A Sialylated Glycan Microarray Reveals Novel Interactions of Modified Sialic Acids with Proteins and Viruses

Abstract

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 ␤-linked galactose at the nonreducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate ␣2-3-and ␣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 ␣2-6-and ␣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 ␣2-6-linked Neu5Ac9Lt or ␣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.

Automated Motif Discovery from Glycan Array Data

“…353 (Kdna2-6Galb1-4GlcNAc) and 256 [Neu5Aca2-6Galb1-4(6OSO3) GlcNAc] are the highest ranking glycans; this suggests the possibility that SNA might prefer sulfated glycans or other sialic acid derivatives, but these motifs do not arise from analysis by GLYMMR, since they are represented fewer than four times on the array. Interestingly, in other work on arrays comprised of derivatives of sialic acid, we observed that SNA preferred Kdn and its acetylated derivatives to Neu5Ac and Neu5Gc, the more common mammalian sialic acids (Song et al, 2011). These results also illustrate the need in glycan microarray studies for presenting glycan determinants in a variety of formats and backbones on multiple glycans to facilitate identification of motifs.…”

N -Glycolylneuraminic Acid on Human Epithelial Cells Prevents Entry of Influenza A Viruses That Possess N -Glycolylneuraminic Acid Binding Ability

“…5). These fluorescent intensities contain both Neu5Ac and Neu5Gc as terminal sialic acids because of recognition of both Neu5Ac and Neu5Gc by the lectins SNA and MAL-I (27). However, MAL-I cannot detect SA␣2,3Gal linkage of O-glycan, which is recognized by MAL-II (28).…”

“…(iv) The difference is derived from reactivities of SNA and MAL-I. According to supplemental material of Song et al (27), SNA bound to Neu5Gc-and Neu5Ac-␣2,6Gal␤1, 4GlcNAc␤1,2Man␣1,3(Neu5Gc-and Neu5Ac-␣2,6Gal␤1, 4GlcNAc␤1,2Man␣1,6)Man␤1,4GlcNAc␤1,4GlcNAcitol (with binding signals being 26,327 and 22,218, respectively, for 10 g/mlSNA)andtoNeu5Gc-andNeu5Ac-␣2,6Gal␤1,4GlcNAc␤1, 3Gal␤1,4Glcitol (with binding signals being 8,544 and 9,187, respectively, for 10 g/ml SNA) but not to Neu5Gc-and Neu5Ac-␣2,6Gal␤1,3GlcNAc␤1,3Gal␤1,4Glcitol (with binding signals being 56 and 96, respectively, for 10 g/ml SNA). Similar structures containing Neu5Gc or Neu5Ac are suggested to be recognized equivalently by SNA.…”

Analysis of Influenza Virus Hemagglutinin Receptor Binding Mutants with Limited Receptor Recognition Properties and Conditional Replication Characteristics

“…Although most influenza virus receptor binding research has focused on Neu5Ac and Neu5Gc glycans, sialic acid on cells is expressed with a highly diverse group of modifications (7). In an effort to address the stereochemical nature of potential receptor species for our viruses, we utilized a recently developed glycan microarray that contains chemically derived sialic acid species (65). This array presents 77 sialic acid glycans with 16 different sialic acid modifications in ␣2,3 and ␣2,6 linkages to 3 different precursor glycans.…”

“…Both glycans are present on the current CFG array, but a plethora of sialic acid modifications exist in nature which are not found on the current CFG array (72). Therefore, we examined binding of the pseudorevertant viruses to a recently generated modified sialic acid glycan microarray (65). The rH3N1 WT, Y98F/S145I, and Y98F/K238N viruses all bound to sialic acid derivatives, albeit very poorly to some.…”