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. 2021 Jun 11;12(1):3573.
doi: 10.1038/s41467-021-23428-x.

Chemoenzymatic modular assembly of O-GalNAc glycans for functional glycomics

Shuaishuai Wang #  1 , Congcong Chen #  1   2   3 , Madhusudhan Reddy Gadi #  1 , Varma Saikam #  1 , Ding Liu  1 , He Zhu  1 , Roni Bollag  4 , Kebin Liu  5 , Xi Chen  6 , Fengshan Wang  7 , Peng George Wang  8   9 , Peixue Ling  10   11   12 , Wanyi Guan  13 , Lei Li  14
Affiliations

Chemoenzymatic modular assembly of O-GalNAc glycans for functional glycomics

Shuaishuai Wang et al. Nat Commun. .

Abstract

O-GalNAc glycans (or mucin O-glycans) play pivotal roles in diverse biological and pathological processes, including tumor growth and progression. Structurally defined O-GalNAc glycans are essential for functional studies but synthetic challenges and their inherent structural diversity and complexity have limited access to these compounds. Herein, we report an efficient and robust chemoenzymatic modular assembly (CEMA) strategy to construct structurally diverse O-GalNAc glycans. The key to this strategy is the convergent assembly of O-GalNAc cores 1-4 and 6 from three chemical building blocks, followed by enzymatic diversification of the cores by 13 well-tailored enzyme modules. A total of 83 O-GalNAc glycans presenting various natural glycan epitopes are obtained and used to generate a unique synthetic mucin O-glycan microarray. Binding specificities of glycan-binding proteins (GBPs) including plant lectins and selected anti-glycan antibodies towards these O-GalNAc glycans are revealed by this microarray, promoting their applicability in functional O-glycomics. Serum samples from colorectal cancer patients and healthy controls are assayed using the array reveal higher bindings towards less common cores 3, 4, and 6 than abundant cores 1 and 2, providing insights into O-GalNAc glycan structure-activity relationships.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Structures of O-GalNAc glycan cores and their common glycan epitope extensions.
a O-GalNAc glycan cores 1–8. b Common glycan epitopes found on O-GalNAc glycans. LN, N-acetyllactosamine (LacNAc); LDN, N,N’-diacetyllactosamine (LacdiNAc); 3SLN, 3’-sialyl LacNAc; 6SLN, 6’-sialyl LacNAc; LeX, Lewis X; SLeX, sialyl-Lewis X; LeY, Lewis Y. Abbreviations: Gal, galactose; Fuc, L-fucose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; Neu5Ac, N-acetylneuraminic acid; Ser, serine; Thr, threonine.
Fig. 2
Fig. 2. Retrosynthetic analysis of O-GalNAc glycans.
a Retrosynthetic analysis of O-GalNAc cores with three synthetic building blocks. b Retrosynthetic analysis of O-GalNAc epitopes with 13 enzyme modules. Enzyme modules: G stands for galactosylation, S stands for sialylation, N stands for N-acetylhexosaminylation, F stands for fucosylation. G1: β1-4 galactosylation with Neisseria meningitidis β1-4 galactosyltransferase (NmLgtB) and donor uridine 5′-diphosphogalactose (UDP-Gal); G2: α1–3 galactosylation with human GTB and UDP-Gal; G3: α1-3 galactosylation with bovine α1-3 GalT (Bα3GalT) and UDP-Gal; S1: α2-3 sialylation with Pasteurella multocida α2-3 sialyltransferase 1 mutant M144D (PmST1-M144D), N. meningitidis CMP-sialic acid synthetase (NmCSS), cytidine 5′-triphosphate (CTP), and N-acetylneuraminic acid (Neu5Ac); S2: α2-6 sialylation with PmST1-P34H/M144L, NmCSS, CTP, and Neu5Ac; S3: α2–6 sialylation with Photobacterium damselae α2–6 sialyltransferase (Pd2,6ST), NmCSS, CTP, and Neu5Ac; S4: α2-6 sialylation with human ST6GalNAc-IV, NmCSS, CTP, and Neu5Ac; N1: β1-4 N-acetylgalactosaminylation with Campylobacter jejuni β1-4 N-acetylgalatosaminyltransferase (CjCgtA) and uridine 5′-diphosphate-N-acetylgalactosamine (UDP-GalNAc); N2: α1-3 N-acetylgalactosaminylation with Helicobacter mustelae α1-3 N-acetyl-galactosaminyltransferase (HmBgtA) and UDP-GalNAc; N3: β1-3 N-acetylglucosaminylation with Helicobacter pylori β1-3 N-acetylglucosaminyltransferase (HpLgtA) and uridine 5’-diphosphate-N-acetylglucosamine (UDP-GlcNAc); N4: β1-4 N-acetylgalactosaminylation with b4GalT-Y289L/C342T (b4GalTm) and UDP-GalNAc; F1: α1-2 fucosylation with H. mustelae α1-2 fucosyltransferase (Hm2FT) and guanosine 5′-diphospho-L-fucose (GDP-Fuc); F2: α1-3 fucosylation with H. pylori α1-3 fucosyltransferase C-terminal 66 amino acid truncation (Hp3FT) and GDP-Fuc. SF, Fmoc protected Ser.
Fig. 3
Fig. 3. Chemical modular assembly of O-GalNAc cores 1–4 and 6 structures.
Reagents and conditions: a NaOMe, MeOH. b TMSOTf, CH2Cl2, −78 °C. c p-toluenesulfonic acid, MeOH. d TMSOTf, CH2Cl2, -40 °C. e i. Zn, AcOH, CH2Cl2; ii. Py, Ac2O. f i. TFA, CH2Cl2; ii. NaOMe, MeOH. g FmocOSu, NaHCO3.
Fig. 4
Fig. 4. Enzymatic modular synthesis of core 1 O-GalNAc glycans.
G1: β1-4 galactosylation with NmLgtB and UDP-Gal; G2: α1-3 galactosylation with human GTB and UDP-Gal; G3: α1-3 galactosylation with Bα3GalT and UDP-Gal; S1: α2-3 sialylation with PmST1-M144D, NmCSS, CTP, and Neu5Ac; S2: α2-6 sialylation with PmST1-P34H/M144L, NmCSS, CTP, and Neu5Ac; S3: α2-6 sialylation with Pd2,6ST, NmCSS, CTP, and Neu5Ac; S4: α2-6 sialylation with human ST6GalNAc-IV, NmCSS, CTP, and Neu5Ac; N1: β1-4 N-acetylgalactosaminylation with CjCgtA and UDP-GalNAc; N2: α1-3 N-acetylgalactosaminylation with HmBgtA and UDP-GalNAc; N3: β1-3 N-acetylglucosaminylation with HpLgtA and UDP-GlcNAc; F1: α1-2 fucosylation with Hm2FT and GDP-Fuc; F2: α1-3 fucosylation with Hp3FT and GDP-Fuc. SF, Fmoc protected Ser.
Fig. 5
Fig. 5. Enzymatic modular synthesis of core 2 O-GalNAc glycans.
G1: β1-4 galactosylation with NmLgtB and UDP-Gal; G3: α1-3 galactosylation with Bα3GalT and UDP-Gal; S1: α2-3 sialylation with PmST1-M144D, NmCSS, CTP, and Neu5Ac; S3: α2-6 sialylation with Pd2,6ST, NmCSS, CTP, and Neu5Ac; N1: β1-4 N-acetylgalactosaminylation with CjCgtA and UDP-GalNAc; F1: α1-2 fucosylation with Hm2FT and GDP-Fuc; F2: α1-3 fucosylation with Hp3FT and GDP-Fuc. SF, Fmoc protected Ser.
Fig. 6
Fig. 6. Binding profiles of lectins towards O-GalNAc glycans.
a The binding profile of STL towards O-GalNAc glycans. b The binding profile of PNA towards O-GalNAc glycans. c The binding profile of Jacalin towards O-GalNAc glycans. The x-axis shows glycans, and the y-axis shows relative fluorescence readout using Cy5-streptavidin (1 μg/mL). PPA = APGS(GalNAcα-)TAPP (100 µM); RPAP = TSAPD(GalNAcα-)TRPAP (100 µM); x-T, Thr-linked counterparts of Ser-linked O-glycans; Biotin = biotinylated PEG amine (0.01 mg/mL); hIgG = human IgG (0.1 mg/mL), mIgG = mouse IgG (0.1 mg/mL); M = Marker (0.01 mg/mL Cy3-conjugated anti-Human IgG + 0.01 mg/mL Alexs647-conjugated anti-Human IgG); NC = printing buffer negative control. n = 3 independent replicates. The individual data points are shown as dots. Data are presented as mean values. Error bars represent standard deviation. Source data are provided as a Source Data file.
Fig. 7
Fig. 7. Heatmap of lgM bindings on the O-GalNAc glycan microarray in sera from colorectal cancer patients and healthy control people.
PPA = APGS(GalNAcα-)TAPP (100 µM); RPAP = TSAPD(GalNAcα-)TRPAP (100 µM); x-T, Thr-linked counterparts of Ser-linked O-glycans; Biotin = biotinylated PEG amine (0.01 mg/mL); NC = printing buffer negative control. n = 3 independent replicates. Data are presented as mean values. Source data are provided as a Source Data file.
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