Pre-B cell receptor acts as a selectivity switch for galectin-1 at the pre-B cell surface.

Galectins are glycan-binding proteins translating the sugar-encoded information of cellular glycoconjugates into physiological activities, including immunity, cell migration, and signaling. Galectins also interact with non-glycosylated partners in the extracellular milieu, among which the pre-B cell receptor (pre-BCR) during B cell development. How these interactions might interplay with the glycan-decoding function of galectins is unknown. Here, we perform NMR experiments on native membranes to monitor Gal-1 binding to physiological cell surface ligands. We show that pre-BCR interaction changes Gal-1 binding to glycosylated pre-B cell surface receptors. At the molecular and cellular levels, we identify α2,3-sialylated motifs as key targeted epitopes. This targeting occurs through a selectivity switch increasing Gal-1 contacts with α2,3-sialylated poly-N-acetyllactosamine upon pre-BCR interaction. Importantly, we observe that this switch is involved in the regulation of pre-BCR activation. Altogether, this study demonstrates that interactions to non-glycosylated proteins regulate the glycan-decoding functions of galectins at the cell surface.

Flagellin is essential for initial attachment to mucosal surfaces by Clostridioides difficile.

IMPORTANCE: Clostridioides difficile is one of the leading causes of hospital-acquired infections worldwide and presents challenges in treatment due to recurrent gastrointestinal disease after treatment with antimicrobials. The mechanisms by which C. difficile colonizes the gut represent a key gap in knowledge, including its association with host cells and mucosa. Our results show the importance of flagellin for specific adhesion to mucosal hydrogels and can help to explain prior observations of adhesive defects in flagellin and pilin mutants.

N-glycosylation modulates enzymatic activity of Trypanosoma congolense trans-sialidase.

Trypanosomes cause the devastating disease trypanosomiasis, in which the action of trans-sialidase (TS) enzymes harbored on their surface is a key virulence factor. TS enzymes are N-glycosylated, but the biological functions of their glycans have remained elusive. In this study, we investigated the influence of N-glycans on the enzymatic activity and structural stability of TconTS1, a recombinant TS from the African parasite Trypanosoma congolense. We expressed the enzyme in Chinese hamster ovary Lec1 cells, which produce high-mannose type N-glycans similar to the TS N-glycosylation pattern in vivo. Our MALDI-TOF mass spectrometry data revealed that up to eight putative N-glycosylation sites were glycosylated. In addition, we determined that N-glycan removal via endoglycosidase Hf treatment of TconTS1 led to a decrease in substrate affinity relative to the untreated enzyme but had no impact on the conversion rate. Furthermore, we observed no changes in secondary structure elements of hypoglycosylated TconTS1 in CD experiments. Finally, our molecular dynamics simulations provided evidence for interactions between monosaccharide units of the highly flexible N-glycans and some conserved amino acids located at the catalytic site. These interactions led to conformational changes, possibly enhancing substrate accessibility and enzyme-substrate complex stability. The here-observed modulation of catalytic activity via N-glycans represents a so-far-unknown structure-function relationship potentially inherent in several members of the TS enzyme family.

Synthetic Approaches to Break the Chemical Shift Degeneracy of Glycans.

NMR spectroscopy is the leading technique for determining glycans' three-dimensional structure and dynamic in solution as well as a fundamental tool to study protein-glycan interactions. To overcome the severe chemical shift degeneracy of these compounds, synthetic probes carrying NMR-active nuclei (e. g., 13 C or 19 F) or lanthanide tags have been proposed. These elegant strategies permitted to simplify the complex NMR analysis of unlabeled analogues, shining light on glycans' conformational aspects and interaction with proteins. Here, we highlight some key achievements in the synthesis of specifically labeled glycan probes and their contribution towards the fundamental understanding of glycans.

The polyfunctional polysialic acid: A structural view.

Polysialic acid (polySia), a homopolymer of α2,8-linked sialic acid residues, modifies a small number of proteins and has central functions in vertebrate signalling. Here, we review the regulatory functions of polySia in signalling processes and the immune system of adult humans, as well as functions based on their chemical properties. The main focus will be on the structure-function relationship of polySia with its interaction partners in humans. Recent studies have indicated that the degree of polymerisation is an important parameter that can guide the regulatory effect of polySia in addition to its binding to target proteins. Therefore, the structures of polySia in solution and bound to interaction partners are compared in order to identify the key factors that define binding specificity.

Automated Glycan Assembly of 19 F-labeled Glycan Probes Enables High-Throughput NMR Studies of Protein-Glycan Interactions.

Protein-glycan interactions mediate important biological processes, including pathogen host invasion and cellular communication. Herein, we showcase an expedite approach that integrates automated glycan assembly (AGA) of 19 F-labeled probes and high-throughput NMR methods, enabling the study of protein-glycan interactions. Synthetic Lewis type 2 antigens were screened against seven glycan binding proteins (GBPs), including DC-SIGN and BambL, respectively involved in HIV-1 and lung infections in immunocompromised patients, confirming the preference for fucosylated glycans (Lex , H type 2, Ley ). Previously unknown glycan-lectin weak interactions were detected, and thermodynamic data were obtained. Enzymatic reactions were monitored in real-time, delivering kinetic parameters. These results demonstrate the utility of AGA combined with 19 F NMR for the discovery and characterization of glycan-protein interactions, opening up new perspectives for 19 F-labeled complex glycans.

Synthesis of asymmetrical multiantennary human milk oligosaccharides.

Despite mammalian glycans typically having highly complex asymmetrical multiantennary architectures, chemical and chemoenzymatic synthesis has almost exclusively focused on the preparation of simpler symmetrical structures. This deficiency hampers investigations into the biology of glycan-binding proteins, which in turn complicates the biomedical use of this class of biomolecules. Herein, we describe an enzymatic strategy, using a limited number of human glycosyltransferases, to access a collection of 60 asymmetric, multiantennary human milk oligosaccharides (HMOs), which were used to develop a glycan microarray. Probing the array with several glycan-binding proteins uncovered that not only terminal glycoepitopes but also complex architectures of glycans can influence binding selectivity in unanticipated manners. N- and O-linked glycans express structural elements of HMOs, and thus, the reported synthetic principles will find broad applicability.

Characterization of a high-affinity sialic acid-specific CBM40 from Clostridium perfringens and engineering of a divalent form.

CBMs (carbohydrate-binding modules) are a class of polypeptides usually associated with carbohydrate-active enzymatic sites. We have characterized a new member of the CBM40 family, coded from a section of the gene NanI from Clostridium perfringens Glycan arrays revealed its preference towards α(2,3)-linked sialosides, which was confirmed and quantified by calorimetric studies. The CBM40 binds to α(2,3)-sialyl-lactose with a Kd of ∼30 µM, the highest affinity value for this class of proteins. Inspired by lectins' structure and their arrangement as multimeric proteins, we have engineered a dimeric form of the CBM, and using SPR (surface plasmon resonance) we have observed 6-11-fold binding increases due to the avidity affect. The structures of the CBM, resolved by X-ray crystallography, in complex with α(2,3)- or α(2,6)-sialyl-lactose explain its binding specificity and unusually strong binding.

Conformational flexibility of N-glycans in solution studied by REMD simulations.

Protein-glycan recognition regulates a wide range of biological and pathogenic processes. Conformational diversity of glycans in solution is apparently incompatible with specific binding to their receptor proteins. One possibility is that among the different conformational states of a glycan, only one conformer is utilized for specific binding to a protein. However, the labile nature of glycans makes characterizing their conformational states a challenging issue. All-atom molecular dynamics (MD) simulations provide the atomic details of glycan structures in solution, but fairly extensive sampling is required for simulating the transitions between rotameric states. This difficulty limits application of conventional MD simulations to small fragments like di- and tri-saccharides. Replica-exchange molecular dynamics (REMD) simulation, with extensive sampling of structures in solution, provides a valuable way to identify a family of glycan conformers. This article reviews recent REMD simulations of glycans carried out by us or other research groups and provides new insights into the conformational equilibria of N-glycans and their alteration by chemical modification. We also emphasize the importance of statistical averaging over the multiple conformers of glycans for comparing simulation results with experimental observables. The results support the concept of "conformer selection" in protein-glycan recognition.