Endostatin phenylalanines 31 and 34 define a receptor binding site.
Endostatin has achieved much attention as a naturally occurring inhibitor of angiogenesis and tumor growth. Endostatin is derived from collagen XVIII's C-terminal domain and deleted or truncated in most patients suffering from Knobloch syndrome blindness. To evaluate the functional significance of two surface-exposed hydrophobic phenylalanines at positions 31 and 34 of endostatin and two human sequence alterations within endostatin, A48T and D104N, we applied the alkaline phosphatase fusion protein method. Replacement of F31 and F34 with alanines led to complete loss of characteristic in situ binding while heparin binding remained intact. In contrast, a non-heparin binding alkaline phosphatase-tagged human endostatin lacking R27 and R139 bound to specific tissue structures. The two Knobloch syndrome-associated endostatin sequence variants did not result in altered in situ binding to murine embryonal tissues, human endothelial cells, heparin and immobilized laminin. However, expression of the endostatin mutant A48T was significantly reduced. This observation may be explained by a lower folding efficiency due to the structural constraints of A48 residing in the hydrophobic core. Our data suggest that residues F31 and F34 form a putative receptor binding site acting independently from heparan sulfate binding and that the A48T mutation destabilizes the endostatin molecule.
Stahl
Sonja
S
Department of Human Genetics, University of Würzburg, Biozentrum, Am Hubland, D-97074 Würzburg, Germany.
Gaetzner
Sabine
S
Mueller
Thomas D
TD
Felbor
Ute
U
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D043170:Collagen Type XVIII / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D043169:Endostatins / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin / Q000737:chemistry; D006801:Humans; D007797:Laminin / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D013045:Species Specificity
D043170:Collagen Type XVIII; D043169:Endostatins; D007797:Laminin; D011993:Recombinant Fusion Proteins; D010649:Phenylalanine; D006493:Heparin; D000469:Alkaline Phosphatase