A recombinant chimeric epidermal growth factor-like module with high binding affinity for integrins.
Integrins are cell surface receptors involved in numerous pathological processes such as metastasis invasion and abnormal angiogenesis. To target these receptors, the epidermal growth factor (EGF)-like domain of human complement protease C1r was used as a natural scaffold to design chimeric modules containing the RGD motif. Here we report a high yield bacterial expression system and its application to the production of two such modules, EGF-RGD and V2, the latter variant mimicking the RGD-containing domain of disintegrins. These modules were characterized chemically, and their biological activity was investigated by cellular assays using various Chinese hamster ovary cell lines expressing beta1 and beta3 integrins and by surface plasmon resonance spectroscopy. Remarkably, the modifications leading to the V2 variant had differential effects on the interaction with beta3 and beta1 integrins. The disintegrin-like V2 module exhibited enhanced binding affinities compared with EGF-RGD, with KD values of 7.2 nm for alpha5beta1 (a 4-fold decrease) and 3.5 nm for alphavbeta3 (a 1.5-fold decrease), comparable with the values determined for natural integrin ligands. Analysis by NMR spectroscopy also revealed a differential dynamic behavior of the RGD motif in the EGF-RGD and V2 variants, providing insights into the structural basis of their relative binding efficiency. These novel RGD-containing EGF modules open the way to the design of improved variants with selective affinity for particular integrins and their use as carriers for other biologically active modules.
Vella
Fanny
F
Laboratoire d'Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel, (Commissariat à l'Energie Atomique CNRS, Université Joseph Fourier), Grenoble 38027 Cedex 1, France.
Thielens
Nicole M
NM
Bersch
Beate
B
Arlaud
Gérard J
GJ
Frachet
Philippe
P
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002448:Cell Adhesion; D006224:Cricetinae; D004815:Epidermal Growth Factor / Q000378:metabolism*; D016023:Integrins / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D016023:Integrins; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor