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J M Aerts

Publications and source records attributed to J M Aerts.

At least 55 records · Page 3Linked to original sources
PubMed1996

The role of the intralysosomal pH in the control of autophagic proteolytic flux in rat hepatocytes.

Current methods to estimate changes in intralysosomal pH in hepatocytes do not discriminate between lysosomes and other intracellular acidic compartments. To obtain selective information on the change in lysosomal function in response to a change in lysosomal pH we have used the liberation of fluorescent 4-methoxy-2-naphthylamide from low concentrations of lysyl-alanyl-4-methoxy-2-naphthylamide, a substrate of lysosomal dipeptidylpeptidase II. Using permeabilized and intact hepatocytes, the activity of this enzyme in response to manipulations meant to increase the intralysosomal pH was compared with intralysosomal protein degradation and with the accumulation of [14C]chloroquine as a pH indicator of intracellular acidic compartments. The data show that changes in intralysosomal pH are indicated by changes in dipeptidylpeptidase II activity and that these are mainly due to a pH-dependent change in substrate accumulation in the lysosomes. Subsequently, the method was applied to establishing the extent to which an increase in intralysosomal pH can contribute to the inhibition of autophagic proteolysis in intact hepatocytes caused by a decrease in intracellular ATP, by an increase in amino acid concentration and by hypo-osmotic cell swelling. The following observations were made. (a) Moderate changes in intracellular ATP do not affect the lysosomal pH. (b) Hypo-osmotic cell swelling, which promotes inhibition of proteolysis by amino acids in freshly isolated hepatocytes, does not affect the lysosomal pH. (c) In addition to their known inhibitory effect on autophagic sequestration, amino acids (leucine in particular) can increase the lysosomal pH and thus inhibit intralysosomal protein degradation directly. (d) Low concentrations of the acidotropic agent methylamine increase the lysosomal pH without having an effect on autophagic proteolytic flux. It is concluded that autophagic proteolysis is not controlled by changes in the lysosomal pH.

2-Naphthylamine↗
PubMed1996

A case of type I Gaucher disease with cardiopulmonary amyloidosis and chitotriosidase deficiency.

Severe cardiopulmonary amyloidosis developed several months after a total splenectomy in a patient with type 1 Gaucher disease and led within a year to his death at 48 years of age. The autopsy findings were dominated by extensive pulmonary and cardiac amyloid infiltration. No Gaucher cells were found in the lungs. Aside from a glucocerebrosidase deficiency the patient was also deficient in chitotriosidase, an enzyme whose activity is usually greatly increased in the serum of Gaucher patients. Analysis of mutations in the glucocerebrosidase gene revealed heterozygosity for N370S and D409H mutations. The normal amount of glucocerebrosidase was found in the spleen by Western blotting. We suggest that amyloidosis should be considered in the differential diagnosis of severe cardiopulmonary disease in Gaucher patients.

Amyloid↗
PubMed1995

Cloning of a cDNA encoding chitotriosidase, a human chitinase produced by macrophages.

We have recently observed that chitotriosidase, a chitinolytic enzyme, is secreted by activated human macrophages and is markedly elevated in plasma of Gaucher disease patients (Hollak, C. E. M., van Weely, S., van Oers, M. H. J., and Aerts, J. M. F. G. (1994) J. Clin. Invest. 93, 1288-1292). Here, we report on the cloning of the corresponding cDNA. The nucleotide sequence of the cloned cDNA predicts a protein with amino acid sequences identical to those established for purified chitotriosidase. Secretion of active chitotriosidase was obtained after transient transfection of COS-1 cells with the cloned cDNA, confirming its identity as chitotriosidase cDNA. Chitotriosidase contains several regions with high homology to those present in chitinases from different species belonging to family 18 of glycosyl hydrolases. Northern blot analysis shows that expression of chitotriosidase mRNA occurs only at a late stage of differentiation of monocytes to activated macrophages in culture. Our results show that, in contrast to previous beliefs, human macrophages can synthesize a functional chitinase, a highly conserved enzyme with a strongly regulated expression. This enzyme may play a role in the degradation of chitin-containing pathogens and can be used as a marker for specific disease states.

Amino Acid Sequence↗
PubMed1995

Individualised low-dose alglucerase therapy for type 1 Gaucher's disease.

Previous studies have shown that enzyme supplementation therapy with alglucerase for type 1 Gaucher's disease is effective at doses of 30-130 U/kg per month. Since both the clinical presentation and the response to therapy in Gaucher's disease are highly variable, individual dosing seems indicated. This notion, as well as the high costs of alglucerase and the unknown long-term side-effects, led us to investigate the efficacy of an individualised very low dose of alglucerase. Twenty-five adults with symptomatic type 1 Gaucher's disease (thirteen splenectomised) received alglucerase 1.15 U/kg three times a week (15 U/kg per month). Every 6 months, the dose was halved, maintained, or doubled, according to the response (based on haematological variables and liver and spleen volume). After 6 months of treatment, eighteen (72%) patients had a response (seventeen moderate, one good). After 12 months (in nineteen patients) and 18 months (in seven patients), all had sustained improvement. Severe splenomegaly resulted in slower haematological responses. Our results are similar to those obtained by others with higher-dose regimens and better than a low-dose regimen of 10U/kg every 2 weeks. We conclude that very low initial doses of alglucerase, when administered frequently, are effective and cost-saving in the treatment of type 1 Gaucher's disease.

Adult↗
PubMed1995

Purification and characterization of human chitotriosidase, a novel member of the chitinase family of proteins.

Recently we noted (Hollak, C.E.M., van Weely, S., van Oers, M.H.J., and Aerts, J.M.F.G. (1994) J. Clin. Invest. 93, 1288-1292) that the clinical manifestation of Gaucher disease is associated with a several hundred-fold increase in chitotriosidase activity in plasma. We report on the purification and characterization of the protein. Two major isoforms of chitotriosidase with isoelectric points of 7.2 and 8.0 and molecular masses of 50 and 39 kDa, respectively, were purified from the spleen of a Gaucher patient. The N-terminal amino acid sequence of the two forms proved to be identical. An antiserum raised against the purified 39-kDa chitotriosidase precipitated all isozymes. Chitotriosidase activity was earlier found to be completely absent in some individuals. These findings in combination suggest that a single gene may encode the different isoforms of chitotriosidase. Both the N-terminal sequence and an internal sequence chitotriosidase proved to be homologous to sequences in proteins that are members of the chitinase family (Hakala, B.E., White,C., and Recklies, A.D. (1993) J. Biol. Chem. 268, 25803-25810). The human chitotriosidase described here showed chitinolytic activity toward artificial substrates as well as chitin and may therefore be considered to be a chitinase.

Amino Acid Sequence↗
PubMed1995

Characterization of glucocerebrosidase in Greek Gaucher disease patients: mutation analysis and biochemical studies.

Gaucher disease is the most frequent lysosomal storage disease in Greece, accounting for 24% of all lysosomal disorders diagnosed during the last 13 years at the Institute of Child Health in Athens. The nature of the defects in glucocerebrosidase in Greek Gaucher patients with non-neuronopathic (type 1) and neuronopathic (types 2 and 3) phenotypes was investigated at the level of the glucocerebrosidase gene and enzyme activity. Mutation analysis performed in 10/23 Gaucher patients with different types of the disorder led to the identification of four mutations, N370S, L444P, R463C and D409H, comprising 75% of the investigated alleles. N370S was only found in association with type 1 disease. The genotype D409H/R463C was identified for the first time and was associated with the severe type 2 disorder. There was no correlation between residual in vitro enzyme activity and either phenotype or genotype. However, in cultured fibroblast of the neuronopathic cases, glucocerebrosidase protein concentration was reduced and the capacity to degrade exogenous C6NBD-glucosylceramide was more severely impaired.

Cells, Cultured↗
PubMed1995

Elevated plasma chitotriosidase activity in various lysosomal storage disorders.

Recently a striking elevation of the activity of chitotriosidase, an endo beta-glucosaminidase distinct from lysozyme, was found in plasma from patients with Gaucher type I disease (McKusick 230800). Plasma chitotriosidase originates from activated macrophages and this elevation is secondary to the basic defect in Gaucher disease. To investigate the specificity of this phenomenon, we have investigated 24 different lysosomal storage diseases. In 11 different diseases increased chitotriosidase activity in plasma was found (in 28% of the patients). None of these diseases showed elevations as high as in Gaucher disease. Chitotriosidase was not significantly elevated in plasma from 20 different non-lysosomal enzymopathies or in plasma from patients with infectious diseases associated with hepatomegaly. The results show that marked elevation of chitotriosidase activity in plasma appears to be specific for Gaucher disease. The data further suggest that elevated levels of chitotriosidase activity in plasma from patients with unexplained diseases may be indicative for a lysosomal disorder.

Adult↗
PubMed1994

Signals on proteins, intracellular targeting and inborn errors of organellar metabolism.

Newly synthesized polypeptides contain signals that direct them to the appropriate intracellular organelles and the organelles contain receptors that recognize the signals. Protein synthesis occurs either on free ribosomes or on ribosomes bound to the endoplasmic reticulum. The proteins synthesized on bound ribosomes are co-translationally translocated into the lumen of the endoplasmic reticulum and contain or acquire targeting information for retention in the endoplasmic reticulum or for sorting to lysosomes and other compartments of the secretory and endocytic pathways. Proteins synthesized on free ribosomes remain in the cytosol or contain signals for import into the nucleus, mitochondria or peroxisomes. The nature of the targeting signals and the mechanisms of import are discussed briefly. Examples are given of inborn errors of metabolism caused by incorrect or impaired incorporation of proteins into mitochondria, lysosomes or peroxisomes.

Biological Transport↗
PubMed1994

Marked elevation of plasma chitotriosidase activity. A novel hallmark of Gaucher disease.

Gaucher disease (GD; glucosylceramidosis) is caused by a deficient activity of the enzyme glucocerebrosidase (GC). Clinical manifestations are highly variable and cannot be predicted accurately on the basis of the properties of mutant GC. Analysis of secondary abnormalities, such as elevated plasma levels of some hydrolases, may help to increase insight into the complicated pathophysiology of the disease and could also provide useful disease markers. The recent availability of enzyme supplementation therapy for GD increases the need for markers as early predictors of the efficacy of treatment. We report the finding of a very marked increase in chitotrisidase activity in plasma of 30 of 32 symptomatic type 1 GD patients studied: the median activity being > 600 times the median value in plasma of healthy volunteers. In three GC-deficient individuals without clinical symptoms, only slight increases were noted. Chitotriosidase activity was absent in plasma of three control subjects and two patients. During enzyme supplementation therapy, chitotriosidase activity declined dramatically. We conclude that plasma chitotriosidase levels can serve as a new diagnostic hallmark of GD and should prove to be useful in assessing whether clinical manifestations of GD are present and for monitoring the efficacy of therapeutic intervention.

Adolescent↗
PubMed1993

Demonstration of the existence of a second, non-lysosomal glucocerebrosidase that is not deficient in Gaucher disease.

In addition to the lysosomal glucocerebrosidase, a distinct beta-glucosidase that is also active towards glucosylceramide could be demonstrated in various human tissues and cell types. Subcellular fractionation analysis revealed that the hitherto undescribed glucocerebrosidase is not located in lysosomes but in compartments with a considerably lower density. The non-lysosomal glucocerebrosidase differed in several respects from lysosomal glucocerebrosidase. The non-lysosomal isoenzyme proved to be tightly membrane-bound, whereas lysosomal glucocerebrosidase is weakly membrane-associated. The pH optimum of the non-lysosomal isoenzyme is less acidic than that of lysosomal glucocerebrosidase. Non-lysosomal glucocerebrosidase, in contrast to the lysosomal isoenzyme, was not inhibited by low concentrations of conduritol B-epoxide, was markedly inhibited by taurocholate, was not stimulated in activity by the lysosomal activator protein saposin C, and was not deficient in patients with Gaucher disease. Non-lysosomal glucocerebrosidase proved to be less sensitive to inhibition by castanospermine or deoxynojirimycin but more sensitive to inhibition by D-gluconolactone than the lysosomal glucocerebrosidase. The physiological function of this second, non-lysosomal, glucocerebrosidase is as yet unknown.

Cells, Cultured↗
PubMed1993

Role of pH in determining the cell-type-specific residual activity of glucocerebrosidase in type 1 Gaucher disease.

The properties of control and 370Asn-->Ser glucocerebrosidase, the frequently encountered mutated form of the enzyme in type 1 Gaucher disease, were studied in vitro as well as in situ. The catalytic properties of purified 370Asn-->Ser glucocerebrosidase were highly dependent on the assay conditions. The enzyme was deficient in activity towards substrate and in reactivity with the irreversible inhibitor conduritol B-epoxide (CBE) when activated by the bile salt taurocholate. In the presence of more physiological activators, the lysosomal activator protein saposin C and phosphatidylserine, the 370Asn-->Ser enzyme was near normal in kinetic properties at pH values approximately 5, but not at higher pH. In intact fibroblasts, the enzymic activity of the 370Asn-->Ser glucocerebrosidase and its reactivity with CBE were found to be clearly deficient. However, in intact lymphoblasts from the same patients, the behavior of the mutant enzyme was near normal. The catalytic efficiency of 370Asn-->Ser glucocerebrosidase in situ was also found to be highly pH dependent. When intact lymphoblasts were cultured in the presence of permeant weak bases, which increase the pH of acidic intracellular compartments, the catalytic efficiency of the mutant enzyme, as assessed by its reactivity with CBE, became markedly impaired. Our findings indicate that the intralysosomal pH in the intact cell can be expected to have a critical influence on the activation state of 370Asn-->Ser glucocerebrosidase and its ability to hydrolyse substrate. This phenomenon may partly underly the marked heterogeneity in clinical manifestation of Gaucher disease among patients with this mutated form of glucocerebrosidase.

Amino Acid Sequence↗
PubMed1991

The identification of type 1 Gaucher disease patients, asymptomatic cases and carriers in The Netherlands using urine samples: an evaluation.

The feasibility of using urine samples for the identification of patients with Gaucher disease and carriers has been investigated. It was found that the pH of a urine sample should be pH 6.0 or lower to ensure stability of lysosomal hydrolases. Two parameters of glucocerebrosidase, which is deficient in Gaucher disease, were studied using urine samples from control subjects, obligate carriers and patients. Firstly, the relative level of glucocerebrosidase activity was measured by relating the activity of the enzyme to that of another lysosomal hydrolase. Secondly, the enzymic activity of glucocerebrosidase per unit of protein was measured using an immunological method. The first method allowed discrimination of nearly all obligate carriers of type 1 Gaucher disease from normal individuals. The second method allowed clear discrimination of the majority of carriers from normal individuals, but some obligate carriers were not distinguishable from normal subjects on the basis of this parameter. However, the combination of both methods allowed discrimination between all obligate carriers examined so far (n = 34) and controls (n = 86). There was variability between healthy individuals in the relative amount of glucocerebrosidase in urine samples. A small proportion of healthy individuals have a relatively high activity of glucocerebrosidase in urine samples, reminiscent of observations made in white blood cells by other investigators. In urine samples from two unrelated parents of Gaucher disease patients a level of glucocerebrosidase activity was present that could not be distinguished from that in samples of patients. These individuals represent cases with subclinical manifestation of Gaucher disease, illustrating once more the remarkable heterogeneity in clinical expression of this disorder.

Enzyme Stability↗
PubMed1991

Characterization of human glucocerebrosidase from different mutant alleles.

Human cDNA was mutagenized to duplicate six naturally occurring mutations in the gene for glucocere-brosidase. The mutant genes were expressed in NIH 3T3 cells. The abnormal human enzymes were purified by immunoaffinity chromatography and characterized. The Asn370----Ser mutant protein differed from normal enzyme in its inhibition by both conduritol B epoxide and glucosphingosine demonstrating that the 370 mutant enzyme has an abnormal catalytic site. In addition, the 370 mutant enzyme is less activated by saposin C, but more stimulated by phosphatidylserine than the wild type enzyme. The Arg463----Cys mutant protein was normal with respect to conduritol B epoxide and glucosphingosine inhibition, but was less activated by both saposin C and phosphatidylserine. The Arg120----Gln mutant protein was catalytically inactive. The Leu444----Pro, the pseudopattern, and the Pro415----Arg mutants appear to have reduced amounts of enzyme protein in cells. The studies demonstrated that mutations in the gene for glucocerebrosidase have different effects on the catalytic activity and stability of the enzyme.

Alleles↗
PubMed1990

Conditions affecting the activity of glucocerebrosidase purified from spleens of control subjects and patients with type 1 Gaucher disease.

Glucocerebrosidase was purified to homogeneity from spleens of control subjects and Type 1 Gaucher disease patients by immunoaffinity chromatography. Activation of the enzyme by taurocholate, phosphatidylserine and sphingolipid activator protein 2 (saposin C; SAP-2) was investigated by titration of combinations of various effectors in the absence and presence of Triton X-100. The specific activity of Type 1 Gaucher glucocerebrosidase was found to be less than 20% of the corresponding control value under most conditions. However, in the presence of optimal amounts of activator protein SAP-2 and phosphatidylserine (and in the absence of Triton X-100 and/or taurocholate), the specific activity of mutant enzyme towards artificial and natural lipid substrates was close to normal when measured at pH 5.0-5.5. At pH values below 5.0, the specific activity of mutant enzyme decreased more rapidly compared to that of control enzyme. The activity of Type 1 Gaucher glucocerebrosidase in the intact cell might, in a comparable manner, be highly dependent on the extent of activation by endogenous activators and on the intralysosomal pH. Values for residual glucocerebrosidase activity, as measured in vitro in extracts of cells and tissues from Type 1 Gaucher disease patients, are indeed highly dependent on the assay conditions employed. Consequently such measurements are of little value in the assessment of the actual capacity for glucosylceramide hydrolysis and for prediction of the clinical severity of the disease.

Detergents↗