Tryptic peptide analysis of the human apolipoprotein E isomorphs

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Apolipoprotein E (apo E) is crucial for lipid metabolism and is found in very low density lipoproteins (VLDL).
• Isoforms of apo E vary in individuals and can be associated with lipid disorders like type III hyperlipoproteinemia.
• Understanding the structural basis of apo E isoforms is key to elucidating their function and role in disease.

Purpose of the Study:

• To investigate the polypeptide backbone differences among human apo E isoforms.
• To compare the apo E profiles in normal individuals versus those with homozygous type III hyperlipoproteinemia.
• To identify the molecular basis of apo E isomorphism.

Main Methods:

• Tryptic cleavage fingerprinting of isolated apo E isoforms.
• Specific chemical modification studies to probe amino acid residues.
• High-performance liquid chromatography (HPLC) for peptide profile analysis.
• Polyacrylamide isoelectric focusing (IEF) for apo E isoform separation.

Main Results:

• Normal apo E resolved into five isoforms (E-I', E-I, E-II, E-III, E-IV) by IEF.
• Type III hyperlipoproteinemic apo E resolved into three isoforms (E-I3', E-I3, E-II3).
• HPLC confirmed peptide identity within normal isoforms (E-I', E-I, E-II) and type III isoforms (E-I3', E-I3, E-II3).
• Distinct HPLC profiles were observed for apo E-II, E-III, E-IV, and E-II3, indicating structural differences.
• Chemical modification indicated that apo E isomorphism is partly due to differences in arginine and cysteine residues, not lysine.

Conclusions:

• Human apo E isomorphism arises from primary amino acid sequence variations and charged carbohydrate heterogeneity.
• The apo E profile in homozygous type III hyperlipoproteinemia shows a deficiency of E-III and E-IV, and an altered E-II isoform (E-II3).
• These structural differences in apo E isoforms contribute to the pathophysiology of hyperlipoproteinemia.