Pneumococcal Neuraminidase Substrates Identified through Comparative Proteomics Enabled by Chemoselective Labeling.

Abstract

Neuraminidases (sialidases) are enzymes that hydrolytically remove sialic acid from sialylated proteins and lipids. Neuraminidases are encoded by a range of human pathogens, including bacteria, viruses, fungi, and protozoa. Many pathogen neuraminidases are virulence factors, indicating that desialylation of host glycoconjugates can be a critical step in infection. Specifically, desialylation of host cell surface glycoproteins can enable these molecules to function as pathogen receptors or can alter signaling through the plasma membrane. Despite these critical effects, no unbiased approaches exist to identify glycoprotein substrates of neuraminidases. Here, we combine previously reported glycoproteomics methods with quantitative proteomics analysis to identify glycoproteins whose sialylation changes in response to neuraminidase treatment. The two glycoproteomics methods-periodate oxidation and aniline-catalyzed oxime ligation (PAL) and galactose oxidase and aniline-catalyzed oxime ligation (GAL)-rely on chemoselective labeling of sialylated and nonsialylated glycoproteins, respectively. We demonstrated the utility of the combined approaches by identifying substrates of two pneumococcal neuraminidases in a human cell line that models the blood-brain barrier. The methods deliver complementary lists of neuraminidase substrates, with GAL identifying a larger number of substrates than PAL (77 versus 17). Putative neuraminidase substrates were confirmed by other methods, establishing the validity of the approach. Among the identified substrates were host glycoproteins known to function in bacteria adherence and infection. Functional assays suggest that multiple desialylated cell surface glycoproteins may act together as pneumococcus receptors. Overall, this method will provide a powerful approach to identify glycoproteins that are desialylated by both purified neuraminidases and intact pathogens.