Non-heparan sulfate-binding interactions of endostatin/collagen XVIII in murine development.
Knobloch syndrome is characterized by a congenital generalized eye disease and cranial defect. Pathogenic mutations preferentially lead to a deletion or functional alteration of collagen XVIII's most C-terminal endostatin domain. Endostatin can be released from collagen XVIII and is a potent inhibitor of angiogenesis and tumor growth. We show differential expression of binding partners for endostatin, vascular endothelial growth factor (VEGF), and the collagen XV endostatin homologue in murine embryonal development using a set of alkaline phosphatase fusion proteins. Consistent with the human phenotype, vascular mesenchyme in the developing eye was identified as endostatin's primary target. While endostatin predominantly bound to blood vessels, the VEGF164 affinity probe labeled nonvascular tissues such as forebrain, hindbrain, the optic nerve, and the surface ectoderm of the future cornea. Strikingly increased staining specificity was observed with a non-heparin/heparan sulfate-binding endostatin probe. In contrast, elimination of the heparan sulfate binding site from VEGF led to complete loss of binding. The collagen XV endostatin homologue showed a highly restricted binding pattern. Oligomerization with endogenous endostatin was ruled out by use of collagen XVIII knockout mice. Our data provide strong evidence that collagen XVIII's C-terminal endostatin domain harbors a prominent tissue-binding site and that binding can occur in the absence of heparan sulfates in situ.
Rychkova
Natalia
N
Department of Human Genetics, University of Würzburg, Biozentrum, Am Hubland, D-97074 Würzburg, Germany.
Stahl
Sonja
S
Gaetzner
Sabine
S
Felbor
Ute
U
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D001921:Brain / Q000196:embryology / Q000378:metabolism*; D003094:Collagen / Q000737:chemistry; D043170:Collagen Type XVIII / Q000378:metabolism*; D003315:Cornea / Q000196:embryology; D004591:Electrophoresis, Polyacrylamide Gel; D043169:Endostatins / Q000378:metabolism*; D005123:Eye / Q000196:embryology; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors; D006497:Heparan Sulfate / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization; D051379:Mice; D018345:Mice, Knockout; D010641:Phenotype; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D000469:Alkaline Phosphatase; D003094:Collagen; D043170:Collagen Type XVIII; D043169:Endostatins; D006497:Heparan Sulfate; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A