Heparin structure and interactions with basic fibroblast growth factor

Science

Volumn 271, Issue 5252, 1996, Pages 1116-1120

  Faham, S ^a   Hileman, R E ^b   Fromm, J R ^b   Linhardt, R J ^b   Rees, D C ^a  

^a CALIFORNIA INSTITUTE OF TECHNOLOGY   (United States)

^b UNIVERSITY OF IOWA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ARGININE; ASPARAGINE; BASIC FIBROBLAST GROWTH FACTOR; DISACCHARIDE; GLUTAMINE; HEPARIN; LYSINE;

ARTICLE; BINDING SITE; CONFORMATIONAL TRANSITION; CRYSTAL STRUCTURE; PRIORITY JOURNAL; PROTEIN BINDING; PROTEIN PROTEIN INTERACTION; PROTEIN STRUCTURE; SIGNAL TRANSDUCTION;

EID: 0029866647     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5252.1116     Document Type: Article

References (47)

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14. Tetrasacchande and hexasaccharide fragments of porcine mucosal heparin were prepared by partial digestion with heparin tyase I, followed by preparative strong anion exchange high-performance liquid chromatography [A Pervin, C. Gallo, K. A. Jandik, X.-J. Man, R J. Linhardt, Glycobiology 5, 83 (1995)]. As a product of this procedure, the heparin fragments contain an unsaturated uronic acid group at the nonreducing end and a mixture of α and β anomers at the reducing end of the oligosaccharide. Consequently, the tetra- and hexasacchande fragments contain one and two intact units of the heparin disaccharides, respectively, in their interior. Microtitration calorimetry (16) provided dissociation constants for binding of bFGF to the tetrasacchande and hexasaccharide of 6 ± 2 μM and 101 ± 1 nM, respectively, in 10 mM sodium phosphate and 1 mM dithiothreitol, pH 7.4. These fragments compete for FGF binding with heparin (10). Although heparin fragments with at least 12 to 14 residues are required for optimal FGF activity [A. Walker, J. E Turnbull, J. T. Gallagher, J Biol. Chem 269, 931 (1994)], FGF-mediated mitogenic activity of heparin hexasaccharide fragments has been demonstrated [T. Barzu, J. C Lormeau, M. Petitou, S. Michelson, J Choay, J. Cell. Physiol. 140, 538 (1989); A. G. Gambanni, C. A. Miyamoto, G. A. Lima, H. B Nader, C. P. Dietrich, Mol Cell Biochem. 124, 121 (1993)].
15. Tetrasacchande and hexasaccharide fragments of porcine mucosal heparin were prepared by partial digestion with heparin tyase I, followed by preparative strong anion exchange high-performance liquid chromatography [A Pervin, C. Gallo, K. A. Jandik, X.-J. Man, R J. Linhardt, Glycobiology 5, 83 (1995)]. As a product of this procedure, the heparin fragments contain an unsaturated uronic acid group at the nonreducing end and a mixture of α and β anomers at the reducing end of the oligosaccharide. Consequently, the tetra- and hexasacchande fragments contain one and two intact units of the heparin disaccharides, respectively, in their interior. Microtitration calorimetry (16) provided dissociation constants for binding of bFGF to the tetrasacchande and hexasaccharide of 6 ± 2 μM and 101 ± 1 nM, respectively, in 10 mM sodium phosphate and 1 mM dithiothreitol, pH 7.4. These fragments compete for FGF binding with heparin (10). Although heparin fragments with at least 12 to 14 residues are required for optimal FGF activity [A. Walker, J. E Turnbull, J. T. Gallagher, J Biol. Chem 269, 931 (1994)], FGF-mediated mitogenic activity of heparin hexasaccharide fragments has been demonstrated [T. Barzu, J. C Lormeau, M. Petitou, S. Michelson, J Choay, J. Cell. Physiol. 140, 538 (1989); A. G. Gambanni, C. A. Miyamoto, G. A. Lima, H. B Nader, C. P. Dietrich, Mol Cell Biochem. 124, 121 (1993)].
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19. 2, and 0.1 M ADA, pH 7.0] This corresponds to about a 6:1 molar ratio of hexasaccharide to bFGF.
20. 79 in the tetrasaccharide, and hence are not included in the refined models. The terminal sugar unit at the non-reducing end of the tetrasaccharide has weaker electron density, indicating a somewhat disordered crystal Coordinates have been deposited with the Brookhaven Protein Data Bank (entries 1BFB and 1BFC for the tetra- and hexasaccharide complexes of bFGF, respectively)
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47. We thank T. Arakawa and G M. Fox of Amgen for discussions and a generous supply of bFGF and D. Bar-Shalom for stimulating conversations. This work was funded in part by a gift from Amgen and U.S. Public Health Service (USPHS) grant GM38060 (R.J.L.), wrth partial support of the x-ray facility provided by the Beckman Institute at California Institute of Technology and USPHS grant GM45162 (D C.R.). S.F. was supported by USPHS Biotechnology Training grant T32 GM08346