Processing of rat liver apoprotein E primary translation product

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Apolipoprotein E (apoE) plays a crucial role in lipid metabolism and transport.
• Understanding the synthesis and post-translational modifications of apoE is essential for elucidating its function.

Purpose of the Study:

• To characterize the primary translation product of rat liver apoE mRNA.
• To investigate the processing and potential glycosylation of rat apoE.

Main Methods:

• Wheat germ cell-free translation system to synthesize rat apoE.
• SDS-polyacrylamide gel electrophoresis and partial proteolytic peptide mapping for protein comparison.
• Heparin-Sepharose chromatography to assess binding properties.
• Analysis of microsome-processed product for oligosaccharide content.

Main Results:

• The primary translation product of rat apoE mRNA was isolated and characterized.
• Rat apoE is initially synthesized with an 18 amino acid N-terminal extension.
• This extension is cotranslationally removed by microsomes with signal peptidase activity.
• The processed rat apoE lacks endoglycosidase H-sensitive oligosaccharides, indicating O-glycosylation.

Conclusions:

• Rat apoE is synthesized as a precursor with an N-terminal signal peptide.
• Signal peptidase activity in microsomes removes the signal peptide cotranslationally.
• Rat apoE is likely O-glycosylated rather than N-glycosylated.