Abstract
Cell surface-associated heparan sulfate proteoglycans, predominantly perlecan, are involved in the process of binding and endocytosis of thrombospondin-1 (TSP-1) by vascular endothelial cells. To investigate the structural properties of heparan sulfate (HS) side chains that mediate this interaction, the proteoglycans were isolated from porcine endothelial cells and HS chains obtained thereof by beta-elimination. To characterize the structural composition of the HS chains and to identify the TSP-1-binding sequences, HS was disintegrated by specific chemical and enzymatic treatments. Cell layer-derived HS chains revealed the typical structural heterogeneity with domains of non-contiguously arranged highly sulfated disaccharides separated by extended sequences containing predominantly N-acetylated sequences of low sulfation. Affinity chromatography on immobilized TSP-1 demonstrated that nearly all intact HS chains possessed binding affinity, whereas after heparinase III treatment only a small proportion of oligosaccharides were bound with similar affinity to the column. Size fractioning of the bound and unbound oligosaccharides revealed that only a specific portion of deca- to tetradecasaccharides possessed TSP-1-binding affinity. The binding fraction contained over 40% di- and trisulfated disaccharide units and was enriched in the content of the trisulfated 2-O-sulfated L-iduronic acid-N-sulfated-6-O-sulfated glucosamine disaccharide unit. Comparison with the disaccharide composition of the intact HS chains and competition experiments with modified heparin species indicated the specific importance of N- and 6-O-sulfated glucosamine residues for binding. Further depolymerization of the binding oligosaccharides revealed that the glucosamine residues within the TSP-1-binding sequences are not continuously N-sulfated. The present findings implicate specific structural properties for the HS domain involved in TSP-1 binding and indicate that they are distinct from the binding sequence described for basic fibroblast growth factor, another HS ligand and a potential antagonist of TSP-1.
Highlights
Cell surface-associated heparan sulfate proteoglycans, predominantly perlecan, are involved in the process of binding and endocytosis of thrombospondin-1 (TSP-1) by vascular endothelial cells
Structure of Cell Layer-derived heparan sulfate (HS)—In previous studies it was shown that porcine endothelial cells synthesize proteoglycans containing HS and CS/DS glycosaminoglycan chains [33]
In view of the involvement of HS chains of the cell layer during binding and endocytosis of TSP-1 [31, 32], structural features of cell layer-derived HS required for the TSP/HS interactions were investigated
Summary
(Received for publication, September 24, 1999, and in revised form, December 20, 1999). Cell surface-associated heparan sulfate proteoglycans, predominantly perlecan, are involved in the process of binding and endocytosis of thrombospondin-1 (TSP-1) by vascular endothelial cells. The N-terminal globular domain possesses binding sequences for heparan sulfate proteoglycans [28], a sequence containing SVTCG within type 1 repeats, which interacts with CD36 and a 50-kDa tumor cell. In several studies the receptor-like function of heparan sulfate proteoglycans has been demonstrated during the process of binding, uptake, and degradation of TSP-1 by cultured cells [26, 28, 31, 32]. We report the isolation and characterization of distinct heparan sulfate oligosaccharides from endothelial cell-associated HSPGs that exhibit specific affinity for TSP-1
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