SEMINARS IN LIVER DISEASE-VOL.
12, NO. 4, 1992
Hepatic and Plasma Lipases
The liver is the primary organ for synthesis and degradation of plasma lipoproteins.' In addition, the liver acts as the major source of lipolytic enzyme^.^' Two of three principal lipases involved in intravascular lipoprotein metabolism are synthesized exclusively by the liver. One of these lipases, hepatic lipase (HL), is synthesized by hepatocytes and functions at the surface of endothelial cells lining the sinusoids. A second liis secreted by the pase, lecithin:cholesterol acyl tran~ferase,~ liver and functions in the plasma. Another lipolytic enzyme, lysosomal acid lipase, is synthesized by hepatocytes and Kupffer cells and by various extrahepatic cell types5 The major extrahepatic enzyme involved in lipoprotein metabolism, lipoprotein lipase (LPL), is synthesized by parenchymal cells, mainly of muscle and adipose t i ~ s u e . "LPL ~ hydrolyzes triglycerides of circulating chylomicrons and very low density lipoproteins (VLDL) and is the rate-limiting enzyme of extrahepatic lipolysis. Presumably, the liver degrades LPL that has been released into the plasma from its location on the luminal surface of the endothelial cells. The primary structures of a number of lipases have now been elucidated by molecular cloning. Further elucidation of the molecular biology of HL and LPL has been achieved from genomic a n a l y ~ i s . ~The - ~ ~similarity in gene organization between these two li~asesindicates that thev are members of a lipase gene family. Using the genomic structure, the molecular basis of inherited lipase deficiencies is now being directly assessed. This review addresses some of the recent advances in the biochemistry, molecular biology, and genetics of several lipolytic enzymes, including hepatic lipase, LPL, and ly sosoma1 acid lipase and their relationship to inherited metabolic disturbances that are characterized by alterations of plasma lipid and lipoprotein levels.
HEPATIC LIPASE HL is synthesized and secreted by hepatocytes and transported to the sinusoidal surface of the l i ~ e r . ' . Catalytic ~ . ~ ~ activity for HL has also been detected in the ovary and adrenal gland.I5 However, no HL protein synthesis was observed in these tissues. The enzyme has been purified from a number of
From the Medizinische Kernklinik und Poliklinik, UniversitatsKrankenhaus Eppendorf, Hamburg, Germany, and Research, VA Wadsworth Medical Center and Department oj'Medicine, University of California, Los Angeles, California.
Reprint requests: Dr. Ameis, Medizinische Kernklinik, Universitats-Krankenhaus Eppendorf, Hamburg, Germany.
sources, including human,16 canine,17 and rat li~er.l~-~O During denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, rat HL migrates with an apparent molecular mass of 53 kDa.20Like most proteins synthesized by the liver and secreted into plasma, HL is glyco~ylated.'~ cDNA clones for h ~ m a n , ~ ' rat,24 -~' and rabbit HL2%ave been obtained and analyzed. The predicted human HL protein consists of 476 amino acids, preceded by a 23 residue signal peptide. Similarly, rat HL cDNA encodes for a 472 amino acid protein and a hydrophobic signal peptide of 22 amino acids. Mouse HL cDNA encodes for a mature protein of 488 amino acids preceded by a signal peptide of 22 amino acids.2s Rabbit HL cDNA encodes for a mature protein of 477 amino acids and a signal peptide of 20 amino acids.26The unglycosylated human HL protein has a predicted molecular mass of 53,500 DA," a value corresponding very closely to the molecular mass of purified rat HL on SDS-polyacrylamide gel analysis. l 5 Comparison of cDNA sequences of human, rat, and rabbit HL reveals a high degree of similarity, with 75 and 79% of identical amino acids between the human and rat and the rat and rabbit proteins, respectively. Human HL has four potential N-linked glycosylation sites; two correspond to homologous sites on rat HL. Site-directed mutagenesis has shown that rat HL lacking one or both of these N-linked glycosylation sites is still enzymatically active. Although secretion of the enzyme is dependent on glycosylation, glycosylation is not absolutely essential for lipolytic acti~ity.~' The catalvtic domains of the HL have also been studied by site-directed mutagenesis. Replacement of serine14' by glycine in rat HL abolished enzymatic activity, suggesting that this serine resides at the catalytic center of HL.28Recently, a chimeric lipase was constructed, containing the 329 N-terminal amino acids of rat HL linked to the C-terminal 136 residues of human LPL.29The chimera hydrolyzed both short-chain and long-chain triacylglycerol substrates with enzymatic characteristics very similar to those of native HL. Examination of the HL chimera with monoclonal antibodies to LPL showed that triolein, or lipase hydrolysis, was inhibited, whereas tributyrin, or esterase hydrolysis, was unaffected by these antibodies. The studies with the HL chimera have led to the conclusion that the active center of HL resides in the N-terminal domain, whereas the C-terminal domain appears to be crucial for the interaction and hydrolysis of long-chain fatty acid substrate^.^^ The overall gene structure of HL was determined from isolated phage and cosmid clone^.'^^^ The HL gene is located on chromosome 15q2I3Oand consists of nine exons, ranging in size from 118 to 243 bp, separated by eight introns varying in length from 0.7 to >8 kB.I0 Comparison of the LPL and HL gene structures firmly established them as members of a lipase gene family."-l3 This gene family also includes pancreatic lipase3' and Drosophila yolk proteins,32suggesting that a common ancestral gene preceded the evolution of these genes." Intron loss, due to recombination and gene sliding events, is
Copyright O 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, N Y 10016. All rights reserved.
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presumed to be responsible for differences in the pancreatic lipase gene organization compared with HL and LPL." An inherited deficiency of human HL has been observed in three independent pedigrees and is associated with premature atheroscler~sis.~'-~ The disorder is characterized by various lipoprotein alterations, including low density lipoprotein (LDL) and high density lipoprotein (HDL) particles significantly enriched in triglyceride. P-VLDL, isolated from a patient with HL deficiency, displaced human LDL from the apoprotein B,E receptor on fibroblasts and effectively stimulated acyl: coenzyme A (CoA) cholesterol acyl transferase a~tivity.'~ Trypsin-treated P-VLDL particles, which are unable to interact with the apoprotein B,E receptor, also stimulated acyl:CoA cholesterol acyl transferase activity, suggesting a role for this lipoprotein fraction in cholesterol accumulation in fibroblast^.'^ HDL-cholesterol in HL-deficient individuals varies between normal and profoundly increased levels.'' Generally, HL-deficient subjects show decreased HDL, levels with an increase in the HDLZfraction size range, consistent with the concept that HL affects the interconversion of HDL, to HDL,.'8 A natural HL deficiency in the rabbit also supports a role for HL in the metabolism of circulating HDL lipoproteins." In this animal model, in vitro incubation of whole plasma or total lipoprotein fraction with various amounts of dog HL resulted in the preferential hydrolysis of HDL triglycerides, and to a lesser extent VLDL triglycerides. The degradation of HDL triglycerides resulted in a profound reduction in particle size, yielding particles similar to human HDL,." Since HDL, particles are thought to act in reverse cholesterol transport, HL may reduce cholesterol accumulation in peripheral cells and thus prevent atherogenesis. Recently, the molecular basis of familial HL deficiency has been explored, utilizing single-stranded conformation polymorphism analysis" and subsequent DNA sequencing. Genomic DNA analysis of six individuals from two unrelated Ontario and Quebec families with complete HL deficiency revealed a C + T substitution producing a thre~nine'~' + methionine missense m ~ t a t i o n . ~All ' six individuals showed the threonine"' -t methionine mutation, whereas 50 random control subjects showed no alteration at this position. Detailed analysis of all nine coding exons of the HL gene of just the Ontario HL-deficient family demonstrated another mutation, a ~erine'~'-- phenylalanine substitution." This mutation cosegregated with the previously described threonine"' + methionine mutation.'' The three HL-deficient individuals from the Ontario family are thus compound heterozygotes for the two mutations, suggesting a causal relationship between the .~~ mutagenesis and mutations and HL d e f i ~ i e n c y Site-directed subsequent in vitro expression of variant HL in eucaryotic cells, as accomplished for human" and rat HL,24could confirm the effects of the reported mutation on HL structural and functional integrity. Clinically, the prevalence of HL variants may be higher than previously assumed. In particular, HL levels of individuals with hypertriglyceridemia or hypercholesterolemia associated with elevated HDLz levels should be evaluated. The implications of HL variants in the population at large have not been clearly defined. However, a recent study using restriction fragment length polymorphism (RFLP) markers of the HL gene locus failed to detect a significant association of HL polymorphism~with accelerated coronary artery disease.42
LIPOPROTEIN LIPASE LPL, a major lipolytic enzyme in the regulation of lipid transuort and metabolism, is located on the luminal surface of the capillary endothelial cells. The lipase is attached to the en-
12, NUMBER 4, 1992
~ ~ . ~1). ~ dothelium via ionic interaction with p r o t e o g l y c a n ~(Fig. In the presence of the required cofactor apoprotein C-11, LPL hydrolyzes the core triglycerides of circulating chylomicrons and VLDL. Liberated free fatty acids are taken up by the surrounding tissues as a source of metabolic energy or for reesterification and storage as triglycerides (Fig. 1). In this gatekeeping function, LPL is directly responsible for the metabolism of 30 to 40% of the daily caloric intake in the form of dietary fat. With the removal of core triglycerides, chylomicrons and VLDL are converted to chylomicron remnants and intermediate density lipoprotein particles. In this conversion, the volume of the particles is significantly reduced (Fig. 2). These denser lipoproteins are cleared by hepatic chylomicron remnant receptors and LDL receptors. LPL is synthesized in extrahepatic parenchymal cells, secreted, and transported through the extracellular space to the ~ , ~ ~active enzyme is glycosylated vascular e n d o t h e l i ~ m . The and appears to be catalytically competent as a noncovalent homodimer6.' with an apparent molecular mass of approximately 60 ~ D AThe . functional ~ ~ enzyme resides on the luminal surface of endothelial cells.47The enzyme is readily released into the circulation by intravenous administration of heparin, thus allowing the detection of lipolytic activity in plasma. A small proportion of LPL adhering to chylomicrons and VLDL may also be released into the circulation. In vitro incubation of LPL with chylomicrons or P-VLDL particles significantly enhances binding and uptake of these particles in HepG2 cells.48 This finding suggests a ligand-like role for LPL in the binding of apoprotein E-containing lipoproteins to the LDL receptorrelated protein, mediating hepatocellular uptake of the particles by its structural features rather than by its lipolytic activity.48 LPL activity responds to tissue energy requirements and is inversely regulated in hearttskeletal muscle and adipose tissue." In heart and skeletal muscle LPL activity is relatively low in fed animals and high during fasting, whereas the opposite is the case in adipose tissue. Hormonal control of LPL is particularly evident in adipose tissue. Both insulin and glucocorticoids affect LPL activity, although the mechanism by which LPL synthesis and release are promoted remains unclear.
FIG. 1. Physiologic role of LPL in the metabolism of circulating triglyceride-rich lipoproteins. A longitudinal section
through a capillary is shown. Chylomicron particles derived from intestinal sources are converted to chylornicron remnants (CR), whereas hepatic-derived VLDL particles are degraded to intermediate density lipoprotein (IDL) and LDL. he resultant lipoproteins are mainly internalized by the liver.
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SEMINARS IN LIVER DISEASE-VOLUME
GRETEN, SCHOTZ
CHYLOMICRON CHYLOMICRON REMNANT
DIAMETER
2.5p
VOLUME
100%
-
0.8pm
3.3%
FIG. 2. Reduction in lipoprotein particle size during triglyceride hydrolysis by LPL. An average diameter of a chylomicron particle is assumed a s 2.5 pm. After hydrolysis of triglycerides, the particle size decreases to 0.8 pm. The overall volume of the particle is progressively reduced to 3.3%, illustrating the highly significant contribution of LPL in the metabolism of circulating chylomicrons and VLDL particles.
Human LPL cDNA clones have been obtained and their DNA sequences determined.so." The overlapping cDNA clones of 3550 bp code for 174 bp of 5' untranslated sequence, an open reading frame of 1425 bp, and a 3' untranslated region of 1556 bp. In the 3' untranslated region, two independent polyadenylation signals are found at 3126 and 3522 bp. The predicted protein consists of 475 amino acids, preceded by a 27 amino acid signal peptide. This prepeptide contains a typical hydrophobic core sequence bounded by charged residues. The N-terminus of the mature human LPL protein was defined by comparing the protein sequence based on cDNA clones with 19 amino acids derived from microsequencing of purified human LPL.52The mature protein consists of 448 amino acids with a predicted molecular weight of 50,394. The reported molecular weight of 60,00W6 suggests that glycosylation contributes about 8% to the molecular weight of LPL. The study of LPL has been profoundly stimulated by the observation of individuals showing deficient LPL activity associated with characteristic alterations in lipoprotein metabol i ~ m . ~Classic ? familial LPL deficiency was originally described by Havel and Gordon in 1960.54Subsequent studies have characterized this disorder as a rare form of lipase deficiency displaying a massive increase in plasma chylomicrons and triglycerides in fasting subjeck5' This chylomicronemia is associated with a variety of symptoms, including recurrent abdominal pain, pancreatitis, hepatosplenomegaly, eruptive cutaneous xanthomas, and lipemia retinalis. The disease is usually, but not always, detected in childhood. In one review of 43 cases, 35 patients (81%) were diagnosed as LPL deficient before age 10 years.55A case of LPL deficiency diagnosed at age 75 years illustrates that the disorder should be considered in all age groups, even in geriatric patients.56The mode of inheritance of LPL deficiency is assumed to be autosomal recessive, and the frequency in the general population is approximately 1 in I m i l l i ~ nimplying ~ ~ . ~ ~ a carrier frequency of 1 in 500. In certain areas of Quebec, Canada, a founder effect has increased the carrier frequency to 1 in 40 individual^.^^ Although LPL deficiency does not seem to predispose to premature atherosclerosis, repeated episodes of acute pancreatitis may threaten the patient's life.59A restriction of the daily dietary fat intake to 20 gm or less is usually sufficient to control triglyceride levels and related clinical symptoms. Analysis of plasma lipoproteins reveals chylomicronemia alone or together with increased levels of VLDL in the fasted state. Plasma triglycerides are usually above 17 mmollliter, whereas total
399
plasma cholesterol is moderately elevated and cholesterol levels in the HDL and LDL fractions are decreased. Inherited chylomicronemia is diagnosed by a reduced or absent LPL activity in postheparin plasma or adipose tissue biopsies. Defects in LPLS4or apoprotein C-II".6' have been established as causes of this syndrome. A rare form of chylomicronemia results from the presence of a plasma inhibitor of LPL.62Also, postheparin plasma lipolytic activity in type I glycogen storage disease (glucose-6-phosphatase deficiency) is significantly reduced by a circulating inhibit~r.~) However, the underlying mechanism is not known in detail. Immunologic alterations have been observed in a Japanese pedigree with chylomicronemia; in that study, plasma immunoglobulin A autoantibodies to LPL and HL resulted in functional LPL defi~iency.~ Apoprotein C-I1 is a 79 amino acid polypeptide synthesized and secreted by the liver, with plasma concentrations in the range of 0.03 to 0.05 mglml. Apoprotein C-I1 is a constituent of chylomicrons, VLDL, and HDL and comprises about 10% of VLDL protein. The physiologic significance of apoprotein C-I1 as an activator of LPL has been established in patients with congenital apoprotein C-I1 deficiency." Two-dimensional polyacrylamide gel electrophoresis of apoproteins or activation assays for LPL are used to detect apoprotein C-I1 defi~iency.~' In these patients, severe hypertriglyceridemia due to functional LPL deficiency occurs. Apoprotein C-I1 cDNA clones have been characteri~ed~~ and utilized to establish the structure of the apoprotein C-I1 Based on this information, molecular defects of apoprotein C-I1 have been elucidated in a number of pedigrees with deficient postheparin lipolytic a ~ t i v i t y . ~ ' . ~For ~ - ~instance, O aG to C transition at the donor splice site of exon 2 of apoprotein C-I1 was shown to affect the normal splicing of apoprotein C-I1 mRNA, resulting in a drastic reduction of mRNA levels and apoprotein C-I1 defi~iency.~' The genomic structure of the human LPL gene has been allowing a direct assessment of the molecular basis of familial LPL deficiency. RFLP analysis indicates that major alterations in the form of insertionldeletion type changes and duplication events are the cause of primary LPL deficiency in a significant number of type I hyperlip~proteinemias.'~.'~AS illustrated in Figure 3, polymerase chain reaction amplification in conjunction with DNA sequencing has located various point mutations along the LPL gene causing LPL defi~iency.~' The mutations result in amino acid changes or premature termination in the LPL protein in different pedigree^.^^-^^ These studies indicate a considerable heterogeneity in the molecular pathol-
FIG. 3. Mutations associated with familial LPL deficiency. The horizontal line represents the LPL protein consisting of 448 amino acid residues. Filled circles indicate the position of the amino acid residues comprising the catalytic triad, ~ e r i n e ' ~ ~ , aspartic acidfs6, and histidinez41.Solid vertical arrowheads above the line locate nonsense mutations resulting in a premature stop codon or a truncated LPL protein. Arrows below the line indicate missense mutations resulting in amino acid substitutions producing catalytically incompetent LPL.
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HEPATIC AND PLASMA LIPASES-AMEIS,
SEMINARS IN LIVER DISEASE-VOLUME
ogy of LPL deficiency, suggesting that LPL is very sensitive to single amino acid substitutions, frequently resulting in loss of enzymatic activity. Subtle changes in enzyme structure only marginally altering enzymatic function, such as enzyme halflife and substrate affinity, are also conceivable. Deficient interaction of LPL with heparan sulfate proteoglycans of endothelial cell surfaces has been described in hypertriglyceridemic ~ a t s . ~In~ this . ' ~ animal model of LPL deficiency, LPL protein was catalytically inactive and failed to bind to endothelial cells. No major alterations appeared in the LPL gene, and LPL mRNA levels were in the normal range.86 Definition of this molecular defect awaits the cloning and sequencing of the LPL gene in these cats. Gene amplification techniques linked to direct screening for mutations and DNA sequence analysis now facilitate the determination of structural variants of LPL. High-level expression in eucaryotic cells will allow biochemical studies of various mutations, for example, with respect to enzyme-substrate interactions or binding to heparan sulfate or apoprotein C-11. Currently, the crystallization and determination of the threedimensional structure of LPL is being pursued. This will aid in defining the effect of genetic mutations on LPL tertiary structure and provide the basis for an improved understanding of their role in familial chylomicronemia and related disorders of lipid metabolism.
LYSOSOMAL ACID LIPASE
12, NUMBER 4, 1992
ing suggested that proteolytic processing of a precursor lipase releases the mature hepatic enzyme of 41 DA.~'These data are in contrast to previous reports of two human hepatic acid li~ ~ , ~ ~ pases with a molecular mass of 29 and 58 ~ D A .Further structural information on these acid lipases has not been available. Recently, human fibroblast acid lipase cDNA clones have been obtained and analyzed.99The predicted fibroblast acid lipase protein consists of 378 amino acids with a molecular mass of 43,95 1 DA. Sequence comparison to known lipases revealed significant amino acid homologies with human gastric lipase and rat lingual lipase, establishing the three enzymes as members of a gene family of acid l i p a ~ e s . ~ ~
CONCLUSION The elucidation of cDNA and genomic structures of various lipolytic enzymes has greatly facilitated the detailed investigation of inherited disorders of lipid metabolism associated with lipase deficiencies. The molecular basis of HL and LPL deficiencies has now been studied in a number of affected kindreds. These naturally occurring mutants have guided studies of structure-function relationships and have aided in delineating functional domains of lipases. A detailed biochemical characterization of lipase variants and chimeric lipases will further contribute to our understanding of their role in lipid metabolism and in the pathogenesis of atherosclerosis.
Acknowledgment. This work was supported in part by the Deutsche A hydrolase essential for intracellular degradation of choForschungsgemeinschaft (Am 6513-I), the Veterans Administration, lesteryl esters and triglycerides has been localized to the lysoand the National Institutes of Health (HL 28481). soma1 ~ompartment.'~It is characterized by a pH optimum of 4.0 to 5.5, and is therefore called lysosomal acid lipase.' This enzyme is synthesized in virtually all cells and tissues of the REFERENCES human body, including fibroblasts, macrophages, lymphocytes, and liver cells. The highest enzymatic activity is in the Cooper AD: Hepatic lipoprotein and cholesterol metabolism. liver where Kupffer cells have a tenfold higher specific activity In: Zakim D, Boyer TD (Eds): Hepatology. Philadelphia, than hepatocytes. Kupffer cells possess a well-developed lyW.B. Saunders, 1990, pp 96-123. sosomal system, reflecting their role in the clearance of various Nilsson-Ehle P, Garfinkel AS, Schotz MC: Lipolytic enzymes molecules. and plasma lipoprotein metabolism. Annu Rev Biochem The physiologic significance of lysosomal acid lipase in 49:667-693, 1980. Kinnunen PKJ: Hepatic endothelial lipase. In: Borgstrom B, the intracellular metabolism of neutral triglycerides and choBrockman HL ( ~ d s )Lipases. : ~msterdam,~lsevier,1984, pp lesteryl esters is illustrated by an inborn error of metabolism 307-328. affecting this en~yrne.'~ In 1956, Wolman and associates studJonas A: Lecithin cholesterol acyltransferase. In: Gotto AM Jr ied an infant whose clinical course was characterized by severe (Ed): Plasma Lipoproteins. Amsterdam, Elsevier, 1987, pp vomiting, abdominal distension, hepatosplenomegaly, and ad299-333. renal calcifications and who died at the age of 2 r n ~ n t h s . 'A~ Fowler SD, Brown WJ: Lysosomal acid lipase. In: Borgstrom marked reduction in lysosomal acid lipase activity was subseB, Brockman HL (Eds): Lipases. Amsterdam, Elsevier, 1984, quently found to be responsible for the clinical p h e n ~ t y p e . ~ pp 329-364. The mode of inheritance is thought to be autosomal recessive Augustin J, Greten H: The role of lipoprotein lipase-molecular properties and clinical relevance. Atheroscl Rev 5:91with variable penetrance, leading to another clinically distinct 124, 1979. phenotype, cholesteryl ester storage d i s e a ~ e . ~In~ .this ~ ' disorGarfinkel AS, Schotz MC: Lipoprotein lipase. In: Gotto AM, der reduced hepatic acid lipase activityy2results in a marked Jr (Ed): Plasma Lipoproteins. Amsterdam, Elsevier, 1987, pp intracellular increase in cholesteryl esters. In contrast to Wol335-357. man's disease, affected individuals reach adulthood. The clinOlivecrona T, Bengtsson-Olivecrona G: Lipoprotein lipase and ical presentation includes a progressive hepatomegaly, in some hepatic lipase. Curr Opinion Lipidol 1:222-230, 1990. cases evolving to hepatic fibrosis. Plasma lipoprotein analysis Cai S-J, Wong DM, Chen S-H, Chan L: Structure of the hureveals elevated total plasma cholesterol and LDL levels, perman hepatic triglyceride lipase gene. Biochemistry 28:8966haps accounting for the premature atherosclerosis observed in 8971, 1989. Ameis D, Stahnke G, Kobayashi J, et al: Isolation and char~.~' some patients with cholesteryl ester storage d i ~ e a s e . ~There acterization of the human hepatic lipase gene. J Biol Chem is no specific therapy available for Wolman's disease or cho265:6552-6555, 1990. lesteryl ester storage disease. Deeb SS, Peng R: Structure of the human lipoprotein lipase Purification of lysosomal acid lipase has been attempted gene. Biochemistry 28:413 1-4135, 1989. from various human tissues, including placenta," cultured fiKirchgessner TG, Chuat J-C, Heinzmann C, et al: Organiza~ ~ . ~ hepatic ~ lysosomal acid lipase b r o b l a s t ~and , ~ ~l i ~ e r . Human tion of the human lipoprotein lipase gene and evolution of the was purified to apparent homogeneity and showed an apparent lipase gene family. Proc Natl Acad Sci USA 86:9647-9651, molecular mass of 41 ~ D on A SDS-polyacrylamide gel electro1989. phoresis. Results from partial amino-terminal peptide sequencOka K, Tkalcevic GT, Nakano T, et al: Structure and poly-
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gaarden JB, Fredrickson DS, Goldstein JL, Brown MS (Eds): The Metabolic Basis of Inherited Disease. New York, McGraw-Hill, 1983, pp 622-642. Breckenridge WC, Little JA, Steiner G , et al: Hypertriglyceridemia associated with deficiency of apolipoprotein C-11. N Engl J Med 298: 1265-1273, 1978. Fojo SS, Beisiegel W, Beil U , et al: Donor splice site mutation in the apolipoprotein (apo) C-I1 (apo C-I1 Hamburg) of a patient with apo C-I1 deficiency. J Clin Invest 82:1489-1494, 1988. Brunzell JD, Miller NE, Alaupovic P, et al: Familial chylomicronemia due to a circulating inhibitor of lipoprotein lipase activity. J Lipid Res 24:12-19, 1983. Muller DPR, Gamlen TR: The activity of hepatic lipase and lipoprotein lipase in glycogen storage disease: Evidence for a circulating inhibitor of postheparin lipolytic activity. Pediatr Res 18:881-885, 1984. Kihara S, Matsuzawa Y, Kubo M, et al: Autoimmune hyperchylomicronemia. N Engl J Med 320:1255-1259, 1989. Fojo SS, Law SW, Brewer HB Jr, et al: Human apolipoprotein C-11: Complete nucleic acid sequence of preapolipoprotein C11. Proc Natl Acad Sci USA 81:6354-6357, 1984. Wei C-F, Tsao Y-K, Robberson DL, et al: The structure of the human apolipoprotein C-I1 gene. J Biol Chem 260:1521115221, 1985. Das HK, Jackson CL, Miller DA, et al: The human apolipoprotein C-I1 gene sequence contains a novel chromosome 19specific minisatellite in its third intron. J Biol Chem 262: 4787-4793, 1987. Fojo SS, Stanelhoef AFH, Marr K, et al: A deletion mutation in the apoC-11 gene (apoC-I1 Nijmegen) of a patient with a deficiency of apolipoprotein C-11. J Biol Chem 263:1791317916, 1988. Fojo SS, de Gennes JL, Chapman J , et al: An initiation codon mutation in the apoC-I1 gene (apoC-I1 Paris) of a patient with a deficiency of apolipoprotein C-11. J Biol Chem 264:2083920842, 1989. Fojo SS, Lohse P, Parrott C, et al: A nonsense mutation in the apolipoprotein C-11 Padova gene in a patient with apolipoprotein C-11 deficiency. J Clin Invest 84: 1215-1219, 1989. Langlois S , Deeb S, Brunzell JD, et al: A major insertion accounts for a significant proportion of mutations underlying human lipoprotein lipase deficiency. Proc Natl Acad Sci USA 86:948-952, 1989. Devlin RH, Deeb S, Brunzell J , Hayden MR: Partial gene duplication involving exon-Alu interchange results in lipoprotein lipase deficiency. Am J Hum Genet 46: 112-1 19, 1990. Hayden MR, Ma Y, Brunzell JD, Henderson HE: Genetic variants affecting human lipoprotein and hepatic lipases. Curr Opinion Lipidol 2:104-109, 1991. Emi M, Wilson DE, Iverius P-H, et al: Missense mutation (Gly to GIu- 188) of human lipoprotein lipase imparting functional deficiency. J Biol Chem 265:5Y 10-5916, 1990. Beg OU, Meng MS, Skarlatos SI, et al: Lipoprotein lipase (Bethesda): A single amino acid substitution (Ala-176 to Thr) leads to abnormal heparin binding and loss of enzymatic activity. Proc Natl Acad Sci USA 87:3474-3478, 1990. Ameis D, Kobayashi J, Davis RC, et al: Familial chylomicronemia (type I hyperlipoproteinemia) due to a single missense mutation in the lipoprotein lipase gene. J Clin Invest 87: 11651170, 1991. Henderson HE, Ma Y, Hassan MF, et al: Amino acid substitution (IIe-I94 + Thr) in exon 5 of the lipoprotein lipase gene causes lipoprotein lipase deficiency in three unrelated probands: Support for a multicentric origin. J Clin lnvest 87: 2005-2011, 1991. Henderson HE, Devlin R, Peterson J , et al: Frameshift mutation in exon 3 of the lipoprotein lipase gene causes a premature stop codon and lipoprotein lipase deficiency. Mol Biol Med 7 5 1 1-517, 1990. Paulweber B, Wiebusch H, Miesenboeck G, et al: Molecular
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basis of lipoprotein lipase deficiency in two Austrian families with type I hyperlipoproteinemia. Atherosclerosis 86:239250, 1991. Gotoda T, Yamada N, Kawamura M, et al: Heterogeneous mutations in the human lipoprotein lipase gene in patients with familial lipoprotein lipase deficiency. J Clin Invest 88: 18561864, 1991. Gotoda T, Yamada N, Murase T, et al: Occurrence of multiple aberrantly spliced mRNAs upon a donor splice site mutation that causes familial lipoprotein lipase deficiency. J Biol Chem 266:24757-24762, 199 1. Kobayashi J, Nishida T, Ameis D, et al: A heterozygous mutation (Ser-447 + a stop codon) in lipoprotein lipase contributes to a defect in lipid interface recognition in a case with type I hyperlipidemia. Biochem Biophys Res Commun 182: 70-77, 1992. Takagi A, Ikeda Y, Tsutsumi Z, et al: Molecular studies on primary lipoprotein lipase (LPL) deficiency. One base deletion (G916) in exon 5 of LPL gene causes no detectable LPL protein due to the absence of LPL mRNA transcript. J Clin Invest 89:581-591, 1992. Sprecher DL, Kobayashi J, Rymaszewski M, et al: A Trp -, nonsense mutation in the lipoprotein lipase gene. J Lipid Res 33:854-866, 1992. Johnstone AC, Jones BR, Thompson JC, Hancock WS: The pathology of an inherited hyperlipoproteinaemia of cats. J Comp Path01 102: 125-137, 1990. Peritz LN, Brunzell JD, Harvey-Clarke C , et al: Characterization of a lipoprotein lipase class 111 type defect in hypertriglyceridemic cats. Clin Invest Med 13:259-263, 1990. Goldstein JL, Dana SE, Faust JR, et al: Role of lysosomal acid lipase in the metabolism of plasma low density lipoproteins. J Biol Chem 250:8487-8495, 1975. Schmitz G , Assmann G: Acid lipase deficiency: Wolman disease and cholesteryl ester storage disease. In: Scriver CR, Beaudet AL, Sly WS, Valle D (Eds): The Metabolic Basis of Inherited Disease. New York, McGraw Hill, 1989, pp 16231644. Abramov S, Schorr S , Wolman M: Generalized xanthomatosis with calcified adrenals. J Dis Child 91:282, 1956. Patrick AD, Lake BD: Deficiency of an acid lipase in Wolman's disease. Nature 222: 1067-1068, 1969. Sloan HR, Fredrickson DS: Enzyme deficiency in cholesteryl ester storage disease. J Clin Invest 51: 1923-1926, 1972. Burke JA, Schubert WK: Deficient activity of hepatic acid lipase in cholesterol ester storage disease. Science 176:309310, 1972. Beaudet AL, Ferry GD, Nichols BD Jr, et al: Cholesterol ester storage disease: Clinical, biochemical, and pathological studies. J Pediatr 90:910, 1977. Burton BK, Mueller HW: Purification and properties of human placental acid lipase. Biochim Biophys Acta 618:449460, 1980. Sando GN, Rosenbaum LM: Human lysosomal acid lipasei cholesteryl ester hydrolase: Purification and properties of the form secreted by fibroblasts in microcarrier culture. J Biol Chem 260:15186-15193, 1985. Warner TG, Dambach LM, Shin JJ, O'Brien JS: Purification of the lysosomal acid lipase from human liver and its role in lysosomal lipid hydrolysis. J Biol Chem 256:2952-2957, 1981. Sjoberg ER, Hatton JD, O'Brien JS: Purification and characterization of a second form of acid lipase in human liver. Biochem J 248:139-144, 1987. Ameis D, Merkel M, Eckerson C, Greten H: Purefication and characterization of a human hepatic lysosomal acid lipase. Submitted for publication. Anderson RA, Sando GN: Cloning and expression of cDNA encoding human lysosomal acid lipase/cholesteryl ester hydrolase. J Biol Chem 266:22479-22484, 1991.
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SEMINARS IN LIVER DISEASE-VOLUME
12, NO. 4, 1992
Hepatic and Plasma Lipases
The liver is the primary organ for synthesis and degradation of plasma lipoproteins.' In addition, the liver acts as the major source of lipolytic enzyme^.^' Two of three principal lipases involved in intravascular lipoprotein metabolism are synthesized exclusively by the liver. One of these lipases, hepatic lipase (HL), is synthesized by hepatocytes and functions at the surface of endothelial cells lining the sinusoids. A second liis secreted by the pase, lecithin:cholesterol acyl tran~ferase,~ liver and functions in the plasma. Another lipolytic enzyme, lysosomal acid lipase, is synthesized by hepatocytes and Kupffer cells and by various extrahepatic cell types5 The major extrahepatic enzyme involved in lipoprotein metabolism, lipoprotein lipase (LPL), is synthesized by parenchymal cells, mainly of muscle and adipose t i ~ s u e . "LPL ~ hydrolyzes triglycerides of circulating chylomicrons and very low density lipoproteins (VLDL) and is the rate-limiting enzyme of extrahepatic lipolysis. Presumably, the liver degrades LPL that has been released into the plasma from its location on the luminal surface of the endothelial cells. The primary structures of a number of lipases have now been elucidated by molecular cloning. Further elucidation of the molecular biology of HL and LPL has been achieved from genomic a n a l y ~ i s . ~The - ~ ~similarity in gene organization between these two li~asesindicates that thev are members of a lipase gene family. Using the genomic structure, the molecular basis of inherited lipase deficiencies is now being directly assessed. This review addresses some of the recent advances in the biochemistry, molecular biology, and genetics of several lipolytic enzymes, including hepatic lipase, LPL, and ly sosoma1 acid lipase and their relationship to inherited metabolic disturbances that are characterized by alterations of plasma lipid and lipoprotein levels.
HEPATIC LIPASE HL is synthesized and secreted by hepatocytes and transported to the sinusoidal surface of the l i ~ e r . ' . Catalytic ~ . ~ ~ activity for HL has also been detected in the ovary and adrenal gland.I5 However, no HL protein synthesis was observed in these tissues. The enzyme has been purified from a number of
From the Medizinische Kernklinik und Poliklinik, UniversitatsKrankenhaus Eppendorf, Hamburg, Germany, and Research, VA Wadsworth Medical Center and Department oj'Medicine, University of California, Los Angeles, California.
Reprint requests: Dr. Ameis, Medizinische Kernklinik, Universitats-Krankenhaus Eppendorf, Hamburg, Germany.
sources, including human,16 canine,17 and rat li~er.l~-~O During denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, rat HL migrates with an apparent molecular mass of 53 kDa.20Like most proteins synthesized by the liver and secreted into plasma, HL is glyco~ylated.'~ cDNA clones for h ~ m a n , ~ ' rat,24 -~' and rabbit HL2%ave been obtained and analyzed. The predicted human HL protein consists of 476 amino acids, preceded by a 23 residue signal peptide. Similarly, rat HL cDNA encodes for a 472 amino acid protein and a hydrophobic signal peptide of 22 amino acids. Mouse HL cDNA encodes for a mature protein of 488 amino acids preceded by a signal peptide of 22 amino acids.2s Rabbit HL cDNA encodes for a mature protein of 477 amino acids and a signal peptide of 20 amino acids.26The unglycosylated human HL protein has a predicted molecular mass of 53,500 DA," a value corresponding very closely to the molecular mass of purified rat HL on SDS-polyacrylamide gel analysis. l 5 Comparison of cDNA sequences of human, rat, and rabbit HL reveals a high degree of similarity, with 75 and 79% of identical amino acids between the human and rat and the rat and rabbit proteins, respectively. Human HL has four potential N-linked glycosylation sites; two correspond to homologous sites on rat HL. Site-directed mutagenesis has shown that rat HL lacking one or both of these N-linked glycosylation sites is still enzymatically active. Although secretion of the enzyme is dependent on glycosylation, glycosylation is not absolutely essential for lipolytic acti~ity.~' The catalvtic domains of the HL have also been studied by site-directed mutagenesis. Replacement of serine14' by glycine in rat HL abolished enzymatic activity, suggesting that this serine resides at the catalytic center of HL.28Recently, a chimeric lipase was constructed, containing the 329 N-terminal amino acids of rat HL linked to the C-terminal 136 residues of human LPL.29The chimera hydrolyzed both short-chain and long-chain triacylglycerol substrates with enzymatic characteristics very similar to those of native HL. Examination of the HL chimera with monoclonal antibodies to LPL showed that triolein, or lipase hydrolysis, was inhibited, whereas tributyrin, or esterase hydrolysis, was unaffected by these antibodies. The studies with the HL chimera have led to the conclusion that the active center of HL resides in the N-terminal domain, whereas the C-terminal domain appears to be crucial for the interaction and hydrolysis of long-chain fatty acid substrate^.^^ The overall gene structure of HL was determined from isolated phage and cosmid clone^.'^^^ The HL gene is located on chromosome 15q2I3Oand consists of nine exons, ranging in size from 118 to 243 bp, separated by eight introns varying in length from 0.7 to >8 kB.I0 Comparison of the LPL and HL gene structures firmly established them as members of a lipase gene family."-l3 This gene family also includes pancreatic lipase3' and Drosophila yolk proteins,32suggesting that a common ancestral gene preceded the evolution of these genes." Intron loss, due to recombination and gene sliding events, is
Copyright O 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, N Y 10016. All rights reserved.
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DETLEV AMEIS, M.D., HEINER GRETEN, M.D., and MICHAEL C. SCHOTZ, Ph.D.
presumed to be responsible for differences in the pancreatic lipase gene organization compared with HL and LPL." An inherited deficiency of human HL has been observed in three independent pedigrees and is associated with premature atheroscler~sis.~'-~ The disorder is characterized by various lipoprotein alterations, including low density lipoprotein (LDL) and high density lipoprotein (HDL) particles significantly enriched in triglyceride. P-VLDL, isolated from a patient with HL deficiency, displaced human LDL from the apoprotein B,E receptor on fibroblasts and effectively stimulated acyl: coenzyme A (CoA) cholesterol acyl transferase a~tivity.'~ Trypsin-treated P-VLDL particles, which are unable to interact with the apoprotein B,E receptor, also stimulated acyl:CoA cholesterol acyl transferase activity, suggesting a role for this lipoprotein fraction in cholesterol accumulation in fibroblast^.'^ HDL-cholesterol in HL-deficient individuals varies between normal and profoundly increased levels.'' Generally, HL-deficient subjects show decreased HDL, levels with an increase in the HDLZfraction size range, consistent with the concept that HL affects the interconversion of HDL, to HDL,.'8 A natural HL deficiency in the rabbit also supports a role for HL in the metabolism of circulating HDL lipoproteins." In this animal model, in vitro incubation of whole plasma or total lipoprotein fraction with various amounts of dog HL resulted in the preferential hydrolysis of HDL triglycerides, and to a lesser extent VLDL triglycerides. The degradation of HDL triglycerides resulted in a profound reduction in particle size, yielding particles similar to human HDL,." Since HDL, particles are thought to act in reverse cholesterol transport, HL may reduce cholesterol accumulation in peripheral cells and thus prevent atherogenesis. Recently, the molecular basis of familial HL deficiency has been explored, utilizing single-stranded conformation polymorphism analysis" and subsequent DNA sequencing. Genomic DNA analysis of six individuals from two unrelated Ontario and Quebec families with complete HL deficiency revealed a C + T substitution producing a thre~nine'~' + methionine missense m ~ t a t i o n . ~All ' six individuals showed the threonine"' -t methionine mutation, whereas 50 random control subjects showed no alteration at this position. Detailed analysis of all nine coding exons of the HL gene of just the Ontario HL-deficient family demonstrated another mutation, a ~erine'~'-- phenylalanine substitution." This mutation cosegregated with the previously described threonine"' + methionine mutation.'' The three HL-deficient individuals from the Ontario family are thus compound heterozygotes for the two mutations, suggesting a causal relationship between the .~~ mutagenesis and mutations and HL d e f i ~ i e n c y Site-directed subsequent in vitro expression of variant HL in eucaryotic cells, as accomplished for human" and rat HL,24could confirm the effects of the reported mutation on HL structural and functional integrity. Clinically, the prevalence of HL variants may be higher than previously assumed. In particular, HL levels of individuals with hypertriglyceridemia or hypercholesterolemia associated with elevated HDLz levels should be evaluated. The implications of HL variants in the population at large have not been clearly defined. However, a recent study using restriction fragment length polymorphism (RFLP) markers of the HL gene locus failed to detect a significant association of HL polymorphism~with accelerated coronary artery disease.42
LIPOPROTEIN LIPASE LPL, a major lipolytic enzyme in the regulation of lipid transuort and metabolism, is located on the luminal surface of the capillary endothelial cells. The lipase is attached to the en-
12, NUMBER 4, 1992
~ ~ . ~1). ~ dothelium via ionic interaction with p r o t e o g l y c a n ~(Fig. In the presence of the required cofactor apoprotein C-11, LPL hydrolyzes the core triglycerides of circulating chylomicrons and VLDL. Liberated free fatty acids are taken up by the surrounding tissues as a source of metabolic energy or for reesterification and storage as triglycerides (Fig. 1). In this gatekeeping function, LPL is directly responsible for the metabolism of 30 to 40% of the daily caloric intake in the form of dietary fat. With the removal of core triglycerides, chylomicrons and VLDL are converted to chylomicron remnants and intermediate density lipoprotein particles. In this conversion, the volume of the particles is significantly reduced (Fig. 2). These denser lipoproteins are cleared by hepatic chylomicron remnant receptors and LDL receptors. LPL is synthesized in extrahepatic parenchymal cells, secreted, and transported through the extracellular space to the ~ , ~ ~active enzyme is glycosylated vascular e n d o t h e l i ~ m . The and appears to be catalytically competent as a noncovalent homodimer6.' with an apparent molecular mass of approximately 60 ~ D AThe . functional ~ ~ enzyme resides on the luminal surface of endothelial cells.47The enzyme is readily released into the circulation by intravenous administration of heparin, thus allowing the detection of lipolytic activity in plasma. A small proportion of LPL adhering to chylomicrons and VLDL may also be released into the circulation. In vitro incubation of LPL with chylomicrons or P-VLDL particles significantly enhances binding and uptake of these particles in HepG2 cells.48 This finding suggests a ligand-like role for LPL in the binding of apoprotein E-containing lipoproteins to the LDL receptorrelated protein, mediating hepatocellular uptake of the particles by its structural features rather than by its lipolytic activity.48 LPL activity responds to tissue energy requirements and is inversely regulated in hearttskeletal muscle and adipose tissue." In heart and skeletal muscle LPL activity is relatively low in fed animals and high during fasting, whereas the opposite is the case in adipose tissue. Hormonal control of LPL is particularly evident in adipose tissue. Both insulin and glucocorticoids affect LPL activity, although the mechanism by which LPL synthesis and release are promoted remains unclear.
FIG. 1. Physiologic role of LPL in the metabolism of circulating triglyceride-rich lipoproteins. A longitudinal section
through a capillary is shown. Chylomicron particles derived from intestinal sources are converted to chylornicron remnants (CR), whereas hepatic-derived VLDL particles are degraded to intermediate density lipoprotein (IDL) and LDL. he resultant lipoproteins are mainly internalized by the liver.
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SEMINARS IN LIVER DISEASE-VOLUME
GRETEN, SCHOTZ
CHYLOMICRON CHYLOMICRON REMNANT
DIAMETER
2.5p
VOLUME
100%
-
0.8pm
3.3%
FIG. 2. Reduction in lipoprotein particle size during triglyceride hydrolysis by LPL. An average diameter of a chylomicron particle is assumed a s 2.5 pm. After hydrolysis of triglycerides, the particle size decreases to 0.8 pm. The overall volume of the particle is progressively reduced to 3.3%, illustrating the highly significant contribution of LPL in the metabolism of circulating chylomicrons and VLDL particles.
Human LPL cDNA clones have been obtained and their DNA sequences determined.so." The overlapping cDNA clones of 3550 bp code for 174 bp of 5' untranslated sequence, an open reading frame of 1425 bp, and a 3' untranslated region of 1556 bp. In the 3' untranslated region, two independent polyadenylation signals are found at 3126 and 3522 bp. The predicted protein consists of 475 amino acids, preceded by a 27 amino acid signal peptide. This prepeptide contains a typical hydrophobic core sequence bounded by charged residues. The N-terminus of the mature human LPL protein was defined by comparing the protein sequence based on cDNA clones with 19 amino acids derived from microsequencing of purified human LPL.52The mature protein consists of 448 amino acids with a predicted molecular weight of 50,394. The reported molecular weight of 60,00W6 suggests that glycosylation contributes about 8% to the molecular weight of LPL. The study of LPL has been profoundly stimulated by the observation of individuals showing deficient LPL activity associated with characteristic alterations in lipoprotein metabol i ~ m . ~Classic ? familial LPL deficiency was originally described by Havel and Gordon in 1960.54Subsequent studies have characterized this disorder as a rare form of lipase deficiency displaying a massive increase in plasma chylomicrons and triglycerides in fasting subjeck5' This chylomicronemia is associated with a variety of symptoms, including recurrent abdominal pain, pancreatitis, hepatosplenomegaly, eruptive cutaneous xanthomas, and lipemia retinalis. The disease is usually, but not always, detected in childhood. In one review of 43 cases, 35 patients (81%) were diagnosed as LPL deficient before age 10 years.55A case of LPL deficiency diagnosed at age 75 years illustrates that the disorder should be considered in all age groups, even in geriatric patients.56The mode of inheritance of LPL deficiency is assumed to be autosomal recessive, and the frequency in the general population is approximately 1 in I m i l l i ~ nimplying ~ ~ . ~ ~ a carrier frequency of 1 in 500. In certain areas of Quebec, Canada, a founder effect has increased the carrier frequency to 1 in 40 individual^.^^ Although LPL deficiency does not seem to predispose to premature atherosclerosis, repeated episodes of acute pancreatitis may threaten the patient's life.59A restriction of the daily dietary fat intake to 20 gm or less is usually sufficient to control triglyceride levels and related clinical symptoms. Analysis of plasma lipoproteins reveals chylomicronemia alone or together with increased levels of VLDL in the fasted state. Plasma triglycerides are usually above 17 mmollliter, whereas total
399
plasma cholesterol is moderately elevated and cholesterol levels in the HDL and LDL fractions are decreased. Inherited chylomicronemia is diagnosed by a reduced or absent LPL activity in postheparin plasma or adipose tissue biopsies. Defects in LPLS4or apoprotein C-II".6' have been established as causes of this syndrome. A rare form of chylomicronemia results from the presence of a plasma inhibitor of LPL.62Also, postheparin plasma lipolytic activity in type I glycogen storage disease (glucose-6-phosphatase deficiency) is significantly reduced by a circulating inhibit~r.~) However, the underlying mechanism is not known in detail. Immunologic alterations have been observed in a Japanese pedigree with chylomicronemia; in that study, plasma immunoglobulin A autoantibodies to LPL and HL resulted in functional LPL defi~iency.~ Apoprotein C-I1 is a 79 amino acid polypeptide synthesized and secreted by the liver, with plasma concentrations in the range of 0.03 to 0.05 mglml. Apoprotein C-I1 is a constituent of chylomicrons, VLDL, and HDL and comprises about 10% of VLDL protein. The physiologic significance of apoprotein C-I1 as an activator of LPL has been established in patients with congenital apoprotein C-I1 deficiency." Two-dimensional polyacrylamide gel electrophoresis of apoproteins or activation assays for LPL are used to detect apoprotein C-I1 defi~iency.~' In these patients, severe hypertriglyceridemia due to functional LPL deficiency occurs. Apoprotein C-I1 cDNA clones have been characteri~ed~~ and utilized to establish the structure of the apoprotein C-I1 Based on this information, molecular defects of apoprotein C-I1 have been elucidated in a number of pedigrees with deficient postheparin lipolytic a ~ t i v i t y . ~ ' . ~For ~ - ~instance, O aG to C transition at the donor splice site of exon 2 of apoprotein C-I1 was shown to affect the normal splicing of apoprotein C-I1 mRNA, resulting in a drastic reduction of mRNA levels and apoprotein C-I1 defi~iency.~' The genomic structure of the human LPL gene has been allowing a direct assessment of the molecular basis of familial LPL deficiency. RFLP analysis indicates that major alterations in the form of insertionldeletion type changes and duplication events are the cause of primary LPL deficiency in a significant number of type I hyperlip~proteinemias.'~.'~AS illustrated in Figure 3, polymerase chain reaction amplification in conjunction with DNA sequencing has located various point mutations along the LPL gene causing LPL defi~iency.~' The mutations result in amino acid changes or premature termination in the LPL protein in different pedigree^.^^-^^ These studies indicate a considerable heterogeneity in the molecular pathol-
FIG. 3. Mutations associated with familial LPL deficiency. The horizontal line represents the LPL protein consisting of 448 amino acid residues. Filled circles indicate the position of the amino acid residues comprising the catalytic triad, ~ e r i n e ' ~ ~ , aspartic acidfs6, and histidinez41.Solid vertical arrowheads above the line locate nonsense mutations resulting in a premature stop codon or a truncated LPL protein. Arrows below the line indicate missense mutations resulting in amino acid substitutions producing catalytically incompetent LPL.
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HEPATIC AND PLASMA LIPASES-AMEIS,
SEMINARS IN LIVER DISEASE-VOLUME
ogy of LPL deficiency, suggesting that LPL is very sensitive to single amino acid substitutions, frequently resulting in loss of enzymatic activity. Subtle changes in enzyme structure only marginally altering enzymatic function, such as enzyme halflife and substrate affinity, are also conceivable. Deficient interaction of LPL with heparan sulfate proteoglycans of endothelial cell surfaces has been described in hypertriglyceridemic ~ a t s . ~In~ this . ' ~ animal model of LPL deficiency, LPL protein was catalytically inactive and failed to bind to endothelial cells. No major alterations appeared in the LPL gene, and LPL mRNA levels were in the normal range.86 Definition of this molecular defect awaits the cloning and sequencing of the LPL gene in these cats. Gene amplification techniques linked to direct screening for mutations and DNA sequence analysis now facilitate the determination of structural variants of LPL. High-level expression in eucaryotic cells will allow biochemical studies of various mutations, for example, with respect to enzyme-substrate interactions or binding to heparan sulfate or apoprotein C-11. Currently, the crystallization and determination of the threedimensional structure of LPL is being pursued. This will aid in defining the effect of genetic mutations on LPL tertiary structure and provide the basis for an improved understanding of their role in familial chylomicronemia and related disorders of lipid metabolism.
LYSOSOMAL ACID LIPASE
12, NUMBER 4, 1992
ing suggested that proteolytic processing of a precursor lipase releases the mature hepatic enzyme of 41 DA.~'These data are in contrast to previous reports of two human hepatic acid li~ ~ , ~ ~ pases with a molecular mass of 29 and 58 ~ D A .Further structural information on these acid lipases has not been available. Recently, human fibroblast acid lipase cDNA clones have been obtained and analyzed.99The predicted fibroblast acid lipase protein consists of 378 amino acids with a molecular mass of 43,95 1 DA. Sequence comparison to known lipases revealed significant amino acid homologies with human gastric lipase and rat lingual lipase, establishing the three enzymes as members of a gene family of acid l i p a ~ e s . ~ ~
CONCLUSION The elucidation of cDNA and genomic structures of various lipolytic enzymes has greatly facilitated the detailed investigation of inherited disorders of lipid metabolism associated with lipase deficiencies. The molecular basis of HL and LPL deficiencies has now been studied in a number of affected kindreds. These naturally occurring mutants have guided studies of structure-function relationships and have aided in delineating functional domains of lipases. A detailed biochemical characterization of lipase variants and chimeric lipases will further contribute to our understanding of their role in lipid metabolism and in the pathogenesis of atherosclerosis.
Acknowledgment. This work was supported in part by the Deutsche A hydrolase essential for intracellular degradation of choForschungsgemeinschaft (Am 6513-I), the Veterans Administration, lesteryl esters and triglycerides has been localized to the lysoand the National Institutes of Health (HL 28481). soma1 ~ompartment.'~It is characterized by a pH optimum of 4.0 to 5.5, and is therefore called lysosomal acid lipase.' This enzyme is synthesized in virtually all cells and tissues of the REFERENCES human body, including fibroblasts, macrophages, lymphocytes, and liver cells. The highest enzymatic activity is in the Cooper AD: Hepatic lipoprotein and cholesterol metabolism. liver where Kupffer cells have a tenfold higher specific activity In: Zakim D, Boyer TD (Eds): Hepatology. Philadelphia, than hepatocytes. Kupffer cells possess a well-developed lyW.B. Saunders, 1990, pp 96-123. sosomal system, reflecting their role in the clearance of various Nilsson-Ehle P, Garfinkel AS, Schotz MC: Lipolytic enzymes molecules. and plasma lipoprotein metabolism. Annu Rev Biochem The physiologic significance of lysosomal acid lipase in 49:667-693, 1980. Kinnunen PKJ: Hepatic endothelial lipase. In: Borgstrom B, the intracellular metabolism of neutral triglycerides and choBrockman HL ( ~ d s )Lipases. : ~msterdam,~lsevier,1984, pp lesteryl esters is illustrated by an inborn error of metabolism 307-328. affecting this en~yrne.'~ In 1956, Wolman and associates studJonas A: Lecithin cholesterol acyltransferase. In: Gotto AM Jr ied an infant whose clinical course was characterized by severe (Ed): Plasma Lipoproteins. Amsterdam, Elsevier, 1987, pp vomiting, abdominal distension, hepatosplenomegaly, and ad299-333. renal calcifications and who died at the age of 2 r n ~ n t h s . 'A~ Fowler SD, Brown WJ: Lysosomal acid lipase. In: Borgstrom marked reduction in lysosomal acid lipase activity was subseB, Brockman HL (Eds): Lipases. Amsterdam, Elsevier, 1984, quently found to be responsible for the clinical p h e n ~ t y p e . ~ pp 329-364. The mode of inheritance is thought to be autosomal recessive Augustin J, Greten H: The role of lipoprotein lipase-molecular properties and clinical relevance. Atheroscl Rev 5:91with variable penetrance, leading to another clinically distinct 124, 1979. phenotype, cholesteryl ester storage d i s e a ~ e . ~In~ .this ~ ' disorGarfinkel AS, Schotz MC: Lipoprotein lipase. In: Gotto AM, der reduced hepatic acid lipase activityy2results in a marked Jr (Ed): Plasma Lipoproteins. Amsterdam, Elsevier, 1987, pp intracellular increase in cholesteryl esters. In contrast to Wol335-357. man's disease, affected individuals reach adulthood. The clinOlivecrona T, Bengtsson-Olivecrona G: Lipoprotein lipase and ical presentation includes a progressive hepatomegaly, in some hepatic lipase. Curr Opinion Lipidol 1:222-230, 1990. cases evolving to hepatic fibrosis. Plasma lipoprotein analysis Cai S-J, Wong DM, Chen S-H, Chan L: Structure of the hureveals elevated total plasma cholesterol and LDL levels, perman hepatic triglyceride lipase gene. Biochemistry 28:8966haps accounting for the premature atherosclerosis observed in 8971, 1989. Ameis D, Stahnke G, Kobayashi J, et al: Isolation and char~.~' some patients with cholesteryl ester storage d i ~ e a s e . ~There acterization of the human hepatic lipase gene. J Biol Chem is no specific therapy available for Wolman's disease or cho265:6552-6555, 1990. lesteryl ester storage disease. Deeb SS, Peng R: Structure of the human lipoprotein lipase Purification of lysosomal acid lipase has been attempted gene. Biochemistry 28:413 1-4135, 1989. from various human tissues, including placenta," cultured fiKirchgessner TG, Chuat J-C, Heinzmann C, et al: Organiza~ ~ . ~ hepatic ~ lysosomal acid lipase b r o b l a s t ~and , ~ ~l i ~ e r . Human tion of the human lipoprotein lipase gene and evolution of the was purified to apparent homogeneity and showed an apparent lipase gene family. Proc Natl Acad Sci USA 86:9647-9651, molecular mass of 41 ~ D on A SDS-polyacrylamide gel electro1989. phoresis. Results from partial amino-terminal peptide sequencOka K, Tkalcevic GT, Nakano T, et al: Structure and poly-
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SEMINARS IN LIVER DISEASE-VOLUME
