Overexpression of Different Isoforms of Glucosaminyl N-Deacetylase/N-Sulfotransferase Results in Distinct Heparan Sulfate N-Sulfation Patterns

Abstract

Functional interactions of heparan sulfate (HS) with selected proteins depend on distinct saccharide sequences which are generated during biosynthesis of the polysaccharide. Glucosaminyl N-deacetylase/N-sulfotransferases (NDSTs) catalyze both the N-deacetylation and N-sulfation reactions that initiate the modification of the (GlcNAc-GlcA)(n) polysaccharide backbone. The N-acetyl/N-sulfate exchange is restricted to certain regions of the polysaccharide chains, and only these can be further modified by glucuronyl C5-epimerization and O-sulfation at various positions. To investigate whether NDST isoforms influenced differently the structure of HS, murine NDST-1 was overexpressed in human kidney 293 cells, and the structure of the HS produced was compared to HS from NDST-2 overexpressing cells [Cheung, W. F., Eriksson, I., Kusche-Gullberg M., Lindahl, U., and Kjellén, L. (1996) Biochemistry 35, 5250-5256]. The level of N-sulfation increased from 40% in control cells to 60% and 80%, respectively, in NDST-1 and NDST-2 transfected cells. Interestingly, the increase in N-sulfation was accompanied by an increased chain length, while no effect on IdoA content or O-sulfation was seen. The most extended N-sulfated domains were found in HS synthesized by NDST-2 transfected cells. Since both the N-deacetylase and the N-sulfotransferase activities were lower in these cells than in the NDST-1 overexpressing cells, we conclude that, in addition to the level of enzyme expression, the NDST isoform also is important in determining the N-sulfation pattern in HS.

Endothelial inflammation: the role of differential expression of N-deacetylase/N-sulphotransferase enzymes in alteration of the immunological properties of heparan sulphate

“…Previous studies have shown that overexpression of NDSTs can result in the production of HS with increased N-and O-sulphation (Cheung et al, 1996;Pikas et al, 2000), suggesting a direct correlation between NDST expression and the level of HS sulphation. It is possible that these changes are not limited to HS; indeed, a recent study has shown that oversulphation of HS in cells which express high levels of NDST-1 is associated with decreased sulphation of chondroitin sulphate (Bengtsson, 2003).…”

Overexpression of Heparan Sulfate 6-O-Sulfotransferases in Human Embryonic Kidney 293 Cells Results in Increased N-Acetylglucosaminyl 6-O-Sulfation

“…It is tempting to speculate that 6-O-sulfation of different target sequences would be catalyzed by different 6-OST isoforms. Because expression of other HS biosynthetic enzymes (HS 2-OST (26) and HS NDST1 and 2 (32,33)) in HEK 293 cells has given important information regarding their roles in HS biosynthesis we decided to express the three murine isoforms of 6-OST in the same system. The structural changes induced by overexpression of 6-OSTs in HEK 293 cells were distinctive but not isoformspecific.…”

Heparan sulfate biosynthesis enzymes EXT1 and EXT2 affect NDST1 expression and heparan sulfate sulfation

“…EXT1 was cloned into the MCS with the cytomegalovirus (CMV) immediate-early promotor and EXT2 into the MCS with the human elongation factor 1␣ (EF1␣) promotor. The three constructs and empty pBudCE4.1 vector alone were transfected into human embryonic kidney (HEK) 293 cells stably expressing NDST1 from the pCDNA3 vector (25), by using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Stable clones were selected by using 0.4 mg/ml Zeocin and 0.8 mg/ml G-418 and were grown in DMEM ϩ L-glutamine, containing 10% FCS, 2.5 units/ml fungizone, and 100 units/ml per 100 g/ml penicillin/streptomycin.…”