Interaction of heparin with human angiogenin.
HT-29 human colon adenocarcinoma cells adhere rapidly to human angiogenin (Ang) via interactions with cell-surface heparan sulfate moieties (Soncin, F., Shapiro, R., and Fett, J. W. (1994) J. Biol. Chem. 269, 8999-9005). Soluble heparin inhibits adhesion, and Ang itself binds tightly to heparin-Sepharose. In the present study, the interaction of Ang with heparin has been further characterized. The basic cluster Arg-31/Arg-32/Arg-33 has been identified as an important component of the heparin binding site. Mutations of these residues, and of Arg-70 as well, decrease both the affinity of Ang for heparin-Sepharose and the capacity of Ang to support cell adhesion. Replacements of four other basic residues do not affect heparin binding. Heparin partially protects Ang from cleavage by trypsin at Lys-60, suggesting that heparin also binds to the region of Ang that contains this residue. The map here determined indicates that the heparin recognition site on Ang lies outside the catalytic center; indeed, heparin has no significant effect on the ribonucleolytic activity of Ang. It also does not influence the angiogenic activity of this protein. Light scattering measurements on Ang-heparin mixtures suggest that 1 heparin chain (mass of 16.5 kDa) can accommodate approximately 9 Ang molecules. The minimum size required for a heparin fragment to effectively inhibit HT-29 cell adhesion to Ang was determined to be 6 disaccharide units. The implications of these findings for inhibition of Ang-mediated tumor establishment in vivo are discussed.
Soncin
F
F
Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.
Strydom
D J
DJ
Shapiro
R
R
D043925:Angiogenesis Inducing Agents / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002448:Cell Adhesion; D002846:Chromatography, Affinity; D003460:Crystallization; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000235:genetics / Q000378:metabolism*; D012542:Scattering, Radiation; D014357:Trypsin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D043925:Angiogenesis Inducing Agents; D009363:Neoplasm Proteins; D011506:Proteins; D006493:Heparin; C046366:angiogenin; D012259:Ribonuclease, Pancreatic; D014357:Trypsin