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. 2024 Jul 26;4(8):2966-2978.
doi: 10.1021/jacsau.4c00307. eCollection 2024 Aug 26.

Chemoenzymatic Synthesis of Sulfated N-Glycans Recognized by Siglecs and Other Glycan-Binding Proteins

Kun Huang  1 , Eleanor E Bashian  2 , Guanghui Zong  1 , Corwin M Nycholat  2 , Ryan McBride  2 , Margaryta Gomozkova  1 , Shengyang Wang  2 , Chin Huang  3   4 , Digantkumar G Chapla  3 , Edward N Schmidt  5 , Matthew Macauley  5 , Kelley W Moremen  3   4 , James C Paulson  2 , Lai-Xi Wang  1
Affiliations

Chemoenzymatic Synthesis of Sulfated N-Glycans Recognized by Siglecs and Other Glycan-Binding Proteins

Kun Huang et al. JACS Au. .

Abstract

Sulfated N-glycans are present in many glycoproteins, which are implicated in playing important roles in biological recognition processes. Here, we report the systematic chemoenzymatic synthesis of a library of sulfated and sialylated biantennary N-glycans and assess their binding to Siglecs and glycan-specific antibodies that recognize them as glycan ligands. The combined use of three human sulfotransferases, GlcNAc-6-O-sulfotransferase (CHST2), Gal-3-O-sulfotransferase (Gal3ST1), and keratan sulfate Gal-6-O-sulfotransferase (CHST1), resulted in asymmetric and symmetric branch-selective sulfation of the GlcNAc and/or Gal moieties of N-glycans. The extension of the sugar chain using α-2,3- and α-2,6-sialyltransferases afforded the sulfated and sialylated N-glycans. These synthetic glycans with different patterns of sulfation and sialylation were evaluated for binding to selected Siglecs and sulfoglycan-specific antibodies using glycan microarrays. The results confirm previously documented glycan-recognizing properties and further reveal novel specificities for these glycan-binding proteins, demonstrating the utility of the library for assessing the specificity of glycan-binding proteins recognizing sulfated and sialylated glycans.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Structures of the synthetic sulfated N-glycans and related reference compounds.
Scheme 1
Scheme 1. Enzymatic Synthesis of GlcNAc-6-O-Sulfated Biantennary N-Glycans (R = Asn-Fmoc)
Scheme 2
Scheme 2. Enzymatic Synthesis of Gal-6-O-Sulfated Biantennary N-Glycans (R = Asn-Fmoc)
Scheme 3
Scheme 3. Enzymatic Synthesis of Gal-3-O-Sulfated Biantennary N-Glycans (R = Asn-Fmoc)
Scheme 4
Scheme 4. Enzymatic Synthesis of a Homogeneous Monosulfated Biantennary N-Glycan (R = Asn-Fmoc)
Scheme 5
Scheme 5. Installation of a Free Glycine at the Asn Moiety of the Sulfated N-Glycans for the Construction of Glycan Microarray
Figure 2
Figure 2
Glycan array analysis of the specificity of several lectins, monoclonal antibodies, and Siglecs for sulfated, sialylated N-glycans. Analysis of the specificity of selected glycan-binding proteins was conducted with a glycan array of 81 glycans, as described in Experimental Procedures. Briefly, glycan-binding proteins and corresponding detection reagents were overlaid on printed glycan microarrays for 50 min at 22 °C in a humidified chamber. Biotinylated lectins ECL (A) and SNA (B) were overlaid at 10 and 1 μg/mL, respectively, in PBST and detected with AF488-conjugated streptavidin. Monoclonal antibodies S2 (C) and KN343 (D) were run at 10 and 1 μg/mL, respectively, in phosphate-buffered saline (PBS) and were detected with Phycoerythrin (PE)-conjugated αIgM secondary (5 and 0.5 μg/mL, respectively). Human Siglec-1 (E), murine Siglec-1 (F), human CD22 (G), murine CD22 (H), human CD33 (I), Siglec-8 (J), and Siglec-F (K) were analyzed at 50 μg/mL in PBS and detected with AF488-conjugated αIgG secondary (25 μg/mL). The human IgG1 isotype control (L) was also detected with AF488-conjugated αIgG secondary. Graphs depict the mean ± SEM of the relative fluorescence signal (RFU). Glycan structures corresponding to glycan numbers can be found in Supporting Information, Table S1. Controls for sialic acid dependence were assessed with corresponding conserved Arg mutants of each Siglec in Supporting Information, Figure S1. (The glycan array MIRAGE data for Figure S1 are given in the Supporting Information, Table S4).
Figure 2
Figure 2
Glycan array analysis of the specificity of several lectins, monoclonal antibodies, and Siglecs for sulfated, sialylated N-glycans. Analysis of the specificity of selected glycan-binding proteins was conducted with a glycan array of 81 glycans, as described in Experimental Procedures. Briefly, glycan-binding proteins and corresponding detection reagents were overlaid on printed glycan microarrays for 50 min at 22 °C in a humidified chamber. Biotinylated lectins ECL (A) and SNA (B) were overlaid at 10 and 1 μg/mL, respectively, in PBST and detected with AF488-conjugated streptavidin. Monoclonal antibodies S2 (C) and KN343 (D) were run at 10 and 1 μg/mL, respectively, in phosphate-buffered saline (PBS) and were detected with Phycoerythrin (PE)-conjugated αIgM secondary (5 and 0.5 μg/mL, respectively). Human Siglec-1 (E), murine Siglec-1 (F), human CD22 (G), murine CD22 (H), human CD33 (I), Siglec-8 (J), and Siglec-F (K) were analyzed at 50 μg/mL in PBS and detected with AF488-conjugated αIgG secondary (25 μg/mL). The human IgG1 isotype control (L) was also detected with AF488-conjugated αIgG secondary. Graphs depict the mean ± SEM of the relative fluorescence signal (RFU). Glycan structures corresponding to glycan numbers can be found in Supporting Information, Table S1. Controls for sialic acid dependence were assessed with corresponding conserved Arg mutants of each Siglec in Supporting Information, Figure S1. (The glycan array MIRAGE data for Figure S1 are given in the Supporting Information, Table S4).
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