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Cell Biology International (2003) 27, 887–895 (Printed in Great Britain)
Sensitivity of lysosomal enzymes to the plant alkaloid sanguinarine: comparison with other SH-specific agents
T. Belyaeva1*, E. Leontieva1, A. Shpakov2, T. Mozhenok1 and M. Faddejeva1
1Institute of Cytology, Russian Academy of Sciences, St Petersburg, Russia
2Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St Petersburg, Russia


Abstract

The influence of the benzo[c]phenanthridine alkaloid sanguinarine on some lysosomal enzyme activities was investigated. Sanguinarine inhibits lysosomal hydrolases in homogenates of cultured mouse fibroblasts. After incubation of mouse fibroblasts in culture with 100μM sanguinarine an approximately 50% decrease in the activities of N-acetyl,d-glucosaminidase (NAGA), β-galactosidase (GAL), arylsulfatase and acid lipase was observed. Because the biological activity of sanguinarine might arise from the interaction of its iminium cation with enzyme thiol groups, we compared its effect on NAGA, GAL and acid phosphatase (AcP) activities with the effects of SH-specific reagents p-chloromercuribenzoic acid (CPMA) and N-ethylmaleimide (NEM). Treatment of lysosomal fractions with millimolar concentrations of sanguinarine induces a dose-dependent inhibition of the enzymes; for example, 0.6mM sanguinarine causes approximately a 40% decrease in AcP and NAGA activities. NEM has similar effects, and increasing the preincubation temperature from 0°C to 37°C intensifies the inhibition due to both agents. CPMA also inhibits the activity of GAL (IC500.7μM), AcP (IC5012.5μM) and NAGA (IC506.8μM) in a dose-dependent manner but is more potent than sanguinarine or NEM. Comparative analysis of the primary structures of these enzymes using the program BLAST reveals the presence of highly conserved cysteine residues, which confirms the importance of thiol-groups for their activities. Thus, both the experimental observations obtained in this study and the literature data imply a significant role of redox-based mechanisms in regulating lysosomal functional activity.


Keywords: Sanguinarine, Lysosomes, N-acetyl-β,d-glucosaminidase, Acid phosphatase, p-chloromercuribenzoic acid, N-ethylmaleimide, Dithiothreitol.

*Corresponding author


1 Introduction

The biological activities of sanguinarine and other quaternary benzo[c]phenanthridine alkaloids are of particular interest in molecular biology and medicine. Sanguinarine, found in plants of Papaveraceae and Rutaceae families (Suffness and Cordell, 1985), has antimicrobial, protistocidal, antifungal, anti-inflammatory and antitumor activities. It affects eukaryotic cells in many ways and several cellular targets for its action have now been established. First, due to the ability of sanguinarine molecules to intercalate between base pairs in double helical DNA and RNA, they alter nucleic acid structure and metabolism (Faddejeva and Belyaeva, 1997). Second, sanguinarine inhibits ATP synthesis in mitochondria by means of neutralization of the negative charges of external side in energized internal mitochondrial membrane (Belyaeva and Faddejeva, 1995). Third, the iminium cation of the alkaloid interacts with nucleophilic groups, especially SH-groups, in enzymes and other proteins (Walterova et al., 1981). For example, it inhibits Na+/K+and Ca2+-ATPases by blocking the SH-groups essential for their activities (Faddejeva and Belyaeva, 1997). It is also a potent inhibitor of protein kinase C (Wang et al., 1997). In addition, sanguinarine and other quaternary benzo[c]phenanthridine alkaloids can block microtubule assembly (Wolff and Knipling, 1993). Importantly,sanguinarine is also capable of inducing a selective apoptotic response in human tumor cells (Ahmad et al., 2000). Micromolar doses exert this effect on cultured human epidermoid carcinoma cells (A431). However, normal human epidermal keratinocytes (NHEKs) in culture do not become apoptotic after treatment with higher doses. The molecular mechanisms by which sanguinarine induces apoptosis specifically in tumor cells are being investigated (Weerasinghe et al., 2001a,b,c).

The interaction of biologically active compounds with organelles and other intracellular targets depends on both cellular permeability and intracellular distribution. Molecules may enter cells by endocytosis or by crossing the plasma membrane (by passive diffusion or by active transport) (Steinberg, 1994). Biologically active molecules that penetrate into the cell by endocytosis enter lysosomal compartment. Substances that enter the cytosol by transmembrane passage and are lysosomotropic (such as weak bases) also accumulate in lysosomes. Sanguinarine is a lysosomotropic agent and might therefore react with the lysosomal enzymes after accumulation. Thus, we have established that sanguirythrine, a mixture of benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine, accumulates in lysosomes and inhibits the lysosomal enzymatic activities (Belyaeva et al., 1990).

The main purpose of this study was to investigate the sanguinarine influence on the activity of some lysosomal enzymes, which regulate many metabolic processes and play a key role in cellular homeostasis. Impaired functioning of one or more hydrolases causes intralysosomal accumulation of undigested material, leading to lysosomal storage diseases. For example, genetic disorders of lysosomal enzymes cause mucopolysaccharidosis, oligosaccharidosis and lipidosis (Scriver et al., 1995). In the present study we have investigated the SH-sensitivity of the main lysosomal enzymes to well known SH-agents CPMA and NEM in comparison with their sensitivity to sanguinarine. Finally, we have carried out the theoretical analysis of primary structures of these enzymes in order to reveal cysteine-containing highly conserved sites.

2 Materials and methods

2.1 Chemicals

p-Nitrophenylphosphate and 2-naphtylcaprylate were obtained from Merck, Germany, p-nitrophenyl-N-acetyl,d-glucosaminide, p-chloromercuribenzoic acid, N-ethylmaleimide, dithiothreitol and 4-nitrocatechol sulfate were obtained from Sigma, USA, p-nitrophenyl,d-galactopyranoside and p-nitrophenyl α,d-mannopyranoside were obtained from Chemapol, Czech Republic, and sanguinarine was obtained from Aldrich, Germany, [3H] acetic anhydride (500 mCi/mmol) was obtained from Amersham, England.

2.2 Cell culture

A suspension subline LS of mouse fibroblast line L was adapted to monolayer growth (subline LSM) (Semenova et al., 1984) (Russian culture collection, St Petersburg). LSM cells were cultured in Eagle's medium (Gibco BRL, Scotland) supplemented with 10% fetal calf serum (Gibco BRL, Scotland) without antibiotics in a 5% CO2atmosphere at 37°C.

Experiments were performed on the cells in logarithmic growth phase. The cells were washed twice with cold 150mM KCl, mechanically removed from glass surface and homogenized in a Dounce homogenizer in cold 150mM KCl, containing 0.1% Triton X-100.

2.3 Isolation of purified lysosome fraction

Liver lysosomes were purified by the method of Yamada et al. (1984)from male Wistar rats (80–120g) that had been starved for 12h. The liver was homogenized in cold 0.25M sucrose, 10mM Tris/HCl, pH 7.4, and resuspended in four volumes of sucrose solution. The nuclear fraction was discarded after centrifugation of the homogenate for 10min at 340×g. The supernatant was supplemented with 0.01 volume of 100mM CaCl2and incubated at 37°C for 5min to swell the mitochondria. The equilibrium densities of other organelles, including lysosomes, remain unchanged by Ca2+. The incubated mixture was layered on iso-osmotic (0.25M sucrose) Percoll at a density of 1.08g/ml and centrifuged for 15min at 60,000×g in a Beckman L7-55 ultracentrifuge, type 65 rotor. Fractions were collected from the bottom of the tubes. The activities of NAGA and succinate dehydrogenase, marker enzymes of lysosomes and mitochondria correspondingly, were determined in each fraction. The fractions that contained NAGA but not succinate dehydrogenase were pooled and centrifuged for 1h at 100,000×g in a Beckman L7-55 ultracentrifuge, type 65 rotor. The turbid layer in the middle of each tube was collected and diluted with two volumes of 0.25M sucrose. The diluted suspension was centrifuged at 10,000×g for 30min several times in order to remove the remaining Percoll. The washed pellet was resuspended in 0.25M sucrose and used as the purified lysosomal fraction. The preparation of lysosomes in the presence of 0.1% Triton X-100 was preincubated for 15min at 0°C or 37°C with the reagents studied, and then the lysosomal enzyme activities were determined.

2.4 Enzyme assays

The activities of NAGA (EC 3.2.1.30), AcP (EC 3.1.3.2) and GAL (EC 3.2.1.23) were determined spectrophotometrically, using the appropriate derivatives of p-nitrophenol as substrates (Barrett, 1972). NAGA activity was determined using 7.5mM p-nitrophenyl-N-acetyl,d-glucosaminide and sodium citrate buffer, pH 4.0; AcP activity using 32mM p-nitrophenylphosphate (disodium salt) and sodium acetate buffer, pH 5.0; GAL activity using 7.5mM p-nitrophenyl,d-galactopyranoside and sodium citrate buffer, pH 4.5. Units of enzymatic activity were defined as the amount (nmol) of p-nitrophenol released in 1min per 1mg of protein. Acid lipase (EC 3.1.1) activity was measured by the hydrolysis of 2-naphtylcaprylate in the medium containing 1.5mM substrate suspension and sodium acetate buffer pH 5.2. The enzyme activity was recorded as nmoles of 2-naphtylcaprylate released in 1min per 1mg of protein. Arylsulfatase (EC 3.1.6.1) activity was determined by the method of Milsom et al. (1972), using 15mM 4-nitrocatechol sulfate in a sodium acetate buffer, pH 5.6, as a substrate. α-Mannosidase (EC 3.2.1.24) activity was determined using 4mM p-nitrophenyl,d-mannopyranoside in sodium acetate buffer, pH 4.0 (Tulsiani and Touster, 1987). Cathepsin D (EC 2.4.23.7) was assayed by measuring the radioactivity released after hydrolysis of [3H]-acetylhemoglobin (Evans and Bosmann, 1977). [3H]-acetylhemoglobin was obtained from crystal bovine hemoglobin (Reanal) and [3H]-acetic anhydride (Barrett, 1972) and purified by gel-filtration on Sephadex G-50. The reaction was performed in sodium acetate buffer, pH 3.5. The activity of cathepsin D is expressed in cpm per mg of protein. The determination of succinate dehydrogenase activity was based on the reduction of K3[Fe(CN)6] in sodium phosphate buffer, pH 7.4, using succinic acid and 25mM K3[Fe(CN)6] (Keilin and King, 1960).

2.5 Determination of protein concentration

Protein was determined by the method of Lowry et al. (1951)using bovine serum albumin as a standard. The protein content of purified lysosomes from rat liver was determined by the method of Bradford (1976).

2.6 Comparative analysis of primary structures of lysosomal hydrolases

Similarity searches and sequence retrieval was performed via the server at the National Institute of Health, Bethesda, MD, USA (program BLAST).

2.7 Statistical analysis

Data analysis and statistical comparisons were made between groups using Student's test and ANOVA.Results are presented as mean±SE. A P value of <0.05 was considered to be statistically significant.

3 Results

3.1 The effect of sanguinarine on the activity of mouse fibroblast lysosomal hydrolases

Mouse fibroblasts (subline LSM) were incubated for 1–4h in a medium containing sanguinarine. Enzyme activities were determined in cellular homogenates.Table 1shows that 100–400μM sanguinarine markedly inhibited the activities of all enzymes tested (up to 50% at 100μM). Thus, NAGA showed 59%, GAL 40%, arylsulfatase 50% and acid lipase 59% of the control activities. Cathepsin D and AcP activities declined to about 50% (58% for cathepsin D and 61% for AcP) after the sanguinarine concentration was increased to 200μM. α-Mannosidase was most sensitive to 100μM sanguinarine, decreasing to 25% of the control activity. According to our unpublished data sanguinarine is accumulated by LSM cells; for instance, these cells concentrate sanguinarine more than 250-fold when its concentration in the medium is 40 μM. For this reason it is very difficult to determine the intracellular alkaloid concentration, responsible for inhibiting the lysosomal enzymes. Investigating the effects of sanguinarine in purified lysosomal fractions allowed us to circumvent this difficulty.


Table 1. The activity of lysosomal enzymes in mouse fibroblasts (subline LSM) under the influence of sanguinarine at different medium concentrations. The activity of cathepsin D is expressed in cpm per mg of protein. The activities of other enzymes are expressed in nmol of reaction products released in 1 min per mg of protein

Image

The incubation time is given in brackets (mean±SE; n=4).

3.2. The effect of sanguinarine on lysosomal enzymatic activities in purified rat liver lysosomes

Purified rat liver lysosomes were preincubated for 15 min with sanguinarine concentrations ranging from 0.3 mM to 2 mM at 0 °C or 37 °C and the activities of GAL, AcP and NAGA were determined (Fig. 1). Sanguinarine inhibited the activities of all enzymes tested, the most pronounced effect being on NAGA. The inhibition percentage was directly proportional to the sanguinarine concentration after preincubation at 0 °C. Preincubation at 37 °C decreased NAGA activity at all sanguinarine concentrations studied, maximal inhibitionoccurring at 0.6 mM (Fig. 1).



Full-size image (13K) - Opens new windowFull-size image (13K)
* P<0.05 versus controls.

3.2 The effect of sanguinarine on lysosomal enzymatic activities in purified rat liver lysosomes

Purified rat liver lysosomes were preincubated for 15min with sanguinarine concentrations ranging from 0.3mM to 2mM at 0°C or 37°C and the activities of GAL, AcP and NAGA were determined (Fig. 1). Sanguinarine inhibited the activities of all enzymes tested, the most pronounced effect being on NAGA. The inhibition percentage was directly proportional to the sanguinarine concentration after preincubation at 0°C. Preincubation at 37°C decreased NAGA activity at all sanguinarine concentrations studied, maximal inhibitionoccurring at 0.6mM (Fig. 1).


Fig. 1

Effect of sanguinarine on the activities of GAL, AcP and NAGA. Sanguinarine concentrations range from 0.3mM to 2mM, the time of preincubation of lysosomal preparation with sanguinarine was 15min, temperature −0°C or 37°C. The activity of the enzymes is expressed in nmol of p-nitrophenol released in 1min per mg of protein. The basal activities of GAL, AcP and NAGA are respectively: 374±35, 510±55 and 5090±540. Results are expressed as percentage of basal activity values (mean±SE; n=4).


AcP was inhibited by higher sanguinarine concentrations than were required to inhibit NAGA, but an increase in preincubation temperature from 0°C to37°C intensified the inhibition. GAL appeared less sensitive to sanguinarine: only a 20% decrease in activity was observed at 2 mM.

The functional activity of lysosomes during the accumulation of lysosomotropic substances depends at least both on the interaction of these substances with lysosomal hydrolases and the impairment of the proton pump that maintains low pH in the organelles. Increase in intralysosomal pH is an important factor in the inhibition of lysosomal function. In the experiments on the purified lysosomes, such pH changes are excluded. Therefore, the results focus directly on the interactions between sanguinarine and enzymemolecules.

3.3 The inhibition of lysosomal enzyme activity by the selective SH-blocker CPMA

Being hydrophobic, CPMA penetrates the internal pockets of many enzyme molecules and selectively binds SH-groups. Treatment of lysosomes with CPMA at concentrations ranging from 1μM to 20μM resulted in a dose-dependent inhibition of GAL, NAGA and AcP (Fig. 2). GAL was most sensitive: even at 1μM concentration CPMA decreased this activity up to 40%. AcP activity was inhibited by 50% at 12.5μM, and the basal NAGA activity decreased by 50% at 6.8μM CPMA.


Fig. 2

Effect of CPMA on the activity of GAL, AcP and NAGA. Concentrations of CPMA range from 1 to 20μM, the time of preincubation of lysosomal preparation with CPMA was 15min, temperature −0°C. The activity of the enzymes is expressed in nmol p-nitrophenol released in 1min per mg of protein. The basal activities of GAL, AcP and NAGA are respectively: 374±35, 510±55 and 5090±540. Results are expressed as percentage of basal activity values (mean±SE; n=4).


3.4 The inhibitory effect of the alkylating agent NEM on lysosomal enzyme activities

NEM is hydrophilic and interacts with SH-groups situated on protein surfaces. The alkylation reaction strongly depends on the temperature, which prompted the idea of investigating the effects of NEM at 0°C and 37°C. We used 15min preincubation of lysosomes with NEM (5–20mM) at these two temperatures. NEM inhibited only GAL and NAGA activities in a dose-dependent manner at 0°C. The basal activities of these two enzymes decreased by about 20% at 20mM NEM concentration (Fig. 3). AcP proved almost insensitive to NEM in these conditions. The increase in temperature to 37°C discernibly enhanced the inhibitory action of NEM on both GAL and NAGA activities and caused a decline in AcP activity (up to 64% of the basal activity at 20mM NEM).


Fig. 3

Effect of NEM on the activity of GAL, AcP and NAGA. Concentrations of NEM range from 5 to 20mM; the time of preincubation of lysosomal preparation with NEM was 15min, temperature −0°C (A) or 37°C (B). The activity of the enzymes is expressed in nmol of p-nitrophenol released in 1 min per mg of protein. The basal activities of GAL, AcP and NAGA are respectively: 374±35, 510±55 and 5090±540. Results are expressed as percentage of basal activity values (mean±SE; n=4).


3.5 The influence of the thiol-containing reagent DTT on lysosomal enzymes

DTT reduces S-S bonds in protein molecules and thus restores SH-bonds. Inhibition of an enzyme by DTT suggests that S-S bonds are functionally important. The reduction of S-S bonds by DTT is sensitive to temperature. Therefore, we again used preincubation at 0°C and 37°C. The basal activities of GAL and AcP were not changed at either temperature after 15min preincubation (Fig. 4), indicating that there are no functionally important S-S-bonds in these enzymes. In contrast, NAGA activity decreased in a dose-dependent manner, and DTT inhibition was enhanced by increasing the preincubation temperature to 37°C (IC501.8mM). DTT almost completely inactivated the enzyme activity at concentrations above 10mM.


Fig. 4

Effect of DTT on the activity of NAGA. DTT was used in 0.5–20mM concentrations; the time of preincubation of lysosomal preparation with DTT was 15min, temperature −0°C or 37°C. The activity of the enzymes is expressed in nmol of p-nitrophenol released in 1min per mg of protein. The basal activity of NAGA is 5090±540. Results are expressed as percentage of basal activity value (mean±SE; n=4).


4 Discussion

In aqueous solution at physiological pH, sanguinarine can form both iminium cations and pseudobases (pKR+=7.9; Walterova et al., 1980). Both formscontribute to its biological activity: the pseudobase form enables sanguinarine to penetrate the cell through the membrane, and the cation can form adducts with nucleophilic groups, notably the SH- and OH-groups in proteins. The OH-groups of serine and threonine are not likely to be sufficiently strong nucleophiles to interact with the iminium group of the alkaloid (Wolff and Knipling, 1993), but protein cysteine SH-groups constitute important biological targets. Sanguinarine specifically inhibits alanine aminotransferase (Walterova et al., 1981) because of the reaction between its iminium group and enzymatic SH-groups (Fig. 5). Thiol-containing compounds partially restored the enzyme activity in the presence of sanguinarine. Further studies demonstrated a similar mechanism of sanguinarine action was involved in the inhibition of other enzymes.


Fig. 5

The reactions of SH-groups with the cation of sanguinarine.


The present study showed the inhibiting effect of sanguinarine on the activities of main lysosomal enzymes. This effect probably depends on the ability of the alkaloid to modify their cysteine SH-groups. To test this possibility we compared the effects of sanguinarine on GAL, AcP and NAGA activities with those of known SH-specific agents (CPMA and NEM) and S-S-specific agent (DTT). Our data on the sensitivities of lysosomal enzymes to CPMA, NEM, and DTT, are consistent with our suggestion about the importance of thiol groups for the activities of these enzymes and are in agreement with the conservation of many cysteine residues in lysosomal enzymes. A comparative analysis of GAL primary structures in chordates, insects, nematodes, fungi, plants and bacteria revealed the presence of highly conserved cysteine residues corresponding to Cys-127 and Cys-230 of human GAL (Table 2A). Cys-127 is present in the primary structures of all the GALs investigated, excluding the AF077544 protein of Caenorhabditis elegans. In addition to the cysteine residues, the surrounding amino acids are also highly conserved and form a consensus motif RxGPYIC(A/G)EWaxGG(L/F)PxWL, where a is a negatively charged amino acid or amide and x is a variable residue. A mutation in the locus (R121S and G123R) leads to GM1-gangliosidosis (Silva et al., 1999; Yoshida et al., 1991). Cys-230 is also present in vertebrate and invertebrate GALs as well as in the plant Carica papaya. Glu-268, the nucleophilic centre in the reaction of β-galactosides hydrolysis, is localized near Cys-230 (McCarter et al., 1997). However, the region containing Cys-230 is not conserved. These observations suggest that Cys-230 is less important for the functional activity of GAL than Cys-127.


Table 2A. Conservation of cysteine residues in the primary structures of lysosomal enzyme, β-galactosidase (comparison with human enzyme)

Image

1—cat; 2—dog; 3—mouse; 4—Drosophila melanogaster; 5—Caenorhabditis elegans (type 1); 6—C. elegans (type 2); 7—Arabidopsis thaliana; 8—Asparagus officinalis; 9—Lycopersicon esculentum; 10—Carica papaya; 11—Bacillus circulans; 12—Streptomyces coelicolor. (+)—conserved cysteine residue; (−)—variable cysteine residue; (*)—near this position presents cysteine residue.

Cys-195, located after the highly conserved motif GGP(V/I)(I/L)xxQ(I/V)ENEYG(S/P), is present in animal GALs but is replaced by other residues in plants, fungi and bacteria enzymes (Table 2A). Glu-188, localized in this motif, is the proton donor in the hydrolysis of β-Image -galactosides by GALs. The replacement of Arg-201 near Cys-195 leads to GM1-gangliosidosis ([Nishimoto et al., 1991]; [Yoshida et al., 1991]). On the basis of this analysis we can suggest that Cys-127, Cys-230 and Cys-195 are involved in the formation of three-dimensional structure of enzyme catalytic site which includes Glu-188 and Glu-268. These last two residues are also important for catalysis. Cys-426, Cys-626 and Cys-634 are conserved in both mammals and nematodes. Replacements of other amino acid residues near the cysteines, in particular Glu-632, induce GM1-gangliosidosis ( [Boustany et al., 1993]).

Functionally active GAL is a high molecular weight complex (approximately 670 kDa), consisting of the protective protein cathepsin A, the polypeptide—the product of C-terminal proteolysis of the 85 kDa GAL precursor, N-acetyl-a-neuraminidase, and (N-acetyl)galactose(amine)-6-sulfate sulfatase ([D'Azzo et al., 1982]; [Hiraiwa et al., 1997]; [Itoh et al., 1998]; [Potier et al., 1990]; [Van Der Spoel et al., 2000]; [Verheijen et al., 1982]). Disruption of the GAL complex by detergents and other agents impairs catalytic activity ( [Hiraiwa et al., 1997]). It has been established earlier that thecovalent bonds are not involved in the complex formation ([D'Azzo et al., 1982]). Our results on the insensitivity of GAL to DTT are in agreement with the above mentioned literature data.

Table 2B. Conservation of cysteine residues in primary structures of lysosomal enzyme, acid phosphatase (comparison with human acid phosphatase type 1)

Image

1—AcP, rat; 2—AcP, mouse; 3—prostatic AcP, mouse; 4—prostatic AcP, rat; 5—prostatic AcP, human; 6—AcP2, human; 7—AcP3, human; 8—AcP, Drosophila melanogaster; 9—AcP, D. subobscura. (+)—conserved cysteine residue; (−)—variable cysteine residue; (*)—near this position presents cysteine residue.

Table 2C. Conservation of cysteine residues in primary structures of lysosomal enzyme, N-acetyl,Image -glucosaminidase (comparison with human α-chain of N-acetyl,Image -glucosaminidase)

Image

1—mouse, α-chain; 2—human, β-chain; 3—cat, β-chain; 4—mouse, β-chain; 5—pig, β-chain; 6—Caenorhabditis elegans; 7—Drosophila melanogaster (Hexo2); 8—Bombyx mori;9—B. mandarina; 10—D. melanogaster (Hexo1); 11—Entamoeba histolytica; 12—Arabidopsis thaliana; 13—Dictyostelium discoideum; 14—Candida albicans; 15—Penicillium chrysogenum; 16—Trichoderma harzianum (strain P1); 17—Porphyromonas gingivalis; 18—Vibrio vulnificus. (+)—conserved cysteine residue; (−)—variable cysteine residue; (*)—near this position presents cysteine residue.

Mutations in GALs that induce inherited gangliosidoses involve the replacement of different amino acid residues by cysteine residues ([Boustany et al., 1993]; [Ishii et al., 1995]; [Morrone et al., 1997]; [Nishimoto et al., 1991]; [Silva et al., 1999]; [Yoshida et al., 1991]). Additional cysteines in the amino acid sequence of GAL cause critical changes in catalytic site. The molecular mechanism of the process includes the involvement of the additional cysteine residues and the formation of disulfide bonds between the cysteines present in the wild type of enzyme. These disulfide bonds might be stable to reduction during enzyme activity. Thus, our results as well as those cited in literature reveal the important role of cysteine residues for GAL activity.

In AcP molecules of vertebrate and invertebrate animals the cysteine residues corresponding Cys-159, 310, 345 and 349 of human AcP are highly conserved. Cys-370 is replaced by other amino acids only in human AcP2. Cys-212 is replaced in human AcP2 and insect enzymes (Table 2B). It has been suggested that the cysteine residues form three disulfide bonds: Cys-159–Cys-370, Cys-212–Cys-310 and Cys-345–Cys-349 participating in the stabilization of three-dimensional structure of AcP catalytic site which includes His-284 (proton donor), Asp-285 and positively charged residues in the N-terminal region of enzyme molecule (His-40, Arg-82) ( [Ostanin et al., 1994]). Our data on the sensitivity of the lysosomal enzymes to CPMA show the importance of SH-groups for AcP activity. However, NEM has a weaker effect, so the data suggest the existence of hydrophobic regions in the AcP catalytic site.

Comparative analysis of NAGA α- and β-chains from different species of animals, fungi, bacteria and plants shows that Cys-277, 328 and 522 (the numbers of the cysteine residues correspond to those of the α-chain of human NAGA) are highly conserved (Table 2C). However, Cys-58, Cys-104, Cys-458 and Cys-505 are conserved only in mammalian enzymes. The replacement of mammalian Cys-309 in the NAGA β-chain (which corresponds to Cys-277 in the α-chain) by tyrosine leads to type II GM2-gangliosidosis (Sandhoff disease) ([Gomez-Lira et al., 1995]). The replacement of Cys-458 in human NAGA α-chain by tyrosine induces type I GM2-gangliosidosis (Tay-Sachs disease) ([Tanaka et al., 1994]). The replacement of Cys-534 in human β-chain (which corresponds to Cys-505 in the α-chain) by tyrosine leads to type II GM2-gangliosidosis ([Kuroki et al., 1995]). At present it has been shown that NAGA catalytic site includes negatively charged residues—Asp-258, residues pair Asp-322—Glu-323 and Glu-462 ( [Fernandes et al., 1997]; [Hou et al., 2001]). Cys-277 and Cys-328 are localized near the enzyme catalytic site.

Our data on the high sensitivity of NAGA to CPMA, NEM and DTT provide strong evidence of the SH-dependence of this enzyme. We suppose that CPMA and NEM alter the SH-groups of NAGA and DTT reduce the S-S bonds of this enzyme that causes the changes of the functionally active form of NAGA oligomeric complex, including some subunits stabilized by S-S bonds, so far as various heteromeric forms of this enzyme have been determined ([Mahuran et al., 1988]).

Comparative analysis of lysosomal enzyme sensitivities to sanguinarine NEM and CPMA revealed an essential difference in their mechanisms of action. The strongest effect was exerted by CPMA. In addition to the enzyme surface SH-groups CPMA molecules can penetrate into internal hydrophobic pockets and react with SH-groups localized in these regions. The effect of this agent is significant even at micromolar concentrations. In contrast, NEM inhibits at millimolar concentrations and is more effective when the preincubation temperature is increased. Sanguinarine, like NEM, inhibits in millimolar doses, but it is more potent than NEM. Increase in preincubation temperature further enhances the sensitivity of the lysosomal enzymes to sanguinarine. This effect is probably determined by kinetic factors, in particular, by the increase in the rate of sanguinarine transfer to the cavity of enzyme catalytic site. Our preliminary studies about the partial elimination of sanguinarine inhibiting effect on AcP and GAL activities in the presence of DTT confirms the ability of alkaloid to interact with SH-groups of enzymes. In the case of NAGA the protective effect of DTT appear can not be manifested because of the strong inhibitory action on the enzyme activity of the reagent itself.

These data indicate that studies of the SH-sensitivity of the lysosomal enzymes and the state of SH-groups in their active sites represent a promising approach to understanding the mechanisms and the treatment of lysosomal storage diseases. In many cellular processes, redox-based regulation has emerged as an important regulatory mechanism. For example, oxidative stress is a potent stimulus for apoptotic cell death ([Loeffler and Kroemer, 2000]). The state of SH-groups in proteins depends on the redox-potential of the whole cell and the relevant organelles. Even a slight change in this potential may lead to a shift either towards free SH-groups or towards the formation of disulfide bonds. Such changes directly influence the three-dimensional structures of proteins and/or protein complexes, and thus the activities of enzymes. Both the experimental observations obtained in this study and the literature data show that the principal lysosomal enzymes are SH-sensitive and their enzymatic activities probably depend on the redox-potential of the cell.

Acknowledgements

This work was supported by the Russian Foundation for Basic Research (grant N 0104-49428).

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Boustany et al., 1993. R.M. Boustany, W.H. Qian and K. Suzuki, Mutations in acid β-galactosidase cause GM1 gangliosidosis in American patients. Am J Hum Genet 53 (1993), pp. 881–888. View Record in Scopus | Cited By in Scopus (17)

Bradford, 1976. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 (1976), pp. 248–254. Abstract | PDF (402 K) | View Record in Scopus | Cited By in Scopus (78877)

Evans and Bosmann, 1977. J. Evans and H.B. Bosmann, Bromodixyuridine (BudR) treatments of melanoma cells decrease cellular proteolytic activity. Exp Cell Res 108 (1977), pp. 151–155.

Faddejeva and Belyaeva, 1997. M.D. Faddejeva and T.N. Belyaeva, Sanguinarine and ellipticine: cytotoxic alkaloids isolated from well-known antitumor plants. Intracellular targets of their action. Cytology (Russia) 39 (1997), pp. 181–207.

Fernandes et al., 1997. M.J. Fernandes, S. Yew, D. Leclerc, B. Henrissat, C.E. Vorgias, R.A. Gravel et al., Identification of candidate active site residues in lysosomal β-hexosaminidase A. J Biol Chem 272 (1997), pp. 814–820. View Record in Scopus | Cited By in Scopus (30)

Hiraiwa et al., 1997. M. Hiraiwa, M. Saitoh, N. Arai, T. Shiraishi, S. Odani, Y. Uda et al., Protective protein in the bovine lysosomal β-galactosidase complex. Biochim Biophys Acta 1341 (1997), pp. 189–199. Abstract | PDF (774 K) | View Record in Scopus | Cited By in Scopus (10)

Hou et al., 2001. Y. Hou, D.J. Vocadlo, A. Leung, S.G. Withers and D. Mahuran, Characterization of the Glu and Asp residues in the active site of human β-hexosaminidase B. Biochemistry 40 (2001), pp. 2201–2209. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (18)

Gomez-Lira et al., 1995. M. Gomez-Lira, A. Sangalli, M. Mottes, C. Perusi, P.F. Pignatti, N. Rizzuto et al., A common β-hexosaminidase gene mutation in adult Sandhoff disease patients. Hum Genet 96 (1995), pp. 417–422. View Record in Scopus | Cited By in Scopus (14)

Ishii et al., 1995. N. Ishii, T. Oohira, A. Oshima, H. Sakuraba, F. Endo, I. Matsuda et al., Clinical and molecular analysis of a Japanese boy with Morquio B disease. Clin Genet 48 (1995), pp. 103–108. View Record in Scopus | Cited By in Scopus (18)

Itoh et al., 1998. K. Itoh, Y. Naganawa, S. Kamei, M. Shimmoto and H. Sakuraba, Stabilizing effect of lysosomal β-galactosidase on the catalytic activity of protective protein/cathepsin A secreted by human platelets. Biochem Biophys Res Commun 253 (1998), pp. 228–234. Abstract | PDF (476 K) | View Record in Scopus | Cited By in Scopus (2)

Keilin and King, 1960. D. Keilin and T.E. King, Effect of inhibitors on the activity of soluble succinate dehydrogenase and the reconstitution of the SDH-cytochrome system from its components. Proc Natl Acad Sci U S A 152 (1960), pp. 163–187. Full Text via CrossRef

Kuroki et al., 1995. Y. Kuroki, K. Itoh, Y. Nadaoka, T. Tanaka and H. Sakuraba, A novel missense mutation (C522Y) is present in the β-hexosaminidase beta-subunit gene of a Japanese patient with infantile Sandhoff disease. Biochem Biophys Res Commun 212 (1995), pp. 564–571. Abstract | PDF (398 K) | View Record in Scopus | Cited By in Scopus (20)

Loeffler and Kroemer, 2000. M. Loeffler and G. Kroemer, The mitochondrion in cell death control: certainties and incognita. Exp Cell Res 256 (2000), pp. 19–26. Abstract | PDF (135 K) | View Record in Scopus | Cited By in Scopus (217)

Lowry et al., 1951. O.H. Lowry, N.J. Roserbrough, A.J. Farr and R.J. Randall, Protein measurement with Folin phenol reagent. J Biol Chem 193 (1951), pp. 265–275.

Mahuran et al., 1988. D.J. Mahuran, K. Neote, M.H. Klavins, A. Leung and R.A. Gravel, Proteolytic processing of pro-α and pro-β precursors from human β-hexosaminidase. Generation of the mature α and βAβB subunits. J Biol Chem 263 (1988), pp. 4612–4618. View Record in Scopus | Cited By in Scopus (19)

McCarter et al., 1997. J.D. McCarter, D.L. Burgoyne, S. Miao, S. Zhang, J.W. Callahan and S.G. Withers, Identification of Glu-268 as the catalytic nucleophile of human lysosomal β-glactosidase precursor by mass spectrometry. J Biol Chem 272 (1997), pp. 396–400. View Record in Scopus | Cited By in Scopus (23)

Milsom et al., 1972. D.W. Milsom, F.A. Rose and K.S. Dodgson, The specific assay of arylsulfatase C, a rat liver microsomal marker enzyme. Biochem J 128 (1972), pp. 331–336.

Morrone et al., 1997. A. Morrone, T. Bardelli, M.A. Donati, M. Giorgi, R. Di Rocco, R. Gatti et al., Identification of new mutations in six Italian patients affected by a variant form of infantile GM1-gangliosidosis with severe cardiomyopathy. Am J Hum Genet 61 (1997), p. A258.

Nishimoto et al., 1991. J. Nishimoto, E. Nanba, K. Inui, S. Okada and K. Suzuki, GM1-gangliosidosis (genetic β-galactosidase deficiency): identification of four mutations in different clinical phenotypes among Japanese patients. Am J Hum Genet 49 (1991), pp. 566–574. View Record in Scopus | Cited By in Scopus (20)

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Cell Biology International
Volume 27, Issue 11, November 2003, Pages 887-895
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Cys-195, located after the highly conserved motif GGP(V/I)(I/L)xxQ(I/V)ENEYG(S/P), is present in animal GALs but is replaced by other residues in plants, fungi and bacteria enzymes (Table 2A). Glu-188, localized in this motif, is the proton donor in the hydrolysis of β-d-galactosides by GALs. The replacement of Arg-201 near Cys-195 leads to GM1-gangliosidosis (Nishimoto et al., 1991; Yoshida et al., 1991). On the basis of this analysis we can suggest that Cys-127, Cys-230 and Cys-195 are involved in the formation of three-dimensional structure of enzyme catalytic site which includes Glu-188 and Glu-268. These last two residues are also important for catalysis. Cys-426, Cys-626 and Cys-634 are conserved in both mammals and nematodes. Replacements of other amino acid residues near the cysteines, in particular Glu-632, induce GM1-gangliosidosis (Boustany et al., 1993).

Functionally active GAL is a high molecular weight complex (approximately 670kDa), consisting of the protective protein cathepsin A, the polypeptide—the product of C-terminal proteolysis of the 85kDa GAL precursor, N-acetyl-a-neuraminidase, and (N-acetyl)galactose(amine)-6-sulfate sulfatase (D'Azzo et al., 1982; Hiraiwa et al., 1997; Itoh et al., 1998; Potier et al., 1990; Van Der Spoel et al., 2000; Verheijen et al., 1982). Disruption of the GAL complex by detergents and other agents impairs catalytic activity (Hiraiwa et al., 1997). It has been established earlier that thecovalent bonds are not involved in the complex formation (D'Azzo et al., 1982). Our results on the insensitivity of GAL to DTT are in agreement with the above mentioned literature data.


Table 2B. Conservation of cysteine residues in primary structures of lysosomal enzyme, acid phosphatase (comparison with human acid phosphatase type 1)


Table 2C. Conservation of cysteine residues in primary structures of lysosomal enzyme, N-acetyl,Image -glucosaminidase (comparison with human α-chain of N-acetyl,Image -glucosaminidase)

Image

1—mouse, α-chain; 2—human, β-chain; 3—cat, β-chain; 4—mouse, β-chain; 5—pig, β-chain; 6—Caenorhabditis elegans; 7—Drosophila melanogaster (Hexo2); 8—Bombyx mori;9—B. mandarina; 10—D. melanogaster (Hexo1); 11—Entamoeba histolytica; 12—Arabidopsis thaliana; 13—Dictyostelium discoideum; 14—Candida albicans; 15—Penicillium chrysogenum; 16—Trichoderma harzianum (strain P1); 17—Porphyromonas gingivalis; 18—Vibrio vulnificus. (+)—conserved cysteine residue; (−)—variable cysteine residue; (*)—near this position presents cysteine residue.

Mutations in GALs that induce inherited gangliosidoses involve the replacement of different amino acid residues by cysteine residues ([Boustany et al., 1993]; [Ishii et al., 1995]; [Morrone et al., 1997]; [Nishimoto et al., 1991]; [Silva et al., 1999]; [Yoshida et al., 1991]). Additional cysteines in the amino acid sequence of GAL cause critical changes in catalytic site. The molecular mechanism of the process includes the involvement of the additional cysteine residues and the formation of disulfide bonds between the cysteines present in the wild type of enzyme. These disulfide bonds might be stable to reduction during enzyme activity. Thus, our results as well as those cited in literature reveal the important role of cysteine residues for GAL activity.

In AcP molecules of vertebrate and invertebrate animals the cysteine residues corresponding Cys-159, 310, 345 and 349 of human AcP are highly conserved. Cys-370 is replaced by other amino acids only in human AcP2. Cys-212 is replaced in human AcP2 and insect enzymes (Table 2B). It has been suggested that the cysteine residues form three disulfide bonds: Cys-159–Cys-370, Cys-212–Cys-310 and Cys-345–Cys-349 participating in the stabilization of three-dimensional structure of AcP catalytic site which includes His-284 (proton donor), Asp-285 and positively charged residues in the N-terminal region of enzyme molecule (His-40, Arg-82) ( [Ostanin et al., 1994]). Our data on the sensitivity of the lysosomal enzymes to CPMA show the importance of SH-groups for AcP activity. However, NEM has a weaker effect, so the data suggest the existence of hydrophobic regions in the AcP catalytic site.

Comparative analysis of NAGA α- and β-chains from different species of animals, fungi, bacteria and plants shows that Cys-277, 328 and 522 (the numbers of the cysteine residues correspond to those of the α-chain of human NAGA) are highly conserved (Table 2C). However, Cys-58, Cys-104, Cys-458 and Cys-505 are conserved only in mammalian enzymes. The replacement of mammalian Cys-309 in the NAGA β-chain (which corresponds to Cys-277 in the α-chain) by tyrosine leads to type II GM2-gangliosidosis (Sandhoff disease) ([Gomez-Lira et al., 1995]). The replacement of Cys-458 in human NAGA α-chain by tyrosine induces type I GM2-gangliosidosis (Tay-Sachs disease) ([Tanaka et al., 1994]). The replacement of Cys-534 in human β-chain (which corresponds to Cys-505 in the α-chain) by tyrosine leads to type II GM2-gangliosidosis ([Kuroki et al., 1995]). At present it has been shown that NAGA catalytic site includes negatively charged residues—Asp-258, residues pair Asp-322—Glu-323 and Glu-462 ( [Fernandes et al., 1997]; [Hou et al., 2001]). Cys-277 and Cys-328 are localized near the enzyme catalytic site.

Our data on the high sensitivity of NAGA to CPMA, NEM and DTT provide strong evidence of the SH-dependence of this enzyme. We suppose that CPMA and NEM alter the SH-groups of NAGA and DTT reduce the S-S bonds of this enzyme that causes the changes of the functionally active form of NAGA oligomeric complex, including some subunits stabilized by S-S bonds, so far as various heteromeric forms of this enzyme have been determined ([Mahuran et al., 1988]).

Comparative analysis of lysosomal enzyme sensitivities to sanguinarine NEM and CPMA revealed an essential difference in their mechanisms of action. The strongest effect was exerted by CPMA. In addition to the enzyme surface SH-groups CPMA molecules can penetrate into internal hydrophobic pockets and react with SH-groups localized in these regions. The effect of this agent is significant even at micromolar concentrations. In contrast, NEM inhibits at millimolar concentrations and is more effective when the preincubation temperature is increased. Sanguinarine, like NEM, inhibits in millimolar doses, but it is more potent than NEM. Increase in preincubation temperature further enhances the sensitivity of the lysosomal enzymes to sanguinarine. This effect is probably determined by kinetic factors, in particular, by the increase in the rate of sanguinarine transfer to the cavity of enzyme catalytic site. Our preliminary studies about the partial elimination of sanguinarine inhibiting effect on AcP and GAL activities in the presence of DTT confirms the ability of alkaloid to interact with SH-groups of enzymes. In the case of NAGA the protective effect of DTT appear can not be manifested because of the strong inhibitory action on the enzyme activity of the reagent itself.

These data indicate that studies of the SH-sensitivity of the lysosomal enzymes and the state of SH-groups in their active sites represent a promising approach to understanding the mechanisms and the treatment of lysosomal storage diseases. In many cellular processes, redox-based regulation has emerged as an important regulatory mechanism. For example, oxidative stress is a potent stimulus for apoptotic cell death ([Loeffler and Kroemer, 2000]). The state of SH-groups in proteins depends on the redox-potential of the whole cell and the relevant organelles. Even a slight change in this potential may lead to a shift either towards free SH-groups or towards the formation of disulfide bonds. Such changes directly influence the three-dimensional structures of proteins and/or protein complexes, and thus the activities of enzymes. Both the experimental observations obtained in this study and the literature data show that the principal lysosomal enzymes are SH-sensitive and their enzymatic activities probably depend on the redox-potential of the cell.

Acknowledgements

This work was supported by the Russian Foundation for Basic Research (grant N 0104-49428).

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Fernandes et al., 1997. M.J. Fernandes, S. Yew, D. Leclerc, B. Henrissat, C.E. Vorgias, R.A. Gravel et al., Identification of candidate active site residues in lysosomal β-hexosaminidase A. J Biol Chem 272 (1997), pp. 814–820. View Record in Scopus | Cited By in Scopus (30)

Hiraiwa et al., 1997. M. Hiraiwa, M. Saitoh, N. Arai, T. Shiraishi, S. Odani, Y. Uda et al., Protective protein in the bovine lysosomal β-galactosidase complex. Biochim Biophys Acta 1341 (1997), pp. 189–199. Abstract | PDF (774 K) | View Record in Scopus | Cited By in Scopus (10)

Hou et al., 2001. Y. Hou, D.J. Vocadlo, A. Leung, S.G. Withers and D. Mahuran, Characterization of the Glu and Asp residues in the active site of human β-hexosaminidase B. Biochemistry 40 (2001), pp. 2201–2209. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (18)

Gomez-Lira et al., 1995. M. Gomez-Lira, A. Sangalli, M. Mottes, C. Perusi, P.F. Pignatti, N. Rizzuto et al., A common β-hexosaminidase gene mutation in adult Sandhoff disease patients. Hum Genet 96 (1995), pp. 417–422. View Record in Scopus | Cited By in Scopus (14)

Ishii et al., 1995. N. Ishii, T. Oohira, A. Oshima, H. Sakuraba, F. Endo, I. Matsuda et al., Clinical and molecular analysis of a Japanese boy with Morquio B disease. Clin Genet 48 (1995), pp. 103–108. View Record in Scopus | Cited By in Scopus (18)

Itoh et al., 1998. K. Itoh, Y. Naganawa, S. Kamei, M. Shimmoto and H. Sakuraba, Stabilizing effect of lysosomal β-galactosidase on the catalytic activity of protective protein/cathepsin A secreted by human platelets. Biochem Biophys Res Commun 253 (1998), pp. 228–234. Abstract | PDF (476 K) | View Record in Scopus | Cited By in Scopus (2)

Keilin and King, 1960. D. Keilin and T.E. King, Effect of inhibitors on the activity of soluble succinate dehydrogenase and the reconstitution of the SDH-cytochrome system from its components. Proc Natl Acad Sci U S A 152 (1960), pp. 163–187. Full Text via CrossRef

Kuroki et al., 1995. Y. Kuroki, K. Itoh, Y. Nadaoka, T. Tanaka and H. Sakuraba, A novel missense mutation (C522Y) is present in the β-hexosaminidase beta-subunit gene of a Japanese patient with infantile Sandhoff disease. Biochem Biophys Res Commun 212 (1995), pp. 564–571. Abstract | PDF (398 K) | View Record in Scopus | Cited By in Scopus (20)

Loeffler and Kroemer, 2000. M. Loeffler and G. Kroemer, The mitochondrion in cell death control: certainties and incognita. Exp Cell Res 256 (2000), pp. 19–26. Abstract | PDF (135 K) | View Record in Scopus | Cited By in Scopus (217)

Lowry et al., 1951. O.H. Lowry, N.J. Roserbrough, A.J. Farr and R.J. Randall, Protein measurement with Folin phenol reagent. J Biol Chem 193 (1951), pp. 265–275.

Mahuran et al., 1988. D.J. Mahuran, K. Neote, M.H. Klavins, A. Leung and R.A. Gravel, Proteolytic processing of pro-α and pro-β precursors from human β-hexosaminidase. Generation of the mature α and βAβB subunits. J Biol Chem 263 (1988), pp. 4612–4618. View Record in Scopus | Cited By in Scopus (19)

McCarter et al., 1997. J.D. McCarter, D.L. Burgoyne, S. Miao, S. Zhang, J.W. Callahan and S.G. Withers, Identification of Glu-268 as the catalytic nucleophile of human lysosomal β-glactosidase precursor by mass spectrometry. J Biol Chem 272 (1997), pp. 396–400. View Record in Scopus | Cited By in Scopus (23)

Milsom et al., 1972. D.W. Milsom, F.A. Rose and K.S. Dodgson, The specific assay of arylsulfatase C, a rat liver microsomal marker enzyme. Biochem J 128 (1972), pp. 331–336.

Morrone et al., 1997. A. Morrone, T. Bardelli, M.A. Donati, M. Giorgi, R. Di Rocco, R. Gatti et al., Identification of new mutations in six Italian patients affected by a variant form of infantile GM1-gangliosidosis with severe cardiomyopathy. Am J Hum Genet 61 (1997), p. A258.

Nishimoto et al., 1991. J. Nishimoto, E. Nanba, K. Inui, S. Okada and K. Suzuki, GM1-gangliosidosis (genetic β-galactosidase deficiency): identification of four mutations in different clinical phenotypes among Japanese patients. Am J Hum Genet 49 (1991), pp. 566–574. View Record in Scopus | Cited By in Scopus (20)

Ostanin et al., 1994. K. Ostanin, A. Saeed and R.L. Van Etten, Heterologous expression of human prostatic acid phosphatase and site-directed mutagenesis of the enzyme active site. J Biol Chem 269 (1994), pp. 8971–8978. View Record in Scopus | Cited By in Scopus (44)

Potier et al., 1990. M. Potier, L. Michaud, J. Tranchemontagne and L. Thauvette, Structureof lysosomal neuraminidase—β-galactosidase—carboxypeptidase multienzymic complex. Biochem J 267 (1990), pp. 197–202. View Record in Scopus | Cited By in Scopus (20)

Scriver et al., 1995. R.S. Scriver, A.L. Beaudet, W.S. Sly and D. Valle, Lysosomal enzymes. In: The metabolic and molecular bases of inherited disease (7th edn ed.),, McGraw-Hill, New York (USA) (1995), pp. 2425–2879.

Semenova et al., 1984. E.G. Semenova, A.V. Khomenko and S.E. Mamaeva, Fluctuations in the cell cycle span and karyotype of murine L cells depending on the mode of cultivation. Cytology (Russia) 26 (1984), pp. 1156–1160. View Record in Scopus | Cited By in Scopus (4)

Silva et al., 1999. C.M. Silva, M.H. Severini, A. Sopelsa, J.C. Coelho, A. Zaha, A. D'Azzo et al., Six novel β-galactosidase gene mutations in Brazilian patients with GM1-gangliosidosis. Hum Mutat 13 (1999), pp. 401–409. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (16)

Steinberg, 1994. T.H. Steinberg, Cellular transport of drugs. Clin Infect Dis 19 (1994), pp. 916–921. View Record in Scopus | Cited By in Scopus (22)

Suffness and Cordell, 1985. M. Suffness and G.A. Cordell, Benzo[c]phenanthridine alkaloids. In: The alkaloids. Chemistry and pharmacology 25, Academic Press, Orlando (1985), pp. 178–188.

Tanaka et al., 1994. A. Tanaka, H. Sakazaki, H. Murakami, G. Isshiki and K. Suzuki, Molecular genetics of Tay-Sachs disease in Japan. J Inherit Metab Dis 17 (1994), pp. 593–600. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (12)

Tulsiani and Touster, 1987. D.R. Tulsiani and O. Touster, Substrate specificities of rat kidney lysosomal and cytosolic α-Image -mannosidases and effects of swainsonine suggest a role of the cytosolic enzyme in glycoprotein catabolism. J Biol Chem 262 (1987), pp. 6506–6514. View Record in Scopus | Cited By in Scopus (24)

Van Der Spoel et al., 2000. A. Van Der Spoel, E. Bonten and A. d'Azzo, Processing of lysosomal β-galactosidase. The C-terminal precursor fragment is an essential domain of the mature enzyme. J Biol Chem 275 (2000), pp. 10035–10040. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (13)

Verheijen et al., 1982. F. Verheijen, R. Brossmer and H. Galjaard, Purification of acid β-galactosidase and acid neuraminidase from bovine testis: evidence for an enzyme complex. Biochem Biophys Res Commun 108 (1982), pp. 868–875. Abstract | Article | PDF (710 K) | View Record in Scopus | Cited By in Scopus (25)

Walterova et al., 1980. D. Walterova, V. Preininger, F. Granbal, V. Simanek and F. Santavy, Fluorescence spectra and cation-pseudobase equilibria of some benzophenanthridine alkaloids. Heterocycles 14 (1980), pp. 597–600.

Walterova et al., 1981. D. Walterova, J. Ulrichova, V. Preininger and V. Simanek, Inhibition of liver alanine aminotransferase activity by benzophenanthridine alkaloids. J Med Chem 24(1981), pp. 1100–1103. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (28)

Wang et al., 1997. B.H. Wang, Z.X. Lu and G.M. Polya, Inhibition of eukaryotic protein kinases by isoquinoline and oxazine alkaloids. Planta Med 63 (1997), pp. 494–498. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (31)

Weerasinghe et al., 2001a. P. Weerasinghe, S. Hallock, S.C. Tang and A. Liepins, Role of Bcl-2 family proteins and caspase-3 in sanguinarine-induced bimodal cell death. Cell Biol Toxicol 17 (2001a), pp. 371–381. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (23)

Weerasinghe et al., 2001b. P. Weerasinghe, S. Hallock, S.C. Tang and A. Liepins, Sanguinarine induces bimodal cell death in K562 but not in high Bcl-2-expressing JM1 cells. Pathol Res Pract 197 (2001b), pp. 717–726. Abstract | PDF (644 K) | Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (16)

Weerasinghe et al., 2001c. P. Weerasinghe, S. Hallock and A. Liepins, Bax, Bcl-2, and NF-kappaB expression in sanguinarine induced bimodal cell death. Exp Mol Pathol 71 (2001c), pp. 89–98. Abstract | PDF (633 K) | View Record in Scopus | Cited By in Scopus (24)

Wolff and Knipling, 1993. J. Wolff and L. Knipling, Antimicrotubule properties of benzophenanthridine alkaloids. Biochemistry 32 (1993), pp. 13334–13339. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (74)

Yamada et al., 1984. H. Yamada, H. Hayashi and Y. Natori, Simple procedure for the isolation of highly purified lysosomes from normal rat liver. J Biochem 95 (1984), pp. 1155–1160. View Record in Scopus | Cited By in Scopus (30)

Yoshida et al., 1991. K. Yoshida, A. Oshima, M. Shimmoto, Y. Fukuhara, H. Sakuraba, N. Yanagisawa et al., Human β-galactosidase gene mutations in GM1-gangliosidosis: a common mutation among Japanese adult/chronic cases. Am J Hum Genet 49 (1991), pp. 435–442. View Record in Scopus | Cited By in Scopus (35)

Corresponding Author Contact InformationCorresponding author


Cell Biology International
Volume 27, Issue 11, November 2003, Pages 887-895
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Image

1—AcP, rat; 2—AcP, mouse; 3—prostatic AcP, mouse; 4—prostatic AcP, rat; 5—prostatic AcP, human; 6—AcP2, human; 7—AcP3, human; 8—AcP, Drosophila melanogaster; 9—AcP, D. subobscura. (+)—conserved cysteine residue; (−)—variable cysteine residue; (*)—near this position presents cysteine residue.

Table 2C. Conservation of cysteine residues in primary structures of lysosomal enzyme, N-acetyl,Image -glucosaminidase (comparison with human α-chain of N-acetyl,Image -glucosaminidase)

Image

1—mouse, α-chain; 2—human, β-chain; 3—cat, β-chain; 4—mouse, β-chain; 5—pig, β-chain; 6—Caenorhabditis elegans; 7—Drosophila melanogaster (Hexo2); 8—Bombyx mori;9—B. mandarina; 10—D. melanogaster (Hexo1); 11—Entamoeba histolytica; 12—Arabidopsis thaliana; 13—Dictyostelium discoideum; 14—Candida albicans; 15—Penicillium chrysogenum; 16—Trichoderma harzianum (strain P1); 17—Porphyromonas gingivalis; 18—Vibrio vulnificus. (+)—conserved cysteine residue; (−)—variable cysteine residue; (*)—near this position presents cysteine residue.

Mutations in GALs that induce inherited gangliosidoses involve the replacement of different amino acid residues by cysteine residues ([Boustany et al., 1993]; [Ishii et al., 1995]; [Morrone et al., 1997]; [Nishimoto et al., 1991]; [Silva et al., 1999]; [Yoshida et al., 1991]). Additional cysteines in the amino acid sequence of GAL cause critical changes in catalytic site. The molecular mechanism of the process includes the involvement of the additional cysteine residues and the formation of disulfide bonds between the cysteines present in the wild type of enzyme. These disulfide bonds might be stable to reduction during enzyme activity. Thus, our results as well as those cited in literature reveal the important role of cysteine residues for GAL activity.

In AcP molecules of vertebrate and invertebrate animals the cysteine residues corresponding Cys-159, 310, 345 and 349 of human AcP are highly conserved. Cys-370 is replaced by other amino acids only in human AcP2. Cys-212 is replaced in human AcP2 and insect enzymes (Table 2B). It has been suggested that the cysteine residues form three disulfide bonds: Cys-159–Cys-370, Cys-212–Cys-310 and Cys-345–Cys-349 participating in the stabilization of three-dimensional structure of AcP catalytic site which includes His-284 (proton donor), Asp-285 and positively charged residues in the N-terminal region of enzyme molecule (His-40, Arg-82) ( [Ostanin et al., 1994]). Our data on the sensitivity of the lysosomal enzymes to CPMA show the importance of SH-groups for AcP activity. However, NEM has a weaker effect, so the data suggest the existence of hydrophobic regions in the AcP catalytic site.

Comparative analysis of NAGA α- and β-chains from different species of animals, fungi, bacteria and plants shows that Cys-277, 328 and 522 (the numbers of the cysteine residues correspond to those of the α-chain of human NAGA) are highly conserved (Table 2C). However, Cys-58, Cys-104, Cys-458 and Cys-505 are conserved only in mammalian enzymes. The replacement of mammalian Cys-309 in the NAGA β-chain (which corresponds to Cys-277 in the α-chain) by tyrosine leads to type II GM2-gangliosidosis (Sandhoff disease) ([Gomez-Lira et al., 1995]). The replacement of Cys-458 in human NAGA α-chain by tyrosine induces type I GM2-gangliosidosis (Tay-Sachs disease) ([Tanaka et al., 1994]). The replacement of Cys-534 in human β-chain (which corresponds to Cys-505 in the α-chain) by tyrosine leads to type II GM2-gangliosidosis ([Kuroki et al., 1995]). At present it has been shown that NAGA catalytic site includes negatively charged residues—Asp-258, residues pair Asp-322—Glu-323 and Glu-462 ( [Fernandes et al., 1997]; [Hou et al., 2001]). Cys-277 and Cys-328 are localized near the enzyme catalytic site.

Our data on the high sensitivity of NAGA to CPMA, NEM and DTT provide strong evidence of the SH-dependence of this enzyme. We suppose that CPMA and NEM alter the SH-groups of NAGA and DTT reduce the S-S bonds of this enzyme that causes the changes of the functionally active form of NAGA oligomeric complex, including some subunits stabilized by S-S bonds, so far as various heteromeric forms of this enzyme have been determined ([Mahuran et al., 1988]).

Comparative analysis of lysosomal enzyme sensitivities to sanguinarine NEM and CPMA revealed an essential difference in their mechanisms of action. The strongest effect was exerted by CPMA. In addition to the enzyme surface SH-groups CPMA molecules can penetrate into internal hydrophobic pockets and react with SH-groups localized in these regions. The effect of this agent is significant even at micromolar concentrations. In contrast, NEM inhibits at millimolar concentrations and is more effective when the preincubation temperature is increased. Sanguinarine, like NEM, inhibits in millimolar doses, but it is more potent than NEM. Increase in preincubation temperature further enhances the sensitivity of the lysosomal enzymes to sanguinarine. This effect is probably determined by kinetic factors, in particular, by the increase in the rate of sanguinarine transfer to the cavity of enzyme catalytic site. Our preliminary studies about the partial elimination of sanguinarine inhibiting effect on AcP and GAL activities in the presence of DTT confirms the ability of alkaloid to interact with SH-groups of enzymes. In the case of NAGA the protective effect of DTT appear can not be manifested because of the strong inhibitory action on the enzyme activity of the reagent itself.

These data indicate that studies of the SH-sensitivity of the lysosomal enzymes and the state of SH-groups in their active sites represent a promising approach to understanding the mechanisms and the treatment of lysosomal storage diseases. In many cellular processes, redox-based regulation has emerged as an important regulatory mechanism. For example, oxidative stress is a potent stimulus for apoptotic cell death ([Loeffler and Kroemer, 2000]). The state of SH-groups in proteins depends on the redox-potential of the whole cell and the relevant organelles. Even a slight change in this potential may lead to a shift either towards free SH-groups or towards the formation of disulfide bonds. Such changes directly influence the three-dimensional structures of proteins and/or protein complexes, and thus the activities of enzymes. Both the experimental observations obtained in this study and the literature data show that the principal lysosomal enzymes are SH-sensitive and their enzymatic activities probably depend on the redox-potential of the cell.

Acknowledgements

This work was supported by the Russian Foundation for Basic Research (grant N 0104-49428).

References

Ahmad et al., 2000. N. Ahmad, S. Gupta, M.M. Husain, K.M. Heiskanen and H. Mukhtar, Differential antiproliferative and apoptotic response of sanguinarine for cancer cells versus normal cells. Clin Cancer Res 6 (2000), pp. 1524–1528. View Record in Scopus | Cited By in Scopus (83)

D'Azzo et al., 1982. A. D'Azzo, A. Hoogeveen, A.J. Reuser, D. Robinson and H. Galjaard, Molecular defect in combined β-galactosidase and neuraminidase deficiency in man. Proc Natl Acad Sci U S A 79 (1982), pp. 4535–4539. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (110)

Barrett, 1972. A.J. Barrett, Lysosomal enzymes. Assay methods. In: Lysosomes: a laboratory handbook, Elsevier, Amersham (1972), pp. 110–125.

Belyaeva et al., 1990. T.N. Belyaeva, A.G. Bulychev, O.E. Lasskaya and E.G. Semenova, Effects of sanguirytrine on the functional activity of lysosomes in fibroblasts. Cytology (Russia) 36 (1990), pp. 16–18. View Record in Scopus | Cited By in Scopus (2)

Belyaeva and Faddejeva, 1995. T.N. Belyaeva and M.D. Faddejeva, Disturbance of energy transduction in rat liver mitochondria by sanguinarine and AphMA. Cytology (Russia) 37 (1995), pp. 237–248.

Boustany et al., 1993. R.M. Boustany, W.H. Qian and K. Suzuki, Mutations in acid β-galactosidase cause GM1 gangliosidosis in American patients. Am J Hum Genet 53 (1993), pp. 881–888. View Record in Scopus | Cited By in Scopus (17)

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Faddejeva and Belyaeva, 1997. M.D. Faddejeva and T.N. Belyaeva, Sanguinarine and ellipticine: cytotoxic alkaloids isolated from well-known antitumor plants. Intracellular targets of their action. Cytology (Russia) 39 (1997), pp. 181–207.

Fernandes et al., 1997. M.J. Fernandes, S. Yew, D. Leclerc, B. Henrissat, C.E. Vorgias, R.A. Gravel et al., Identification of candidate active site residues in lysosomal β-hexosaminidase A. J Biol Chem 272 (1997), pp. 814–820. View Record in Scopus | Cited By in Scopus (30)

Hiraiwa et al., 1997. M. Hiraiwa, M. Saitoh, N. Arai, T. Shiraishi, S. Odani, Y. Uda et al., Protective protein in the bovine lysosomal β-galactosidase complex. Biochim Biophys Acta 1341 (1997), pp. 189–199. Abstract | PDF (774 K) | View Record in Scopus | Cited By in Scopus (10)

Hou et al., 2001. Y. Hou, D.J. Vocadlo, A. Leung, S.G. Withers and D. Mahuran, Characterization of the Glu and Asp residues in the active site of human β-hexosaminidase B. Biochemistry 40 (2001), pp. 2201–2209. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (18)

Gomez-Lira et al., 1995. M. Gomez-Lira, A. Sangalli, M. Mottes, C. Perusi, P.F. Pignatti, N. Rizzuto et al., A common β-hexosaminidase gene mutation in adult Sandhoff disease patients. Hum Genet 96 (1995), pp. 417–422. View Record in Scopus | Cited By in Scopus (14)

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McCarter et al., 1997. J.D. McCarter, D.L. Burgoyne, S. Miao, S. Zhang, J.W. Callahan and S.G. Withers, Identification of Glu-268 as the catalytic nucleophile of human lysosomal β-glactosidase precursor by mass spectrometry. J Biol Chem 272 (1997), pp. 396–400. View Record in Scopus | Cited By in Scopus (23)

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Morrone et al., 1997. A. Morrone, T. Bardelli, M.A. Donati, M. Giorgi, R. Di Rocco, R. Gatti et al., Identification of new mutations in six Italian patients affected by a variant form of infantile GM1-gangliosidosis with severe cardiomyopathy. Am J Hum Genet 61 (1997), p. A258.

Nishimoto et al., 1991. J. Nishimoto, E. Nanba, K. Inui, S. Okada and K. Suzuki, GM1-gangliosidosis (genetic β-galactosidase deficiency): identification of four mutations in different clinical phenotypes among Japanese patients. Am J Hum Genet 49 (1991), pp. 566–574. View Record in Scopus | Cited By in Scopus (20)

Ostanin et al., 1994. K. Ostanin, A. Saeed and R.L. Van Etten, Heterologous expression of human prostatic acid phosphatase and site-directed mutagenesis of the enzyme active site. J Biol Chem 269 (1994), pp. 8971–8978. View Record in Scopus | Cited By in Scopus (44)

Potier et al., 1990. M. Potier, L. Michaud, J. Tranchemontagne and L. Thauvette, Structureof lysosomal neuraminidase—β-galactosidase—carboxypeptidase multienzymic complex. Biochem J 267 (1990), pp. 197–202. View Record in Scopus | Cited By in Scopus (20)

Scriver et al., 1995. R.S. Scriver, A.L. Beaudet, W.S. Sly and D. Valle, Lysosomal enzymes. In: The metabolic and molecular bases of inherited disease (7th edn ed.),, McGraw-Hill, New York (USA) (1995), pp. 2425–2879.

Semenova et al., 1984. E.G. Semenova, A.V. Khomenko and S.E. Mamaeva, Fluctuations in the cell cycle span and karyotype of murine L cells depending on the mode of cultivation. Cytology (Russia) 26 (1984), pp. 1156–1160. View Record in Scopus | Cited By in Scopus (4)

Silva et al., 1999. C.M. Silva, M.H. Severini, A. Sopelsa, J.C. Coelho, A. Zaha, A. D'Azzo et al., Six novel β-galactosidase gene mutations in Brazilian patients with GM1-gangliosidosis. Hum Mutat 13 (1999), pp. 401–409. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (16)

Steinberg, 1994. T.H. Steinberg, Cellular transport of drugs. Clin Infect Dis 19 (1994), pp. 916–921. View Record in Scopus | Cited By in Scopus (22)

Suffness and Cordell, 1985. M. Suffness and G.A. Cordell, Benzo[c]phenanthridine alkaloids. In: The alkaloids. Chemistry and pharmacology 25, Academic Press, Orlando (1985), pp. 178–188.

Tanaka et al., 1994. A. Tanaka, H. Sakazaki, H. Murakami, G. Isshiki and K. Suzuki, Molecular genetics of Tay-Sachs disease in Japan. J Inherit Metab Dis 17 (1994), pp. 593–600. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (12)

Tulsiani and Touster, 1987. D.R. Tulsiani and O. Touster, Substrate specificities of rat kidney lysosomal and cytosolic α-Image -mannosidases and effects of swainsonine suggest a role of the cytosolic enzyme in glycoprotein catabolism. J Biol Chem 262 (1987), pp. 6506–6514. View Record in Scopus | Cited By in Scopus (24)

Van Der Spoel et al., 2000. A. Van Der Spoel, E. Bonten and A. d'Azzo, Processing of lysosomal β-galactosidase. The C-terminal precursor fragment is an essential domain of the mature enzyme. J Biol Chem 275 (2000), pp. 10035–10040. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (13)

Verheijen et al., 1982. F. Verheijen, R. Brossmer and H. Galjaard, Purification of acid β-galactosidase and acid neuraminidase from bovine testis: evidence for an enzyme complex. Biochem Biophys Res Commun 108 (1982), pp. 868–875. Abstract | Article | PDF (710 K) | View Record in Scopus | Cited By in Scopus (25)

Walterova et al., 1980. D. Walterova, V. Preininger, F. Granbal, V. Simanek and F. Santavy, Fluorescence spectra and cation-pseudobase equilibria of some benzophenanthridine alkaloids. Heterocycles 14 (1980), pp. 597–600.

Walterova et al., 1981. D. Walterova, J. Ulrichova, V. Preininger and V. Simanek, Inhibition of liver alanine aminotransferase activity by benzophenanthridine alkaloids. J Med Chem 24(1981), pp. 1100–1103. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (28)

Wang et al., 1997. B.H. Wang, Z.X. Lu and G.M. Polya, Inhibition of eukaryotic protein kinases by isoquinoline and oxazine alkaloids. Planta Med 63 (1997), pp. 494–498. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (31)

Weerasinghe et al., 2001a. P. Weerasinghe, S. Hallock, S.C. Tang and A. Liepins, Role of Bcl-2 family proteins and caspase-3 in sanguinarine-induced bimodal cell death. Cell Biol Toxicol 17 (2001a), pp. 371–381. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (23)

Weerasinghe et al., 2001b. P. Weerasinghe, S. Hallock, S.C. Tang and A. Liepins, Sanguinarine induces bimodal cell death in K562 but not in high Bcl-2-expressing JM1 cells. Pathol Res Pract 197 (2001b), pp. 717–726. Abstract | PDF (644 K) | Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (16)

Weerasinghe et al., 2001c. P. Weerasinghe, S. Hallock and A. Liepins, Bax, Bcl-2, and NF-kappaB expression in sanguinarine induced bimodal cell death. Exp Mol Pathol 71 (2001c), pp. 89–98. Abstract | PDF (633 K) | View Record in Scopus | Cited By in Scopus (24)

Wolff and Knipling, 1993. J. Wolff and L. Knipling, Antimicrotubule properties of benzophenanthridine alkaloids. Biochemistry 32 (1993), pp. 13334–13339. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (74)

Yamada et al., 1984. H. Yamada, H. Hayashi and Y. Natori, Simple procedure for the isolation of highly purified lysosomes from normal rat liver. J Biochem 95 (1984), pp. 1155–1160. View Record in Scopus | Cited By in Scopus (30)

Yoshida et al., 1991. K. Yoshida, A. Oshima, M. Shimmoto, Y. Fukuhara, H. Sakuraba, N. Yanagisawa et al., Human β-galactosidase gene mutations in GM1-gangliosidosis: a common mutation among Japanese adult/chronic cases. Am J Hum Genet 49 (1991), pp. 435–442. View Record in Scopus | Cited By in Scopus (35)

Corresponding Author Contact InformationCorresponding author


Cell Biology International
Volume 27, Issue 11, November 2003, Pages 887-895
Result list | previous < 2 of 10 > next 


Mutations in GALs that induce inherited gangliosidoses involve the replacement of different amino acid residues by cysteine residues (Boustany et al., 1993; Ishii et al., 1995; Morrone et al., 1997; Nishimoto et al., 1991; Silva et al., 1999; Yoshida et al., 1991). Additional cysteines in the amino acid sequence of GAL cause critical changes in catalytic site. The molecular mechanism of the process includes the involvement of the additional cysteine residues and the formation of disulfide bonds between the cysteines present in the wild type of enzyme. These disulfide bonds might be stable to reduction during enzyme activity. Thus, our results as well as those cited in literature reveal the important role of cysteine residues for GAL activity.

In AcP molecules of vertebrate and invertebrate animals the cysteine residues corresponding Cys-159, 310, 345 and 349 of human AcP are highly conserved. Cys-370 is replaced by other amino acids only in human AcP2. Cys-212 is replaced in human AcP2 and insect enzymes (Table 2B). It has been suggested that the cysteine residues form three disulfide bonds: Cys-159–Cys-370, Cys-212–Cys-310 and Cys-345–Cys-349 participating in the stabilization of three-dimensional structure of AcP catalytic site which includes His-284 (proton donor), Asp-285 and positively charged residues in the N-terminal region of enzyme molecule (His-40, Arg-82) (Ostanin et al., 1994). Our data on the sensitivity of the lysosomal enzymes to CPMA show the importance of SH-groups for AcP activity. However, NEM has a weaker effect, so the data suggest the existence of hydrophobic regions in the AcP catalytic site.

Comparative analysis of NAGA α- and β-chains from different species of animals, fungi, bacteria and plants shows that Cys-277, 328 and 522 (the numbers of the cysteine residues correspond to those of the α-chain of human NAGA) are highly conserved (Table 2C). However, Cys-58, Cys-104, Cys-458 and Cys-505 are conserved only in mammalian enzymes. The replacement of mammalian Cys-309 in the NAGA β-chain (which corresponds to Cys-277 in the α-chain) by tyrosine leads to type II GM2-gangliosidosis (Sandhoff disease) (Gomez-Lira et al., 1995). The replacement of Cys-458 in human NAGA α-chain by tyrosine induces type I GM2-gangliosidosis (Tay-Sachs disease) (Tanaka et al., 1994). The replacement of Cys-534 in human β-chain (which corresponds to Cys-505 in the α-chain) by tyrosine leads to type II GM2-gangliosidosis (Kuroki et al., 1995). At present it has been shown that NAGA catalytic site includes negatively charged residues—Asp-258, residues pair Asp-322—Glu-323 and Glu-462 (Fernandes et al., 1997; Hou et al., 2001). Cys-277 and Cys-328 are localized near the enzyme catalytic site.

Our data on the high sensitivity of NAGA to CPMA, NEM and DTT provide strong evidence of the SH-dependence of this enzyme. We suppose that CPMA and NEM alter the SH-groups of NAGA and DTT reduce the S-S bonds of this enzyme that causes the changes of the functionally active form of NAGA oligomeric complex, including some subunits stabilized by S-S bonds, so far as various heteromeric forms of this enzyme have been determined (Mahuran et al., 1988).

Comparative analysis of lysosomal enzyme sensitivities to sanguinarine NEM and CPMA revealed an essential difference in their mechanisms of action. The strongest effect was exerted by CPMA. In addition to the enzyme surface SH-groups CPMA molecules can penetrate into internal hydrophobic pockets and react with SH-groups localized in these regions. The effect of this agent is significant even at micromolar concentrations. In contrast, NEM inhibits at millimolar concentrations and is more effective when the preincubation temperature is increased. Sanguinarine, like NEM, inhibits in millimolar doses, but it is more potent than NEM. Increase in preincubation temperature further enhances the sensitivity of the lysosomal enzymes to sanguinarine. This effect is probably determined by kinetic factors, in particular, by the increase in the rate of sanguinarine transfer to the cavity of enzyme catalytic site. Our preliminary studies about the partial elimination of sanguinarine inhibiting effect on AcP and GAL activities in the presence of DTT confirms the ability of alkaloid to interact with SH-groups of enzymes. In the case of NAGA the protective effect of DTT appear can not be manifested because of the strong inhibitory action on the enzyme activity of the reagent itself.

These data indicate that studies of the SH-sensitivity of the lysosomal enzymes and the state of SH-groups in their active sites represent a promising approach to understanding the mechanisms and the treatment of lysosomal storage diseases. In many cellular processes, redox-based regulation has emerged as an important regulatory mechanism. For example, oxidative stress is a potent stimulus for apoptotic cell death (Loeffler and Kroemer, 2000). The state of SH-groups in proteins depends on the redox-potential of the whole cell and the relevant organelles. Even a slight change in this potential may lead to a shift either towards free SH-groups or towards the formation of disulfide bonds. Such changes directly influence the three-dimensional structures of proteins and/or protein complexes, and thus the activities of enzymes. Both the experimental observations obtained in this study and the literature data show that the principal lysosomal enzymes are SH-sensitive and their enzymatic activities probably depend on the redox-potential of the cell.

Acknowledgements

This work was supported by the Russian Foundation for Basic Research (grant N 0104-49428).

References

Ahmad N, Gupta, S, Husain, MM, Heiskanen, KM, Mukhtar, H. Differential antiproliferative and apoptotic response of sanguinarine for cancer cells versus normal cells. Clin Cancer Res 2000:6:1524-8
Medline   

D'Azzo A, Hoogeveen, A, Reuser, AJ, Robinson, D, Galjaard, H. Molecular defect in combined β-galactosidase and neuraminidase deficiency in man. Proc Natl Acad Sci U S A 1982:79:4535-9
Crossref   Medline   

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Received 10 December 2002/12 May 2003; accepted 8 July 2003

doi:10.1016/S1065-6995(03)00161-6


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)