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Related Experiment Videos

Alteration in apolipoprotein A-I 22-mer repeat order results in a decrease in lecithin:cholesterol acyltransferase

M G Sorci-Thomas1, L Curtiss, J S Parks

  • 1Department of Comparative Medicine, La Jolla, California 92034, USA. msthomas@isnet.is.bgsm.edu

The Journal of Biological Chemistry
|March 14, 1997
PubMed
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This study investigates how altering apolipoprotein A-I structure affects its function in lipid binding and enzyme activation. Replacing a key repeat region significantly reduced lecithin:cholesterol acyltransferase activation, impacting lipid metabolism.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Lipid Metabolism

Background:

  • Apolipoprotein A-I (apoA-I) contains tandem repeats crucial for lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation.
  • Previous studies showed deleting repeat 6 or 7 drastically reduces LCAT activation.
  • Variations in hydrophobic and amphipathic character exist among apoA-I tandem repeats.

Purpose of the Study:

  • To investigate the functional impact of substituting a hydrophobic repeat into the apoA-I structure.
  • To analyze the biophysical and biochemical properties of a mutant apoA-I (10F6 apoA-I) where repeat 6 is replaced by repeat 10.
  • To understand how structural modifications in apoA-I affect LCAT activity and lipid interactions.

Main Methods:

  • Construction and expression of a mutant apoA-I (10F6 apoA-I) using a baculoviral system.

Related Experiment Videos

  • Purification of recombinant 10F6 apoA-I and formation of lipid-cholesterol complexes.
  • Analysis of complex size using nondenaturing polyacrylamide gel electrophoresis.
  • Biochemical assays to determine LCAT activation kinetics (Vmax/Km).
  • Monoclonal antibody epitope mapping to assess structural changes in lipid-free and lipid-bound apoA-I.

    • Main Results:

    • Recombinant complexes with 10F6 apoA-I showed similar Stokes diameters to wild-type apoA-I complexes.
    • 10F6 apoA-I complexes exhibited a 5-6 fold lower apparent Vmax/apparent Km for LCAT activation compared to wild-type.
    • Epitope mapping confirmed the absence of the repeat 6 domain (residues 143-165) and reduced antibody binding in the 119-144 region of 10F6 apoA-I.

    Conclusions:

    • Substituting repeat 6 with the more hydrophobic repeat 10 in apoA-I significantly impairs LCAT activation.
    • The reduced LCAT reactivity is likely due to increased stabilization within the 121-165 amino acid domain and conformational changes in adjacent repeats.
    • These findings highlight the critical role of specific repeat structures in apoA-I's function in lipid metabolism and LCAT activation.