Molecular modeling of the dimeric structure of human lipoprotein lipase and functional studies of the carboxyl‐terminal domain

Abstract

Lipoprotein lipase (LPL) plays a key role in lipid metabolism. Molecular modeling of dimeric LPL was carried out using INSIGHT II based upon the crystal structures of human, porcine, and horse pancreatic lipase. The dimeric model reveals a saddle-shaped structure and the key heparinbinding residues in the amino-terminal domain located on the top of this saddle. The models of two dimeric conformations -a closed, inactive form and an open, active form -differ with respect to how surface-loop positions affect substrate access to the catalytic site. In the closed form, the surface loop covers the catalytic site, which becomes inaccessible to solvent. Large conformational changes in the open form, especially in the loop and carboxyl-terminal domain, allow substrate access to the active site. To dissect the structure-function relationships of the LPL carboxylterminal domain, several residues predicted by the model structure to be essential for the functions of heparin binding and substrate recognition were mutagenized. Arg405 plays an important role in heparin binding in the active dimer. Lys413/Lys414 or Lys414 regulates heparin affinity in both monomeric and dimeric forms. To evaluate the prediction that LPL forms a homodimer in a Ôhead-to-tailÕ orientation, two inactive LPL mutants -a catalytic site mutant (S132T) and a substrate-recognition mutant (W390A/W393A/ W394A) -were cotransfected into COS7 cells. Lipase activity could be recovered only when heterodimerization occurred in a head-to-tail orientation. After cotransfection, 50% of the wild-type lipase activity was recovered, indicating that lipase activity is determined by the interaction between the catalytic site on one subunit and the substraterecognition site on the other.Keywords: lipoprotein lipase; dimeric model structure; heparin binding; substrate recognition; catalytic activity.Lipoprotein lipase (LPL) belongs to a mammalian lipase family that includes pancreatic lipase (PL), hepatic lipase (HL), gastric lipase, and endothelial lipase [1,2]. The primary function of LPL is triglyceride hydrolysis in triglyceride-rich lipoproteins, such as chylomicron and very low density lipoprotein (VLDL) particles, which are converted to remnants. LPL is secreted from a variety of tissues, such as adipocyte, macrophage, and muscle cells, and is bound to the capillary bed of endothelium via cellular surface heparan sulfate proteoglycans (HSPG), a function reflected in LPL's strong affinity for heparin. LPL deficiencies in humans are manifested as severe hypertriglyceridemia [3-5] and arteriosclerosis [6]. Genetically engineered mice lacking LPL also exhibit hypertriglyceridemia. In addition to lipolytic activity, LPL functions as a ligand for lipoprotein receptors, such as low density lipoprotein (LDL) receptor, LDL receptor related protein (LRP), GP330/ LRP-2, and VLDL receptor [7][8][9][10][11].A model structure of LPL had previously been constructed, based on the crystal structure of human PL as a template [12]. The model structure exhibited two domainsa large N-t...

Angiopoietin-like Protein 4 Inhibition of Lipoprotein Lipase

“…There is no crystal structure for LPL, but due to high sequence similarity with pancreatic lipase, its crystal structure can serve as a model to provide information about the structure-function relationships that govern LPL activity (9,10). The LPL protein is composed of two structurally distinct domains that are connected by a short linker.…”

“…The LPL protein is composed of two structurally distinct domains that are connected by a short linker. The N-terminal domain includes the active site, and homology modeling suggests that it takes on an ␣/␤-hydrolase fold (9). The C-terminal domain is modeled as a ␤-sandwich and is responsible for binding the cell surface protein glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) (9 -11).…”

Isolation and Characterization of Low Sulfated Heparan Sulfate Sequences with Affinity for Lipoprotein Lipase

“…Models of the LPL dimer in complex with heparin have been proposed (15,19). Yet the exact combination of the dimer with its oligosaccharide ligand awaits further refinement.…”

“…In LPL, four clusters of basic amino acids are exposed on the opposite side of the molecule compared with the entrance to the catalytic site, and at least two, located in the N-terminal domain, seem to be important for HS binding (14,16,17). Participation of positive regions also in the C-terminal domain of LPL have been demonstrated (17)(18)(19). Heparin fragments of 8 -10 monomer units have been shown to bind to LPL and to displace the enzyme from endothelial sites, from synthetic heparin-covered surfaces and from surfaces covered by lipoprotein receptors (20 -22).…”

“…4D). A loss of a sulfate group shifted the elution position of an oligosaccharide considerably (15)(16)(17)(18)(19)(20) min in the applied gradient), while the loss of an uronic acid residue had much less impact on the elution behavior of the remaining fragment due to the protonated form of the carboxyl groups at the pH used. All fragments that originally contained a 2-O-sulfated uronic acid residue in the nonreducing end shifted position (indicated by "-2S" appended to the original peak identification) as compared with the deaminated sample in Fig.…”