Binding of ApoA-I Mutants (L144R, L159P) to Haptoglobin.

INTRODUCTION: ApoA-1, the major apolipoprotein of the high density lipoprotein (HDL), plays a key role in the removal of cholesterol excess from peripheral cells in a recognized anti-atherosclerotic process called “reverse cholesterol transport (RCT)”. Among known ApoA-1 variants two, with one amino acid change (Zavalla, L159P; Zaragoza, L144R), are associated to poor RCT. Both mutations are located within the domain of ApoA1 (Leu141 – Ala164) binding Haptoglobin (Hpt) 1, a protein of the acute phase of inflammation. Hpt binding to ApoA1 was suggested to impair the role of HDL in RCT1. AIMS: Our aims were to study whether peptides containing the mutations of Zavalla (Pep Zav) or Zaragoza (Pep Zara) are able to bind Hpt or compete with native ApoA1, exposed on HDL, for binding Hpt. METHODS: The binding of biotinylated peptides to Hpt and the experiments of competition of acetylated peptides with HDL for Hpt binding were performed by ELISA, as previously published1. RESULTS: Both Pep-Zav and Pep-Zara bound to Hpt with efficiency similar to that of the peptide harbouring the native sequence of ApoA1 (pep 141–164). Nevertheless, when the same peptides were analysed for their ability to influence Hpt binding to HDL, only Pep Zav effectively displaced (50% at 15μM) Hpt from HDL (fig), as was the case for the pep. 141-164. CONCLUSIONS: We suggest that the L>P conversion, in the Zavalla variant, enhances hydrophilicity in ApoA1, improving the binding to Hpt. Therefore, as ApoA-1 binding to Hpt negatively affects RCT, individuals with Zavalla mutation might be exposed to higher risk of cardiovascular disease. 1.Spagnuolo M.S. et al (2005) J. Biol. Chem. 280, 1193-1198