Chondroitin 4-sulphotransferase-1 and chondroitin 6-sulphotransferase-1 are affected differently by uronic acid residues neighbouring the acceptor GalNAc residues

Abstract

C4ST-1 (chondroitin 4-sulphotransferase-1) and C6ST-1 (chondroitin 6-sulphotransferase-1) transfer sulphate from PAPS (adenosine 3'-phosphate 5'-phosphosulphate) to positions 4 and 6 respectively of the GalNAc residues of chondroitin. We showed previously that C4ST-1 purified from rat chondrosarcoma and recombinant C4ST-1 both transfer sulphate efficiently to position 4 of the GalNAc residues of DSDS (desulphated dermatan sulphate). We report here the specificity of C4ST-1 and C6ST-1 in terms of uronic acid residue recognition around the GalNAc residue to which sulphate is transferred. When [35S]glycosaminoglycans formed from DSDS after incubation with [35S]PAPS and C4ST-1 were digested with chondroitinase ACII, a major part of the radioactivity was recovered in disaccharide fractions and the remainder distributed to tetrasaccharides and larger fractions, indicating that C4ST-1 mainly transferred sulphate to position 4 of the GalNAc residue located at the GlcA-GalNAc-GlcA sequence. Structural analysis of tetrasaccharide and larger oligosaccharide fractions indicated that C4ST-1 mainly transferred sulphate to the GalNAc residue adjacent to the reducing side of the GlcA residue. On the other hand, when [35S]glycosaminoglycans formed from DSDS after incubation with [35S]PAPS and C6ST-1 were digested with chondroitinase ACII, a major part of the radioactivity was recovered in fractions larger than hexasaccharides, indicating that C6ST-1 transferred sulphate to the GalNAc residues located in the L-iduronic acid-rich region. Structural analysis of the tetrasaccharide and larger oligosaccharide fractions indicated that C6ST-1 showed very little preference for the GalNAc residue neighbouring the GlcA residue. These results indicate that C4ST-1 and C6ST-1 differ from each other in the recognition of uronic acid residues adjacent to the targeted GalNAc residue.

Figure 1. Digestion of DS and DSDS with chondroitinase ABC or ACII

DS (A) and DSDS (B, C) were digested with chondroitinase ABC (A, B) or ACII (C). The digested materials were separated with a SAX-HPLC. The column was monitored at 232 nm. The arrows indicate the elution position of ΔDi-0S (1), ΔDi-6S (2), ΔDi-2S (3), ΔDi-4S (4), ΔDi-diS_D (5), ΔDi-diS_B (6) and ΔDi-diS_E (7).

Figure 2. Western-blot analysis of the affinity-purified C4ST-1 and C6ST-1

The FLAG–C4ST-1 and FLAG–C6ST-1 fusion proteins were extracted from COS-7 cells that were transfected with the respective cDNA and purified with an anti-FLAG monoclonal antibody-conjugated column as described under the Experimental section. The affinity-purified C4ST-1 (lanes 1 and 3) and C6ST-1 (lanes 2 and 4) were detected with anti-FLAG antibody before (lanes 1 and 2) or after (lanes 3 and 4) the N-glycosidase F digestion. Molecular-mass standards were as follows: myosin, 205 kDa; β-galactosidase, 116 kDa; phosphorylase b, 97 kDa; BSA, 66 kDa; egg albumin, 45 kDa; carbonic anhydrase, 29 kDa.

Figure 3. Superdex 30 chromatography of chondroitinase ACII or ABC digests of ³⁵S-labelled glycosaminoglycans derived from DSDS by incubation with [³⁵S]PAPS and the affinity-purified C4ST-1 or C6ST-1

The sulphotransferase reaction was carried out as described under the Experimental section. The ³⁵S-labelled glycosaminoglycans formed from DSDS after the reaction with C4ST-1 (A–C) or C6ST-1 (D–F) were applied to the Superdex 30 column before (A, D) or after digestion with chondroitinase ABC (B, E) or ACII (C, F). The arrows indicate the elution position of Blue Dextran (Vo), ΔDi-4S (Di), CS tetrasaccharide (Tet), chondroitin tetrasaccharide (Ch-Tet), CS hexasaccharide (Hex), CS octasaccharide (Oct), CS decasaccharide (Dec), CS dodecasaccharide (12) and CS tetradecasaccharide (14).

Figure 4. Separation of tetrasaccharide fractions by SAX-HPLC

The C4-tetra (A) and C6-tetra (B) fractions shown in Figures 3(C) and 3(F) respectively were pooled separately, freeze-dried and applied to SAX-HPLC as described under the Experimental section. The broken line depicts the concentration of KH₂PO₄. The arrows indicate the elution position of ΔDi-0S (1), GalNAc(6SO₄) (2), GalNAc(4SO₄) (3), ΔDi-6S (4), ΔDi-4S (5), GalNAc(4,6-SO₄) (6), ΔDi-diS_D (7) and ΔDi-diS_E (8). The elution positions of Tet-40, Tet-44, Tet-60 and Tet-64 are indicated.

Figure 5. Chondroitinase ABC digestion of the trisaccharides derived from C4-tetra after mercuric acetate treatment

The trisaccharides derived from peak a (A, B) or peak b (C, D) shown in Figure 4(A) were separated with SAX-HPLC before (A, C) or after (B, D) digestion with chondroitinase ABC. The standards were the same as those described in the legend to Figure 4. The elution positions of Tri-40 and Tri-44 are indicated.

Scheme 1. Structural analysis of monosulphated tetrasaccharides formed after chondroitinase ACII digestion

³⁵S-labelled glycosaminoglycans formed from DSDS after the reaction with C4ST-1 (A) or C6ST-1 (B) were digested with chondroitinase ACII. The monosulphated tetrasaccharide fractions (peak a in Figure 4A and peak c in Figure 4B) were purified and treated with mercuric acetate. The resulting trisaccharides (peak e in Figure 5A and peak g in Figure 6A) were digested with chondroitinase ABC. GalNAc(4SO₄) was obtained from the non-reducing terminal of peak e, and ΔDi-6S was obtained from the reducing terminal of peak g.

Figure 6. Chondroitinase ABC digestion of the trisaccharides derived from C6-tetra after mercuric acetate treatment

The trisaccharides derived from peak c (A, B) or peak d (C, D) shown in Figure 4(B) were separated with SAX-HPLC before (A, C) or after (B, D) digestion with chondroitinase ABC. The standards were the same as those described in the legend to Figure 4. The elution positions of Tri-60 and Tri-64 are indicated.

Figure 7. β-Glucuronidase digestion of a disaccharide (Di-X) derived from peak g after β-N-acetylhexosaminidase digestion

The monosulphated trisaccharide (peak g in Figure 6A) was digested with β-N-acetylhexosaminidase and separated by Superdex 30 chromatography. The resulting disaccharide (Di-X) was mixed with Di-6S and separated with SAX-HPLC before (B, D) or after (C, E) digestion with β-glucuronidase. The column was eluted with 40 mM KH₂PO₄ isocratically and monitored by the absorption at 210 nm (A–C) and the ³⁵S-radioactivity (D, E). The broken line depicts the concentration of KH₂PO₄. The elution profile of standard GalNAc(6SO₄) (1) and Di-6S (2) is indicated in (A). A large background before 5 min and a small peak at approx. 17 min in (C) are derived from the β-glucuronidase preparation.

Figure 8. Chondroitinase ABC digestion of the oligosaccharides derived from C4- or C6-oligo after mercuric acetate treatment

Oligosaccharides derived from C4-oligo (A) and C6-oligo (B) after mercuric acetate treatment were separated with SAX-HPLC after digestion with chondroitinase ABC. The standards were the same as those described in the legend to Figure 4.