Abstract
The binding of purified fibrinogen receptor alpha IIb beta 3, vitronectin receptor alpha V beta 3, and fibronectin receptor alpha 5 beta 1 to their corresponding ligands in solid-phase binding assays was used to examine the inhibitory activity of various linear and cyclic penta- and hexapeptides of different conformation containing RGD or RAD sequences. Cyclic peptides with different defined backbone conformations were designed by introducing a single D-amino acid or a proline at different positions in the ring. The data were calibrated for alpha IIb beta 3 integrin incorporated into a planar lipid bilayer by a physical method (total internal reflection fluorescence microscopy) which yielded KD = 1.7 microM for a linear RGD peptide and KD = 0.03 microM for fibrinogen. With this integrin, three cyclic hexapeptides ([GRGDFL], [ARGDFV], [GRGDFV]) were 2-4-fold more inhibitory than the linear GRGDS peptide in solid-phase assays and showed similar inhibition as the fibrinogen ligand. Six peptides had the same or a 2-fold lower activity as the linear reference peptide, and three peptides were up to 7-fold less active. Replacement of Arg or Asp by their stereoisomers or Gly by Ala resulted in a 100-1000-fold reduction in activity. With the two other integrins, a single cyclic pentapeptide [RGDFV] was 10-fold more active (alpha V beta 3) or equal in activity (alpha 5 beta 1) to linear GRGDS, while all of the other cyclic peptides were moderately or distinctly less active. Changes in the RGD sequence caused a less dramatic reduction in binding strength for alpha V beta 3 and alpha 5 beta 1 than for alpha II beta 3. Inhibitory activity was compared with the distance between the C beta atoms of Arg and Asp residues as determined by NMR and indicated that the optimum distance is in the range of 0.75-0.85 nm for alpha IIb beta 3 and at or below 0.67 nm for alpha V beta 3 and alpha 5 beta 1. This indicates that alpha IIb beta 3 less sensitive to variations in the RGD backbone structure and can accommodate a larger distance than the integrins alpha V beta 3 and alpha 5 beta 1.
Highlights
From the $Max-Planck-Znstitut fur Biochemie, 082152 Martinsried, Federal Republic of Germany, BBiozentrum, University of Basel, CH-4056 Basel, Switzerland, a n d the llorganisch-Chemisches Znstitut, nchnische Universitat, 085748 Munchen, Federal Republic of Germany
Severalof the a6P1 to theircorrespondingligands in solid-phasebind- ligands, such as fibrinogen, vitronectin,and fibronectin, bindto ing assayswas usedto examine the inhibitory activity of individual RGD-dependent integrins with distinctly different various linear and cyclic penta- and hexapeptides of dif- affinities in spite of sharing this common recognition motif
The data were calibrated for aIIbp3 integrin incorporated into a planar lipid bilayer by a physical method hich yielded KD= 1.7 p~for a linear RGD peptide and KD= 0.03 p~ for fibrinogen
Summary
Extracellular Matrix Proteins and Antibodies-Vitronectin was prepared from human serum by heparin chromatographyin 8 M urea [22]. Bound integrins were eluted with mM EDTA into 1.5-ml vials containing 25 pl of 1 M MgCI, and concentrated in Centricon 100 microconcentrators. They were storedat 4 "C in neutralbuffer containing (Eq 1). The purified integrins were characterizebdy SDS-gel electrophoresis in which III,is the measured ratio of fluorescence with and without followed by protein staining and by immunoblotting [24], which dem- unlabeled peptide, [P"] and K,' are the concentration and dissociation onstrated the purity and identity of the two subunits.' Chain-specific equilibrium constant of labeled peptideand [PI and KDthe correspondmonoclonal antibodies were used to detecptossible contaminations and ing quantitiesof the unlabeled peptide. The preparations of aVp3 were essentially freeof aIIb and/35 subunits.'
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