A peptide inhibiting the collagen binding function of integrin alpha2I domain.

  • Ivaska J
  • Käpylä J
  • Pentikäinen O
 et al. 
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Abstract

Integrin alpha2 subunit forms in the complex with the beta1 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 alpha2 subunit. Venom from a pit viper, Bothrops jararaca, has been shown to inhibit the interaction of platelet alpha2beta1 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 ralpha2I 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 ralpha2I 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 alpha2I 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 ralpha2I 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 alpha2I 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 alpha2beta1 integrin, and the attachment was inhibited by anti-integrin antibodies.

Author-supplied keywords

  • Amino Acid Sequence
  • Antigens
  • Base Sequence
  • CD
  • CD: chemistry
  • CD: drug effects
  • CD: metabolism
  • Cell Membrane
  • Cell Membrane: metabolism
  • Collagen
  • Collagen: metabolism
  • Crotalid Venoms
  • Crotalid Venoms: chemistry
  • Cyclic
  • Cyclic: pharmacology
  • DNA Primers
  • Europium
  • Europium: chemistry
  • Humans
  • Integrin alpha2
  • Metalloendopeptidases
  • Metalloendopeptidases: chemistry
  • Peptides
  • Recombinant Proteins
  • Recombinant Proteins: chemistry
  • Recombinant Proteins: metabolism

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Authors

  • Johanna Ivaska

  • Jarmo Käpylä

  • O Pentikäinen

  • A M Hoffrén

  • J Hermonen

  • Pasi Huttunen

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