Formation of mitogenically active PDGF-B dimer does not require interchain disulfide bonds

Area of Science:

• Molecular Biology
• Protein Chemistry
• Cell Signaling

Background:

• Platelet-Derived Growth Factor (PDGF) is a mitogen for mesenchymal cells, existing as disulfide-bonded dimers (AA, BB, AB).
• The dimeric structure was presumed essential for PDGF's biological activity.
• Reduction of PDGF dimers leads to loss of activity and monomer formation, questioning the role of specific disulfide bonds.

Purpose of the Study:

• To investigate the role of intrachain versus interchain disulfide bonds in PDGF-B activity and dimerization.
• To determine if disulfide bonds are critical for PDGF's correct protein conformation and biological function.

Main Methods:

• Site-directed mutagenesis of PDGF-B to alter cysteine residues involved in disulfide bonds.
• Analysis of mitogenic activity using cell proliferation assays.
• SDS-polyacrylamide gel electrophoresis to assess monomeric/dimeric state.
• Light scattering and sedimentation equilibrium to determine molecular weight and dimerization state.
• Disulfide bond analysis to confirm bond formation.

Main Results:

• Mutants lacking all or key cysteines showed reduced dimerization and significantly less mitogenic activity.
• A mutant (PDGF-B(C2,4S)) lacking only interchain disulfide bonds retained significant mitogenic activity, forming noncovalent dimers at physiological pH.
• This mutant's activity was sensitive to low pH, dissociating into monomers, unlike wild-type PDGF-BB.

Conclusions:

• Intrachain disulfide bonds are critical for maintaining the correct conformation of PDGF monomers.
• Properly folded PDGF monomers can form active noncovalent dimers even without interchain disulfide bonds.
• Interchain disulfide bonds primarily enhance PDGF dimer stability rather than being essential for its existence or activity.