Structure-function studies of human cholesteryl ester transfer protein by linker insertion scanning mutagenesis.

Area of Science:

• Biochemistry
• Molecular Biology
• Lipid Metabolism

Background:

• Human plasma cholesteryl ester transfer protein (CETP) facilitates the exchange of cholesteryl ester (CE) and triglyceride (TG) between lipoproteins.
• Understanding the molecular regions responsible for CETP's neutral lipid transfer activities is crucial for elucidating its function.

Purpose of the Study:

• To identify specific molecular regions within CETP that are essential for its cholesteryl ester (CE) and triglyceride (TG) transfer activities.
• To investigate the structural requirements for neutral lipid transfer by CETP.

Main Methods:

• Construction and expression of 27 linker insertion mutants at 18 sites in the CETP molecule in a mammalian cell line (COS).
• Assay of cholesteryl ester (CE) and triglyceride (TG) transfer activities of the expressed mutant CETP proteins.
• Hydrophobicity analysis and secondary structure predictions to assess protein folding.

Main Results:

• Most CETP mutants exhibited normal cholesteryl ester transfer activity, with reduced secretion.
• Insertions in three specific regions (amino acids 48-53, 165, and 373-379) severely impaired CE transfer activity.
• TG transfer activity was similarly affected, suggesting shared structural requirements for both lipid transfer activities.

Conclusions:

• Specific regions of CETP, particularly around amino acids 48-53, 165, and 373-379, are critical for its neutral lipid transfer functions.
• The region near amino acid 379 may be involved in essential steps like binding to phosphatidylcholine on lipoprotein surfaces.
• CETP likely possesses similar structural requirements for both CE and TG transfer activities.