Proline hydroxylation in collagen supports integrin binding by two distinct mechanisms

Kalle H Sipilä,Kati Drushinin,Pekka Rappu,Johanna Jokinen,Tiina A Salminen,Antti M Salo,Jarmo Käpylä,Johanna Myllyharju,Jyrki Heino

doi:10.1074/jbc.ra118.002200

Abstract

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.

Highlights

Collagens are the most abundant extracellular matrix proteins in vertebrates and have a characteristic triple-helix structure
We report that hydroxyprolines generated by Collagen prolyl 4-hydroxylases (C-P4Hs) can affect integrin binding by two distinct mechanisms: stabilization of the collagenous helix at body temperature and by strengthening the direct interaction between specific integrin ␣I domains
We could not detect any significant differences between binding to skin collagen derived from the WT mice (n ϭ 5) and collagen from P4ha1ϩ/Ϫ;P4ha2Ϫ/Ϫ mice (n ϭ 6), when the experiments were performed at room temperature (Fig. 1A)

Summary

Present address

Collagens are the most abundant extracellular matrix proteins in vertebrates. They have a characteristic triple-helical structure that is composed of three collagen ␣ chains. C-P4H-I and C-P4H-II are likely to have distinct differences in their substrate-binding sites because the Km values of C-P4H-II for synthetic collagen-like (Pro-Pro-Gly) and Gly-Val-Pro-Gly-Val peptides and a full-length procollagen chain are 3– 6-fold higher than in the case of C-P4H-I [5, 6]. Studies with synthetic peptide substrates have shown that the rate of hydroxylation of a proline residue by C-P4H-I is affected by the nature of amino acids in the X position and in other parts of the peptide substrate [3]. Studies with gene-modified mice have shown that homozygous inactivation of the P4ha gene, which encodes the catalytic ␣(I) subunit of the main isoenzyme, C-P4H-I, leads to an 80% decrease in total C-P4H activity and

The abbreviations used are

Results

Discussion

Experimental procedures