Binding of endostatin to endothelial heparan sulphate shows a differential requirement for specific sulphates.
Endostatin is a naturally occurring proteolytic fragment of the C-terminal domain of collagen XVIII. It inhibits angiogenesis by a mechanism that appears to involve binding to HS (heparan sulphate). We have examined the molecular interaction between endostatin and HS from micro- and macrovessel endothelial cells. Two discrete panels of oligosaccharides were prepared from metabolically radiolabelled HS, using digestion with either heparinase I or III, and then examined for their endostatin affinity using a sensitive filter-binding assay. Two types of endostatin-binding regions were identified: one comprising sulphated domains of five or more disaccharides in length, enriched in 6-O-sulphate groups, and the other contained long heparinase I-resistant fragments. In the latter case, evidence from the present study suggests that the binding region encompasses a sulphated domain fragment and a transition zone of intermediate sulphation. The contribution to binding of specific O-sulphate groups was determined using selectively desulphated HS species, namely HS from Hs2st-/- mutant cells, and by comparing the compositions of endostatin-binding and non-binding oligosaccharides. The results indicate that 6-O-sulphates play a dominant role in site selectivity and 2-O-sulphates are not strictly essential.
Blackhall
Fiona H
FH
Department of Medical Oncology, University of Manchester, Cancer Research UK, Christie Hospital NHS Trust, Manchester M20 4BX, UK.
Merry
Catherine L R
CL
Lyon
Malcolm
M
Jayson
Gordon C
GC
Folkman
Judah
J
Javaherian
Kashi
K
Gallagher
John T
JT
D001665:Binding Sites; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003094:Collagen / Q000378:metabolism*; D043170:Collagen Type XVIII; D004187:Disaccharides / Q000032:analysis; D043169:Endostatins; D004730:Endothelium, Vascular / Q000737:chemistry*; D006497:Heparan Sulfate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D013431:Sulfates / Q000737:chemistry
D003094:Collagen; D043170:Collagen Type XVIII; D004187:Disaccharides; D043169:Endostatins; D006497:Heparan Sulfate; D009844:Oligosaccharides; D010446:Peptide Fragments; D013431:Sulfates