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Biofunctionalization of a generic collagenous triple helix with the α2β1 integrin binding site allows molecular force measurements

Author:
Niland, Stephan, Westerhausen, Christoph, Schneider, Stefan W., Eckes, Beate, Schneider, Matthias F., Eble, Johannes A.
Source:
international journal of biochemistry & cell biology 2011 v.43 no.5 pp. 721-731
ISSN:
1357-2725
Subject:
atomic force microscopy, binding sites, biomedical materials, crosslinking, fibroblasts, homeostasis, integrins, pseudopodia, receptors
Abstract:
The integrin α2β1 plays an important role in force-transmitting cell–matrix interactions. It recognizes the peptide sequence GFOGER (O=4-hydroxy-proline) presented as trimer within a collagenous triple-helical framework. We produced the recombinant non-hydroxylated mini-collagen, termed FC3, which harbors the α2β1 integrin recognition site. FC3 consists of a foldon-stabilized host triple helix of three chains with 10 GPP-repeats, into which the integrin binding motif was inserted. The triple-helical structure could further be stabilized by covalently cross-linking the three chains. Unlike collagen-I, FC3 lacks binding sites for matrix proteins and cellular receptors other than the collagen-binding integrins. It showed a preference for α2β1 over α1β1 integrin, especially when the chains were neither cross-linked nor prolyl-hydroxylated. Using FC3 as substratum for primary skin fibroblasts, we showed that the loss of α2β1 integrin could not be compensated by other collagen-binding integrins, suggesting a major role of α2β1 integrin in exerting sufficient mechanical force to induce or sustain cell spreading. Atomic force microscopy revealed that a single α2β1 integrin can withstand tensile forces of up to approximately 160pN before it releases FC3. Moreover, FC3 is fully competent to agonistically elicit α2β1 integrin-induced cell reactions, such as recruitment of α2β1 integrin into focal adhesions and lamellipodia formation. The biofunctionalized mini-collagen sheds light on the molecular forces of the α2β1 integrin–collagen interaction, which affects tissue homeostasis by contracting the connective tissue and by contributing to interstitial tissue pressure regulation. Additionally, biofunctionalized mini-collagens can be useful in force-resistant cell attachment to biomedical materials.
Agid:
996051