High Affinity Binding between Lipoprotein Lipase and Lipoproteins Involves Multiple Ionic and Hydrophobic Interactions, Does Not Require Enzyme Activity, and Is Modulated by Glycosaminoglycans

M Mahmood Hussain,M Jawaad Hussain,Joseph C Obunike,Afsar Shaheen,Gregory S Shelness,Ira J Goldberg

doi:10.1074/jbc.m005317200

M Mahmood Hussain, M Jawaad Hussain + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m005317200

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Abstract

Lipoprotein lipase (LPL) physically associates with lipoproteins and hydrolyzes triglycerides. To characterize the binding of LPL to lipoproteins, we studied the binding of low density lipoproteins (LDL), apolipoprotein (apo) B17, and various apoB-FLAG (DYKDDDDK octapeptide) chimeras to purified LPL. LDL bound to LPL with high affinity (K(d) values of 10(-12) m) similar to that observed for the binding of LDL to its receptors and 1D1, a monoclonal antibody to LDL, and was greater than its affinity for microsomal triglyceride transfer protein. LDL-LPL binding was sensitive to both salt and detergents, indicating the involvement of both hydrophobic and hydrophilic interactions. In contrast, the N-terminal 17% of apoB interacted with LPL mainly via ionic interactions. Binding of various apoB fusion peptides suggested that LPL bound to apoB at multiple sites within apoB17. Tetrahydrolipstatin, a potent enzyme activity inhibitor, had no effect on apoB-LPL binding, indicating that the enzyme activity was not required for apoB binding. LDL-LPL binding was inhibited by monoclonal antibodies that recognize amino acids 380-410 in the C-terminal region of LPL, a region also shown to interact with heparin and LDL receptor-related protein. The LDL-LPL binding was also inhibited by glycosaminoglycans (GAGs); heparin inhibited the interactions by approximately 50% and removal of trace amounts of heparin from LPL preparations increased LDL binding. Thus, we conclude that the high affinity binding between LPL and lipoproteins involves multiple ionic and hydrophobic interactions, does not require enzyme activity and is modulated by GAGs. It is proposed that LPL contains a surface exposed positively charged amino acid cluster that may be important for various physiological interactions of LPL with different biologically important molecules. Moreover, we postulate that by binding to this cluster, GAGs modulate the association between LDL and LPL and the in vivo metabolism of LPL.

Highlights

Lipoprotein lipase (LPL) physically associates with lipoproteins and hydrolyzes triglycerides
low density lipoproteins (LDL) bound with similar affinity to LPL and 1D1. These studies indicated that the LDL-LPL binding involves high affinity interactions that are greater than that found for MTP and are similar to the binding of LDL to its monoclonal antibody
The high affinity binding between LDL and LPL is due to both hydrophobic and hydrophilic interactions between these molecules

Summary

EXPERIMENTAL PROCEDURES

Materials—Bovine milk LPL was purified using heparin affinity chromatography as described before [14]. ELISA plates were coated with 0.5 ␮g of purified, homodimeric bovine LPL in 100 ␮l of 0.05 M sodium borate, pH 9.5, in triplicate wells by incubating at 4 °C for 18 h. A standard curve for the apoB was generated by coating wells with a monoclonal antibody, 1D1, and incubating with different concentrations of LDL (0 –14 ng/well) as described before [15, 16]. Binding of Different apoB-FLAG Chimeras to LPL—COS-7 cells were transiently transfected with various FLAG chimeras (1 ␮g DNA/ml) containing C-terminally truncated apoB cDNAs using Fugene-6 transfection reagent (Roche Molecular Biochemicals) and conditioned media (72 h) were used to study the binding of different secreted apoB polypeptides to immobilized LPL as described above [12]. The molecular masses used for LDL, LPL, Triton X102, and heparin were 512, 120, 0.757, and 16.5 kDa, respectively

RESULTS

LPL binding

DISCUSSION