Influence of saccharide modifications on heparin lyase III substrate specificities

Abstract

A library of 23 synthetic heparan sulfate (HS) oligosaccharides, varying in chain length, types, and positions of modifications, was used to analyze the substrate specificities of heparin lyase III enzymes from both Flavobacterium heparinum and Bacteroides eggerthii. The influence of specific modifications, including N-substitution, 2-O sulfation, 6-O sulfation, and 3-O sulfation on lyase III digestion was examined systematically. It was demonstrated that lyase III from both sources can completely digest oligosaccharides lacking O-sulfates. 2-O Sulfation completely blocked cleavage at the corresponding site; 6-O and 3-O sulfation on glucosamine residues inhibited enzyme activity. We also observed that there are differences in substrate specificities between the two lyase III enzymes for highly sulfated oligosaccharides. These findings will facilitate obtaining and analyzing the functional sulfated domains from large HS polymer, to better understand their structure/function relationships in biological processes.

Keywords: glycomics; glycosaminoglycans; heparan sulfate; heparin lyase; mass spectrometry.

Fig. 1

Lyase3B digestion of GT1–6. (A–F) Total ion chromatograms, TICs; (G) extracted mass spectrum of GT5 at 27.5 min; (H) extracted mass spectrum of GT5 at 31.5 min; (I) extracted mass spectrum of GT6 at 27.5 min. Peaks between m/z 750 and 800 in G and I correspond to dimerized D0A0 and G0A0, respectively. (A) GT1, A0G0A0G0-PNP; (B) GT2, G0A0G0A0G0-PNP; (C) GT3, A0G0A0G0A0G0-PNP; (D) GT4, G0A0G0A0G0A0G0-PNP; (E) GT5, A0G0A0G0A0G0A0G0-PNP; (F) GT6, G0A0G0A0G0A0G0A0G0-PNP. The y-axis on all panels is relative ion abundance. This figure is available in black and white in print and in colour at Glycobiology online.

Fig. 2

Lyase3B digestion of GT7–12. (A–F) TICs; (G) extracted mass spectrum of GT11 at 25.8 min; (H) extracted mass spectrum of GT11 at 31.5 min; (I) extracted mass spectrum of GT12 at 25.8 min. (A) GT7, S0G0S0G0-PNP; (B) GT8, G0S0G0S0G0-PNP; (C) GT9, S0G0S0G0S0G0-PNP; (D) GT10, G0S0G0S0G0S0G0-PNP; (E) GT11, S0G0S0G0S0G0S0G0-PNP; (F) GT12, G0S0G0S0G0S0G0S0G0-PNP. The y-axis on all panels is relative ion abundance. This figure is available in black and white in print and in colour at Glycobiology online.

Fig. 3

Lyase3B digestion of GT12-16. (A–E) TICs; (F) extracted mass spectrum of GT13 at 25.6 min; (G) extracted mass spectrum of GT14 at 25.6 min; (H) extracted mass spectrum of GT13 at 24.3 min. dp, degree of polymerization. (A) GT12, G0S0G0S0G0S0G0S0G0-PNP; (B) GT13, G0S0G0S0G0S0G0S6G0-PNP; (C) GT14, G0S0G0S0G0S6G0S6G0-PNP; (D) GT15, G0S0G0S6G0S6G0S6G0-PNP; (E) GT16, G0S6G0S6G0S6G0S6G0-PNP. The y-axis on all panels is relative ion abundance. This figure is available in black and white in print and in colour at Glycobiology online.

Fig. 4

Lyase3F digestion of hexasaccharides B10, B12 and B16. (A–C) TICs; (D) extracted mass spectrum of B10 at 25.3 min; (E) extracted mass spectrum of B10 at 26.0 min; (F) extracted mass spectrum of B12 at 22.7 min; (G) extracted mass spectrum of B16 at 23.4 min. (A) B10, G0S6I0S6I0S6-R; (B) B12, G0S6I2S6I0S6-R; (C) B16, G0S6I0S6I2S6-R. The y-axis on all panels is relative ion abundance. This figure is available in black and white in print and in colour at Glycobiology online.

Fig. 5

Comparison of enzymatic digestion of hexasaccharide B7 using lyase3B and lyase3F. (A) TIC of lyase3B digestion; (B) TIC of lyase3F digestion; (C) extracted mass spectrum of A at 22.7 min; (D) extracted mass spectrum of A at 26.2 min; (E) extracted mass spectrum of B at 21.8 min. Hex, intact hexasaccharide. B7, G0S6G0S9I0S6-R. The y-axis on all panels is relative ion abundance. Panels A, C, and D were modified from our previous publication (Chopra et al. 2021). This figure is available in black and white in print and in colour at Glycobiology online.

Fig. 6

Summary of the activities of lyase3B and lyase3F against synthetic heparin saccharides. (A) Unsulfated saccharides were digested completely to disaccharides by both enzymes (Figure 1). (B) Saccharides with N-sulfated glucosamine residues were digested completely to disaccharide by both enzymes (Figure 2). (C) Nonasaccharides with N-sulfated glucosamine residues and up to three 6-O-sulfate groups at the RE were digested differently by both enzymes. X, Y, Z = H, H, SO₃H or H, SO₃H, SO₃H or SO₃H, SO₃H, SO₃H (Figure 3 and Supplementary Figure S8). The short arrow indicated that the sites were less preferable. (D) Nonasaccharides with N-sulfated glucosamine residues and four 6-O-sulfate groups were completely digested by lyase3B but largely remains intact after the treatment of lyase3F (Figure 3 and Supplementary Figure S8). (E) Highly sulfated hexasaccharides show resistance to both lyases depending on the position of 2-O-sulfation (Figure 4). (F) A highly sulfated hexasaccharide was cleaved at the RE side of GlcNS3S6S by lyase3B but not by lyase3F (Figure 5). This figure is available in black and white in print and in colour at Glycobiology online.

Scheme 1

A flowchart showing the data annotation using GT1 as a representation. The products and ions detected are indicated with solid outline.