Multiple low-affinity interactions support binding of human osteopontin to integrin αXβ2

Area of Science:

• Immunology
• Biochemistry
• Molecular Biology

Background:

• Integrin α(X)β(2) (CD11c/CD18) is a myeloid leukocyte receptor known to bind various ligands with polyanionic properties.
• Osteopontin (OPN), an intrinsically disordered protein, mediates phagocytosis via α(X)β(2), but the binding basis is unknown.
• Existing knowledge does not explain how OPN's specific chemical features, beyond general polyanionicity, enable α(X)β(2) binding.

Purpose of the Study:

• To elucidate the molecular basis of osteopontin (OPN) binding by integrin α(X)β(2).
• To identify the specific chemical characteristics of ligands that mediate α(X)β(2) recognition.
• To investigate the role of OPN phosphorylation in α(X)β(2) interaction.

Main Methods:

• Quantitative analysis of monovalent interactions between α(X)β(2) and OPN/fragments/caseins using dissociation constants and apparent stoichiometries.
• Comparison with cell adhesion studies to understand ligand discrimination.
• Synchrotron radiation circular dichroism spectroscopy to assess OPN folding in response to phosphorylation.

Main Results:

• Monovalent interactions are weak (Kd > 10(-5)M), but high apparent stoichiometries drive binding.
• Glutamate side chains, not aspartate, are crucial for discriminating α(X)β(2) ligands.
• OPN phosphorylation does not contribute to α(X)β(2) binding and does not alter OPN's overall folding.

Conclusions:

• Integrin α(X)β(2) recognition relies on high apparent stoichiometry and specific glutamate residues, not OPN phosphorylation.
• This binding mode differs significantly from other OPN receptors, highlighting a unique mechanism.
• The findings provide novel insights into leukocyte integrin-ligand interactions and immune cell function.