Biosynthesis of human preapolipoprotein A-IV

Area of Science:

• Molecular Biology
• Biochemistry
• Lipid Metabolism

Background:

• Apolipoprotein A-IV (apoA-IV) plays a role in lipid transport, but its synthesis and processing are not fully understood.
• Previous studies suggested apoA-IV might undergo post-translational modifications similar to other apolipoproteins.

Purpose of the Study:

• To elucidate the primary translation product and processing of human intestinal apolipoprotein A-IV mRNA.
• To compare the N-terminal sequence of synthesized apoA-IV with mature, chylomicron-associated apoA-IV.
• To investigate the association of apoA-IV and apoA-I with lipoproteins in Hep G2 cells.

Main Methods:

• Purification of primary translation product from ascites and wheat germ cell-free systems.
• NH2-terminal sequencing via Edman degradation.
• Agarose gel chromatography of Hep G2 culture media.
• Incubation of plasma with Hep G2 media to assess lipoprotein transfer.

Main Results:

• Human apoA-IV is synthesized with a 20-amino acid N-terminal signal peptide, which is co-translationally cleaved.
• The N-terminal sequence of the cell-free product matches the mature, chylomicron-associated apoA-IV, indicating no further N-terminal processing.
• Neither apoA-IV nor apoA-I are secreted from Hep G2 cells in association with particles resembling plasma lipoproteins.
• ApoA-I, but not apoA-IV, transfers to lipoproteins when plasma is incubated with Hep G2 media.

Conclusions:

• The 20-amino acid N-terminal extension of apoA-IV functions as a signal peptide and is removed during translation.
• ApoA-IV does not undergo N-terminal post-translational proteolysis, unlike proapoA-II or proapoA-I.
• The interaction of apoA-IV with lipoproteins is not regulated by N-terminal proteolytic processing.