Functional topology of a surface loop shielding the catalytic center in lipoprotein lipase

Faustinella, Fabrizia; Smith, Louis C.; Chan, Lawrence

doi:10.1021/bi00147a002

Cited by 40 publications

(18 citation statements)

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“…Ser 267 , although not the acyl-accepting serine residue within the catalytic site, is nonetheless highly conserved, being found in all members of the lipase gene family in all species. 31 Faustinella et al 32 demonstrated that mutagenesis of this serine to alanine in lipoprotein lipase resulted in a protein that was expressed but had greatly reduced activity. Indeed, Ser 267 is adjacent to the a-helical "flap" region (or lid domain) present in all lipases, which is presumably responsible for lipase interfacial activation 33 and is only 10 residues away from the histidine residue contributing to the serine-histidine-aspartate catalytic triad.…”

Section: Discussionmentioning

confidence: 99%

Molecular characterization of human hepatic lipase deficiency. In vitro expression of two naturally occurring mutations.

Durstenfeld

Ben-Zeev

Reue

et al. 1994

Arterioscler Thromb

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Individuals with hepatic lipase (HL) deficiency are often characterized by elevated levels of triglycerides and cholesterol and may be subject to premature atherosclerosis. Missense mutations in the HLgene have been identified in two affected families: substitutions of serine for phenylalanine at amino acid 267 and threonine for methionine at amino acid 383 (S267F and T383M, respectively). To confirm the role of S267F and T383M in HL deficiency, we introduced these mutations separately into human HL cDNA by site-directed mutagenesis, and the resulting constructs were independently expressed in COS cells. HL activity and mass were measured and compared with wild-type HL transfectants to determine the effect of these mutations on lipase activity and secretion. Although similar amounts of HL protein were detected intracellularly after transfection with the wild-type and mutant constructs, S267F and T383M HL activity levels were markedly decreased: in S267F, no HL activity was detected, and H epatic lipase (HL) is synthesized and secreted by hepatocytes and functions as an endothelium-bound enzyme within liver sinusoids. 13The primary function of HL is hydrolysis of core triglycerides and surface phospholipids of chylomicron remnants and high-density lipoproteins (HDL) and intermediate-density lipoproteins.2 -3 The enzyme has been proposed to augment the uptake of HDL cholesterol by liver (ie, reverse cholesterol transport) and to be involved in the conversion of HDL 2 to HDL3. 2 -3 HL is also thought to increase the hepatic clearance of chylomicron remnants, possibly by aiding in the exposure of crucial apolipoprotein E epitopes.4 These functions are important components of the lipid transport system that ultimately determines levels of circulating cholesterol and triglyceride. Indeed, individuals with reduced HL activity in postheparin plasma generally exhibit elevated levels of triglyceride and cholesterol and may be subject to premature atherosclerosis. 5 " 7 Heritable HL deficiency has been identified in several families.6 -813 Hegele et al 1415 analyzed genomic DNA from two of these families to determine whether the cause of HL deficiency was due to mutations occurring within the structural gene. They identified several base changes, three of which resulted in amino acid substitutions: an A-»G change located in exon 5 (nucleotide activity levels in T383M were 38% of wild-type HL. Heparininduced secretion of the two HL mutants was also severely affected: no detectable activity could be measured in the media of S267F, although some inactive mass (12% of wild-type HL) was secreted; mutant T383M secreted 4% and 20% of wild-type activity and mass, respectively. These results indicate that the single amino acid substitution present in HL S267F is sufficient to render the enzyme completely nonfunctional; in contrast, the T383M mutant retains partial activity but is poorly secreted. Thus, these defects appear capable of accounting for the HL-deficient phenotypes exhibited by individuals carrying the T383...

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Section: Discussionmentioning

confidence: 99%

Molecular characterization of human hepatic lipase deficiency. In vitro expression of two naturally occurring mutations.

Durstenfeld

Ben-Zeev

Reue

et al. 1994

Arterioscler Thromb

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“…Refs. [17][18][19][20][21][22]. However, the structural basis of LPL in its noncatalytic role to act as a bridge for circulating lipoproteins and their receptors is poorly understood.…”

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confidence: 99%

“…By taking advantage of these fibroblast-like cells that can be readily induced to produce wild-type and mutant LPLs by transfection (17)(18)(19)24), we have analyzed the structural requirement for the effect of LPL in LDL receptor-mediated uptake and catabolism of normal human VLDL. Our results indicate that in these cells, the LDL receptor can bind to and catabolize VLDL in the presence of LPL.…”

mentioning

confidence: 99%

Structure-Function Relationship of Lipoprotein Lipase-mediated Enhancement of Very Low Density Lipoprotein Binding and Catabolism by the Low Density Lipoprotein Receptor

Salinelli

Mims

et al. 1996

Journal of Biological Chemistry

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118

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We examined the structure-function relationship of human lipoprotein lipase (hLPL) in its ability to enhance the binding and catabolism of very low density lipoproteins (VLDL) in COS cells. Untransfected COS cells did not bind to or catabolize normal VLDL. Expression of wild-type hLPL by transient transfection enhanced binding, uptake, and degradation of the VLDL (a property of LPL that we call bridge function). Heparin pretreatment and a monoclonal antibody ID7 that blocks LDL receptor-binding domain of apoE both inhibited binding, and apoE2/E2 VLDL from a Type III hyperlipidemic subject did not bind. However, LDL did not reduce 125 I-VLDL binding to the hLPL-expressing cells, whereas rabbit ␤-VLDL was an effective competitor. By contrast, LDL reduced uptake and degradation of 125 I-VLDL to the same extent as excess unlabeled VLDL or ␤-VLDL. These data suggest that binding occurs by direct interaction of VLDL with LPL but the subsequent catabolism of the VLDL is mediated by the LDL receptor. Mutant hLPLs that were catalytically inactive, S132A, S132D, as well as the partially active mutant, S251T, and S172G, gave normal enhancement of VLDL binding and catabolism, whereas the partially active mutant S172D had markedly impaired capacity for the process; thus, there is no correlation between bridge function and lipolytic activity. A naturally occurring genetic variant hLPL, S4473 Ter, has normal bridge function. The catalytic center of LPL is covered by a 21-amino acid loop that must be repositioned before a lipid substrate can gain access to the active site for catalysis. We studied three hLPL loop mutants (LPL-cH, an enzymatically active mutant with the loop replaced by a hepatic lipase loop; LPL-cP, an enzymatically inactive mutant with the loop replaced by a pancreatic lipase loop; and C216S/C239S, an enzymatically inactive mutant with the pair of Cys residues delimiting the loop substituted by Ser residues) and a control double Cys mutant, C418S/C438S. Two of the loop mutants (LPL-cH and LPL-cP) and the control double Cys mutant C418S/ C438S gave normal enhancement of VLDL binding and catabolism, whereas the third loop mutant, C216S/ C239S, was completely inactive. We conclude that although catalytic activity and the actual primary sequence of the loop of LPL are relatively unimportant (wild-type LPL loop and pancreatic lipase loops have little sequence similarity), the intact folding of the loop, flanked by disulfide bonds, must be maintained for LPL to express its bridge function.

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“…Although these two loops differ in primary sequence, they both show a similar predicted (by the Chou-Fasman algorithm) alternation of a-helical and P-sheet regions. However, replacement of the human LPL loop with the loop from pancreatic lipase yields an inactive human LPL [17].…”

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confidence: 99%

Movable lobes and flexible loops in proteins Structural deformations that control biochemical activity

Kempner

1993

FEBS Letters

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Two classes of protein whose structure is modified by small ligands are reviewed. Proteins of one group contain two massive domains joined by a flexible link; in response to small molecules, the two lobes approach and enclose the ligand. In the other, a short segment of amino acids moves as a flexible loop over the ligand which often is trapped in a non-aqueous environment. Biochemical reaction rates are altered dramatically by these movements.

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Functional topology of a surface loop shielding the catalytic center in lipoprotein lipase

Cited by 40 publications

References 23 publications

Molecular characterization of human hepatic lipase deficiency. In vitro expression of two naturally occurring mutations.

Molecular characterization of human hepatic lipase deficiency. In vitro expression of two naturally occurring mutations.

Structure-Function Relationship of Lipoprotein Lipase-mediated Enhancement of Very Low Density Lipoprotein Binding and Catabolism by the Low Density Lipoprotein Receptor

Movable lobes and flexible loops in proteins Structural deformations that control biochemical activity

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