Sipilä, K., Drushinin, K., Rappu, P., Jokinen, J., Salminen, T., Salo, A., Käpylä, J., Myllyharju, J., Heino, J. (2018) Proline hydroxylation in collagen supports integrin binding by two distinct mechanisms. Journal of Biological Chemistry, 293 (20), 7645-7658. doi:10.1074/jbc.RA118.002200
Proline hydroxylation in collagen supports integrin binding by two distinct mechanisms
|Author:||Sipilä, Kalle H.1; Drushinin, Kati2; Rappu, Pekka1;|
1Department of Biochemistry, University of Turku
2Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu
3Structural Bioinformatics Laboratory, Biochemistry, Faculty of Science and Engineering, Åbo Akademi University
|Online Access:||PDF Full Text (PDF, 2.8 MB)|
|Persistent link:|| http://urn.fi/urn:nbn:fi-fe201902225964
American Society for Biochemistry and Molecular Biology,
|Publish Date:|| 2019-04-03
Collagens are the most abundant extracellular matrix proteins in vertebrates and have a characteristic triple-helix structure. Hydroxylation of proline residues is critical for helix stability, and diminished prolyl hydroxylase activity causes wide-spread defects in connective tissues. Still, the role of proline hydroxylation in the binding of collagen receptors such as integrins is unclear. Here, we isolated skin collagen from genetically modified mice having reduced prolyl 4-hydroxylase activity. At room temperature, the reduced proline hydroxylation did not affect interactions with the recombinant integrin α2I domain, but at 37 °C, collagen hydroxylation correlated with the avidity of α2I domain binding. Of note, LC–MS/MS analysis of isolated skin collagens revealed no major changes in the hydroxyproline content of the main integrin-binding sites. Thus, the disrupted α2I domain binding at physiological temperatures was most likely due to structural destabilization of the collagenous helix. Integrin α2I binding to the triple-helical GFPGER motif was slightly weaker than to GFOGER (O = hydroxyproline). This phenomenon was more prominent when α1 integrin was tested. Integrin α1β1 expressed on CHO cells and recombinant α1I domain showed remarkably slower binding velocity and weaker avidity to GFPGER when compared with GFOGER. Structural modeling revealed the critical interaction between Arg-218 in α1I and the hydroxyproline residue in the integrin-binding motif. The role of Arg-218 was further validated by testing a variant R218D α1I domain in solid-phase binding assays. Thus, our results show that the lack of proline hydroxylation in collagen can affect integrin binding by a direct mechanism and via structural destabilization of the triple helix.
Journal of biological chemistry
|Pages:||7645 - 7658|
|Type of Publication:||
A1 Journal article – refereed
|Field of Science:||
1182 Biochemistry, cell and molecular biology
This work was supported by Academy of Finland Project Grants 259769 (to J. H.) and 296498 (to J. M.), Academy of Finland Center of Excellence 2012–2017 Grant 251314 (to J. M.), grants from the Sigrid Jusélius Foundation (to J. H., J. M., and T. A. S.), the Jane and Aatos Erkko Foundation (to J. M.), Tor, Joe, and Pentti Borg's Foundation (to T. A. S.), and the Turku Doctoral Programme of Biomedical Science. J. M. owns equity in FibroGen Inc., which develops hypoxia-inducible factor P4H inhibitors as potential therapeutics. This company supports hypoxia response research in the J. M. group.
|Academy of Finland Grant Number:||
296498 (Academy of Finland Funding decision)
251314 (Academy of Finland Funding decision)
This research was originally published in the Journal of Biological Chemistry. Kalle H. Sipilä, Kati Drushinin, Pekka Rappu, Johanna Jokinen, Tiina A. Salminen, Antti M. Salo, Jarmo Käpylä, Johanna Myllyharju, and Jyrki Heino. Proline hydroxylation in collagen supports integrin binding by two distinct mechanisms. J. Biol. Chem. 2018; 293:7645-7658. © the Author(s).