Wilma E. Donker-Koopman

Elevated globotriaosylsphingosine is a hallmark of Fabry disease.

Fabry disease is an X-linked lysosomal storage disease caused by deficiency of α-galactosidase A that affects males and shows disease expression in heterozygotes. The characteristic progressive renal insufficiency, cardiac involvement, and neuropathology usually are ascribed to globotriaosylceramide accumulation in the endothelium. However, no direct correlation exists between lipid storage and clinical manifestations, and treatment of patients with recombinant enzymes does not reverse several key signs despite clearance of lipid from the endothelium. We therefore investigated the possibility that globotriaosylceramide metabolites are a missing link in the pathogenesis. We report that deacylated globotriaosylceramide, globotriaosylsphingosine, and a minor additional metabolite are dramatically increased in plasma of classically affected male Fabry patients and plasma and tissues of Fabry mice. Plasma globotriaosylceramide levels are reduced by therapy. We show that globotriaosylsphingosine is an inhibitor of α-galactosidase A activity. Furthermore, exposure of smooth muscle cells, but not fibroblasts, to globotriaosylsphingosine at concentrations observed in plasma of patients promotes proliferation. The increased intima-media thickness in Fabry patients therefore may be related to the presence of this metabolite. Our findings suggest that measurement of circulating globotriaosylsphingosine will be useful to monitor Fabry disease and may contribute to a better understanding of the disorder.

Transglycosidase Activity of Chitotriosidase IMPROVED ENZYMATIC ASSAY FOR THE HUMAN MACROPHAGE CHITINASE

Chitotriosidase is a chitinase that is massively expressed by lipid-laden tissue macrophages in man. Its enzymatic activity is markedly elevated in serum of patients suffering from lysosomal lipid storage disorders, sarcoidosis, thalassemia, and visceral Leishmaniasis. Monitoring of serum chitotriosidase activity in Gaucher disease patients during progression and therapeutic correction of their disease is useful to obtain insight in changes in body burden on pathological macrophages. However, accurate quantification of chitotriosidase levels by enzyme assay is complicated by apparent substrate inhibition, which prohibits the use of saturating substrate concentrations. We have therefore studied the catalytic features of chitotriosidase in more detail. It is demonstrated that the inhibition of enzyme activity at excess substrate concentration can be fully explained by transglycosylation of substrate molecules. The potential physiological consequences of the ability of chitotriosidase to hydrolyze as well as transglycosylate are discussed. The novel insight in transglycosidase activity of chitotriosidase has led to the design of a new substrate molecule, 4-methylumbelliferyl-(4-deoxy)chitobiose. With this substrate, which is no acceptor for transglycosylation, chitotriosidase shows normal Michaelis-Menten kinetics, resulting in major improvements in sensitivity and reproducibility of enzymatic activity measurements. The novel convenient chitotriosidase enzyme assay should facilitate the accurate monitoring of Gaucher disease patients receiving costly enzyme replacement therapy.

Ultrasensitive in situ visualization of active glucocerebrosidase molecules

Deficiency of glucocerebrosidase (GBA) underlies Gaucher disease, a common lysosomal storage disorder. Carriership for Gaucher disease has recently been identified as major risk for parkinsonism. Presently, no method exists to visualize active GBA molecules in situ. We here report the design, synthesis and application of two fluorescent activity-based probes allowing highly specific labeling of active GBA molecules in vitro and in cultured cells and mice in vivo. Detection of in vitro labeled recombinant GBA on slab gels after electrophoresis is in the low attomolar range. Using cell or tissue lysates, we obtained exclusive labeling of GBA molecules. We present evidence from fluorescence-activated cell sorting analysis, fluorescence microscopy and pulse-chase experiments of highly efficient labeling of GBA molecules in intact cells as well as tissues of mice. In addition, we illustrate the use of the fluorescent probes to study inhibitors and tentative chaperones in living cells.

Identification of the Non-lysosomal Glucosylceramidase as β-Glucosidase 2

The primary catabolic pathway for glucosylceramide is catalyzed by the lysosomal enzyme glucocerebrosidase that is defective in Gaucher disease patients. A distinct non-lysosomal glucosylceramidase has been described but its identity remained enigmatic for years. We here report that the non-lysosomal glucosylceramidase is identical to the earlier described bile acid β-glucosidase, being β-glucosidase 2 (GBA2). Expressed GBA2 is identical to the native non-lysosomal glucosylceramidase in various enzymatic features such as substrate specificity and inhibitor sensitivity. Expression of GBA2 coincides with increased non-lysosomal glucosylceramidase activity, and GBA2-targeted RNA interference reduces endogenous non-lysosomal glucosylceramidase activity in cells. GBA2 is found to be located at or close to the cell surface, and its activity is linked to sphingomyelin generation. Hydrophobic deoxynojirimycins are extremely potent inhibitors for GBA2. In mice pharmacological inhibition of GBA2 activity is associated with impaired spermatogenesis, a phenomenon also very recently reported for GBA2 knock-out mice (Yildiz, Y., Matern, H., Thompson, B., Allegood, J. C., Warren, R. L., Ramirez, D. M., Hammer, R. E., Hamra, F. K., Matern, S., and Russell, D. W. (2006) J. Clin. Invest. 116, 2985–2994). In conclusion, GBA2 plays a role in cellular glucosylceramide metabolism.

Treatment of Fabry disease with different dosing regimens of agalsidase: effects on antibody formation and GL-3

Two different enzyme preparations are used for the treatment of Fabry disease patients, agalsidase alpha (Replagal, Shire) and agalsidase beta (Fabrazyme, Genzyme). Therapeutic efficacy of both products has been variable probably due to differences in gender, severity, age and other patient characteristics. We studied the occurrence of alpha-Gal A antibodies and their effect on urinary and plasma globotriaosylceramide (GL-3), plasma chitotriosidase and clinical outcome in 52 patients after 12 months of treatment with either 0.2mg/kg agalsidase alppha (10 males, 8 females) or beta (8 males, 5 females) or 1.0mg/kg agalsidase beta (10 males, 11 females). Antibodies were detected in 18/28 male patients after 6 months. None of the females developed antibodies. Following 12 months of 0.2mg/kg treatment, urinary GL-3 decreased in antibody negative (AB-) but increased in antibody positive (AB+) patients. Treatment with 1.0mg/kg gave a reduction in urinary GL-3 in both AB- and AB+ patients. Levels of plasma GL-3 and chitotriosidase decreased in all patient groups. Twelve months of 0.2mg/kg treatment did not change renal function or left ventricular mass. Further, no change in renal function was seen following 1.0mg/kg treatment and left ventricular mass decreased in both AB- and AB+ patients. In summary, alpha-Gal A antibodies frequently develop in male Fabry disease patients and interfere with urinary GL-3 excretion. Infusion of a dose of 1.0mg/kg results in a more robust decline in GL-3, less impact, if any of antibodies, stable renal function and reduction of LVMass.

Long-Term Effect of Antibodies against Infused Alpha-Galactosidase A in Fabry Disease on Plasma and Urinary (lyso)Gb3 Reduction and Treatment Outcome

Introduction Enzyme replacement therapy (ERT) with alpha-Galactosidase A (aGal A) may cause antibody (AB) formation against aGal A in males with Fabry disease (FD). Anti agalsidase ABs negatively influence globotriaosylceramide (Gb3) reduction. We investigated the impact of agalsidase AB on Gb3 and lysoGb3 and clinical outcome in Fabry patients on ERT. Methods Adult male and female patients on ERT for at least one year were included. Urinary Gb3 was measured by HPLC, plasma lysoGb3 by LC-ESI-MS/MS and AB with a neutralization assay. Results Of the 59 patients evaluable patients, 0/30 females and 17/29 males developed anti-agalsidase antibodies (AB+). Only 3/17 males had transient (low) titers (tolerized). All AB+ patients developed antibodies during the first year of treatment. Change of agalsidase preparation (or dose) did not induce antibody formation. AB+ males had significant less decline in plasma lysoGb3 compared to AB− males (p = 0.04). Urinary Gb3 levels decreased markedly in AB− but remained comparable to baseline in AB+ males (p<0.01). (Lyso)Gb3 reduction in plasma and urine on ERT was correlated with LVmass reduction in females and development white matter lesions and stroke. Conclusion In male patients antibodies against aGal A remained present up to 10 years of ERT. The presence of these antibodies is associated with a less robust decrease in plasma lysoGb3 and a profound negative effect on urinary Gb3 reduction, which may reflect worse treatment outcome.

A procedure for the rapid purification in high yield of human glucocerebrosidase using immunoaffinity chromatography with monoclonal antibodies

A novel chromatographic immunoaffinity procedure is described for the purification of Form I glucocerebrosidase (see J. M. F. G. Aerts, W. E. Donker-Koopman, M. K. Van der Vliet, L. M. V. Jonsson, E. I. Ginns, G. J. Murray, J. A. Barranger, J. M. Tager, and A. W. Schram, 1985, Eur. J. Biochem. 150, 565-574) from extracts of human tissues. The affinity support consists of two monoclonal anti-(glucocerebrosidase) antibodies immobilized by covalent coupling to CNBr-activated Sepharose 4B. After adsorption of the enzyme from a crude detergent extract, the column is washed successively with 30% ethylene glycol in citrate buffer (pH 6), 1% Triton X-100 in citrate phosphate buffer (pH 5.2), and 50% ethylene glycol in citrate buffer. The enzyme is eluted with 90% ethylene glycol in citrate buffer. After dilution to 30% ethylene glycol, the immunoaffinity purification is repeated. The procedure can be completed within less than 18 h. The final preparations have a high specific activity (50 U/mg protein (n = 4) for the placental enzyme) and contain no detectable impurities after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The yield is high (81 +/- 8% for the placental enzyme). The immunoaffinity column has a high capacity, can be regenerated easily, and can be utilized repeatedly without loss of activity.

Efficient routing of glucocerebrosidase to lysosomes requires complex oligosaccharide chain formation

The biosynthesis and intracellular transport of the membrane-associated lysosomal enzyme glucocerebrosidase was studied in the monoblast cell line U937. Addition to the cultures of the oligosaccharide trimming inhibitors swainsonine or deoxymannojirimycin led to an increased intracellular activity of glucocerebrosidase. This was due to prevention of the lysosomal degradation of the enzyme. When homogenates of control cells were fractionated on Percoll gradients glucocerebrosidase, like beta-hexosaminidase, was distributed in two peaks, one at low density and one at high density. When homogenates of cells cultured in the presence of oligosaccharide trimming inhibitors were fractionated beta-hexosaminidase was still distributed in two peaks but glucocerebrosidase was found mainly in low density fractions also containing galactosyltransferase activity. It is concluded that complex type oligosaccharide chain formation is required for efficient routing of glucocerebrosidase to the lysosomes in U937 cells.

Deficient activity of glucocerebrosidase in urine from patients with type 1 Gaucher disease

Glucocerebrosidase is present in considerable amounts in human urine. The enzyme is stable in concentrated urine for several days when stored at 0 degrees C. Like tissue glucocerebrosidase, the urinary enzyme is inhibited by conduritol B-epoxide and hydrolyses not only glucocerebroside but also the synthetic substrate 4-methyl-umbelliferyl-beta-D-glucoside. The enzyme is deficient in urine from patients with Gaucher disease (type 1). It is possible to discriminate completely between patients with type 1 Gaucher disease and control subjects by measuring the ratio glucocerebrosidase/beta-hexosaminidase in urine. The value of this ratio (mean +/- SE) with the synthetic substrates 4-methylumbelliferyl-beta-glucoside and p-nitrophenyl-beta-N-acetylglucosaminide, respectively, was 34.2 +/- 3.7 (n = 24) in the controls and 2.1 +/- 0.9 (n = 21) in the patients.

Mass spectrometric quantification of glucosylsphingosine in plasma and urine of type 1 Gaucher patients using an isotope standard.

Deficiency of glucocerebrosidase (GBA) leads to Gaucher disease (GD), an inherited disorder characterised by storage of glucosylceramide (GlcCer) in lysosomes of tissue macrophages. Recently, we reported marked increases of deacylated GlcCer, named glucosylsphingosine (GlcSph), in plasma of GD patients. To improve quantification, [5-9] (13)C5-GlcSph was synthesised for use as internal standard with quantitative LC-ESI-MS/MS. The method was validated using plasma of 55 GD patients and 20 controls. Intra-assay variation was 1.8% and inter-assay variation was 4.9% for GlcSph (m/z 462.3). Plasma GlcSph levels with the old and new methods closely correlate (r=0.968, slope=1.038). Next, we analysed GlcSph in 24h urine samples of 30 GD patients prior to therapy. GlcSph was detected in the patient samples (median 1.20nM, range 0.11-8.92nM), but was below the limit of quantification in normal urine. Enzyme replacement therapy led to a decrease of urinary GlcSph of GD patients, coinciding with reductions in plasma GlcSph and markers of Gaucher cells (chitotriosidase and CCL18). In analogy to globotriaosylsphingsone in urine of Fabry disease patients, additional isoforms of GlcSph differing in structure of the sphingosine moiety were identified in GD urine samples. In conclusion, GlcSph can be sensitively detected by LC-ESI-MS/MS with an internal isotope standard. Abnormalities in urinary GlcSph are a hallmark of Gaucher disease allowing biochemical confirmation of diagnosis.