Variable expression of leukocyte cytosolic broad-specificity β-glucosidase activity

Ciinica Chimica Acta, 216 (1993) 1I-3,3 © 1993 Elsevier Science Publishers B.V. All fights reserved. 0009-8981/93/g06.00 CCA 05514

Variable expression of leukocyte cytosolic broadspecificity fl-glucosidase activity G W. F o r s y t h a, K . M . R o m e r o b, J. A l v e r s o n b, D.J. V a n d e r J a g t b and R . H . Glew b aVeterinary Physiological Sciences, University of Saskatchewan, Saskatoon, Saskatchewan. S7N OWO (Canada) and bDepartment of Biochemistry, School of Medicine. University of New Mexico, Albuquerque. N M 87131 (USA)

(Received 27 July 1992, revision received 19 January 1993; accepted 20 January 1993)

Key words: Glucocerebrosidase; Gaucher disease; Glucocerebrosidase mutations; Gaucher heterozygotes

Summary The cytosolic/~-glucosidase activity that is found in a variety of mammalian tissues has no clearly defined function. In vitro assay conditions under which the broad-specificity ~-glucosidase hydrolyzes glucocerebroside at a significant rate have not been described. Nonetheless, it has been suggested that this enzyme might play an accessory role with lysosomal glucocerebrosidase in catalyzing the hydrolysis of glucosylceramide. However, this hypothesis would require that activity of both enzymes be low in severe cases of Gaucher disease in which there are pathological accumulations of glucosylceramide in one or more of the affected organs, i.e. spleen, liver and bone marrow. Information is lacking regarding the normal range of cytosolic /3-glucosidase activity in humans, p.Nitrophenyl-/3-D-mannoside was found to be a potent inhibitor (Ki = 0.068 mM) of cytosolic (3-glucosidase. In parallel studies, the activity of glucocerebrosidase was found to be minimally affected by p-nitrophenyl-/~-D-mannoside at concentrations as high as 2.5 mM. This information was used to design an assay system that would allow us to estimate glucocerebrosidase and cytosolic/~-glucosidase activities in extracts of human leukocytes. Average cytosolic ~-glucosidase activity with 4-heptyl-umbelliferyl-/3-Dglucoside as a substrate was 8.8 nmol/h/mg protein in leukocytes from 356 subjects (range, 0.2-28). Average leukocyte glucocerebrosidase specific activity was 16 nmol/h/mg protein (range 5.3-45.7). No correlation was observed between cytosolic ~-glucosidase and glucocerebrosidase activity for control and Gaucher heterozygote Correspondence to: R.H. Glew, Department of Biochemistry, School of Medicine, University of New Mexico, Albuquerque, NM 87131, USA.

12

populatio~ls (r = 0,19 and 0.25, respectively). The wide range of leukocyte cytosolic /3-glucosidase activity in individuals tested in this study indicates that a substantial proportion of ~he population may lack sufficient cytosolic ~-glucosidase activity to assist a defective lysosomal glucocerebrosidase in hydrolyzing glucosylceramide.

Introduction

/3-Glucosidase activity in mammalian liver, spleen and kidney resides in at least two separate enzymes. The lysosomal/3-glucosidase, glucocerebrosidase, has an acidic pH optimum, and is an efficient catalyst for the degradation of glucosylceramide. Reductions in glucocerebrosidase activity lead to increased tissue levels of glucosylceramide arid the clinical symptoms of Gaucher disease [1,2]. The other prominent/3-glucosid~se is a cytosolic broad-specificity enzyme that functions optimally at or near a neutral pH [3,4]. The cytosolic/3-glucosidase catalyzes the hydrolysis of several naturally occurring plant glucosides in vitro, but it has a relatively low in vitro activity against glucosylceramide [5-7]. In spite of this low activity in cleaving glucosylceramide, several authors have speculated that the cytosolic ~-glucosidase enzyme could function to reduce the rate of glucosylceramide accumulation in individuals having marked glucocerebrosidase deficiency [8-10]. This could account for the milder clinical phenotype of some individuals who are known to be homozygous for the mutations that usually produce severe type II neuropathic Ga:ucher disease [11]. This hypothesis assumes t,hat coexisting mutations in glucocerebrosidase and in the cytosolic/~-glucosidase would be necessary to produce severe clinical phenotypes of Gaucher disease. The low probability of independent mutations arising at two functionally related genetic loci in one individual makes this an unlikely event. However, low cytosolic ~-glucosidase activity could also arise from natural variability in the expression of this enzyme in different individuals. The cytosolic ~-glucosidase, which has no known role i:a intermediary metabolism, could have variable levels of expression in the human r;opulation. In the absence of mutations affecting the function of glucocerebrosidase, low expression of cytosolic B-glucosidase activity might produce a benign phe,iotype, or may even avoid the release of potentially toxic aglycones from dietary cyanogenic glycosides [12]. This study was carried out to assess the normal distribution of cytosolic /3glucosidase actMty and to determine whether there is any evidence for an association between the activity of the cytosolic and lysosomal forms of the/3-glucosidase enzyme in human leukocytes. Materials and Methods

Reagents p-Nitrophenyl-~-D-mannoside (pNP-/~-Ma~o, pNP-/3-D.gala~se (pNP-/3-Gal), pNP43-galactosyl(6 - I)galactose (pNP-/3-GalGal) were pu,:/~ased from Sigma

Chemical Co. 4-Heptylumbelliferyl-/3-D-glucoside (C7UG) was synthesized according to published methods [13]. Human p!acental glucocerebrosidase (Ceredase) was a gift of Dr. S. Furbish, Genzyme, Boston, MA.

Preparation of leukocytes ~ Leukocytes were prepared ?i'rom whole blood collected in EDTA by a dextran flotation procedure [13]. Th¢/mixed white cell pellets obtained from 20 ml of blood were suspended in 50 ~! of physiological saline and frozen at -70°C until they were analyzed for B-glucosidase activity. Leukocyte {3-glucosidase assays Cell suspensions were thawed and immediately returned to ice prior to cell disruption by ultrasonication for 5 s. Disrupted cell suspensions were assayed for total/3glucosidase activity in a medium containing 2.5 mM C7UG [14], 0.4% (w/v) sodium taurodeoxycholate, 0.25% (w/v) Lubrol, buffered with 0.2 M sodium acetate (pH 5.5). Glucocerebrosidase activity was determined by measuring the rate of release of 4-heptylumbelliferone in the presence of 6.0 mM pNP-/3-Man. Enzyme reactions were stopped by dilution with NH4OH-glycine buffer (pH 10.5) and the release of the fluorescent 4-heptylumbelliferone was quantitated using excitation and emission wavelengths of 360 and 550 nm, respectively. Cytosolic/3-glucosidase activity was calculated as the difference between total/~glucosidase and glucocerebrosidase activities. Assays were performed in triplicate and results are expressed as specific /3-glucosidase activity (nmol 4-heptyiumbelliferone produced/h/mg protein). )

The transglycosylation assay The assays for transglycosylation were carried out in the absence and presence of 10 mM pNP-/3-Man. The incubation mixture contained 5 mM pNP-/3-Gal, 20 mM sodium acetate (pH 6.0) and an appropriate amount of purified guinea pig liver cytosolic ~3-glucosidase. After a 30-min incubation at 37°C, the reactions were terminated by the addition of 10 ~l of 50% (w/v) trifluoroacetic acid (TFA). The reaction products were analyzed using a Waters 600E gradient controller, equipped with an absorbance detector (Model 484) and a reversed-phase column (DeltaPak Cis, 8 mm x 100 rnm, Waters, Milford, MA). The mobile phase consisted of 0.1% (v/v) TFA in water (A) and 0.1% TFA in 95% (v/v) acetonitrile (B). The gradient controller was set for 5 min of 100% solvent A, 20 min of linear gradient from 0-20% B, 8 min of linear gradient from 20-80% B, followed by 2 rain from 80-100% B. The column was then washed with 100% B for 3 min. The flow rate was 1.0 ml/min and the eluent was monitored at 310 nm. The products of the enzymatic reaction were identified using pNP-~-Gai, pNP-~3-Man, and pNP-GalGai standards. {3-Glucosidase inhibition Cytosolic ~-glucosidase was purified to homogeneity from guinea pig liver [4,14]. The purified enzyme catalyzed the hydrolysis of approximately 3.4 × 105 nmol of 4-methylumbelliferyl-/~-D-glucoside (MUG) per h/mg protein at pH 6.0 and 37°C. The effect of addition of varying amounts of pNP-/~-Man on the activity of the

cytosolic/3-glucosidase was determined. The effect of pNP-/3-Man on the hydrolysis of the C7UG substrate by glucocerebrosidase was measured in the presence of 6.0 mM pNP-/3-Man.

Subjects Leukocytes were obtained from a population known to be at risk as carriers of the common glucocerebrosidase mutations. The group included 77 known heterozygotes for mutations at positions 84 (G -- GG), 1226 (A -- G), or 1448 (T -- C) in the glucocerebrosidase cDNA, and 279 individuals found to have normal DNA sequences at these specific loci within the glucosidase gene.

Statistical analyses Rate data from kinetic studies were analyzed using Enzfitter software (Elsevier-BIOSOFT). Regression analyses were calculated using the NCSS statistical program (Dr. J.L. Hintze, Kaysville, UT). Results

Inhibition of cytosolic [3-glucosidase-catalyzedtransglycosylation by pNP-{3-Man The authors first became aware of the inhibitory effects of pNP-/3-Man on the cytosolic/~-glucosidase when they attempted to use it as a substrate in a transglycosylation reaction that is catalyzed by the enzyme [15]. Glucosidases have the general property of catalyzing the hydrolytic cleavage of glycosidic bonds. However, primary alcohols are better nucleophiles than water, and a number of glucosidases will catalyze the transfer of glucose to acceptor alcohols in preference to water [15]. Such transglycosylation reactions can lead to the synthesis of disaccharides when the acceptor alcohol is part of a second monosaccharide. The low specificity of the mammalian cytosolic/3-glucosidase provides unique opportunities to examine the favored products of these transglycosylation reactions. For example, with pNP-/3-Gal as a substrate, it has been shown that the cytosolic ~-glucosidase can synthesize pNP-/3GalGal [I 5], In examining a series of potential glycone acceptors for galactose resi-. dues in this transglycosylation reaction pNP-/3-Man was added to the reaction medium. Instead of acting as a galactose acceptor, pNP-/3-Man caused nearly complete inhibition of the synthesis of pNP-/3-GalGal by the cytosolic/3-glucosidase (Fig. I). Inhibition of pNP-/~-GalGal synthesis was accompanied by almost total inhibition of pNP release. The discovery of this potent inhibitory effect gave encouragement for pNP-B-Man to be tested for its inhibitory effects in the hydrolytic cleavage of umbelliferyl glucoside substrate by the cytosolic/~-glucosidase.

pNP-{3-Man inhibition of CTUG hydrolysis by cytosolic {J-glucosidase Quantitation of glucocerebrosidase activity in cell homogenates is complicated by the presence of the cytosolic/~-glucosidase. MUG is an excellent substrate for both enzymes, and considerable residual/3-glucosidase activity is expressed at pH 5.5, the optimal pH for the glucocerebrosidase assay. C7UG has been demonstrated to be a more specific substrate for glucocerebrosidase in tissue homogenates, having a lower Km than MUG [13]. The authors examined the inhibitory effects of pNP-/3-

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16 Man on C7UG hydrolysis by the cytosolic/~-glucosidase with the aim of obtaining an improved separation of the two enzyme activities when an in vitro tissue homogenate serves as the enzyme source, In the absence of pNP-/3-Man the cytosolic/3glucosidase had a Km of 5.5 mM for C7UG. Analysis of this inhibitory effect yielded a constant slope (Km///max) in the plot of l/S versus I/V for each inhibitor concentration tested (Fig. 2). This result is indicative of uncompetitive inhibition, in which the inhibitor combines with the enzymesubstrate complex to give an unproductive enzyme-inhibitor substrate complex. It is characteristic of uncompetitive inhibition that saturating inhibitor concentration will drive the velocity to zero [16]. Replots of l/Km app versus [/] yielded a Ki for pNP-/3-Man of 0.017 mM in the C7UG assay system (Fig. 3).

Assay conditions for determination of cytosolic 13-glucosidase activity Due to the uncompetitive nature of the inhibition noted above, the addition of suitable concentrations of pNP-/~-Man to the leukocyte assay medium for glucocerebrosidase has the potential to completely eliminate the contribution of cytosolic/3-glucosidase activity to the hydrolysis of C7UG. The activity of purified glucocerebrosidase in hydrolyzing C7UG in the presence of 6.0 mM pNP-/3-Man was 93% of the control activity measured without added pNP-/3-Man (Table I). Under the same assay conditions the cytosolic B-glucosidase had only 8% of the activity measured without added pNP-/~-Man. These results support the use of the difference between the total /3-glucosidase activity measured in the absence of pNP-B-Man and the remaining activity measured in the presence of pNP-/3-Man as an estimate of cytosolic/~-glucosidase activity in tissue homogenates. .

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TABLE I The effect of pNP-~-mannoside on the activity of cytosolic guinea pig ~-glucosidase and lysosomal glucocerebrosidase Enzyme

Inhibitor addeda

Specific activityb

Relative activity (%)

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1620 ! 25 371 346

100 8 !00 93

The cytosolic fl-glucosidase was purified from guinea pig liver. Pure lysosomai/3-glucosidase (glucocerebrosidase) was derived from placenta. All enzyme assays were performed using CTUG as the substrate in the presence of Lubrol (0.25%, w/v) and TDC (0.2%, w/v). apNP-/~-Man, 6 raM. bSpecific activity, ~moi 4-heptylumbelliferone released/h/mg/protein.

Distribution of leukocyte cytosolic (3-glucosidase activity Leukocyte extracts from 356 individuals were assayed in triplicate for total/3glucosidase activity and for glucocerebrosidase activity by adding pNP-/~-Man to the assay system as described above. Figure 4 gives a frequency distribution o f cytosolic /3-glucosidase activity, estimated as the difference between total activity and residual

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18

activity measured in the presence of pNP-/3-Man. These leukocyte homogenates exhibited a wide range of cytosolic/~-glucosidase activity, with the largest number of individuals falling in the activity interval of 4-8 nmol/h/mg protein. The enzyme activity within this interval is relatively low in comparison with a maximum relative activity of 28 nmol/h/mg protein observed in one individual. Known heterozygotes for one of the three common mutant forms of glucocerebrosidase (cDNA position 1226 A --- G) [17] had a similar frequency distribution of cytosolic/3, glucosidase activity, although there were a larger number of these subjects in the lowest activity interval.

Correlation of cytosolic {J-glucosidase and glucocerebrosidase activity Two related enzymes that have the same or two related functions might be expected to be coordinately expressed.This question was investigated by determining the degree of correlation between the cytosolic/3-glucosidase and glucocerebrosidase activities. For the control population, the correlation coefficient for these two enzyme activities was 0.19, indicating essentially total independence of expression of the two/~-glucosidases in leukocy~es (Fig. 5). Individuals who are heterozygous for common mutations in the glucocerebrosidase gene usually have a significantly lower leukocyte glucocerebrosidase activity than individuals who are homozygous for the wild type form of the gene. Reductions in glucocerebrosidase activity could potentially cause subclinical accumulations of glucosylceramide, especially if the mutation lies at a locus such as cDNA position 85, causing chain termination, or cDNA position 1448, interfering in the function of the active site of the enzyme. If glucocerebroside were an inducer 40 y . 7,3622 + 0.13288x

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for synthesis of the cytosolic enzyme, then this enzyme activity could be increased in individuals lacking normal glucocerebrosidase activity. This type of induction could produce a negative correlation between the activities of the two enzymes in affected tissues. Although there were three individuals with relatively low glucocerebrosidase activity and high ~-glucosidase activity, the overall population of 77 heterozygotes had a correlation coefficient of 0.25 between the two enzyme activities (Fig. 6), indicating that the level of expression of the two enzymes is totally independent within the study population. Discussion

It is inconvenient to use the natural substrate, glucosylceramide, in screening for ~-glucosidase activity in large population studies. Nevertheless, fluorescent watersoluble umbeiliferone glucosides provide sensitive reagents that are suitable for the rapid screening of enzyme activity when one is dealing with a large number of tissue samples. However, since these water-soluble ~-glucosides are also cleaved by the cytosolic, broad-specificity/3-glucosidase, their use ~ves.rise to estimates of total/3glucosidase activity. It has been demonstrated that cytosolic ~-glucosidase activity is inhibited by pNP-~-Man. In the present paper, cytosolic ~-glucosidase activity was calculated by subtracting the activity obtained in the presence of pNP-/~-Man from the activity measured in the absence of inhibitor. The lack of correlation between cytosolic j3-glucosidase and lysosomal glucocerebrosidase activities provides evidence against linkage of the association between the two enzymes.

20

Cheetham et al. reported that cytosolic/~-glucosidase exhibited a pronounced bimodai activity distribution in post-mortem liver samples [ 18]. In their population they found six of 21 liver samples to be devoid of cytosolic/3-glucosidase activity. Although the leukocyte data from the current study are distributed through a range of activities similar to that reported for the post-mortem liver samples, there appears to be no clear separation of individuals into two populations. The distinct bimodal distribution seen by Cheetham et al. could be unique to the liver, or it might be an artifact arising from post-mortem losses of enzyme activity. It has been demonstrated that pNP-/3-Man is an effective inhibitor of both the glycosyltransferase and hydrolytic activities of the guinea pig liver cytosolic /3glucosidase. The uncompetitive inhibition caused by pNP-/3-Man with CTUG as substrate in the presence of Lubrol-sodium taurodeoxycholate activators may represent some type of interaction between one of the detergents and one of the known hydrophobic substrate binding sites present on the cytosolic enzyme [15]. It has been shown that the cytosolic /3-glucosidase has separate binding sites for glycoside substrates and for alkyl-alcohol acceptor molecules such as n-butanol or arylglycosides [ 15]. Evidence has been presented indicating that a cytosolic enzyme is unlikely to play a role in the breakdown of a glucosylceramide contained in membranous structures [61. Inhibition of cytosolic/3-glucosidase activity in vitro by natural breakdown products of glucocerebroside, such as sphingosine [7], have also been cited as evidence against a role for the cytosolic enzyme in glycolipid catabolism. However, it should be noted that sphingosine is an intermediate and not a final end-product of lipid catabolism. Such an intermediate would be expected to accumulate in vitro in assays using purified/3-glucosidase. However, an inhibitory intermediate could be removed in vivo by other catabolic enzymes, or by salvage pathways which function in the normal glycolipid metabolism that takes place within cells. Thus, it is possible that end-product or 'intermediate' inhibition may not be a significant obstacle to in vivo glycolipid hydrolysis by the cytosolic ~-glucosidase. The rate of glucosyisphingosine hydrolysis by the cytosolic/3-glucosidase, as determined in in vitro assays by Legler and Bieberich, was only 4% of the rate of MUG hydrolysis [7], This rate appears to be quite low when comparing in vitro activities of the two enzymes in leukocyte homogenates for catalyzing the hydrolysis of C7UG. Some in vivo rate enhancement of the activity of the cytosolic/3-glucosidase toward glucocerebroside may be necessary for this enzyme to play a significant backup role to glucocerebrosidase in glucosylceramide catabolism. There are Gaucher patients who are homozygous for the 1448 ( T - C) transversion but who fail to show the predicted neuropathic phenotype associated with this mutation [11]. A comparison of the levels of cytosolic/~-glucosidase activity in the tissues of these individuals with the activity of the enzyme in homozygous 1448 (T - C) subjects with severe neuropathic disease might help to clarify the involvement of the cytosolic B-glucosidase in the clinical phenotype presented by Gaucher patients. Fhe findings on the natural variability of leucocyte B-glucosidase activity indicate that the normal range of expression of the enzyme may be wide enough to contribute to the phenotypic variations that are seen in Gaucher patients with identical glucocerebroside genotypes.

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Acknowledgements This work was supported by grants from the Research Allocation Committee, University of New Mexico School of Medicine, and Dor Yeshorim Committee for the Prevention of Jewish Genetic Diseases, Brooklyn, New York. References 1 Glew RH, Basu A, LaMarco KL, Prence EM. Mammalian glucocerebrosidase: implications for Gaucher's disease. Lab Invest 1988;58:5-25. 2 Grabowski GA, Gatt S, Horowitz M. Acid B-glucosidase: enzymology and molecular biology of Gaucher disease. Crit Rev Biochem Mol Biol 1990:25:385-414. 3 Daniels LB, Glew RH. /~-Glucosidase assays in the diagnosis of Gaucher's disease. Clin Chem 1982;28:569-577. 4 Glew RH, Gopalan V, Forsyth GW, VanderJagt DJ. The cytosolic broad-specificity/~-glucosidase. Am Chem Soc 1993;in press. 5 Distler JJ, Jourdian GW. The purification and properties of an aryl ~-hexosidase from bovine liver. Arch Biochem Biophys 1977;i 78:631-643. 6 Daniels LB, Coyle PJ, Chiao Y-B, Glew RH, Labow RS. Purification and characterization of a cytosolic broad specificity/3-glucosidase from human liver. J Biol Chem 198 !;256:i3004-13013. 7 Legler G, Bieberich E. Isolation of a cytosolic/3-glucosidase from calf liver, and characterization of its active site with alkylglucosides and basic glucosyl derivatives. Arch Biochem Biophys 1988;260:429-436. 8 Owada M, Sakiyama T, Kitagawa T. Neuropathic Gaucher's disease with normal 4methylumbelliferyl-O-glucosidase activity in the liver. Pediatr Res 1977;I 1:641-646. 9 Burton BK, Ben-Youseph Y, Nadler HL. Lactosyl ceramidosis: deficient activity of a neutral/3glucosidase in liver and cultivated fibroblasts. Clin Chim Acta 1978;88:483-493. 10 Chiao Y-B, Hoysen GM, Peters SP et al. Multiple glycosidase deficiencies in a case of juvenile (type 3) Oaucher disease. Proc Nat! Acad Sci USA 1978;75:2448-2452. I ! Glew RH, Gopalan V, Hubbell CA, Beutler E, Geil JD, Lee RE. A case ofnonneurologic Gaucher's disease that biochemically resembles the neurologic types. J Neuropathol Exp Neurol 1991:50:i08-117. 12 Gopalan V, Pastuszyn AP, Galey WR, Jr, Glew RH. Exolytic hydrolysis of toxic plant glucosides by guinea pig liver cytosolic/~-glucosidase. J Biol Chem 1992;267:14027-32. 13 Butcher BA, Gopalan V, Lee RE, Richards TC, Waggoner AS, Glew RH. Use of 4heptylumbelliferyI.B-~glucoside to identify Gaucher's disease heterozygotes. Ciin Chim Acta 1989:184:235-242. 14 DePetro JJ. MSc Thesis, 1987, University of Pittsburgh, PA. 15 Gopalan V, VanderJagt DL, Libell DP, Glew RH. Transglucosylation as a probe of the mechanism of action of mammalian cytosolic B-glucosidase. J Biol Chem 1992;267:9629-9638. 16 Segel IH. Enzyme Kinetics, New York: Wiley lnt~rscience, 1975;227-,'72. 17 Beutler E. Gaucher disease, new molecular approaches to diagnosis and treatment. Science 1992;256:794-799. lg Cheetham PSJ, Dance NE, Robinson D. A benign deficiency of type B B.galactosidase in human liver. Clin Chim Acta 1978,83:67-74