Glycoproteomics-Compatible MS/MS-Based Quantification of Glycopeptide Isomers.

Glycosylation is an essential protein modification occurring on the majority of extracellular human proteins, with mass spectrometry (MS) being an indispensable tool for its analysis, that not only determines glycan compositions, but also the position of the glycan at specific sites via glycoproteomics. However, glycans are complex branching structures with monosaccharides interconnected in a variety of biologically relevant linkages, isomeric properties that are invisible when the readout is mass alone. Here, we developed an LC-MS/MS-based workflow for determining glycopeptide isomer ratios. Making use of isomerically defined glyco(peptide) standards, we observed marked differences in fragmentation behavior between isomer pairs when subjected to collision energy gradients, specifically in terms of the galactosylation/sialylation branching and linkage. These behaviors were developed into component variables that allowed for relative quantification of isomerism within mixtures. Importantly, at least for small peptides, the isomer quantification appeared to be largely independent from the peptide portion of the conjugate, allowing a broad application of the method.

Receptor Density-Dependent Motility of Influenza Virus Particles on Surface Gradients.

Influenza viruses can move across the surface of host cells while interacting with their glycocalyx. This motility may assist in finding or forming locations for cell entry and thereby promote cellular uptake. Because the binding to and cleavage of cell surface receptors forms the driving force for the process, the surface-bound motility of influenza is expected to be dependent on the receptor density. Surface gradients with gradually varying receptor densities are thus a valuable tool to study binding and motility processes of influenza and can function as a mimic for local receptor density variations at the glycocalyx that may steer the directionality of a virus particle in finding the proper site of uptake. We have tracked individual influenza virus particles moving over surfaces with receptor density gradients. We analyzed the extracted virus tracks first at a general level to verify neuraminidase activity and subsequently with increasing detail to quantify the receptor density-dependent behavior on the level of individual virus particles. While a directional bias was not observed, most likely due to limitations of the steepness of the surface gradient, the surface mobility and the probability of sticking were found to be significantly dependent on receptor density. A combination of high surface mobility and high dissociation probability of influenza was observed at low receptor densities, while the opposite occurred at higher receptor densities. These properties result in an effective mechanism for finding high-receptor density patches, which are believed to be a key feature of potential locations for cell entry.

Hydrolytic (In)stability of Methacrylate Esters in Covalently Cross-Linked Hydrogels Based on Chondroitin Sulfate and Hyaluronic Acid Methacrylate.

Chondroitin sulfate (CS) and hyaluronic acid (HA) methacrylate (MA) hydrogels are under investigation for biomedical applications. Here, the hydrolytic (in)stability of the MA esters in these polysaccharides and hydrogels is investigated. Hydrogels made with glycidyl methacrylate-derivatized CS (CSGMA) or methacrylic anhydride (CSMA) degraded after 2-25 days in a cross-linking density-dependent manner (pH 7.4, 37 °C). HA methacrylate (HAMA) hydrogels were stable over 50 days under the same conditions. CS(G)MA hydrogel degradation rates increased with pH, due to hydroxide-driven ester hydrolysis. Desulfated chondroitin MA hydrogels also degrade, indicating that sulfate groups are not responsible for CS(G)MA's hydrolytic sensitivity (pH 7.0-8.0, 37 °C). This sensitivity is likely because CS(G)MA's N-acetyl-galactosamines do not form hydrogen bonds with adjacent glucuronic acid oxygens, whereas HAMA's N-acetyl-glucosamines do. This bond absence allows CS(G)MA higher chain flexibility and hydration and could increase ester hydrolysis sensitivity in CS(G)MA networks. This report helps in biodegradable hydrogel development based on endogenous polysaccharides for clinical applications.

Hierarchical Multivalent Effects Control Influenza Host Specificity.

Understanding how emerging influenza viruses recognize host cells is critical in evaluating their zoonotic potential, pathogenicity, and transmissibility between humans. The surface of the influenza virus is covered with hemagglutinin (HA) proteins that can form multiple interactions with sialic acid-terminated glycans on the host cell surface. This multivalent binding affects the selectivity of the virus in ways that cannot be predicted from the individual receptor-ligand interactions alone. Here, we show that the intrinsic structural and energetic differences between the interactions of avian- or human-type receptors with influenza HA translate from individual site affinity and orientation through receptor length and density on the surface into virus avidity and specificity. We introduce a method to measure virus avidity using receptor density gradients. We found that influenza viruses attached stably to a surface at receptor densities that correspond to a minimum number of approximately 8 HA-glycan interactions, but more interactions were required if the receptors were short and human-type. Thus, the avidity and specificity of influenza viruses for a host cell depend not on the sialic acid linkage alone but on a combination of linkage and the length and density of receptors on the cell surface. Our findings suggest that threshold receptor densities play a key role in virus tropism, which is a predicting factor for both their virulence and zoonotic potential.

Identification of a secondary binding site in human macrophage galactose-type lectin by microarray studies: Implications for the molecular recognition of its ligands.

The human macrophage galactose-type lectin (MGL) is a C-type lectin characterized by a unique specificity for terminal GalNAc residues present in the tumor-associated Tn antigen (αGalNAc-Ser/Thr) and its sialylated form, the sialyl-Tn antigen. However, human MGL has multiple splice variants, and whether these variants have distinct ligand-binding properties is unknown. Here, using glycan microarrays, we compared the binding properties of the short MGL 6C (MGLshort) and the long MGL 6B (MGLlong) splice variants, as well as of a histidine-to-threonine mutant (MGLshort H259T). Although the MGLshort and MGLlong variants displayed similar binding properties on the glycan array, the MGLshort H259T mutant failed to interact with the sialyl-Tn epitope. As the MGLshort H259T variant could still bind a single GalNAc monosaccharide on this array, we next investigated its binding characteristics to Tn-containing glycopeptides derived from the MGL ligands mucin 1 (MUC1), MUC2, and CD45. Strikingly, in the glycopeptide microarray, the MGLshort H259T variant lost high-affinity binding toward Tn-containing glycopeptides, especially at low probing concentrations. Moreover, MGLshort H259T was unable to recognize cancer-associated Tn epitopes on tumor cell lines. Molecular dynamics simulations indicated that in WT MGLshort, His259 mediates H bonds directly or engages the Tn-glycopeptide backbone through water molecules. These bonds were lost in MGLshort H259T, thus explaining its lower binding affinity. Together, our results suggest that MGL not only connects to the Tn carbohydrate epitope, but also engages the underlying peptide via a secondary binding pocket within the MGL carbohydrate recognition domain containing the His259 residue.