Tetrasulfated disaccharide unit in heparan sulfate: enzymatic formation and tissue distribution

Abstract

We previously reported that the heparan sulfate 3-O-sulfotransferase (3OST)-5 produces a novel component of heparan sulfate, i.e. the tetrasulfated disaccharide (Di-tetraS) unit ( Mochizuki, H., Yoshida, K., Gotoh, M., Sugioka, S., Kikuchi, N., Kwon, Y.-D., Tawada, A., Maeyama, K., Inaba, N., Hiruma, T., Kimata, K., and Narimatsu, H. (2003) J. Biol. Chem. 278, 26780-26787 ). In the present study, we investigated the potential of other 3OST isoforms to produce Di-tetraS with heparan sulfate and heparin as acceptor substrates. 3OST-2, 3OST-3, and 3OST-4 produce Di-tetraS units as a major product from both substrates. 3OST-5 showed the same specificity for heparin, but the production from heparan sulfate was very low. Di-tetraS production by 3OST-1 was negligible. We then investigated the presence of Di-tetraS units in heparan sulfates from various rat tissues. Di-tetraS was detected in all of the tissues analyzed. Liver and spleen contain relatively high levels of Di-tetraS, 1.6 and 0.95%, respectively. However, the content of this unit in heart, large intestine, ileum, and lung is low, less than 0.2%. We further determined the expression levels of 3OST transcripts by quantitative real time PCR. The 3OST-3 transcripts are highly expressed in spleen and liver. The 3OST-2 and -4 are specifically expressed in brain. These results indicate that the Di-tetraS unit is widely distributed throughout the body as a rare and unique component of heparan sulfate and is synthesized by tissue-specific 3OST isoforms specific for Di-tetraS production.

FIGURE 1.

Disaccharide units of heparan sulfate. Twelve sulfation patterns of disaccharide units are illustrated. The abbreviations are shown under the substance.

FIGURE 2.

Western blot analysis of recombinant 3OST isoforms. The FLAG-tagged 3OST proteins were expressed in insect cells, purified with an anti-FLAG agarose gel, and subjected to Western blot analysis as described under “Experimental Procedures.” Lanes 1-5 are 3OST-1, 3OST-2, 3OST-3, 3OST-4, and 3OST-5, respectively. The positions of molecular size standards (in kDa) are indicated on the left.

FIGURE 3.

HPLC analysis of ³⁵S-labeled products derived from heparan sulfate. Heparan sulfate was incubated with [³⁵S]PAPS and one of the recombinant 3OST isoforms. The ³⁵S-labeled products were digested with a mixture of heparin lyases, and then the digests were separated by HPLC on a SAX column as described under “Experimental Procedures.” The fractions were collected, and the radioactivity was determined by liquid scintillation counting. The five panels (A-E), show the elution profiles of radiolabeled products derived from the reactions of 3OST-1, 3OST-2, 3OST-3, 3OST-4, and 3OST-5, respectively. The five peaks of panel E have been identified as ΔTetra-1 (peak 1), ΔDi-(N,3,6)S (peak 2), ΔTetra-2 (peak 3), ΔDi-(2,N,3)S (peak 4), and ΔDi-tetraS (peak 5) in a previous study (27). These positions are also indicated above the figure. For the abbreviations, see Fig. 1.

FIGURE 4.

HPLC analysis of ³⁵S-labeled products derived from heparin. Heparin was incubated with [³⁵S]PAPS and one of the recombinant 3OST isoforms. The ³⁵S-labeled products were digested with a mixture of heparin lyases and analyzed by HPLC on a SAX column as described under “Experimental Procedures.” The five panels(A-E) show the elution profile of radio labeled products derived from the reactions of 3OST-1, 3OST-2, 3OST-3, 3OST-4, and 3OST-5, respectively. The arrows indicate the elution positions of ΔTetra-1 (peak 1), ΔDi-(N,3,6)S (peak 2), ΔTetra-2 (peak 3), ΔDi-(2,N,3)S (peak 4), and ΔDi-tetraS (peak 5). For the abbreviations, see Fig. 1.

FIGURE 5.

A comparison of ³⁵S-labeled products derived from heparan sulfate and heparin by the reaction of 3OST isoforms. Percentages of radiolabeled products were calculated from Figs. 3 and 4. A, compositions of radiolabeled products derived from heparan sulfate by the reactions of 3OST isoforms, as indicated on the left. B, compositions of radiolabeled products derived from heparin by the reactions of 3OST isoforms.

FIGURE 6.

Typical chromatograms of unsaturated disaccharides prepared from rat tissue heparan sulfate. A, heparan sulfate isolated from rat liver was digested with a mixture of heparin lyases, and the resultant disaccharides were subjected to reverse-phase ion pair chromatography with post-column fluorescence labeling as described under “Experimental Procedures.” B, nine disaccharide standards are ΔDi-0S (peak 1), ΔDi-NS (peak 2), ΔDi-6S (peak 3), ΔDi-2S (peak 4), ΔDi-(N,6)S (peak 5), ΔDi-(2,N)S (peak 6), ΔDi-(2,6)S (peak 7), ΔDi-(2,N,6)S (peak 8), and ΔDi-tetraS (peak 9). For the abbreviations, see Fig. 1.

FIGURE 7.

Quantitative analysis of 3OST isoform transcripts in human tissues using real time PCR. Standard curves for 3OST isoforms and GAPDH were generated by serial dilution of plasmid DNA or control DNA as described under “Experimental Procedures.” The expression levels of 3OST isoforms were normalized to that of the GAPDH transcript, which was measured in the same cDNAs. The data were obtained from triplicate experiments and given as the means ± S.D.

FIGURE 8.

Analysis of heparan sulfate and heparin sulfated by excess amounts of 3OST-3. Ten micrograms of heparan sulfate or heparin were incubated with nonradioactive PAPS and an excess amount of 3OST-3 in the standard reaction mixture and digested with a mixture of heparin lyases, as described under “Experimental Procedures.” The resultant disaccharides were subjected to reverse-phase ion pair chromatography with post-column fluorescence labeling. A and C show the analysis of heparan sulfate and heparin, without sulfation reaction, respectively. B and D show the analysis of reaction products from heparan sulfate and heparin, respectively. The elution positions of standard disaccharides are indicated by numbers; see legend to Fig. 6.