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 β-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 α2-3- and α2-6-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 α2-6- and α2-3-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 α2-6-linked Neu5Ac9Lt or α2-6-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.

FIGURE 1.

The design and preparation of an SGM. Fluorescent glycans (GAEABs) terminating in β-linked galactose were acceptors in a one-pot three-enzyme synthetic scheme with pyruvate, CTP and mannose, N-acetylmannosamine, or various derivatives to generate fluorescent sialyl glycans containing α2–3- and α2–6-linked sialic acid forms. The purified fluorescent sialyl glycans were structurally confirmed and printed as an SGM.

FIGURE 2.

HPLC profiles of LNnT-AEAB and several sialylated products, including Neu5Acα2–6-LNnT-AEAB, Neu5Gcα2–6-LNnT-AEAB, Neu5Acα2-3-LNnT-AEAB, and Neu5Gcα2–3-LNnT-AEAB (from bottom to top). The position of elution of the precursor, LNnT-AEAB, is indicated in the bottom profile.

FIGURE 3.

Structures of the sialic acid derivatives. a, 16 different sialic acid forms were obtained. b, four different fluorescent glycans were used as sialyltransferase acceptors. c, the structure of the bifunctional fluorescent tag used as the linker to covalently couple the glycan to the NHS-derivatized glass slide.

FIGURE 4.

Binding of lectins and viruses to the SGM. The y axis represents 77 sialylated glycans and three controls (LNnT, NA2, and Man5), corresponding to chart ID 1–80. All glycans are AEAB conjugates. The glycans are sorted for discrimination of α2–6 and α2–3 linkages; the sialic acids Neu5Ac, Neu5Gc, Kdn, and derivatives; and the underlying structures lactose, LNnT, NA2, and LNT. The x axis represents lectins and viruses interrogated on the SGM, shown in individual lanes. All lectins are biotinylated and detected with cyanine 5- or Alexa488-labeled streptavidin. All viruses are labeled with Alexa488. A heat map format is used to demonstrate the relative binding strength, with red indicating strong and yellow indicating weak. The binding signals of all lectins and viruses are normalized individually and shown in individual lanes, except for the sialidase treatments (lanes 3, 6, and 9) and mild acid treatments (lanes 4, 7, and 10), which are normalized together with corresponding lectins (lanes 2, 5, and 8).

FIGURE 5.

Binding of lectin ConA and RCA-I to the SGM. a, the SGM was interrogated with biotinylated Con A (10 μg/ml) followed by Alexa 488-labeled streptavidin at 1 μg/ml. b, the SGM was interrogated with biotinylated RCA-I (10 μg/ml) followed by Alexa 488-labeled streptavidin at 1 μg/ml. c, as a control, the SGM was digested with A. ureafaciens sialidase (150 milliunits/ml, pH 5.0, 37 °C for 4 h). Then the SGM was interrogated with biotinylated RCA-I (10 μg/ml) followed by Alexa 488-labeled streptavidin at 1 μg/ml. d, as a separate control, after mild acid treatment (0.1 n HCl, 80 °C, 1 h) of the SGM to remove the acid-labile sialic acid, the SGM was interrogated with biotinylated RCA-I (10 μg/ml) followed by Alexa 488-labeled streptavidin at 1 μg/ml.

FIGURE 6.

Binding of sialic acid-binding lectins, SNA and MAL-I, to the SGM. a, the SGM was interrogated with biotinylated SNA (10 μg/ml) followed by Alexa 488-labeled streptavidin at 1 μg/ml. b, the SGM was treated with A. ureafaciens sialidase and then interrogated with biotinylated SNA (10 μg/ml) followed by Alexa 488-labeled streptavidin at 1 μg/ml. c, the SGM was treated with mild acid and then interrogated with biotinylated SNA (10 μg/ml) followed by Alexa 488-labeled streptavidin at 1 μg/ml. d, the SGM was interrogated with biotinylated MAL-I (10 μg/ml) followed by Alexa 488-labeled streptavidin at 1 μg/ml. e, the SGM was treated with A. ureafaciens sialidase and then interrogated with biotinylated MAL-I (10 μg/ml) followed by Alexa 488-labeled streptavidin at 1 μg/ml. f, the SGM was treated with mild acid and then interrogated with biotinylated MAL-I (10 μg/ml) followed by Alexa 488-labeled streptavidin at 1 μg/ml.

FIGURE 7.

Binding of viruses to the SGM. Various preparations of fluorescently labeled virus, hPIV1 (a), hPIV2 (b), hPIV3 (c), influenza A viruses A/Oklahoma/447/08 H1N1 (d), and A/Oklahoma/483/08 H3N2 (e) were suspended in binding buffer and applied to the SGM.

FIGURE 8.

The general structure of a modified sialic acid with 5-position and 9-position substitution group noted.