Localization of the heparin binding site in human apolipoprotein AV

Abstract

Apolipoprotein (apo) A-V is a potent modulator of plasma triacylglycerol (TG) levels. The molecular basis for this activity is not understood. Recent studies have shown that apoA-V possesses heparin binding activity. A stretch of 42 amino acids (residue 186 to 227) in apoA-V has been identified by sequence analysis to contain 5 arginines, 3 lysines, 4 histidines and no negatively charged amino acids. This segment is hypothesized to be the heparin binding region of apoA-V. To test this, site directed mutagenesis was used to generate three mutations in this region, in which positively charged amino acids were replaced by either negatively charged or uncharged polar amino acids. The resulting mutatants (R210E/K211Q-apoA-V, K215Q/K217E-apoA-V, and R210E/K211Q/K215Q/K217E-apoA-V) were complexed with dimyristoylphosphatidylcholine (DMPC) and subjected to heparin Sepharose affinity chromatography and surface plasmon resonance spectroscopy using a heparin-coated sensor chip. With both methods of analysis, compared to WT apoA-V DMPC complexes, the mutants showed decreased affinity for heparin. These results show that positively charged residues between position 186 and 227 of human apoA-V contribute to heparin binding. This region of apoA-V may play an important role in cell surface interactions by binding with heparan sulfate proteogylcans, indirectly affecting lipoprotein lipase activity, possibly contributing to apoA-V’s ability to influence plasma TG levels. Supported by NIH.