Human apolipoprotein A-I. Post-translational modification by fatty acid acylation

Area of Science:

• Biochemistry
• Molecular Biology
• Lipid Metabolism

Background:

• Human apolipoproteins are secreted proteins, some undergoing proteolytic and carbohydrate modifications.
• Apolipoprotein A-I (apo-A-I), central to high-density lipoproteins, experiences co-translational processing and post-translational conversion.
• Previous research indicated acylation primarily in cellular membrane proteins.

Purpose of the Study:

• To investigate post-translational modifications of secreted apolipoprotein A-I.
• To determine if apolipoprotein A-I undergoes fatty acid acylation.
• To explore the implications of lipid-protein covalent linkage in apolipoprotein metabolism.

Main Methods:

• Utilized the human hepatoma cell line HEP-G2 for studying secreted proteins.
• Incorporated radiolabeled [14C]palmitate into apo-A-I, analyzed via SDS-PAGE and autoradiography.
• Confirmed acylation using immunoprecipitation, gas chromatography/mass spectrometry, and hydroxylamine treatment for deacylation.

Main Results:

• Secreted nascent apo-A-I was acylated with palmitate, confirmed by multiple analytical techniques.
• 80% of acylated apo-A-I was found in the proapolipoprotein A-I isoform; oleate was not incorporated.
• Palmitate acylation persisted even after conversion to mature apo-A-I in serum and plasma.

Conclusions:

• Established fatty acid acylation as a modification of human secretory proteins, specifically apo-A-I.
• Demonstrated that palmitoylation of apo-A-I is a specific and stable post-translational modification.
• Suggests that lipid covalent linkage to apolipoproteins is crucial for apolipoprotein and lipoprotein metabolism.