An unusual heparin-binding peptide from the carboxy-terminal hep-2 region of fibronectin.
A synthetic 22 residue peptide, N22W, with sequence NVSPPRRARVTDATETTITISW, derived from the amino terminus of type III module 13 in the carboxy-terminal hep-2 domain of fibronectin, was found to exhibit unusual heparin binding properties. Titration of fluoresceinamine-labeled heparin (FA-heparin) with N22W at 25 degrees C and pH 7.4 in 0.02 M Tris buffer containing 0.15 M NaCl (TBS) produced a cooperative sigmoidal increase in fluorescence polarization anisotropy with half-saturation near 2.5 microM. The increase in anisotropy was even greater than that produced by the much larger 30-kDa hep-2 fragment of fibronectin and saturation was achieved at lower concentration. Simply deleting the C-terminal Trp from the peptide abolished its heparin-binding activity as did deletion of residues TETTITIS or mutation of the RR doublet to SS. Further analysis suggested that peptide-peptide interactions mediated by the carboxy-terminal region of N22W play an important role in its binding to heparin. A branched tetrameric peptide containing four copies of N21S caused a nearly hyperbolic increase in anisotropy of FA-heparin with an apparent Kd of 0.3 microM in TBS, 10-fold lower than that of the monomer or of the parent domain from which the peptide was derived. The results illustrate that peptide-peptide interactions can lead to stronger binding by allowing multiple points of contact with the negatively charged polysaccharide.
Ingham
K C
KC
Holland Laboratory, American Red Cross, Rockville, Maryland 20855.
Brew
S A
SA
Migliorini
M
M
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism*; D005452:Fluoresceins; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006493:Heparin / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship
D005353:Fibronectins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D006493:Heparin