A model of GAG/MIP-2/CXCR2 interfaces and its functional effects

Area of Science:

• Immunology
• Structural Biology
• Biochemistry

Background:

• Chemokines like MIP-2/CXCL2 are crucial for neutrophil chemotaxis.
• Glycosaminoglycans (GAGs) regulate chemokine function by inducing oligomerization.
• Understanding chemokine-receptor-GAG interactions is key to controlling immune responses.

Purpose of the Study:

• To elucidate the structure of MIP-2 and its complex with CXCR2.
• To identify GAG-binding residues on MIP-2 and their role in receptor interaction.
• To investigate the tissue-specific regulation of MIP-2 activity by GAGs.

Main Methods:

• X-ray crystallography to determine MIP-2 structure.
• NMR spectroscopy to identify MIP-2 GAG-binding sites.
• Site-directed mutagenesis to create MIP-2 mutants.
• In vitro neutrophil chemotaxis assays.
• In vivo neutrophil recruitment studies in mouse peritoneum and lung.

Main Results:

• The 1.9 Å structure of MIP-2 was determined, and a model of the MIP-2/CXCR2 complex was created.
• Specific MIP-2 residues interacting with heparin were identified using NMR.
• Mutants lacking GAG-binding ability showed a 10-fold decrease in in vitro neutrophil chemotaxis.
• In vivo, MIP-2 mutants exhibited reduced activity in the lung but not the peritoneum.

Conclusions:

• GAGs play a critical role in modulating MIP-2/CXCL2 mediated neutrophil recruitment.
• The identified GAG-binding residues are essential for MIP-2's in vitro chemotactic activity.
• GAG regulation of chemokine function is demonstrably tissue-dependent, impacting immune cell trafficking.