Interaction of lipoprotein lipase with heparin fragments and with heparan sulfate: Stoichiometry, stabilization, and kinetics

The interaction of lipoprotein lipase (LPL) with heparan sulfate and with size-fractionated fragments of heparin was characterized by several approaches (stabilization, sedimentation, surface plasmon resonance, circular dichroism, fluorescence). The results show that heparin decasaccharides form a 1:1 complex with dimeric LPL and that decasaccharides are the shortest heparin fragments which can completely stabilized dimeric LPL, while shorter fragments (hexa- and tetrasaccharides) were less efficient. Binding of heparin did not induce major rearrangements in the conformation of LPL, supporting the view that the heparin binding region is preformed in the native structure, Interaction of LPL with heparan sulfate, as studied by surface plasmon resonance, was found to be a fast exchange process characterized by a high value for the association rate constant, 1.7 x 10(8) M(-1) s(-1), a relatively high dissociation rate constant, 0.05 s(-1), and as a result a very low equilibrium dissociation constant equal to 0.3 nM at 0.15 M NaCl, The contribution of electrostatics was estimated to be 44% for the binding of LPL to heparan sulfate, 49% for the binding of LPL to unfractionated heparin, and 60% for the binding of LPL to affinity-purified heparin decasaccharides at 0.15 M NaCl. The number of ionic interactions between LPL and high affinity decasaccharides was estimated to be 10, We propose an essential role of electrostatic steering in the association, Monomeric LPL had 6000-fold lower affinity for heparin than dimeric LPL had, expressed as a ratio of equilibrium dissociation constants. A model for binding of LPL to heparan sulfate-covered surfaces is proposed. Due to the fast rebinding, LPL is concentrated to the close proximity of the heparan sulfate surface, As the dissociation is also fast, the enzyme exchanges rapidly between specific binding sites on the immobilized heparan sulfate, without leaving the surface. This model may also apply to LPL at the endothelium of blood vessels.