Biofunctionalization of a generic collagenous triple helix with the α2β1 integrin binding site allows molecular force measurements.
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.
Excellence Cluster Cardio-Pulmonary System, Frankfurt University Hospital, Frankfurt/Main, Germany.
Stephan
S
Niland
Christoph
C
Westerhausen
Stefan W
SW
Schneider
Beate
B
Eckes
Matthias F
MF
Schneider
Johannes A
JA
Eble
D000818:Animals; D001665:Binding Sites; D001696:Biomechanical Phenomena; D002417:Cattle; D045744:Cell Line, Tumor; D024042:Collagen Type I / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D038982:Integrin alpha2beta1 / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid
D024042:Collagen Type I; D038982:Integrin alpha2beta1; D011994:Recombinant Proteins