title: Functional analysis and modeling of a conformationally constrained Arg-Gly-Asp sequence inserted into human lysozyme.
abstract: To examine the effect of a conformational constraint introduced into the Arg-Gly-Asp (RGD) sequence on cell adhesion activity, we have constructed mutant proteins by inserting RGD-containing sequences flanked by two Cys residues between Val74 and Asn75 of human lysozyme. CRGDC-, CRGDSC-, and CGRGDSC-inserted mutant lysozymes were expressed in yeast, purified, and designated as Cys-RGD3, Cys-RGD4, and Cys-RGD5, respectively. In baby hamster kidney cells, these mutants were shown to possess high cell adhesion activity by interaction with vitronectin receptor (integrin alpha v beta 3), and this activity is 2-3-fold higher than that of the RGDS-inserted mutant lysozyme, RGD4. The mutant proteins also inhibited the binding of human fibrinogen to its receptor (integrin alpha IIb beta 3) at a lower concentration than the RGD4 protein. Peptide mapping and mass spectrometric analyses showed that the two inserted Cys residues in these mutants are linked to each other without any effects on the mode of the four disulfide bonds present in native human lysozyme. These results suggest that the introduction of a conformational constraint into the RGD region significantly increases the cell adhesion activity. The conformation of the RGD region in Cys-RGD4 was modeled by a Monte Carlo simulation. Most of the sampled conformations were grouped into three classes; the first is characterized by an extended Gly conformation, the second assumes a type II' beta turn, and the third has a salt bridge between Arg and Asp.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Protein Engineering Research Institute, Osaka, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Yamada
authors.forename: A
authors.initials: A
authors.lastname: Uyeda
authors.forename: A
authors.initials: A
authors.lastname: Kidera
authors.forename: M
authors.initials: M
authors.lastname: Kikuchi
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002448:Cell Adhesion / Q000502:physiology*; D003198:Computer Simulation; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000378:metabolism; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009010:Monte Carlo Method; D009113:Muramidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016254:Mutagenesis, Insertional; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011487:Protein Conformation; D014407:Tumor Cells, Cultured
chemical_list: D004220:Disulfides; D016023:Integrins; D009842:Oligopeptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C047981:arginyl-glycyl-aspartic acid; D005340:Fibrinogen; D009113:Muramidase; D003545:Cysteine