Identifying Function Determining Residues in Neuroimmune Semaphorin 4A.

Area of Science:

• Immunology
• Molecular Biology
• Bioinformatics

Background:

• Semaphorin 4A (Sema4A) stabilizes human regulatory T (Treg) cells via Plexin B1 engagement.
• Sema4A-deficient mice show increased allergic airway inflammation and fewer Treg cells.
• Sema4D-deficient mice exhibit reduced inflammation and more Treg cells, despite both binding Plexin B1.

Purpose of the Study:

• To compare the in vitro effects of Sema4A and Sema4D on human Treg cells.
• To identify critical residues in Sema4A for Plexin B1 binding using homology modeling based on Sema4D.

Main Methods:

• In vitro PBMC cultures to assess Sema4A and Sema4D effects on Treg cells.
• Homology modeling and Bayesian Partitioning with Pattern Selection (BPPS) for sequence analysis.
• Site-directed mutagenesis of Sema4A residues (M198A, F223A) and assessment of Plexin B1 binding and Treg activity.

Main Results:

• Sema4A and Sema4D exhibited opposing effects on human Treg cells in vitro; Sema4D inhibited Treg cell numbers and Foxp3 expression.
• Sema4A and Sema4D competitively bind to Plexin B1.
• Mutating Sema4A residue F223 to Alanine (F223A) abolished Treg stabilization activity, despite intact Plexin B1 binding.

Conclusions:

• Sema4A and Sema4D have distinct functional roles in Treg cell regulation, mediated by differential binding to Plexin B1.
• Specific residues, like F223 in Sema4A, are critical for Treg cell stabilization function.
• This study provides a foundation for developing engineered Sema4A variants as potential immunotherapeutics for conditions like asthma.