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Demonstration of ╬▓-N-acetyl-D-glucosaminidase and ╬▓-N-acetyl-D-hexosaminidase in Drosophila Kc-cells.

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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.3.2.1.30), the other one a P-N-acetyl-D-hexosaminidase (E.C.3.2.1.52).
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 [8], 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
[19], 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 [21], 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 [18].
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. [22].
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 [44] (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 [16].
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 [23]. However,
there is also a report that hexosaminidase A cannot be converted to hexosaminidase B in a vertebrate system [24].
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 [18]. 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.3.2.1.30)whereas hexosaminidase I1 shows about the same affinities
towards the two substrates and is therefore a P-N-acetylhexosaminidase
(E.C.3.2.1.52).
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[8].
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 [32]
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 [38].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.
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glucosaminidase, demonstration, drosophila, hexosaminidase, acetyl, cells
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