Emerging chemical and biochemical tools for studying 3- O-sulfated heparan sulfate

Abstract

Heparan sulfate is a widely expressed polysaccharide in the extracellular matrix and on the cell surface. 3-O-sulfated heparan sulfate represents only a small percentage of heparan sulfate from biological sources. However, this subpopulation is closely associated with biological functions of heparan sulfate. The 3-O-sulfated heparan sulfate is biosynthesized by heparan sulfate 3-O-sulfotransferase, which exists in seven different isoforms. This review article summarizes the recent progress in the substrate specificity studies of different 3-O-sulfotransferase isoforms involving the use of homogeneous oligosaccharide substrates and crystal structural analysis. The article also reviews a newly developed liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method to analyze the level of 3-O-sulfated heparan sulfate with high sensitivity and quantitative capability. This newly emerged technology will provide new tools to study the structure and function relationship of heparan sulfate.

Keywords: glycosaminoglycans; heparan sulfate; heparin; sulfotransferases.

Figure 1.

Substrate specificity of 3-OST isoforms. The site of 3-O-sulfation is highlighted with a blue box. The abbreviated trisaccharide sequences are listed underneath of each saccharide structures. The 3-O-sulfation in the abbreviated trisaccharide is emphasized in both bold face and underlined. 3-OST, 3-O-sulfotransferase.

Figure 2.

Crystal structures of 3-O-sulfotransferase (3-OST) isoforms-1, -3, and -5. A: superpositions of the crystal structures of the catalytic domains for 3-OST-1 (pink, substrate dark pink; PDBcode 3UAN), -3 (gray, substrate green; pdbcode 6XL8), and -5 (light yellow, substrate cyan; pdbcode 7SCE). Positions of saccharides relative to the acceptor glucosamine (0) are labeled (+) for nonreducing end and (−) for the reducing end of the substrate. Black arrows denote largest structural differences between isoforms near the HS binding cleft. The PAP for all three structures is shown in tan and superimpose well. B: the shorthand representations of the central pentasaccharide unit of the oligosaccharide substrates used in the crystal studies are shown. C: conformations of GlcA and IdoA2S found in heparan sulfate/heparin. GlcA, glucuronic acid; HS, heparan sulfate; IdoA2S, 2-O-sulfo iduronic acid; PAP, 3′-phosphoadenosine 5′-phosphate.

Figure 3.

Differences in HS binding between the 3-O-sulfotransferase (3-OST) isoforms -1, -3, and -5. A: Crystal structure of the catalytic domain of 3-OST-3 binding oligosaccharide substrate (protein gray, HS green; pdbcode 6XL8). B: superposition of crystal structures of 3-OST-1 (pink, substrate dark pink; PDBcode 3UAN) and -5 (light yellow, substrate cyan; pdbcode 7SCE). Residues discussed in text are displayed in stick. Red arrows denote the acceptor 3-OH on glucosamine 0. Pink, black, and yellow dashed lines represent interactions between HS and 3-OST-1, -3, and -5, respectively. Solid black lines represent interactions in 3-OST-3 with the sodium ion. Shorthand representation of the substrates in the active site are shown at the bottom of the figure. HS, heparan sulfate.

Figure 4.

Binding by a 6-O-sulfated oligosaccharide substrate to 3-OST-3. A: superposition of 3-OST-3 binding HS without 6-O-sulfo groups (protein gray, HS green; pdbcode 6XL8) with 3-OST-3 binding 6-O-sulfo containing HS in a productive binding mode consistent with catalysis (protein light blue, HS light purple; PDB 6XKG molecule B). Red arrow denotes acceptor 3OH position. B: superposition of the productive binding mode of HS with 6-O-sulfo group (protein light blue, HS light purple/transparent; PDB 6XKG 3-OST-3 molecule B) with 3-OST-3 binding the same 8-mer oligosaccharide in a nonproductive manner (protein and HS in tan; PDB 6XKG 3-OST-3 molecule A). The red asterisk is positioned at the 3OH that would typically be found bound at the active site near the PAP as in A. The structure of the octasaccharide substrate with 6-O-sulfation used in the crystal study is shown at the bottom of the figure. HS, heparan sulfate; PAP, 3′-phosphoadenosine 5′-phosphate; 3-OST, 3-O-sulfotransferase.

Figure 5.

Analysis of 3-O-sulfated HS using a LC-MS/MS method. The HS polysaccharides from biological sources are mixed with known amounts of ¹³C-labeled 8-mer calibrants. The mixture is subjected to the digestion with heparin lyases. The 3-O-sulfated oligosaccharides, presented as Tetra-1, Tetra-2, and Di3S are liberated along with non-3-O-sulfated disaccharides. The products are then analyzed by LC-MS/MS. Comparing the ratio of peak area ¹³C-labeled Tetra-1 and unlabeled Tetra, one can determine the amount of Tetra-1 from the HS polysaccharides. Red dots denote the location of the ¹³C-atoms. HS, heparan sulfate; LC-MS/MS, liquid chromatography coupled with tandem mass spectrometry.