Cellular uptake of C4b-binding protein is mediated by heparan sulfate proteoglycans and CD91/LDL receptor-related...

Area of Science:

• Immunology
• Biochemistry
• Cell Biology

Background:

• C4b-binding protein (C4BP) is a key complement inhibitor and carrier for protein S.
• Previous studies identified CD91 as a receptor involved in C4BP's in vivo clearance.
• A CD91-interactive site on C4BP alpha-chains was found to overlap its heparin-binding site.

Purpose of the Study:

• To further elucidate the molecular mechanisms underlying the C4BP-CD91 interaction.
• To investigate the role of specific residues and heparin-sulfate proteoglycans (HSPG) in C4BP cellular uptake.

Main Methods:

• Site-directed mutagenesis of cationic residues in C4BP alpha-chains.
• Binding affinity assays (Kd determination) for wild-type and mutant C4BP to CD91.
• Cellular degradation assays using CD91-expressing and deficient cells.
• In vivo clearance experiments in mice.
• Experiments assessing C4BP binding and degradation in the presence and absence of HSPG.

Main Results:

• Mutagenesis of cationic residues (R39, R64, R66) in C4BP alpha-chains significantly reduced CD91 binding affinity (20-fold decrease).
• The triple mutant C4BPalpha/R39Q-R64Q-R66Q showed reduced intracellular degradation by CD91-expressing cells and prolonged in vivo survival (3-fold).
• Absence of HSPG nearly abolished C4BP cell binding and intracellular degradation, indicating a critical role for HSPG in C4BP endocytosis.

Conclusions:

• Cellular uptake and degradation of C4BP are dependent on both CD91 and HSPG.
• Positively charged residues within the C4BP alpha-chain are essential for interaction with both HSPG and CD91.
• These findings reveal a dual mechanism for C4BP endocytosis involving electrostatic interactions with cell surface proteoglycans and the CD91 receptor.