Binding of heparin by type III domains and peptides from the carboxy terminal hep-2 region of fibronectin.
The major sites of heparin binding by fibronectin are located in fragments of 30 or 40 kDa that contain type III modules 12 through 14 or 15. Various proteolytic or recombinant subfragments and several synthetic peptides derived from this region have been compared with respect to their binding to fluorescein-labeled heparin in solution. Binding was monitored by the change in fluorescence anisotropy at 25 degrees C and pH 7.4 in 0.02 M Tris buffer, alone (TB) or with 0.15M NaCl (TBS). A 23-kDa fragment containing III13 and III14 but lacking III12 had Kd values of 0.3 and 1.8 microM in TB, and TBS, respectively, indistinguishable from the 30-kDa parent. Fragments containing only module III13 bound 2-3-fold weaker than the parent while those containing only III14 bound 6-50-fold weaker depending on the ionic strength. Fragments containing only III12 or III15 failed to bind at all in TBS. A cationic peptide derived from the amino terminus of III13 and containing the Arg-Arg-Ala-Arg consensus sequence, whose integrity was shown by Barkalow and Schwarzbauer [Barkalow, F. J., & Schwarzbauer, J. E. (1991) J. Biol. Chem. 266, 7812-7818] to be critical, failed to bind in TBS but bound weakly in TB. Two additional cationic peptides derived from the middle and C-terminal regions of III14 showed similar behavior. Thus while the major determinant(s) of heparin binding are located in III13, those determinants are only active when part of a properly folded structure. Furthermore, module III13 when isolated had a slightly lower affinity than fragments containing both III13 and III14.(ABSTRACT TRUNCATED AT 250 WORDS)
Ingham
K C
KC
Holland Laboratory, American Red Cross, Rockville, Maryland 20855.
Brew
S A
SA
Migliorini
M M
MM
Busby
T F
TF
D000595:Amino Acid Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D005353:Fibronectins / Q000737:chemistry* / Q000378:metabolism; D005454:Fluorescence Polarization; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding
D005353:Fibronectins; D010446:Peptide Fragments; D006493:Heparin