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

Abstract

Heparan sulfate (HS) interacts with a variety of proteins and thus mediates numerous complex biological processes. These interactions critically depend on the patterns of O-sulfate groups within the HS chains that determine binding sites for proteins. In particular the distribution of 6-O-sulfated glucosamine residues influences binding and activity of HS-dependent signaling molecules. The protein binding domains of HS show large structural variability, potentially because of differential expression patterns of HS biosynthetic enzymes along with differences in substrate specificity. To investigate whether different isoforms of HS glucosaminyl 6-O-sulfotransferase (6-OST) give rise to differently sulfated domains, we have introduced mouse 6-OST1, 6-OST2, and 6-OST3 in human embryonic kidney 293 cells and compared the effects of overexpression on HS structure. High expression of any one of the 6-OST enzymes resulted in appreciably increased 6-O-sulfation of N-sulfated as well as N-acetylated glucosamine units. The increased 6-O-sulfation was accompanied by a decrease in nonsulfated as well as in iduronic acid 2-O-sulfated disaccharide structures. Furthermore, overexpression led to an altered HS domain structure, the most striking effect was the formation of extended 6-O-sulfated predominantly N-acetylated HS domains. Although the effect was most noticeable in 6-OST3-expressing cells, these results were largely independent of the particular 6-OST isoform expressed and mainly influenced by the level of overexpression.

Structural alteration of cell surface heparan sulfate through the stimulation of the signaling pathway for heparan sulfate 6-O-sulfotransferase-1 in mouse fibroblast cells

“…This suggestion was supported by previous result that overexpression of 6-OST-1 increased the percentage composition of ΔHexUA-GlcNAc6S. 40) In conclusion, the present study shows the artificial control of sulfated structure of cell-surface HS by identifying the signaling pathway for 6-OST-1 for the first time. We found that L-M cells synthesized more 6-O-sulfated disaccharide units in HS in response to adrenaline.…”

Heparan sulfate 6-O-endosulfatases: discrete in vivo activities and functional co-operativity

“…This differential activity of mSulf1 and mSulf2 in the mSulf knock-out MEFs may have important biological implications. Indeed, it is important to note that although no mouse 6-Osulfotransferase knock-outs have yet been published, in vitro experiments suggest that individual isoforms are non-specific and complementary in their sites of action [29,30]. Therefore the predominant control of 6S patterning, as seen by binding ligands, is probably due to mSulf activity.…”

Heparan Sulfate Proteoglycans Biosynthesis and Post Synthesis Mechanisms Combine Few Enzymes and Few Core Proteins to Generate Extensive Structural and Functional Diversity

“…At high concentration, this enzyme, which normally preferentially targets IdoA, will start to increase the 2- O- sulfation of GlcA residues, which are then no longer substrate for the C5-epi and thus cannot be converted back to IdoA [ 271 ]. Overexpression of 6-OSTs increased both GlcNS and GlcNAc 6- O- sulfation, but also reduced the total IdoA content, 2- O- sulfation and modified the distribution of the N- sulfated domains [ 272 ]. HS produced by C5-epi and 2-OST knockout animals are enriched in N- and 6- O- sulfation, which could suggest a compensation mechanism to overcome the loss of IdoA and 2- O- sulfate, but the underlying mechanism remains unknown [ 127 , 179 ].…”