Glycan specificity of neuraminidases determined in microarray format

Carbohydr Res. 2016 Jun 16:428:31-40. doi: 10.1016/j.carres.2016.04.003. Epub 2016 Apr 8.

Abstract

Neuraminidases hydrolytically remove sialic acids from glycoconjugates. Neuraminidases are produced by both humans and their pathogens, and function in normal physiology and in pathological events. Identification of neuraminidase substrates is needed to reveal their mechanism of action, but high-throughput methods to determine glycan specificity of neuraminidases are limited. Here we use two glycan labeling reactions to monitor neuraminidase activity toward glycan substrates. While both periodate oxidation and aniline-catalyzed oxime ligation (PAL) and galactose oxidase and aniline-catalyzed oxime ligation (GAL) can be used to monitor neuraminidase activity toward glycans in microtiter plates, only GAL accurately measured neuraminidase activity toward glycans displayed on a commercial glass slide microarray. Using GAL, we confirm known linkage specificities of three pneumococcal neuraminidases and obtain new information about underlying glycan specificity.

Keywords: Chemoenzymatic labeling; Microarray; Neuraminidase; Sialic acids.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Humans
  • Microarray Analysis / methods*
  • Neuraminidase / genetics
  • Neuraminidase / metabolism*
  • Polysaccharides / chemistry
  • Polysaccharides / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Staining and Labeling
  • Streptococcus pneumoniae / enzymology*
  • Streptococcus pneumoniae / genetics
  • Substrate Specificity

Substances

  • Bacterial Proteins
  • Polysaccharides
  • Recombinant Proteins
  • Neuraminidase