Hypercholesterolaemia and hepatosplenomegaly: two manifestations of cholesteryl ester storage disease.

Cholesteryl ester storage disease (CESD) is a rare autosomal recessive disease caused by mutations in LIPA. Here we describe two different clinical presentations of this disease: one case with a clear phenotype of familial hypercholesterolaemia and one case with hepatosplenomegaly from childhood onwards. These two cases exemplify the diversity of clinical phenotypes of patients with CESD. Knowledge on the phenotypic variability of the disease is of clinical relevance in light of enzyme replacement therapy (sebelipase alpha) for patients with mutations in LIPA, which is currently under development.

Value of plasma chitotriosidase to assess non-neuronopathic Gaucher disease severity and progression in the era of enzyme replacement therapy.

Gaucher disease (GD) is caused by deficiency of the enzyme glucocerebrosidase catalysing the regular lysosomal degradation of glucosylceramide. In the common non-neuropathic variant of GD, glucosylceramide-laden macrophages (Gaucher cells) accumulate in various tissues. Gaucher cells secrete chitotriosidase, an active chitinase, resulting in increased plasma chitotriosidase levels, which can be sensitively monitored by an enzyme activity assay. Plasma chitotriosidase is a rough estimate of body burden of Gaucher cells. Non-neuronopathic GD is presently treated by enzyme replacement therapy (ERT) and substrate reduction therapy (SRT). We addressed the question whether plasma chitotriosidase acts as (predictive) marker of clinical manifestations in non-neuronopathic GD patients receiving treatment. Reductions in plasma chitotriosidase during therapy correlated with corrections in liver and spleen volumes and showed positive trends with improvements in haemoglobin and platelet count and bone marrow composition. The occurrence of long-term complications and associated conditions such as multiple myeloma, bone complications, Parkinson's disease, hepatocellular carcinoma and pulmonary hypertension positively correlated with the plasma chitotriosidase level pre-therapy, the average plasma chitotriosidase during 3 years of ERT and the residual plasma chitotriosidase after 2 years of ERT. In summary, plasma chitotriosidase is a valuable marker in the assessment and follow-up of GD patients.

Acid sphingomyelinase (Asm) deficiency patients in The Netherlands and Belgium: disease spectrum and natural course in attenuated patients.

Niemann-Pick disease (NPD) is a neurovisceral lysosomal storage disorder caused by acid sphingomyelinase (ASM) deficiency, which can be categorized as either Niemann-Pick disease type A [NPD-A], with progressive neurological disease and death in early childhood, or as Niemann-Pick disease type B [NPD-B], with a more variable spectrum of manifestations. Enzyme replacement therapy (ERT) with recombinant sphingomyelinase is currently studied as potential treatment for NPD-B patients. The objective of this study is to characterize the clinical features of patients with ASM deficiency in the Netherlands and Belgium with focus on the natural disease course of NPD-B patients. Prospective and retrospective data on ASM deficient patients were collected in The Netherlands and part of Belgium. Patients with NPD-B that could be followed prospectively were evaluated every 6-12 months for pulmonary function tests, 6 minute walk test (6 MWT), imaging (bone marrow infiltration measured by QCSI, organ volumes by MRI and CT scan of the lungs) and biochemical markers. Twenty-five patients with ASM deficiency were identified (13 males, 12 females, median age 13years, range 1-59 years). Nine patients had died at the time of the study, including four NPD-A patients at the age of 1,1, 2, 3 and five NPDB patents at the age of 5, 6, 43, 56 and 60 years. There was a high prevalence of homozygosity and compound heterozygosity for the common p.Arg608del mutation in 43% and 19% of NPD-B patients, respectively. In NPD-B patients, thrombocytopenia was present in most, while anemia and leucopenia were less common (33% and 6 % respectively). HDL cholesterol was reduced in most patients. Pulmonary disease was severe in several patients. Follow-up up to 11 years revealed a gradual decrease in platelet count. Detailed investigations in 6 NPD-B patients with follow-up in 4 patients revealed remarkable stable disease parameters up to 6 years, with some decline in pulmonary function and 6 MWT. Bone marrow fat fractions were decreased, indicating the presence of storage macrophages. Lung involvement was not related to the extent of visceromegaly, cytopenia or bone marrow involvement. In conclusion, in NPD-B patients pulmonary disease is the most debilitating feature. Disease manifestations are mostly stable in attenuated patients. Bone marrow infiltration is a less prominent feature of the disease.

Markers of bone turnover in Gaucher disease: modeling the evolution of bone disease.

CONTEXT: Gaucher disease (GD) is a lysosomal storage disorder characterized by abundant presence of macrophages. Bone complications and low bone density are believed to arise from enhanced bone resorption mediated through macrophage-derived factors. OBJECTIVE: The objective of the study was to investigate the relationship between bone turnover and bone complications in GD. DESIGN: This was a retrospective cohort study and review of the literature. PATIENTS: Forty adult type I GD patients were included in the study. OUTCOME MEASURES: Levels of the bone-resorption marker, type 1 collagen C-terminal telopeptide, and two bone-formation markers, N-terminal propeptide of type 1 procollagen and osteocalcin, were investigated in relation to clinical bone disease, measures of overall disease severity, and imaging data representing bone marrow infiltration. RESULTS: Osteocalcin was decreased in 50% of our patients (median 0.35 nmol/liter, normal 0.4-4.0), indicating a decrease of bone formation. Type 1 collagen C-terminal telopeptide and N-terminal propeptide of type 1 procollagen were within the normal range for most patients. Osteocalcin concentration was negatively correlated to measures of overall disease severity and positively correlated with imaging data (correlation coefficient 0.423; P = 0.025), suggesting a relation with disease severity. A review of the literature revealed variable outcomes on bone resorption markers but more consistent abnormalities in bone formation markers. Two of three reports conclude that bone-formation parameters increase in response to enzyme therapy, but none describes an effect on bone-resorption markers. CONCLUSIONS: In contrast to earlier hypotheses, we propose that in GD patients, primarily a decrease in bone formation causes an imbalance in bone remodeling.

Plasma globotriaosylsphingosine: diagnostic value and relation to clinical manifestations of Fabry disease.

Fabry disease is an X-linked lysosomal storage disorder due to deficiency of alpha-Galactosidase A, causing accumulation of globotriaosylceramide and elevated plasma globotriaosylsphingosine (lysoGb3). The diagnostic value and clinical relevance of plasma lysoGb3 concentration was investigated. All male and adult female patients with classical Fabry disease could be discerned by an elevated plasma lysoGb3. In young pre-symptomatic Fabry heterozygotes, lysoGb3 levels can be normal. Individuals carrying the R112H and P60L mutations, without classical Fabry symptoms, showed no elevated plasma lysoGb3. Multiple regression analysis showed that there is no correlation of plasma lysoGb3 concentration with total disease severity score in Fabry males. However, plasma lysoGb3 concentration did correlate with white matter lesions (odds ratio: 6.1 per 100 nM lysoGb3 increase (95% CI: 1.4-25.9, p=0.015). In females, plasma lysoGb3 concentration correlated with overall disease severity. Furthermore, plasma lysoGb3 level was related to left ventricular mass (19.5+/-5.5 g increase per 10 nM lysoGb3 increase; p=0.001). In addition, it was assessed whether lifetime exposure to lysoGb3 correlates with disease manifestations. Male Fabry patients with a high lysoGb3 exposure (>10,000 U), were moderately or severely affected, only one mildly. Female patients with a low exposure (<1000 U) were asymptomatic or mildly affected. A large proportion of the females with an exposure >1000 U showed disease complications. Plasma lysoGb3 is useful for the diagnosis of Fabry disease. LysoGb3 is an independent risk factor for development of cerebrovascular white matter lesions in male patients and left ventricular hypertrophy in females. Disease severity correlates with exposure to plasma lysoGb3.

Low HDL cholesterol levels in type I Gaucher disease do not lead to an increased risk of cardiovascular disease.

OBJECTIVE: A low plasma high-density lipoprotein cholesterol (HDL-c) concentration is an important risk factor for the development of atherosclerotic cardiovascular disease. HDL-c levels are abnormally low in type I Gaucher disease (GD) patients. The aim of this study was to determine whether GD is associated with premature atherosclerosis. METHODS: Lipid profiles, apolipoproteins, and carotid artery intima-media thickness (cIMT) were analyzed in 40 type I GD patients, 34 carriers and 41 control subjects. cIMT is a non-invasive validated biomarker for the status of atherosclerosis and present and future cardiovascular disease risk. RESULTS: Compared to control subjects, patients showed decreased HDL-c (1.1+/-0.3 mmol/L) as well as mildly decreased low-density lipoprotein cholesterol (LDL-c) levels (2.8+/-0.7 mmol/L), with an increased ApoB/ApoA1 ratio. In carriers, HDL-c levels were normal, but LDL-c levels were decreased (2.7+/-0.8 mmol/L). Mean cIMT measurements were not different in the three study groups (patients: 0.63+/-0.1mm versus carriers: 0.64+/-0.1mm versus control subjects: 0.65+/-0.1 mm). CONCLUSION: In Gaucher disease low HDL-c levels do not lead to premature atherosclerosis as assessed by cIMT measurement. This indicates that the inverse relationship between levels of HDL-c and risk of cardiovascular disease in the general population may not be present in all conditions characterised by low HDL-c levels.

Plasma glucosylceramide and ceramide in type 1 Gaucher disease patients: correlations with disease severity and response to therapeutic intervention.

The concentrations of plasma glucosylceramide (GlcCer) and ceramide (Cer) were determined in a cohort of type 1 Gaucher disease patients. In plasma of untreated patients, GlcCer concentrations were on average 3-fold increased (median Gaucher: 17.5 nmol/ml, range: 6.5-45.5 (n=27); median control: 5.9 nmol/ml, range 4.0-8.6 (n=15)). Although plasma Cer concentrations were not significantly different between the two groups (median Gaucher: 7.2 nmol/ml, range: 4.2-10.9 (n=27); median control: 7.8 nmol/ml, range 5.7-11.9 (n=15)) in individual patients plasma GlcCer/Cer ratio yields slightly better discrimination between Gaucher disease patients and normal individuals than the GlcCer levels. Positive correlations were detected between plasma GlcCer concentration and GlcCer/Cer ratio and severity of disease, plasma chitotriosidase and CCL18, surrogate markers of storage cells. Gaucher disease is treated by enzyme replacement and substrate reduction therapy. Both therapies were found to result in decreases in plasma GlcCer already within 6 months, without causing abnormal plasma GlcCer or Cer concentrations. The corrections in plasma GlcCer were most robust in patients with a pronounced clinical response. In conclusion, plasma GlcCer concentration and GlcCer/Cer ratio is of value to monitor Gaucher disease manifestation and response to therapeutic intervention.

Short-term manipulation of plasma free fatty acids does not change skeletal muscle concentrations of ceramide and glucosylceramide in lean and overweight subjects.

CONTEXT: Increased plasma free fatty acid (FFA) concentrations may be in part responsible for the increased levels of ceramide in skeletal muscle of obese subjects. OBJECTIVE: We studied the effect of lowering and increasing plasma FFA levels on muscle ceramide and glucosylceramide concentrations in lean and obese subjects. DESIGN: Plasma FFAs were either increased or decreased for 6 h by infusing a lipid emulsion or using Acipimox, respectively. Muscle biopsies were performed before and after the intervention for measurements of ceramide and glucosylceramide. STUDY SUBJECTS: Eight lean [body mass index 21.9 (range, 19.6-24.6) kg/m2] and six overweight/obese [body mass index 34.4 (27.8-42.5) kg/m2] subjects without type 2 diabetes mellitus participated in the study. MAIN OUTCOME MEASURE: Differences in muscle ceramide and glucosylceramide upon manipulation of plasma FFAs were measured. RESULTS: There were no differences in muscle ceramide and glucosylceramide between lean and obese subjects, respectively. Increasing or decreasing plasma FFAs for 6 h had no effect on ceramide [high FFAs: 24 (19-25) vs. 24 (22-27) pmol/mg muscle, P=0.46; and 22 (20-28) vs. 24 (18-26) pmol/mg muscle, P=0.89 in lean and obese, respectively; low FFAs: 26 (24-35) vs. 23 (18-27) pmol/mg muscle, P=0.17 and 24 (15-44) vs. 24 (19-42) pmol/mg muscle, P=0.6 in lean and obese, respectively] and glucosylceramide [high FFAs: 2.0 (1.7-4.3) vs. 3.4 (2.1-4.6) pmol/mg muscle, P=0.17; and 3.0 (1.3-6.7) vs. 2.6 (1.2-3.9) pmol/mg muscle, P=0.89 in lean and obese, respectively; low FFAs: 2.2 (1.0-4.4) vs. 1.7 (1.4-3.0) pmol/mg muscle, P=0.92; and 6.6 (1.0-25.0) vs. 4.3 (1.3-7.6) pmol/mg muscle, P=0.7 in lean and obese, respectively] concentrations in skeletal muscle. CONCLUSION: Short-term manipulation of plasma FFAs has no effect on ceramide and glucosylceramide concentrations in skeletal muscle from lean and obese subjects.

The Dutch Fabry cohort: diversity of clinical manifestations and Gb3 levels.

BACKGROUND: Fabry disease (OMIM 301500) is an X-linked lysosomal storage disorder with characteristic vascular, renal, cardiac and cerebral complications. Globotriaosylceramide (Gb(3)) accumulates in Fabry patients as a result of alpha-galactosidase A deficiency. The phenotypic variability is high, but the relationship between clinical symptoms in individual Fabry patients has not been uniformly documented. Also, the relation between the most prominent biochemical abnormalities, elevated Gb(3) levels in plasma and urine, and clinical symptoms is not firmly established. METHODS: Clinical and biochemical characteristics of 96 (25 deceased) Dutch Fabry patients were collected retrospectively and before the initiation of enzyme therapy. RESULTS: Clinical assessment revealed that median life expectancy was 57 years for male and 72 years for female patients. Cerebral complications, acroparaesthesias and gastrointestinal complications, but not cardiac and auditory complications, were all seen more frequently in male than female patients. Glomerular filtration rate (GFR) was highly variable in male patients, including 2 patients with GFR < 30 ml/min, but median GFR did not differ between males and females (103 and 101 ml/min, respectively). Hyperfiltration was more frequently observed in the female patient group. Microalbuminuria was present in 60% of males and 45% of females. No specific pattern of combined symptoms existed except for a relationship between left ventricular hypertrophy (LVH) and cerebral complications (males 36%, females 32%), or proteinuria (males 35%, females 31%). Gb(3) was found to be more elevated in plasma samples from male (n = 26; median 6.27 micromol/L (1.39-9.74)) than female Fabry patients (n = 37; median 2.16 (0.77-4.18)). This was also observed for urinary Gb(3): males (n = 22) median 1851 nmol/24 h (40-3724); females (n = 29) median 672 (86-2052). Plasma and urinary Gb(3) levels correlated with each other in both males (r = 0.4, p = 0.05) and females (r = 0.4, p = 0.03), but no correlation between elevated Gb(3) levels and clinical symptoms could be detected. CONCLUSION: Analysis of the characteristics of the Dutch Fabry cohort has revealed that a limited relationship between various disease manifestations exists and that individual symptoms do not correlate with elevated urinary or plasma Gb(3) levels, limiting their value as surrogate disease markers.

Plasma chitotriosidase in male Fabry patients: a marker for monitoring lipid-laden macrophages and their correction by enzyme replacement therapy.

Increased plasma chitotriosidase is a well established surrogate marker for the occurrence of lipid-laden macrophages in the glycosphingolipidosis Gaucher disease. The complete lack of surrogate markers for Fabry disease, X-linked globotriaosylceramidosis stemming from deficiency in the lysosomal alpha-galactosidase A (AGA), prompted us to study chitotriosidase in this disorder. In male Fabry patients plasma chitotriosidase is significantly elevated, consistent with the presence of lipid-laden macrophages in several tissues. Increased levels are detectable at very young age and precede clinical manifestations. No strict correlation exists with severity of disease manifestations. Upon therapy with either of the two available recombinant AGA preparations, plasma chitotriosidase levels are nicely normalized in male Fabry patients. However, in patients developing neutralizing antibodies towards AGA, reduction in plasma chitotriosidase is hampered. In sharp contrast to the situation in male patients, females heterozygous for AGA deficiency show no significantly elevated plasma chitotriosidase. This suggests that circulating endogenous AGA in heterozygotes is sufficient to supplement enzyme-deficient macrophages. In conclusion, for the first time a biological marker for lipid-laden cells in Fabry patients is demonstrated; elevated plasma chitotriosidase levels reflecting lipid-laden macrophages. Corrections in this marker illustrate the efficacy of enzyme replacement therapy in clearing the lipid accumulation in this particular cell type.

Identification and use of biomarkers in Gaucher disease and other lysosomal storage diseases.

UNLABELLED: The value of biomarkers in the clinical management of lysosomal storage diseases is best illustrated by the present use of plasma chitotriosidase levels in the diagnosis and monitoring of Gaucher disease. The enzyme chitotriosidase is specifically produced and secreted by the pathological storage macrophages (Gaucher cells). Plasma chitotriosidase levels are elevated on average 1000-fold in symptomatic patients with Gaucher disease and reflect the body burden on storage cells. Changes in plasma chitotriosidase reflect changes in clinical symptoms. Monitoring of plasma chitotriosidase levels is nowadays commonly used in decision making regarding initiation and optimization of costly therapeutic interventions (enzyme replacement therapy or substrate reduction therapy). A novel substrate has been developed that further facilitates the measurement of chitotriosidase in plasma samples. Moreover, an alternative Gaucher-cell marker, CCL18, has been very recently identified and can also be employed to monitor the disease, particularly in those patients lacking chitotriosidase due to a genetic mutation. There is a need for comparable surrogate markers for other lysosomal storage diseases and the search for such molecules is an area of intense investigation. CONCLUSION: The use of biomarkers can provide valuable insight into the molecular pathogenesis of LSDs, such as Gaucher disease and Fabry disease.

Plasma chitotriosidase and CCL18: early biochemical surrogate markers in type B Niemann-Pick disease.

Type B Niemann-Pick disease (NPD) is a nonneuronopathic lysosomal storage disorder which is characterized by accumulation of sphingomyelin-laden macrophages. The availability of plasma markers for storage cells may be of great value in facilitating therapeutic decisions. Given the similarity of the storage cells in NPD and Gaucher disease, we studied Gaucher plasma markers (chitotriosidase and CCL18) in two siblings homozygous for the R228C mutation in acid sphingomyelinase (ASM) and a type B course of NPD. The older sibling, first examined at the age of 9 months, showed marked hepatosplenomegaly and pulmonary involvement. The younger sibling has mild asymptomatic hepatosplenomgaly at the age of 5 months. Analysis of plasma specimens revealed markedly increased levels of chitotriosidase and CCL18 in the older sibling. In the younger child also, plasma chitotriosidase and CCL18 were clearly elevated above normal values almost immediately after birth and rapidly increased further. Histochemistry confirmed production of CCL18 by foam cells. In conclusion, plasma chitotriosidase and CCL18 may also serve as markers for the formation of pathological lipid-laden macrophages in type B NPD, in analogy to Gaucher disease. The availability of sensitive plasma surrogate markers may be of great value for monitoring the efficacy of enzyme supplementation therapy that is currently being developed.

GLUT-1 deficiency without epilepsy--an exceptional case.

The GLUT-1 deficiency is a metabolic disorder caused by a defect in glucose transport across the blood-brain barrier as a result of a defect in the glucose-transport protein. Patients present with epileptic seizures, delayed development, ataxia and hypotonia, and in many cases acquired microcephaly. In most patients, treatment with a ketogenic diet proved to be successful in controlling the epilepsy. We report a 9-year-old boy with retardation and ataxia, but without epilepsy, caused by GLUT-1 deficiency, proven biochemically and by DNA analysis. Treatment with a medium-chain triglyceride ketogenic diet had a beneficial effect.

L-Arabinosuria: a new defect in human pentose metabolism.

A female patient, the first child of healthy non-consanguineous parents, presented at the age of 16 months with delayed motor development and facial dysmorphism. In addition she displayed a palatoschizis and multiple skeletal abnormalities as hypoplastic scapulae, hypoplastic os ilea, and an extreme cervical kyphosis. Biochemical investigation of urine revealed no abnormalities except for the presence of large amounts of reducing sugars. The sugar was identified as L-arabinose, which mainly originated from fruit formula in her diet. In addition highly elevated levels of L-arabitol were found in urine, plasma, and cerebrospinal fluid. Although little is known about human arabinose metabolism, we presume that L-arabitol dehydrogenase is deficient in our patient. As polyols are potentially toxic to the central nervous system there could be deleterious long-term effects of this disorder. Withdrawal of dietary fruit led to normalization of polyol levels. The above-mentioned clinical abnormalities are probably not related to this new inborn error of metabolism and should be considered as a separate entity.

Biosynthesis of phosphatidylcholine from a phosphocholine precursor pool derived from the late endosomal/lysosomal degradation of sphingomyelin.

Previous studies suggest that the steps of the CDP- choline pathway of phosphatidylcholine synthesis are tightly linked in a so-called metabolon. Evidence has been presented that only choline that enters cells through the choline transporter, and not phosphocholine administered to cells by membrane permeabilization, is incorporated into phosphatidylcholine. Here, we show that [(14)C]phosphocholine derived from the lysosomal degradation of [(14)C]choline-labeled sphingomyelin is incorporated as such into phosphatidylcholine in human and mouse fibroblasts. Low density lipoprotein receptor-mediated endocytosis was used to specifically direct [(14)C]sphingomyelin to the lysosomal degradation pathway. Free labeled choline was not found either intracellularly or in the medium, not even when the cells were energy-depleted. Deficiency of lysosomal acid phosphatases in mouse or alkaline phosphatase in human fibroblasts did not affect the incorporation of lysosomal [(14)C]sphingomyelin-derived [(14)C]phosphocholine into phosphatidylcholine, supporting our finding that phosphocholine is not degraded to choline prior to its incorporation into phosphatidylcholine. Inhibition studies and analysis of molecular species showed that exogenous [(3)H]choline and sphingomyelin-derived [(14)C]phosphocholine are incorporated into phosphatidylcholine via a common pathway of synthesis. Our findings provide evidence that, in fibroblasts, phosphocholine derived from sphingomyelin is transported out of the lysosome and subsequently incorporated into phosphatidylcholine without prior hydrolysis of phosphocholine to choline. The findings do not support the existence of a phosphatidylcholine synthesis metabolon in fibroblasts.

Human alpha-N-acetylgalactosaminidase (alpha-NAGA) deficiency: no association with neuroaxonal dystrophy?

Two new individuals with alpha-NAGA deficiency are presented. The index patient, 3 years old, has congenital cataract, slight motor retardation and secondary demyelinisation. Screening of his sibs revealed an alpha-NAGA deficiency in his 7-year-old healthy brother who had no clinical or neurological symptoms. Both sibs are homozygous for the E325K mutation, the same genotype that was found in the most severe form of alpha-NAGA deficiency presenting as infantile neuroaxonal dystrophy. Thus, at the age of 7 years the same genotype of alpha-NAGA may present as a 'non-disease' (present healthy case) and can be associated with the vegetative state (the first two patients described with alpha-NAGA deficiency). The clinical heterogeneity among the 11 known individuals with alpha-NAGA deficiency is extreme, with a 'non-disease' (two cases) and infantile neuroaxonal dystrophy (two cases) at the opposite sides of the clinical spectrum. The broad spectrum is completed by a very heterogeneous group of patients with various degrees of epilepsy/behavioural difficulties/psychomotor retardation (four patients) and a mild phenotype in adults without overt neurological manifestations who have angiokeratoma and clear vacuolisation in various cell types (three cases). These observations are difficult to reconcile with a straightforward genotype-phenotype correlation and suggest that factors or genes other than alpha-NAGA contribute to the clinical heterogeneity of the 11 patients with alpha-NAGA deficiency.

Delayed lysosomal metabolism of lipids in mucolipidosis type IV fibroblasts after LDL-receptor-mediated endocytosis.

We specifically probed the low-density lipoprotein-receptor-dependent endosomal/lysosomal pathway of lipid degradation in control and mucolipidosis type IV fibroblasts using either [choline-methyl-14C]sphingomyelin in complex with apolipoprotein E, or cholesteryl [14C]oleate-labelled low-density lipoprotein as a substrate. Mucolipidosis type IV fibroblasts metabolized [14C]sphingomyelin and cholesteryl [14C]oleate significantly more slowly than controls and fibroblasts from patients with Hurler disease or Niemann-Pick disease type C. So far, no lysosomal enzyme deficiency has been reported for mucolipidosis type IV. Rather, the defect in mucolipidosis type IV cells has recently been suggested to be related to intracellular trafficking. Our results suggest that the defect in mucolipidosis type IV also affects the low-density lipoprotein-receptor-mediated endocytosis pathway.

Difference in substrate specificity between human and mouse lysosomal acid lipase: low affinity for cholesteryl ester in mouse lysosomal acid lipase.

Lysosomal acid lipase (LAL) is essential for the intracellular degradation of cholesteryl esters (CE) and triacylglycerols (TG) that are delivered to lysosomes by low density lipoprotein (LDL) receptor mediated endocytosis. We have analysed the difference in the catalytic properties and substrate specificity of human and mouse LALs. LAL activities were measured in human and mouse fibroblasts and in HeLa cells transiently expressing wild-type or site-directed mutant LALs of the two species using the T7 vaccinia system. Cholesteryl esterase and triacylglycerol lipase activities were determined in cellular homogenates with a phospholipid/detergent vesicle assay, an assay frequently used to diagnose human LAL deficiency syndromes, and with LDL particles, a more physiological substrate. Characterisation of human and mouse LAL using these two assays demonstrated marked differences in their TG and CE hydrolysing activities. Compared to human LAL mouse LAL showed a much lower cholesteryl esterase activity in both assays used. The difference was more pronounced in the vesicle assay. The lower cholesteryl esterase activity of mouse LAL did not affect the LDL-CE degradation in intact fibroblasts. The analysis of site-directed mutants suggests a role of the non-conserved cysteine residue at position 240 in cholesteryl esterase activity in human LAL. Our results show a significant difference between human and mouse LAL in their specificity toward cholesteryl esters. The low cholesteryl esterase activity does not result in reduced LDL-cholesterol ester degradation in mouse fibroblasts in situ. In addition, this work emphasises the importance of the physical state of substrates in studies of the specificity and properties of lipolytic enzymes.