A1 Refereed original research article in a scientific journal
A peptide inhibiting the collagen binding function of integrin alpha I-2 domain
Authors: Ivaska J, Kapyla J, Pentikainen O, Hoffren AR, Hermonen J, Huttunen P, Johnson MS, Heino J
Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Publication year: 1999
Journal: Journal of Biological Chemistry
Journal name in source: JOURNAL OF BIOLOGICAL CHEMISTRY
Journal acronym: J BIOL CHEM
Volume: 274
Issue: 6
First page : 3513
Last page: 3521
Number of pages: 9
ISSN: 0021-9258
DOI: https://doi.org/10.1074/jbc.274.6.3513
Abstract
Integrin alpha(2) subunit forms in the complex with the beta(1) subunit a cell surface receptor binding extracellular matrix molecules, such as collagens and laminin-1. It is a receptor for echovirus-1, as well. Ligands are recognized by the special "inserted" domain (I domain) in the integrin alpha(2) subunit, Venom from a pit viper, Bothrops jararaca, has been shown to inhibit the interaction of platelet alpha(2)beta(1) integrin with collagen because of the action of a disintegrin/metalloproteinase named jararhagin. The finding that crude B. jararaca venom could prevent the binding of human recombinant r alpha(2)I domain to type I collagen led us to study jararhagin further. Synthetic peptides representing hydrophilic and charged sequences of jararhagin, including the RSECD sequence replacing the well known RGD motif in the disintegrin-like domain, were synthesized. Although the disintegrin-like domain derived peptides failed to inhibit r alpha(2)I domain binding to collagen, a basic peptide from the metalloproteinase domain proved to be functional. In an in vitro assay, the cyclic peptide, CTRKKHDNAQC, was shown to bind strongly to human recombinant alpha(2)I domain and to prevent its binding to type I and IV collagens and to laminin-1, Mutational analysis indicated that a sequence of three amino acids, arginine-lysine-lysine (RKK), is essential for r alpha(2)I domain binding, whereas the mutation of the other amino acids in the peptide had little if any effect on its binding function. Importantly, the peptide was functional only in the cyclic conformation and its affinity was strictly dependent on the size of the cysteine-constrained loop. Furthermore, the peptide could not bind to alpha(2)I domain in the absence of Mg2+, suggesting that the conformation of the I domain was critical, as well. Cells could attach to the peptide only if they expressed alpha(2)beta(1) integrin, and the attachment was inhibited by anti-integrin antibodies.
Integrin alpha(2) subunit forms in the complex with the beta(1) subunit a cell surface receptor binding extracellular matrix molecules, such as collagens and laminin-1. It is a receptor for echovirus-1, as well. Ligands are recognized by the special "inserted" domain (I domain) in the integrin alpha(2) subunit, Venom from a pit viper, Bothrops jararaca, has been shown to inhibit the interaction of platelet alpha(2)beta(1) integrin with collagen because of the action of a disintegrin/metalloproteinase named jararhagin. The finding that crude B. jararaca venom could prevent the binding of human recombinant r alpha(2)I domain to type I collagen led us to study jararhagin further. Synthetic peptides representing hydrophilic and charged sequences of jararhagin, including the RSECD sequence replacing the well known RGD motif in the disintegrin-like domain, were synthesized. Although the disintegrin-like domain derived peptides failed to inhibit r alpha(2)I domain binding to collagen, a basic peptide from the metalloproteinase domain proved to be functional. In an in vitro assay, the cyclic peptide, CTRKKHDNAQC, was shown to bind strongly to human recombinant alpha(2)I domain and to prevent its binding to type I and IV collagens and to laminin-1, Mutational analysis indicated that a sequence of three amino acids, arginine-lysine-lysine (RKK), is essential for r alpha(2)I domain binding, whereas the mutation of the other amino acids in the peptide had little if any effect on its binding function. Importantly, the peptide was functional only in the cyclic conformation and its affinity was strictly dependent on the size of the cysteine-constrained loop. Furthermore, the peptide could not bind to alpha(2)I domain in the absence of Mg2+, suggesting that the conformation of the I domain was critical, as well. Cells could attach to the peptide only if they expressed alpha(2)beta(1) integrin, and the attachment was inhibited by anti-integrin antibodies.
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