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Alteration in apolipoprotein a-i 22-mer repeat order results in a decrease in lecithin:Cholesterol acyltransferase reactivity

Academic Article
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Overview

authors

  • SorciThomas, M. G.
  • Curtiss, Linda
  • Parks, J. S.
  • Thomas, M. J.
  • Kearns, M. W.

publication date

  • 1997

journal

  • Journal of Biological Chemistry  Journal

abstract

  • Apolipoprotein A-I contains eight 22-amino acid and two 11-amino acid tandem repeats that comprise 80% of the mature protein. These repeating units are believed to be the basic motif responsible for lipid binding and lecithin:cholesterol acyltransferase (LCAT) activation. Computer analysis indicates that despite a fairly high degree of compositional similarity among the tandem repeats, significant differences in hydrophobic and amphipathic character exist. Our previous studies demonstrated that deletion of repeat 6 (143-164) or repeat 7 (165-186) resulted in a 98-99% reduction of LCAT activation as compared with wild-type apoA-I. To determine the effects of substituting one of these repeats with a more hydrophobic repeat we constructed a mutant apoA-I protein in which residues 143-164 (repeat 6) were replaced with repeat 10 (residues 220-241). The cloned mutant protein, 10F6 apoA-I, was expressed and purified from an Sf-9 cell baculoviral system and then analyzed using a number of biophysical and biochemical techniques. Recombinant complexes prepared at a 100:5:1 molar ratio of L-alpha-dimyristoylphosphatidylcholine:cholesterol:wild-type or 10F6 apoA-I showed a doublet corresponding to Stokes diameters of 114 and 108 A on nondenaturing 4-30% polyacrylamide gel electrophoresis. L-alpha-Dimyristoylphosphatidylcholine 10F6 apoA-I complexes had a 5-6-fold lower apparent Vmax/apparent Km as compared with wild-type apoA-I containing particles. As expected, monoclonal antibody epitope mapping of the lipid-free and lipid-bound 10F6 apoA-I confirmed that a domain expressed between residues 143 and 165 normally found in wild-type apoA-I was absent. The region between residues 119 and 144 in 10F6 apoA-I showed a marked reduction in monoclonal antibody binding capacity. Therefore, we speculate that the 5-6-fold lower LCAT reactivity in 10F6 compared with wild-type apoA-I recombinant particles results from increased stabilization within the 121-165 amino acid domain due to more stable apoprotein helix phospholipid interactions as well as from conformational alterations among adjacent amphipathic helix repeats.

subject areas

  • Amino Acid Sequence
  • Apolipoprotein A-I
  • Enzyme Activation
  • Humans
  • Molecular Sequence Data
  • Phosphatidylcholine-Sterol O-Acyltransferase
  • Recombinant Proteins
  • Repetitive Sequences, Nucleic Acid
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

PubMed ID

  • 9054424
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Additional Document Info

start page

  • 7278

end page

  • 7284

volume

  • 272

issue

  • 11

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