Mark H. Doolittle

Mutations in LMF1 cause combined lipase deficiency and severe hypertriglyceridemia.

Hypertriglyceridemia is a hallmark of many disorders, including metabolic syndrome, diabetes, atherosclerosis and obesity. A well-known cause is the deficiency of lipoprotein lipase (LPL), a key enzyme in plasma triglyceride hydrolysis. Mice carrying the combined lipase deficiency (cld) mutation show severe hypertriglyceridemia owing to a decrease in the activity of LPL and a related enzyme, hepatic lipase (HL), caused by impaired maturation of nascent LPL and hepatic lipase polypeptides in the endoplasmic reticulum (ER). Here we identify the gene containing the cld mutation as Tmem112 and rename it Lmf1 (Lipase maturation factor 1). Lmf1 encodes a transmembrane protein with an evolutionarily conserved domain of unknown function that localizes to the ER. A human subject homozygous for a deleterious mutation in LMF1 also shows combined lipase deficiency with concomitant hypertriglyceridemia and associated disorders. Thus, through its profound effect on lipase activity, LMF1 emerges as an important candidate gene in hypertriglyceridemia.

Maturation of Lipoprotein Lipase in the Endoplasmic Reticulum CONCURRENT FORMATION OF FUNCTIONAL DIMERS AND INACTIVE AGGREGATES

The maturation of lipoprotein lipase (LPL) into a catalytically active enzyme was believed to occur only after its transport from the endoplasmic reticulum (ER) to the Golgi apparatus. To test this hypothesis, LPL located in these two subcellular compartments was separated and compared. Heparin affinity chromatography resolved low affinity, inactive LPL displaying ER characteristics from a high affinity, active fraction exhibiting both ER and Golgi forms. The latter forms were further separated by β-ricin chromatography and were found to have comparable activities per unit of LPL mass. Thus, LPL must reach a functional conformation in the ER. Active LPL, regardless of its cellular location, exhibited the expected dimer conformation. However, inactive LPL, found only in the ER, was highly aggregated. Kinetic analysis indicated a concurrent formation of LPL dimer and aggregate and indicated that the two forms have dissimilar fates. Whereas the dimer remained stable even when confined to the ER, the aggregate was degraded. Degradation rates were not affected by proteasomal or lysosomal inhibitors but were markedly reduced by ATP depletion. Lowering the redox potential in the ER by dithiothreitol caused the dimer to associate with calnexin, BiP, and protein-disulfide isomerase to form large, inactive complexes; dithiothreitol removal induced complex dissociation with restoration of the functional LPL dimer. In contrast, the LPL aggregate was only poorly associated with ER chaperones, appearing to be trapped in an irreversible, inactive conformation destined for ER degradation.

Lipase maturation factor 1: structure and role in lipase folding and assembly

Purpose of review Lipase maturation factor 1 (LMF1) is a membrane-bound protein located in the endoplasmic reticulum. It is essential to the folding and assembly (i.e., maturation) of a selected group of lipases that include lipoprotein lipase, hepatic lipase and endothelial lipase. The purpose of this review is to examine recent studies that have begun to elucidate the structure and function of LMF1 and to place it in the context of lipase folding and assembly. Recent findings Recent studies identified mutations in LMF1 that cause combined lipase deficiency and hypertriglyceridemia in humans. These mutations result in the truncation of a large, evolutionarily conserved domain (DUF1222), which is essential for interaction with lipases and their attainment of enzymatic activity. The structural complexity of LMF1 has been further characterized by solving its topology in the endoplasmic reticulum membrane. Recent studies indicate that in addition to lipoprotein lipase and hepatic lipase, the maturation of endothelial lipase is also dependent on LMF1. Based on its apparent specificity for dimeric lipases, LMF1 is proposed to play an essential role in the assembly and/or stabilization of head-to-tail lipase homodimers. Summary LMF1 functions in the maturation of a selected group of secreted lipases that assemble into homodimers in the endoplasmic reticulum. These dimeric lipases include lipoprotein lipase, hepatic lipase and endothelial lipase, all of which contribute significantly to plasma triglyceride and high-density lipoprotein cholesterol levels in humans. Future studies involving genetically engineered mouse models will be required to fully elucidate the role of LMF1 in normal physiology and diseases.

Hepatic lipase maturation: a partial proteome of interacting factors.

Tandem affinity purification (TAP) has been used to isolate proteins that interact with human hepatic lipase (HL) during its maturation in Chinese hamster ovary cells. Using mass spectrometry and Western blotting, we identified 28 proteins in HL-TAP isolated complexes, 16 of which localized to the endoplasmic reticulum (ER), the site of HL folding and assembly. Of the 12 remaining proteins located outside the ER, five function in protein translation or ER-associated degradation (ERAD). Components of the two major ER chaperone systems were identified, the BiP/Grp94 and the calnexin (CNX)/calreticulin (CRT) systems. All factors involved in CNX/CRT chaperone cycling were identified, including UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT), glucosidase II, and the 57 kDa oxidoreductase (ERp57). We also show that CNX, and not CRT, is the lectin chaperone of choice during HL maturation. Along with the 78 kDa glucose-regulated protein (Grp78; BiP) and the 94 kDa glucose-regulated protein (Grp94), an associated peptidyl-prolyl cis-trans isomerase and protein disulfide isomerase were also detected. Finally, several factors in ERAD were identified, and we provide evidence that terminally misfolded HL is degraded by the ubiquitin-mediated proteasomal pathway. We propose that newly synthesized HL emerging from the translocon first associates with CNX, ERp57, and glucosidase II, followed by repeated posttranslational cycles of CNX binding that is mediated by UGGT. BiP/Grp94 may stabilize misfolded HL during its transition between cycles of CNX binding and may help direct its eventual degradation.

Lipase Maturation Factor LMF1, Membrane Topology and Interaction with Lipase Proteins in the Endoplasmic Reticulum

Lipase maturation factor 1 (LMF1) is predicted to be a polytopic protein localized to the endoplasmic reticulum (ER) membrane. It functions in the post-translational attainment of enzyme activity for both lipoprotein lipase and hepatic lipase. By using transmembrane prediction methods in mouse and human orthologs, models of LMF1 topology were constructed and tested experimentally. Employing a tagging strategy that used insertion of ectopic glycan attachment sites and terminal fusions of green fluorescent protein, we established a five-transmembrane model, thus dividing LMF1 into six domains. Three domains were found to face the cytoplasm (the amino-terminal domain and loops B and D), and the other half was oriented to the ER lumen (loops A and C and the carboxyl-terminal domain). This representative model shows the arrangement of an evolutionarily conserved domain within LMF1 (DUF1222) that is essential to lipase maturation. DUF1222 comprises four of the six domains, with the two largest ones facing the ER lumen. We showed for the first time, using several naturally occurring variants featuring DUF1222 truncations, that Lmf1 interacts physically with lipoprotein lipase and hepatic lipase and localizes the lipase interaction site to loop C within DUF1222. We discuss the implication of our results with regard to lipase maturation and DUF1222 domain structure.

Maturation of hepatic lipase. Formation of functional enzyme in the endoplasmic reticulum is the rate-limiting step in its secretion.

Among three lipases in the lipase gene family, hepatic lipase (HL), lipoprotein lipase, and pancreatic lipase, HL exhibits the lowest intracellular specific activity (i.e. minimal amounts of catalytic activity accompanied by massive amounts of inactive lipase mass in the endoplasmic reticulum (ER)). In addition, HL has a distinctive sedimentation profile, where the inactive mass overlaps the region containing active dimeric HL and trails into progressively larger molecular forms. Eventually, at least half of the HL inactive mass in the ER reaches an active, dimeric conformation (t½ = 2 h) and is rapidly secreted. The remaining inactive mass is degraded. HL maturation occurs in the ER and is strongly dependent on binding to calnexin in the early co-/post-translational stages. Later stages of HL maturation occur without calnexin assistance, although inactive HL at all stages appears to be associated in distinct complexes with other ER proteins. Thus, unlike other lipases in the gene family, HL maturation is the rate-limiting step in its secretion as a functional enzyme.

Lipase maturation factor 1 is required for endothelial lipase activity

Lipase maturation factor 1 (Lmf1) is an endoplasmic reticulum (ER) membrane protein involved in the posttranslational folding and/or assembly of lipoprotein lipase (LPL) and hepatic lipase (HL) into active enzymes. Mutations in Lmf1 are associated with diminished LPL and HL activities (“combined lipase deficiency”) and result in severe hypertriglyceridemia in mice as well as in human subjects. Here, we investigate whether endothelial lipase (EL) also requires Lmf1 to attain enzymatic activity. We demonstrate that cells harboring a (cld) loss-of-function mutation in the Lmf1 gene are unable to generate active EL, but they regain this capacity after reconstitution with the Lmf1 wild type. Furthermore, we show that cellular EL copurifies with Lmf1, indicating their physical interaction in the ER. Finally, we determined that post-heparin phospholipase activity in a patient with the LMF1W464X mutation is reduced by more than 95% compared with that in controls. Thus, our study indicates that EL is critically dependent on Lmf1 for its maturation in the ER and demonstrates that Lmf1 is a required factor for all three vascular lipases, LPL, HL, and EL.

A quantitative assay measuring the function of lipase maturation factor 1.

Newly synthesized lipoprotein lipase (LPL) and related members of the lipase gene family require an endoplasmic reticulum maturation factor for attainment of enzyme activity. This factor has been identified as lipase maturation factor 1 (Lmf1), and mutations affecting its function and/or expression result in combined lipase deficiency (cld) and hypertriglyceridemia. To assess the functional impact of Lmf1 sequence variations, both naturally occurring and induced, we report the development of a cell-based assay using LPL activity as a quantitative reporter of Lmf1 function. The assay uses a cell line homozygous for the cld mutation, which renders endogenous Lmf1 nonfunctional. LPL transfected into the mutant cld cell line fails to attain activity; however, cotransfection of LPL with wild-type Lmf1 restores its ability to support normal lipase maturation. In this report, we describe optimized conditions that ensure the detection of a complete range of Lmf1 function (full, partial, or complete loss of function) using LPL activity as the quantitative reporter. To illustrate the dynamic range of the assay, we tested several novel mutations in mouse Lmf1. Our results demonstrate the ability of the assay to detect and analyze Lmf1 mutations having a wide range of effects on Lmf1 function and protein expression.

Lipase and phospholipase protocols

Part I. Lipase Activity, Purification, and Expression. Phospholipase A2 and Phosphatidylinositol- Specific Phospholipase C Assays by HPLC and TLC with Fluorescent Substrate, H. Stewart Hendrickson. Fluorometric Phospholipase Assays Based on Polymerized Liposome Substrates, Wonhwa Cho, Shih-Kwang Wu, Edward Yoon, and Lenka Lichtenbergova. Triglyceride Lipase Assays Based on a Novel Fluorogenic Alkyldiacyl Glycerol Substrate, Albin Hermetter. Purification and Assay of Mammalian Group I and Group IIa Secretory Phospholipase A2, Wonhwa Cho, Sang Kyou Han, Byung-In Lee, Yana Snitko, and Rajiv Dua. Determination of Plasmalogen-Selective Phospholipase A2 Activity by Radiochemical and Fluorometric Assay Procedures, Akhlaq A. Farooqui, Hsiu-Chiung Yang, Yutaka Hirashima, and Lloyd A. Horrocks. Human Plasma Platelet-Activating Factor Acetylhydrolase, Diana M. Stafforini and Larry W. Tjoelker. Assays for Pancreatic Triglyceride Lipase and Colipase, Mark E. Lowe. Bile Salt-Activated Lipase, Chi-Sun Wang, Azar Dashti, and Deborah Downs. Determining Lipoprotein Lipase and Hepatic Lipase Activity Using Radiolabeled Substrates, Veronique Briquet-Laugier, Osnat Ben-Zeev, and Mark H. Doolittle. Lysosomal Acid Lipase: Assay and Purification, Martin Merkel, Anne-Christine Tilkorn, Heiner Greten, and Detlev Ameis. Hormone-Sensitive Lipase and Neutral Cholesteryl Ester Lipase, Cecilia Holm and Torben Osterlund. Lecithin-Cholesterol Acyltransferase: Assay of Cholesterol Esterification and Phospholipase A2 Activities, John S. Parks, Abraham K. Gebre, and James W. Furbee. Purification of Lipases and Phospholipases by Heparin-Sepharose Chromatography, Akhlaq A. Farooqui and Lloyd A. Horrocks. Large-Scale Lipoprotein Lipase Purification from Adipose Tissue, Andre Bensadoun, Jean Hsu, and Barry Hughes. Purification of Rat Hepatic Lipase Essentially Free of Apolipoprotein E and Apolipoprotein B, Andre Bensadoun, Barry Hughes, Kristan Melford, Jean Hsu, and Dawn L. Braesaemle. High-LevelExpression and Purification of Human Hepatic Lipase from Mammalian Cells, John S. Hill. High-Level Baculoviral Expression of Hormone-Sensitive Lipase, Cecilia Holm and Juan Antonio Contreras. High-Level Baculoviral Expression of Lysosomal Acid Lipase, Anne-Christine Tilkorn, Martin Merkel, Heiner Greten, and Detlev Ameis. One-Step Purification and Biochemical Characterization of Recombinant Pancreatic Lipases Expressed in Insect Cells, Sofiane Bezzine, Francine Ferrato, Veronique Lopez, Alain de Caro, Robert Verger, and Frederic Carriere. Modulation of the Expression Level of Human Acidic Lipases by Various Signal Peptides, Stephane Canaan, Liliane Dupuis, Mireille Riviere, Robert Verger, and Catherine Wicker-Planquart. Part II. Biochemical Characterization. Immunodetection of Lipoprotein Lipase: Antibody Production, Immunoprecipitation, and Western Blotting Techniques, Mark H. Doolittle and Osnat Ben-Zeev. Immunological Characterization of Digestive Lipases, Alain De Caro, Sofiane Bezzine, Veronique Lopez, Mustapha Aoubala, Cecile Daniel, Robert Verger, and Frederic Carriere. Determining Lipase Subunit Structure by Sucrose Gradient Centrifugation, Osnat Ben-Zeev and Mark H. Doolittle. Detecting Ligands Interacting with Lipoprotein Lipase, Sivaram Pillarisetti. Monolayer Techniques for Studying Lipase Kinetics, Stephane Ransac, Margarita Ivanova, Ivan Panaiotov, and Robert Verger. Efficient Immobilization of Phospholipase A2, Wonhwa Cho and Zhen Shen. Part III. Molecular Genetic Characterization. An Introduction to Internet Resources for the Molecular and Genetic Analysis of the Lipases, Karen Reue. Techniques for the Measurement of Lipoprotein Lipase Messenger RNA, Gouri Ranganathan and Philip A. Kern. In Vitro Transcription and Translation of Lipoprotein Lipase, Gouri Ranganathan and Philip A. Kern. Induced Lipase Mutations in the Mouse, Clay F. Semenkovich. Index.

Mechanisms of lipase maturation.

Abstract Lipases are acyl hydrolases that represent a diverse group of enzymes present in organisms ranging from prokaryotes to humans. This article focuses on an evolutionarily related family of extracellular lipases that include lipoprotein lipase, hepatic lipase and endothelial lipase. As newly synthesized proteins, these lipases undergo a series of co- and post-translational maturation steps occurring in the endoplasmic reticulum, including glycosylation and glycan processing, and protein folding and subunit assembly. This article identifies key factors that facilitate this process and discusses mechanisms that direct early and late events in lipase folding and assembly. Lipase maturation employs the two general chaperone systems operating in the endoplasmic reticulum, as well as a recently identified lipase‑specific chaperone termed lipase maturation factor 1. We propose that the two general chaperone systems act in a coordinated manner early in lipase maturation in order to help create partially folded monomers; lipase maturation factor 1 then facilitates final monomer folding and subunit assembly into fully functional homodimers. Once maturation is complete, the lipases exit the endoplasmic reticulum and are secreted to extracellular sites, where they carry out a number of functions related to lipoprotein and lipid metabolism.