Demonstration of ╬▓-N-acetyl-D-glucosaminidase and ╬▓-N-acetyl-D-hexosaminidase in Drosophila Kc-cells.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 17:3-13 (1 991) Demonstration of P-N-Acetyl-DG lucosaminidase and p-N-Acety I-DHexosaminidase in Drosophila K,-Ce IIs Ulrich Sommer and Klaus-Dieter Spindler lnstitut fur Zoologie, Lehrstuhl fur Horrnon- und Entwicklungsphysiologie, Heinrich-Heine-Universitaf Diisseldarf, Diisseldorf, E R. G. K,-cells from Drosophila melanogaster, grown under serum-free conditions, produce two p-hexosaminidases and secrete these enzymes into the medium. The two enzymes were separated by DEAE-exchange chromatography. According to their substrate specificities one enzyme is a f3-N-acetyl-D-glucosarninidase (E.C.126.96.36.199), the other one a P-N-acetyl-D-hexosaminidase (E.C.188.8.131.52). The P-N-acetyl-D-glucosaminidase is predominant i n the medium, the 6-Nacetyl-D-hexosaminidase within the cells. The K, values for the substrates pNP-GlcNAc, pNP-CalNAc, and (GICNAC)~ are 0.8,16.73, and 1.67 m M for the p-N-acetyl-D-glucosaminidase and 0.24,0.44, and 0.2 rnM for the P-N-acetylD-hexosaminidase. Both enzymes are inhibited by the products and the P-N-acetyl-D-glucosarninidase is also inhibited stereospecifically by the substrates pNP-GlcNAc and (GICNAC)~. Both enzymes are inhibited in a partial competitive way by acetamidolactones, the Kis being as low as 0.1 pM. Key words: intra- and extracellular enzymes, kinetic properties, inhibition INTRODUCTION Despite the fact that chitin is the main organic skeletal component in arthropods and has to be degraded several times throughout their life cycle, there is relatively little information available on the chitin-degrading enzymes [ 1-41. Besides, the regulation of the activity of these enzymes is of utmost importance, since, with the presumable exception of the chitin-degrading enzymes that are involved in nutrition, chitin degradation is developmentally regulated. The mechanism of regulation of chitinolytic activity is still not unequivocally explained [1-41. P-Hexosaminidases hydrolyse substrates containing 2-acetamido-2-deoxyD-hexoses like carbohydrates, glycoproteins, and glycolipids. One important Received June4,1990; accepted January14,1991. Address reprint requests to Prof. Dr. K.-D. Spindler, lnstitut fur Zoologie, Lehrstuhl fur Horrnonund Entwicklungsphysiologie, Heinrich-Heine-Universiat Dusseldorf, Universitatsstr. 1, D-4000 Dusseldorf 1, F.R.G. 0 1991 Wiley-Liss, Inc. 4 Sommer and Spindler role of these enzymes is their involvement in chitin degradation which is a necessary step in the molting process of all arthropods [5-7].(3-Hexosaminidase isoenzymes have been demonstrated in Culex quinquefuciufus , Locusfu migvatoriu [l],and fully characterized in Bombyx mori [9,10] and Muizducu sextu [11-14]. The use of established cell lines could be advantageous in solving at least some of the methodological problems: in whole animals especially, there is the problem of enzymatic activity introduced by the food, and from bacteria, fungi, or other symbionts. But so far only two papers exist on chitinolytic enzymes in insect cell lines [8,15], and unfortunately the enyzmes have been characterized only very preliminarily. Aside from this, in both cases vertebrate serum which contains chitin-degrading enzyme activity (14,171 has been used in the growth medium. We therefore decided to study chitinolytic enzymes from a Drosophilu &-cell line which is maintained serum free. A partial characterization of chitinases from this cell line has already been described [MI. MATERIALS AND METHODS Drosophilu Cell Line Culture The Drosophilu k-cell line, originally derived from Echalier and Ohanessian , was kindly supplied by Dr. Wyss (Zurich) and maintained as a suspension culture in a medium (201 at 25°C without serum. Sample Preparation For the determination of the enzymatic activity, appropriate volumes of the suspension culture were centrifuged (500 g, 10 min). The pellet containing the K,-cells was used for the preparation of cytosol, whereas the supernatant medium was used as another enzyme source. The cytosol of the cells was prepared as follows: about lo8 cells/ml were washed in isotonic buffer and sonified (under ice-cooling; Branson sonifier, microtip; 60 W for 10 s) in 0.2 M citrate-phosphate buffer, pH 5.5. The sample was centrifuged (10,000 g, 10 min, 4"C), and the pellet was resuspended and treated again as described. If enzymatic activity of the medium was to be analysed the cells were first separated from the medium, and an ammonium sulfate precipitation (70% saturation at 4°C) of the medium was performed. After 3 h the solution was centrifuged (20,000 g) and the pellet resuspended in 10 mM Na+-K'-phosphate buffer, pH 6.8. If necessary, the enzyme preparations were concentrated, depending on the volume, either by dialysis against polyethylene glycol (MW 20,000, Sigma, St. Louis, MO), by ultrafiltration with Amicon filters (PM 10, PM 30; Amicon, Danvers, MA), or with a Millipore filter PTGC 11K (Millipore, Bedford, MA, USA). Protein was determined according to Bradford , using bovine serum albumin (Sigma) as a standard. Hexosaminidase Assay The standard assay was as follows: 50 pl (3-D-hexosaminidasein 0.2 M citratephosphate buffer, pH 5.5, was incubated between 2 and 30 min, depending on the enzyme concentration with 50 pl of the same buffer and 50 p1 of substrate - @- N D-Hexosarni nidases in Drosophila 5 (either 9 mM pNP-GlcNAc*or 3 mM pNP-GalNAc in the same buffer, both from Sigma) at 37°C. At the end of the incubation period the reaction was stopped with 2.5 ml of 10-mM NaOH and the absorption measured at 410 nm. When the influence of inhibitors was tested, 50 pl of the inhibitor in 0.2 M citratephosphate buffer, pH 5.5 was used instead of the 50 pl buffer. If chitobiose (Sigma)was used as a substrate, the resulting endproduct, GlcNAc, was determined as already described . Cation-Ion Exchange-Chromatography DEAE-Sephadex A50 was prepared according to instructions (Pharmacia, Freiburg, BRD). The column (2.5 X 40 cm) was equilibrated with 10 mM Na+K+-phosphate buffer, pH 6.8. Chromatography was performed at a flow rate of 30 ml/h. Desalted material (desalting by gel filtration on Sephadex G-25) was used as the enzyme source. The desorption of bound proteins was performed with a linear gradient of NaCl(0-0.8 M NaCI, 1liter). The salt gradient was measured by determining the osmolarity of the fractions. Isoelectric Focusing Either preparative (according to LKB) or analytical isoelectric focusing (6% polyacrylamide gels, 6 x 120 mm) was performed. Prior to focusing the samples were dialyzed against 1%glycine in 10% sucrose. After preparative focusing, the gel was fractionated, the fractions eluted with 0.2 M citrate-phosphate buffer pH 5.5, and enzymatic activity determined. The demonstration of hexosaminidase activity on the polyacrylamide gel was performed using the fluorogenic substrate MUF-GlcNAc (0.4 mg/ml). The gels were thoroughly rinsed after the run in this substrate and then incubated for 5 to 15 min at 37°C. Enzymatic activity was visible in UV (254 nm). The pH profile was determined by a surface electrode. RESULTS Dvosophila &-cells are able to produce P-D-hexosaminidaseand to secrete it into the medium. After one day of incubation there is about the same total activity of hexosaminidase within the cells as in the medium. With increasing numbers of cells, this proportion changes and there is about a tenfold higher total activity within the medium than in the cells (Fig. 1).P-D-Hexosaminidaseactivity from the medium (Fig. 2a) and from the cells (Fig. 2b) separates on a DEAE-ion exchange column into two peaks of activity eluting at 85 mM and 0.2 M NaC1. The activity relationship between the two P-D-hexosaminidases is different in the medium than in the cells. In the cells the activity ratio of hexosaminidase I to hexosaminidase I1 is 1:7.3, as compared to 3.7:l in the medium. When rechromatographed, each enzyme elutes at the same salt concentration as it did initially. The different behavior of the two enzymes on *Abbreviations used: acetamidogalactonolactone= 2-acetamido-2-deoxy-D-galactonolactone; acetamidogluconolactone = 2-acetarnido-2-deoxy-D-gluconolactone;Gal NAc = N-acetyl-P-Dgalactosamine; (GICNAC)~ = chitobiose; ManNAc = N-acetyl-f3-D-mannosamine;M W = molecular weight; MUF-ClcNAc = 4-methylumbelliferyl-N-acetyl-~-D-glucosamine; pl = isoelectric point; pNP- = p-nitrophenyl-. 6 Sommer and Spindler 'units (refer to legend)' hours and activity of hexosaminidase in the Fig. 1. Increase in the number of cells ( x 105/ml; 0) The activity i s given as mU/ml cells or medium. = ratio of total cells (m)or in the medium activity in the medium against activity in the cells. . DEAE is also reflected in different isoelectric points. The hexosaminidase I has a PI of 6.0 ? 0.1 (n = 6). The PI of hexosaminidase I1 is 4.8 k 0.1 (n = 8) if a gradient from pH 3-10 is used, but with a smaller pH range (3x 8), three enzymatically active zones at PIS of 4.7,4.8, and 4.9 can be seen (n = 7). The K, values for the two P-D-hexosaminidases and three different substrates were determined under steady-state conditions at substrate saturation. The results of these experiments are shown in Figure 3 and Table 1. The two enzymes differ markedly in their catalytic activity towards the three substrates with hexosaminidase I always having lower hydrolytic rates than hexosaminidase 11. Hexosaminidase I1 has the higher hydrolysis rate towards (GlcNAc)2 and catalyzed the reaction with pNP-GlcNAc at nearly the same rate. With those two substrates hexosaminidase I1 shows a pronounced substrate inhibition, whereas with hexosaminidase I no such substrate inhibition occurs, In order to explain the different kinetic properties, we tested the effects of three different aminosugars (1mM) and three lactones (0.1 mM) on the activities of the two enzymes. The aminosugars either do not (ManNAc) or only weakly (GlcNAc, GalNAc; 3-20%) inhibit the two enzymes, whereas the two acetamidolactones, acetamidoglucono-and acetamidogalactono-lactone,have strong effects on enzymatic activity, especially that of hexosaminidase I. D-Gluconic acid lactone exerts only a weak effect on hexosaminidase I (11to 21%), but not on hexosaminidase 11. The type of inhibition produced by these two lactones was then studied in detail (Fig. 4 and Table 2). The three different inhibitor constants were determined according to Ahlers et al. . p-N- D-Hexosaminidases in Drosophila , 7 AmOamollkn absorbance 1.2 1200 - 1000 1 - 800 0.8 - BOO 0.6 - 400 0.4 0.2 0 0 20 40 60 80 100 120 140 I:"" 180 fractions A moamollkn absorbance 1.6 I b / t / 0 1 0 1200 ~looo 1aoo - 600 0.6 0.2 0'4 I - 400 - 200 1 1 20 40 80 80 0 100 fractions Fig. 2. Chromatography of the hexosaminidases from the medium (a) and the cytosol (b) of Drosophila K,-cells o n DEAE-Sephadex. The hexosaminidases elute at 85 (HEX I ) and 200 (HEX I I ) m M NaCI. * = absorbance at 410 nm; - = absorbance at 276 nm; A = mOsmol/kg. Independent of the substrate, the inhibitor, or the P-D-hexosaminidaseused for those experiments, there is always the following relative magnitude of inhibitor-constants: Ki < Kii < K, < Kiii.There is also a higher sensitivity of hexosaminidase I1 towards both inhibitors. Comparing the inhibitor-constants with the highest affinity, Ki, the two enzymes not only differ in the absolute value of Ki but also with regard to the substrates and inhibitors. Ki for hexosaminidase I and acetamidogluconolactone is about the same for both substrates, but for acetamidogalactonolactone the inhibition is eightfold higher when pNP-P-GalNAc is used as the substrate, whereas for hexosaminidase 11, the Ki for acetamidogalactonolactone is the same for both substrates but the Ki values differ for acetamidogluconolactone. DISCUSSION Our studies on the p-D-hexosaminidases from Drosophila &cells unequivocally demonstrate that insect cell lines are able to produce P-D-hexosaminidase 8 Sommer and Spindler 0.06 b 0,04 0.03 0.02 0,Ol // 0 -6 0 6 10 15 V A [mMl Fig. 3. Determination of kinetic properties of the two hexosaminidases from Drosophila K,cells. The data are given as Lineweaver-Burke-plots.Only representative data for the substrate pNP-p-GlcNAcare demonstrated for hexosaminidase I (a) and hexosaminidase I I (b). TABLE 1. Kinetic Parameters of the Two p-D-HexosaminidasesFrom Drosophila K,-Cells* Substrate Concentration (mM) K,-value (mM) V (mM/rnin x rng protein) V x K,-' Hexosaminidase I PNP-P-GlcNAc pNP-p-GalNAc (G~CNAC)~ 0.08-4.0 0.80-1 .O 1.OO-5.0 0.80 16.73 1.66 84.0 x 10-3 405.0 X 69.5 x 10-3 0.11 0.02 0.04 Hexosaminidase I1 PNP-p-GlcNAc pNP-P-GalNAc (G1cNAc)Z 0.08-0.2 0.08-1.0 0.05-0.4 0.24 0.44 0.20 17.1 x 10-3 13.2 x 0.5 x lo-' 0.07 0.03 0.000003 Values are derived from linear regression lines (correlation coefficients >0.95; P < 0.05). p-N-D-Hexosaminidasesin Drosopbila 0.1 0 0.3 0.2 9 0.5 0.4 I [mMI l l v fmln/uMI 0.8 1 bl "7 I :1 0,1 0 0 -2 4 2 6 8 1 0 1 2 14 1/A [l/mM] 0 02 0.4 0,s I lmMI 0.8 1 1.2 Fig. 4. Effect of acetamido-galactonolactone on the hydrolysis of pNP-p-GlcNAcby the hexosaminidase I (a) and II (b,c) according to Dixon and Webb  (a,c) or in a double-reciprocal plot (b).A = concentration of the substrate: 0.08 0.12 (O),0.4 ( + ) and 0.6 (0) mM. I = concentration of the inhibitor: 0.001 (&), 0.1 (0),0.4 ( + ) and 1.0 (A) rnM. (v), 10 Sommer and Spindler TABLE 2. Inhibitor Constants (Ki, Kii, Kiii in pM) of the Two P-D-Hexosaminidases From Drosophila K,-Cells Acetamidogluconolactone Ki pNP-P-GlcNAc pNP-P-GalNAc Kii Kiii Hexosaminidase I 0.25 8.47 27.1 x lo3 0.30 1.30 72.5 X lo" Acetamidogalactonolactone Ki Kii Kiii 0.80 0.10 8.10 0.13 8.10 X lo3 21.75 x lo3 1.90 2.09 9.90 6.20 1.25 X lo3 Hexosaminidase I1 PNP-P-GIcNAc yNP-p-GalNAc 2.85 10.22 11.42 25.00 0.96 x lo3 1.08 X lo3 1.31 x lo3 isoenzymes and to secrete them into the medium. The use of serum-free media was essential since vertebrate sera contain P-N-acetylglucosaminidases which are still active after the usual procedure of complement inactivation . The P-D-hexosaminidase activity of both cells and medium from the K,cells can be separated on an anion exchanger into distinct forms. These two forms elute at sodium chloride concentrations of 85 mM and 0.2 M. Under our experimental conditions the two P-D-hexosaminidases are not interconvertible, in contrast to the situation in some vertebrate systems . However, there is also a report that hexosaminidase A cannot be converted to hexosaminidase B in a vertebrate system . The two enzymes from the K,-cells of Drosophilu hydrolyse (GlcNAc)2,pNPGlcNAc, and pNP-GalNAc and they both have P-N-acetylglucosaminidase and -galactosidaseactivity. Similar activity has been described for enzymes from a variety of biological systems like fungi, plants, invertebrates, and vertebrates [11,25-281. Neither enzyme has activity against colloidal chitin or Micvococcus luteus, and they are, therefore, neither lysozymes nor chitinases. The latter enzyme is also present both in the K,-cells and in the medium . The two enzymes are therefore true P-D-hexosaminidases. They can be distinguished not only by their electric charge but also by their substrate preferences. Hexosaminidase I has about 20-fold higher affinity towards pNP-GlcNAc as compared with pNP-GlalNAc and is therefore a p-N-acetylglucosaminidase (E.C.184.108.40.206)whereas hexosaminidase I1 shows about the same affinities towards the two substrates and is therefore a P-N-acetylhexosaminidase (E.C.220.127.116.11). The affinity of the two enzymes towards the substrate pNPGlcNAc is about the same as e.g. for the corresponding enzymes fromMunducu sextu [ll],but also for a variety of other arthropod N-acetyl-P-D-glucosaminidases [2,4]. Pronounced differences in the K, values for pNP-GalNAc or (GlcNAc)2 for the two enzymes are no special feature of the Drosophilu K,cells but were also reported for the two P-N-acetylhexosaminidases from Munducu sexfu [ll]or from the medium of a cell line from Culex quinquefaciutus. The inhibition of the two enzymes is very complex and three different types of inhibition were detected: 1)The products GlcNAc and GalNAc inhibit both enzymes. Such a type of inhibition is not absolutely necessary for P-D-hexosaminidases but it has been demonstrated in quite different species from fungi to mammals [11,26,29-351; 2) The P-N-acetylhexosaminidase is also inhibited by the substrates pNP-GlcNAc and (G~CNAC)~ but not by pNP-GalNAc. Puri- P-N-D-Hexosarninidasesin Drosophila 11 fied P-N-acetylhexosaminidases from Bombylr mori [ 101 and Manduca sexta  show this type of inhibition, also; 3) The strongest inhibition is exerted by acetamidolactones,which have been shown to be very potent competitiveinhibitors for P-N-acetylhexosaminidases in other systems [27,32,36,37]. In contrast to those findings, the inhibition in the K,-cells is only partially competitive (mixed), which suggests that in Drosophila &-cells, the inhibitor and the substrate bind simultaneously. The inhibitor may bind to a regulatory center of the enzyme which interacts with the catalytical center. Our results clearly demonstrate that Drosophila K,-cells are capable of producing and secreting two types of hexosaminidases which can be distinguished by their isoelectric points and kinetics when different substrates and inhibitors are used. Knowledge of the differences between isoenzymes is important for studies on the regulation of p-D-hexosaminidases since it has been shown in chicken oviduct that progesterone induces only one of the two isoenzymes of p-N-acetylglucosaminidases .A positive correlation between the titers of ecdysteroids and chitin degrading enzymes has already been demonstrated for several arthropods [1-4,39-411. Since K,-cells respond both morphologically and biochemically to ecdysteroids [42,43], they seem to be well suited for studying the interaction between molting hormones and chitinolytic enzymes. LITERATURE CITED 1. Spindler K-D: Chitin: Its synthesis and degradation in arthropods. In: The Larval Serum Proteins of Insects. Scheller K, ed. Thieme Verlag, Stuttgart, pp 135-150 (1983). 2. Kramer KJ, Dziadik-Turner C, Koga D: Chitin metabolism in insects. In: Comprehensive Insect Physiology Biochemistry and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergammon Press, Oxford. 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