Site-specific immobilization of the endosialidase reveals QSOX2 is a novel polysialylated protein.

Polysialic acid (polySia) is a linear polymer of α2,8-linked sialic acid residues that is of fundamental biological interest due to its pivotal roles in the regulation of the nervous, immune, and reproductive systems in healthy human adults. PolySia is also dysregulated in several chronic diseases, including cancers and mental health disorders. However, the mechanisms underpinning polySia biology in health and disease remain largely unknown. The polySia-specific hydrolase, endoneuraminidase NF (EndoN), and the catalytically inactive polySia lectin EndoNDM, have been extensively used for studying polySia. However, EndoN is heat stable and remains associated with cells after washing. When studying polySia in systems with multiple polysialylated species, the residual EndoN that cannot be removed confounds data interpretation. We developed a strategy for site-specific immobilization of EndoN on streptavidin-coated magnetic beads. We showed that immobilizing EndoN allows for effective removal of the enzyme from samples, while retaining hydrolase activity. We used the same strategy to immobilize the polySia lectin EndoNDM, which enabled the enrichment of polysialylated proteins from complex mixtures such as serum for their identification via mass spectrometry. We used this methodology to identify a novel polysialylated protein, QSOX2, which is secreted from the breast cancer cell line MCF-7. This method of site-specific immobilization can be utilized for other enzymes and lectins to yield insight into glycobiology.

The sweet side of sex as a biological variable.

Glycobiology as a field holds enormous potential for understanding human health and disease. However, few glycobiology studies adequately address the issue of sex differences in biology, which severely limits the conclusions that can be drawn. Numerous CAZymes, lectins, and other carbohydrate-associated molecules have the potential to be differentially expressed and regulated with sex, leading to differences in O-GlcNAc, N-glycan branching, fucosylation, sialylation, and proteoglycan structure, among others. Expression of proteins involved in glycosylation is influenced through hormones, miRNA, and gene dosage effects. In this review, we discuss the benefits of incorporating sex-based analysis in glycobiology research and the potential drivers of sex differences. We highlight examples of where incorporation of sex-based analysis has led to insights into glycobiology. Finally, we offer suggestions for how to proceed moving forward, even if the experiments are already complete. Properly incorporating sex based analyses into projects will substantially improve the accuracy and reproducibility of studies as well as accelerate the rate of discovery in the glycosciences.

Synthetic Strategies for Modified Glycosphingolipids and Their Design as Probes.

The plasma membrane of cells contains a diverse array of lipids that provide important structural and biological features. Glycolipids are typically a minor component of the cell membrane and consist primarily of glycosphingolipids (GSLs). GSLs in vertebrates contain a multifarious assortment of glycan headgroups, which can be important to biological functions based on lipid-lipid and lipid-protein interactions. The design of probes to study these complex targets requires advanced synthetic methodologies. In this Review, we will discuss recent advances in chemical and chemoenzymatic synthesis of GSLs in conjunction with the use of these approaches to design new probes. Examples using either chemical or enzymatic semisynthesis methods starting from isolated GSLs will also be reviewed. Focusing primarily on vertebrate glycolipids, we will highlight examples of radionuclide, fluorophore, photoresponsive, and bioorthogonal tagged GSL probes.

Molecular dynamics simulations of viral neuraminidase inhibitors with the human neuraminidase enzymes: Insights into isoenzyme selectivity.

Inhibitors of viral neuraminidase enzymes have been previously developed as therapeutics. Humans can express multiple forms of neuraminidase enzymes (NEU1, NEU2, NEU3, NEU4) that share a similar active site and enzymatic mechanism with their viral counterparts. Using a panel of purified human neuraminidase enzymes, we tested the inhibitory activity of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), zanamivir, oseltamivir, and peramivir against each of the human isoenzymes. We find that, with the exceptions of DANA and zanamivir, these compounds show generally poor activity against the human neuraminidase enzymes. To provide insight into the interactions of viral inhibitors with human neuraminidases, we conducted molecular dynamics simulations using homology models based on coordinates reported for NEU2. Simulations revealed that an organized water is displaced by zanamivir in binding to NEU2 and NEU3 and confirmed the critical importance of engaging the binding pocket of the C7-C9 glycerol sidechain. Our results suggest that compounds designed to target the human neuraminidases should provide more selective tools for interrogating these enzymes. Furthermore, they emphasize a need for additional structural data to enable structure-based drug design in these systems.

Attenuation of Polysialic Acid Biosynthesis in Cells by the Small Molecule Inhibitor 8-Keto-sialic acid.

Sialic acids are key mediators of cell function, particularly with regard to cellular interactions with the surrounding environment. Reagents that modulate the display of specific sialyl glycoforms at the cell surface would be useful biochemical tools and potentially allow for therapeutic intervention in numerous challenging chronic diseases. While multiple strategies are being explored for the control of cell surface sialosides, none that shows high selectivity between sialyltransferases or that targets a specific sialyl glycoform has yet to emerge. Here, we describe a strategy to block the formation of α2,8-linked sialic acid chains (oligo- and polysialic acid) through the use of 8-keto-sialic acid as a chain-terminating metabolic inhibitor that, if incorporated, cannot be elongated. 8-Keto-sialic acid is nontoxic at effective concentrations and serves to block polysialic acid synthesis in cancer cell lines and primary immune cells, with minimal effects on other sialyl glycoforms.

Human neuraminidases have reduced activity towards modified sialic acids on glycoproteins.

Multiple levels of diversity in sialic acid presentation can influence the substrate activity of sialosides for glycoside hydrolases. Few reports have investigated the specificity of human neuraminidase (hNEU) activity towards modified sialic acid residues that can occur on glycoproteins. Previously, we evaluated hNEU activity towards 9-O-acetylated sialic acid in glycolipid substrates and found that hNEU generally discriminated against 9-O-acetylated sialic acid over Neu5Ac. Here, we have investigated the substrate specificity of hNEU enzymes for a glycoprotein substrate (bovine submaxillary mucin) containing 9-O-acetylated and Neu5Gc residues. Using this model substrate, we observe a general trend for hNEU tolerance of Neu5Ac > Neu5Gc ≫ Neu5,9Ac2, consistent with our previous results with glycolipid substrates. These results expand our understanding of hNEU enzyme specificity and suggest that naturally occurring modifications of sialic acids can play a role in regulating hNEU activity.

A quantitative, high-throughput method identifies protein-glycan interactions via mass spectrometry.

Glycan binding by glycan-binding proteins and processing by carbohydrate-active enzymes is implicated in physiological and pathophysiological processes. Comprehensive mapping of glycan interactions is essential to understanding of glycan-mediated biology and can guide the development of new diagnostics and therapeutics. Here, we introduce the competitive universal proxy receptor assay (CUPRA), which combines electrospray ionization mass spectrometry, competitive binding and heterobifunctional glycan-based ligands to give a quantitative high-throughput method for screening glycan libraries against glycan-binding and glycan-processing proteins. Application of the assay to human (siglec-2), plant (Sambucus nigra and Maackia amurensis lectins) and bacterial (cholera toxin, and family 51 carbohydrate binding module) proteins allowed for the identification of ligands with affinities (Kd) ≤ 1 mM. The assay is unprecedentedly versatile and can be applied to natural libraries and, when implemented in a time-resolved manner, provides a quantitative measure of the activities and substrate specificity of carbohydrate-active enzymes.

Human Neuraminidase Isoenzymes Show Variable Activities for 9- O-Acetyl-sialoside Substrates.

Recognition of terminal sialic acids is central to many cellular processes, and structural modification of sialic acid can disrupt these interactions. A prominent, naturally occurring, modification of sialic acid is 9- O-acetylation (9- O-Ac). Study of this modification through generation and analysis of 9- O-Ac sialosides is challenging because of the lability of the acetate group. Fundamental questions regarding the role of 9- O-Ac sialic acids remain unanswered, including what effect it may have on recognition and hydrolysis by the human neuraminidase enzymes (hNEU). To investigate the substrate activity of 9- O-acetylated sialic acids (Neu5,9Ac2), we synthesized an acetylated fluorogenic hNEU substrate 2'-(4-methylumbelliferyl)-9- O-acetyl-α-d- N-acetylneuraminic acid. Additionally, we generated a panel of octyl sialyllactosides containing modified sialic acids including variation in linkage, 9- O-acetylation, and C-5 group (Neu5Gc). Relative rates of substrate cleavage by hNEU were determined using fluorescence spectroscopy and electrospray ionization mass spectrometry. We report that 9- O-acetylation had a significant, and differential, impact on sialic acid hydrolysis by hNEU with general substrate tolerance following the trend of Neu5Ac > Neu5Gc ≫ Neu5,9Ac2 for NEU2, NEU3, and NEU4. Both NEU2 and NEU3 had remarkably reduced activity for Neu5,9Ac2 containing substrates. Other isoenzymes appeared to be more tolerant, with NEU4 even showing increased activity on Neu5,9Ac2 substrates with an aryl aglycone. The impact of these minor structural changes to sialic acid on hNEU activity was unexpected, and these results provide evidence of the substantial influence of 9- O-Ac modifications on hNEU enzyme substrate specificity. Furthermore, these findings may implicate hNEU in processes governed by 9- O-acetyltransferases and -esterases.