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.
Highlights
Heparan sulfate (HS),3 a linear polysaccharide covalently attached to various core proteins, is expressed ubiquitously in animal cells and found at the cell surface and in the extracellular matrix [1]
Northern blot analyses resulted in hybridization signals of different intensities, indicating that the transfections had resulted in clones with different expression levels of 6-OST1, 6-OST2, or 6-OST3 mRNA
The overexpression resulted in increased O-sulfotransferase activity that varied among different cell clones from 2- to 7-fold increase to Ն29-fold increase in 6-OST1 and 6-OST3 transfectants, whereas for 6-OST2 transfectants, the increased activity ranged from 2- to 7-fold to an ϳ22-fold increase
Summary
Heparan sulfate (HS), a linear polysaccharide covalently attached to various core proteins, is expressed ubiquitously in animal cells and found at the cell surface and in the extracellular matrix [1]. The three mouse 6-OST enzymes are all capable of sulfating all potential target structures in vitro, with different efficiency [24]. The roles of each individual 6-OST in generating specific HS protein-binding sequences and their functions in vivo remain unknown. A strict control of the expression of 6-OSTs may be one of the means for the cell to regulate HS structure and function, and alteration in the relative amounts of the various 6-OST isoforms in a cell could result in distinct 6-O-sulfation patterns. The different isoforms gave rise to similar 6-O-sulfation patterns, but the level of expression had a dramatic influence on the structural properties of the polysaccharide, suggesting a need for strict regulation of 6-OST levels in HS biosynthesis
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