Proline hydroxylation in collagen supports integrin binding by two distinct mechanisms

Area of Science:

• Biochemistry
• Structural Biology
• Cell Biology

Background:

• Collagens are vital extracellular matrix proteins with a triple-helix structure.
• Proline hydroxylation is essential for collagen stability and connective tissue integrity.
• The impact of proline hydroxylation on collagen-integrin interactions remains largely unknown.

Purpose of the Study:

• To investigate the role of proline hydroxylation in collagen binding to integrin receptors.
• To determine how reduced prolyl hydroxylase activity affects integrin-mediated collagen interactions.

Main Methods:

• Isolation of skin collagen from genetically modified mice with reduced prolyl 4-hydroxylase activity.
• Recombinant integrin α2I domain binding assays at varying temperatures.
• LC-MS/MS analysis of collagen hydroxyproline content.
• Binding assays using integrin α1β1 expressed on CHO cells and recombinant α1I domain.
• Structural modeling and site-directed mutagenesis (R218D) of integrin α1I.

Main Results:

• Reduced proline hydroxylation did not affect integrin α2I binding at room temperature but decreased avidity at 37 °C.
• LC-MS/MS showed no significant change in hydroxyproline at main integrin-binding sites, suggesting structural destabilization.
• Integrin α1β1 and α1I domain exhibited weaker and slower binding to non-hydroxylated collagen motifs (GFPGER) compared to hydroxylated ones (GFOGER).
• Structural modeling identified a key interaction between Arg-218 in α1I and hydroxyproline, validated by mutagenesis.

Conclusions:

• Collagen proline hydroxylation influences integrin binding through direct interactions and by maintaining triple-helix structural stability.
• Reduced hydroxylation can impair integrin binding, potentially contributing to connective tissue defects.
• Specific integrin-hydroxyproline interactions, like those involving α1I Arg-218, are critical for binding affinity.