The Morphogenic Properties of Oligomeric Endostatin Are Dependent on Cell Surface Heparan Sulfate

Andrew Clamp,Fiona H Blackhall,Audrey Henrioud,Gordon C Jayson,Kashi Javaherian,Jeff Esko,John T Gallagher,Catherine L.R Merry

doi:10.1074/jbc.m512400200

Abstract

Endostatin has attracted considerable attention because of its ability to inhibit angiogenesis. This property of monomeric endostatin contrasts with that of the trimeric endostatin moiety generated from the intact C-terminal domain of collagen XVIII that induces a promigratory phenotype in endothelial cells. This activity is inhibited by monomeric endostatin. In this study we demonstrate that the effect of oligomeric endostatin can also be inhibited by exogenous glycosaminoglycans in a size-dependent manner, with heparin oligosaccharides containing more than 20 monosaccharide residues having optimal inhibitory activity. Oligomeric endostatin was also found to induce morphological changes in Chinese hamster ovary cells, an epithelial cell line. This novel observation allowed the utilization of a panel of Chinese hamster ovary cell mutants with defined glycosaminoglycan biosynthetic defects. The action of oligomeric endostatin on these cells was shown to be dependent on cell surface glycosaminoglycans, principally heparan sulfate with N- and 6-O-sulfation of glucosamine residues rather than iduronate 2-O-sulfation being important for bioactivity. The responsiveness of a cell line (pgsE-606) with globally reduced heparan sulfate sulfation and shortened S domains, however, indicates that overall heparan sulfate domain patterning is the key determinant of the bioactivity of oligomeric endostatin. Purified heparin-monomeric endostatin constructs generated by zero-length cross-linking techniques were found to be unable to inhibit the action of oligomeric endostatin. This indicates a mechanism for the perturbation of oligomeric endostatin action by its monomeric counterpart via competition for glycosaminoglycan attachment sites at the cell surface.

Highlights

Endostatin is a cryptic C-terminal fragment of collagen XVIII released by matrix metalloproteases, cathepsin L, and elastase [3, 4]
Endothelial cell migration and capillary tubule breakdown are induced by the addition of human endostatin dimer (HED) and this breakdown is inhibited by monomeric endostatin (HEM) in a dose-dependent manner
In this study we provide the first biological evidence that the activity of oligomeric endostatin is dependent on the presence of cell surface GAGs

Summary

EXPERIMENTAL PROCEDURES

Materials—Tinzaparin was from Leo Pharmaceuticals (Princes Risborough, UK). Bio-Gel P-10 columns were obtained from Bio-Rad. Wells were seeded with 50,000 BAE cells in 1 ml of assay medium (Dulbecco’s modified Eagle’s medium with 5% donor calf serum and 300 ␮g/ml glutamine) and the plates were incubated at 37 °C for 16 h allowing the formation of capillary-like endothelial tubules. Human endostatin monomer (HEM) [9] at 3000 nM or variable concentrations of intact heparin or size-defined oligosaccharides were added 30 min prior to the addition of HED. 50,000 CHO cells per well were seeded in standard growth medium and HED was added immediately, or after 16 h incubation. Purification of Heparin Cross-linked Endostatin Monomer—Crosslinked constructs were separated from unreacted HEM by size exclusion high performance liquid chromatography using a 7.5 ϫ 300-mm TSKG4000pw column run at 0.5 ml/min in 1 M sodium chloride, pH 7.0. To determine the effect of the purification procedure on protein integrity, the uncross-linked HEM purified by chromatography was subjected to the same experimental protocol

RESULTS

GAG phenotype

DISCUSSION