Sialidases as regulators of bioengineered cellular surfaces

Abstract

Human sialidases (NEUs) catalyze the removal of N-acetyl neuraminic acids from the glycome of the cell and regulate a diverse repertoire of nominal cellular functions, such as cell signaling and adhesion. A greater understanding of their substrate permissivity is of interest in order to discern their physiological functions in disease states and in the design of specific and effective small molecule inhibitors. Towards this, we have synthesized soluble fluorogenic reporters of mammalian sialidase activity bearing unnatural sialic acids commonly incorporated into the cellular glycocalyx via metabolic glycoengineering. We found cell-surface sialidases in Jurkat capable of cleaving unnatural sialic acids with differential activities toward a variety of R groups on neuraminic acid. In addition, we observed modulated structure-activity relationships when cell-surface sialidases were presented glycans with unnatural bulky, hydrophobic or fluorinated moieties incorporated directly via glycoengineering. Our results confirm the importance of cell-surface sialidases in glycoengineering incorporation data. We demonstrate the flexibility of human NEUs toward derivatized sugars and highlight the importance of native glycan presentation to sialidase binding and activity. These results stand to inform not only metabolic glycoengineering efforts but also inhibitor design.

Enhanced Cross-Linking of Diazirine-Modified Sialylated Glycoproteins Enabled through Profiling of Sialidase Specificities

“…Previous studies have shown that substitutions on the N-acyl side chain of sialic acid can affect sialidase activity in vitro, − although these effects are isoenzyme specific. In addition, while sialic acid analogs bearing larger N-acyl groups are generally poor substrates for purified sialidases, some secreted sialidases appear to be more tolerant of these substitutions in metabolically labeled cells . Thus, addition of diazirine to the N-acyl side chain of sialic acid could result in altered activity of sialidases, such that certain sialidases may be able to selectively cleave Neu5Ac, but not SiaDAz­(2me) or SiaDAz­(4me), from glycoconjugates.…”

Impacting Bacterial Sialidase Activity by Incorporating Bioorthogonal Chemical Reporters onto Mammalian Cell-Surface Sialosides

“…3 The sialidases were selected on the basis of their species and colonization site diversity, as well as their known broad substrate specificity. 6 To screen bacterial sialidases activity against our unnatural sialosides, we developed an in vitro enzymatic 1 H NMR coupled assay which, unlike previous methods, 7,26 is fully compatible with 5-and 9-modified sialic acids, various sialyl linkage specificities, as well as the composition of the underlying glycans. Accordingly, sialosides 1−10 were individually incubated with either C. perfringens, S. pneumoniae, V. cholerae, or S. typhimurium sialidase at 37 °C in acetate buffer made from deuterated water (50 mM, pH 5.5).…”

Low molecular weight fucoidan restores diabetic endothelial glycocalyx by targeting neuraminidase2: A new therapy target in glycocalyx shedding

“…Glycocalyx shedding is regulated by glycocalyx shedding enzymes. Hyaluronidase (HYAL) (Dogne et al, 2016), heparanases (HPSE) (Arfian et al, 2019), matrixmetalloproteinases (MMP) (Ramnath et al, 2014), neuraminidase (NEU) (Mensah et al, 2019; Zamora et al, 2015) and hyaluronic acid synthetase (HAS) (Zhang et al, 2018) are directly involved in glycocalyx degradation. These enzymes can trigger glycocalyx shedding.…”