antigen, a cancer-specific O-glycan on mucins. Two glycoproteins showing high riproximin binding, the NA3-presenting asialofetuin and the clustered Tn-rich asialo-bovine submaxillary mucin, were subsequently chosen as model glycoproteins to mimic the binding interactions of riproximin with the two types of glycans. ELISA analyses were used to relate the two binding specificities of riproximin to its two sugar binding sites. The ability of riproximin to cross-link the two model proteins revealed that binding of the two types of glycoconjugates occurs within different binding sites. The biological implications of these binding properties were analyzed in cellular assays. The cytotoxicity of riproximin was found to depend on its specific and concomitant interaction with the two glycoconjugates as well as on dynamic avidity effects typical for lectins binding to multivalent glycoproteins. The presence of definite, cancer-related structures on the cells to be targeted determines the therapeutic potency of riproximin. Due to its cross-linking ability, riproximin is expected to show a high degree of specificity for cells exposing both NA2/NA3 and clustered Tn structures.