Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 286, Issue 36, Pages 31610-31622Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M111.274217
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Funding
- National Institutes of Health (NIH) [RO1GM085448, GM62116, GM076360]
- Consortium for Functional Glycomics under NIGMS
- Defense Advanced Research Projects Agency [HR0011-10-00]
- Oklahoma Center for the Advancement of Science and Technology [HR09-001]
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Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in beta-linked galactose at the non-reducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate alpha 2-3- and alpha 2-6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting alpha 2-6- and alpha 2-3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-D-glycero-D-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either alpha 2-6-linked Neu5Ac9Lt or alpha 2-6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions.
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