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Effect of 2-deoxyglucose on lysosomal enzymes in cultured human skin fibroblasts
EFFECT
OF 2-DEOXYGLUCOSE CULTURED PH.
G. de
GROOT,’ A.
ON
HUMAN A.
WESTERVELD.:’
SKIN
STRLJLAND.’ M.
R. N.
LYSOSOMAL
IN
FIBROBLASTS
KALSBEEK.’
HAMERS’
ENZYMES
and
P. MEERA J. M.
KHAN.’
TAGER’
SUMMARY Addition of 2-deoxyglucose, an inhibitor of glycosylation of proteins. to the medium of contluent cultures of human skin fibroblasts prevents the increase in specific activity of lysosomal enzymes that normally occurs after confluence. Maximal inhibition is obtained at a concentration of about I mM 2-deoxyglucose. The inhibition by I-deoxyglucose is reversible. The K,,, . pH dependence and electrophoretic mobility of the acid hydrolases tested was the same in cells cultured with or without Z-deoxyglucose. In homogenates of cultured human skin fibroblasts, about 95 “r of the /3hexosaminidase and a-galactosidase activity and about 65% of the acid phosphatase activity with fi-glycerolphosphate as substrate binds to concanavalin A (ConA); 2-deoxyglucose affects only the activity able to bind to ConA. In cells cultured in the presence of ?-deoxyglucose. the specific activity of alkaline phosphodiesterase I. a plasma membrane glycoprotein is lowered. ?-Deoxyglucase has no effect on the specific activity of succinate dehydrogenase. lactate dehydrogenase or total cellular protein.
All lysosomal enzymes investigated so far, including glucocerebrosidase, a membranebound enzyme, are glycoproteins [l-3]. It is believed that the carbohydrate moiety plays an important role in the intracellular transport of the enzymes and their packaging into lysosomes [4]. Indeed, in all current hypotheses about the mechanisms involved in the packaging of lysosomal enzymes [S81, an important role is assigned to the oligosaccharide moiety as a recognition marker for the enzymes. As demonstrated initially by Neufeld and co-workers [9]. the rapid endocytosis of extracellular lysosomal enzymes by cultured human skin fibroblasts is dependent on the presence in the enzymes of a specific oligosaccharide structure, which was subsequently identified by Kaplan et al. [ 10, 1l] as mannoIA-7YIXIX
sylphosphate (see also [ 12-141). According to Neufeld and co-workers the packaging of lysosomal enzymes involves transport of the enzymes to the cell surface followed by their endocytosis via a specific receptor [7] which recognises mannosylphosphate groups [ 15. 161. An alternative hypothesis, according to which lysosomal enzymes are cycled via the plasma membrane without being released into the extracellular space has been advanced by Lloyd [8] and Von Figura & Weber [ 171. Sly et al. [ 181 too, propose that intracellular transport only is involved in the packaging of lysosomal enzymes, and that secretion and recapture seen in cultured fibroblasts is a minor pathway. Novikoff [19] postulated that the glycosylation of lysosomal enzymes occurs
'08
Groat et 111.
The substrates were the methylumbelliferylglyco~ides. final volume 0.5 ml. The reaction mixture 100 mM sodium acetate at the pH indicated. for the substrate (column 2) was measured indicated
contained The K,,, at the pH
Enzyme
R,r, (mM)
Substrate cont. (mM)
PH
/3-Hexosaminidase a-Galactosidase P-Glucuronidase a-Glucosidase Arylsulphatase P-Glucosidase
0.3’ 3.4 OS6 0.44 I.2 0.78
I.5 3.6 1.7 0.44 3.5 1.8
5.0 4.6 3.5 4.6 5.0 5.0
during passage through the smooth endoplasmatic reticulum and the Golgi apparatus, where packaging occurs. Since glycosylation can be inhibited by a number of compounds, including tunicamycin [20], 2deoxyglucose [2 I] and glucosamine [22], it should be possible to investigate the role of the oligosaccharide moiety in the packaging of lysosomal enzymes by making use of these inhibitors. In this paper we show that the addition of 2-deoxyglucose to confluent cultures of human skin fibroblasts prevents the increase in lysosomal enzyme activity that normally occurs after confluence [see refs 23-261.
MATERIALS Cell
AND
carried out on confluent cultures. In t~dt’tto hat-\r\r the cells. they were first washed three time> with Hanks’ balanced sa1t solution IFI~M L.ab\~ and then detached from the flash by incubation with 0.25 c; trvpsin (Flow Labs) in Hank<’ balanced salt \oIution ioll-5 min. depending on the age of the tr\p\in \alution. When the cells were detached. 5 ml of medium containing FCS were added. and the cell> \\el-e centrifuged. The washing was repeated once with a phorphate-free medium containing 130 mM NaC‘I. 2.7 mM KCI, 14.3 mM NaHCO,, and 10 mM glucose. The cells were subsequently lysed hl addmg 0.5 ml of a solution containing 0.05 ci Triton X-100 and 2 mM EDTA. The solutions of 2-deoxyglucose (Sigma) and lactate (Sigma) were sterilized by passing through a Millipore filter (0.X pm) before adding to the medium.
All the acid hydrolases except acid pho\phatase were measured using the methylumbelliferyl glycosides as substrates (Koch-Light). The reaction mixture (final volume 0.5 ml) contained fibroblast homogenate or medium, 100 mM sodium acetate buffer at the pH shown in table 1, and substrate at a concentration at least I .5 times the K,,, value (see table I ). except in the case of a-glucosidase. since methylumbelliferyl-cglucoside is poorly soluble. The reaction was carried out for 0.5-3.0 h at 37°C and stopped by the addition of I.5 ml 300 mM glycine-NaOH (pH 10.6). The fluorescence of the liberated 4-methylumbelliferone wa>
Table 2. .EfTect qf’ 2-deolc?‘gllrc.o.sll otz enzyme actilqities und totrd protein in cultured humnn skin ,fibroblosts Fibroblasts were grown to confluence as described in Materials and Methods and then grown further either in the absence or in the presence of 5 mM ?-deoxyglucose. The cells were harvested 7 days after confluence and the enzyme activities were measured in the homogenates. Values are means + S.D., with the number of observations given in parentheses. or values from individual observations
METHODS Parameter
Activity or amount after culture with 7-deoxyglucose (ci of control)
Protein Succinate dehydrogenase Lactate dehydrogenase P-Hexosaminidase Acid phosphatase cY-Galactosidase Arylsulphatase /3-Glucuronidase cu-Glucosidase Phosphodiesterase I
99?lO (6) 91. 109(Z) 103. I IO II) SO+ IO (6) 78+ll (61 37t Ii (61 4ot 614) 26+ 614) 50,64(2t 43. 36 (2)
crrlt1we
Human skin fibroblasts were grown in Ham’s F-IO medium (Flow Laboratories), supplemented with 15 % heat-inactivated fetal calf serum (FCS) (Flow Labs), I6 mM NaHCO,, penicillin (250 U/ml) and streptomycin (250 pg/ml). If the medium was older than one week, extra glutamine (5 mM) was added. All the experiments described in the tables and figures were carried out with a line of fetal human skin fibroblasts, kindly provided by Mr Nice Leschot (Dept of Anthropogenetics, University of Amsterdam). The cells were grown in 25 cm2 Falcon plastic flasks containing 5 ml medium. The medium was refreshed every 3 days. Unless otherwise stated, all the experiments were
qf 2-deoxyglucose
Effect
on lysosomal
enzymes
209
2. Abscissa: 2-deoxyglucose cont. (mM) in medium; ordinate: activity (5% of that in cells grown in the absence of 2-deoxyglucose). Effect of adding different concentrations of 2-deoxyglucose to the medium of a confluent cell culture on the activity after 7 days of O-O, /3-glucuronidase; A-A. cY-galactosidase; U--O, /3-hexosaminidase; O-O, acid phosphatase; W--m, succinate dehydrogenase; n--n, on total cell protein after 7 days. Fig.
I
I
1
I
I
I
,
I
I
o
2
4
6
0
2
4
6
8
I. Abscissa: time after confluence (days); ordifrufe: (A-E) activity (mU/mg protein); (F) protein (mg/flask). Effect of 5 mM 2-deoxyglucose, added to the medium of a confluent cell culture, on total cellular protein (F) and on the activity of /3-hexosaminidase (A), arylsulphatase (B), o-glucosidase (C), N-acetylru-galactosaminidase (D). and P-glucosidase (E). Culture in O-O, absence; O-O, presence of 2-deoxyglucose. For conditions see text. Fig.
measured in an Eppendorf fluorimeter with a 3 13-366 nm filter for excitation and a 430-3 000 nm secondary filter. Acid phosphatase was measured with /3-glycerophosphate as substrate [27]. N-acetyl-o-galactosaminidase with p-nitrophenyl-a-galactosaminide as substrate [28]. succinate dehydrogenase according to Morre [29], lactate dehydrogenase following Bergmeyer & Bernt [30]. and phosphodiesterase I as described by Razzell [31]. The rates of the enzymic reactions were linear for the time intervals used in the assays. Triton X-100 at the concentration used in the assays had no effect on enzyme activities when tested with a homogenate made by freezing and thawing of the cells. Protein was estimated according to Lowry [32]. All enzyme activities are expressed as nmol product formed per min per mg total protein in the cell extract. The abbreviations for the enzymes are according to the recommendations of the Committee on Human Gene Nomenclature [60]. The electrophoretic characterization of acid phosphatase (ACP). P-hexosaminidase (HEX), /3-glucuronidase (GUSB). and cY-galactosidase (GALA), was carried out on cellogel (Chemetron) as described elsewhere [33-361.
RESULTS The specific activity of acid hydrolases in cultured human skin fibroblasts changes dramatically during growth of the cells [2326]. In the log phase, the specific activity first declines and then increases, and this increase continues for several days after the cells become confluent. The effect of adding 2-deoxyglucose at a concentration of 5 mM to the medium of cultures of confluent fibroblasts is shown in fig. 1. Whereas 2-deoxyglucose had no effect on the protein content of the cells, it prevented the increase in the activity of acid hydrolases that is characteristic of cells cultivated without 2-deoxyglucose. The concentration dependence of the inhibitory effect of 2-deoxyglucose is shown in fig. 2. In this experiment, a half-maximal effect on acid hydrolases was observed at about 0.5 mM 2-deoxyglucose. On the other hand, 2-deoxyglucose had relatively little effect on the total cellular protein, or on the mitochondrial enzyme succinate dehydrogenase, which is apparently not a glycoprotein [37]; the activity of succinate de-
2 I0
(;l~oot
1’1 t/l.
Enryme Il..37 II. I6
a-Galactosidase
Normal +2-deox\gluco~e
1.11 -1.1)
Acid
Not-mal +2-deoxyglucose
4.6 4.6
3 I’)
L.1 II x
Nor-ma1 t 2-deoxyglucose
s.s > .\
7’ 3x
0.3: II..36
phosphatahe
/3Hexosaminidase
hydrogenase varied between 8&l IOf of the control value (see also table 7). In order to eliminate the possibility that the effect of I-deoxyglucose may have been caused by depletion of respiratory substrate, due to inhibition of glucose transport into the cells. 5 mM lactate was routinely added to the culture medium. Furthermore. the glutamine present in the medium may have been the main respiratory substrate [38]. These considerations. together with
F/,6,. 3. r\b.\ci.v.\cr: time after beeding the cells tdayhl: ,~~/i~rtc,: activity (8 of that at confluence (day 71 in cells without 2-deoxyglucose). Reversibility of the effect of 5 mM 2.deoxyglucobe on b-glucuronidase. For incubation conditions. see text. Ceils cultured (S--O. continuously without ?deoxyglucose: O-U. continuously in the presence of 2-deoxyglucose; m-B, for 7 days in the presence of 5 mM 7-deoxyglucose after which the medium VW, changed to one without 2-deoxyglucose.
2.s -.‘-I
the fact that culture of the cell5 in the presence of 2-deoxyglucose had no effect on the morphology of the cells. total cellular protein. the activity of succinate dehydrogenase and of the cytosolic enzyme lactate dehydrogenase (table 21. suggest that 1deosyglucose had no gross effect on thr energy metabolism of the cells. Table 2 summarizes the result5 of different experiments in which cells were grown for 7 days in the presence of 5 mM Z-deosyglucose. The magnitude of the influence of 2-deoxyglucose on the acid hydrolaseb was dependent on the enzyme:, measured: ,!!Lglucuronidase and u-galactosidasr were intluenced most strongly. uhereaa acid phosphatase and n-glucosidaae showed the lowest response. In addition, in cells cultured with 2-deoxyglucose there uas a marked decrease in the activity of alkaline phosphodiesterase I. a glycoprotein [39] located in the plasma membrane [30]. On the other hand. 2-deoxyglucose had no detectable effect on lactate dehydrogenasr. succinate dehydrogenaar (see also fig. 2) or total cellular protein ccf figs I. 2). In the experiment of fig. 3 cell5 Mere cultured for 7 dab5 in the pt-esence or absence of 5 mM 2-deoxyglucose. The control cells uere cultured in the absence of 2-deoxyglucose for ;I further I I days. The
F;q. 4. .Acid phosphatase IACPI, a-galactosidase (GALA), P-glucuronidase (CiUSBI and P-helosaminidase t HEX) electropherograms on Cellogel. Origin at bottom and migration anodal. Channels I and 2 carry the extracts of human fetal skin fibroblasts cultured in the growth medium with and without 2.deosyglucose. respectively. The letter< refer to different iso7ymes (see text).
cells grown in the presence of 2-deoxyglucose were divided into two portions. One half was cultured for I I days in control medium, the other half in a medium containing 2-deoxyglucose. On day 7. the activity of P-glucuronidase in cells grown in the presence of 2-deoxyglucose was about 30 Q? of that in control cells. The activity in cells grown for a further I I days in the presence of 2-deoxyglucose remained at about the same level. When the cells were transferred to control medium on day 7, there was an
almost immediate rise in the activity of pglucuronidase. the extent of which was similar to that seen in cells grown continuously in the control medium. All acid hydrolases tested responded in an analogous manner. Thus the effect of 2-deoxyglucose on acid hydrolases is reversible. In table 3. the characteristics of the acid hydrolase activities of cells grown in the presence and absence of 2-deoxyglucose are compared. 3-Deoxyglucose had no effect on the pH optimum but caused a decrease in V without altering the K,,, for the substrates. The effect of 2-deoxyglucose on the electrophoretic mobility of several acid hydrolases in cellulose acetate gels is shown in fig. 4. The major isozyme of acid phosphatase (fig. 4~) did not exhibit a change in the rate of migration. while the minor isozyme (fig. 417) was not seen in homogenates of cells grown in the presence of 2-deoxyglucose. Regions A and B of (Ygalactosidase activity correspond to (Ygalactosidase A and B, respectively. (The latter activity is identical with N-acetyl-agalactosaminidase [41-43].) The activity band corresponding to a-galactosidase A could not be detected in 2-deoxyglucosegrown cells. The intensity of the band corresponding to a-galactosidase B was reduced in 2-deoxyglucose-grown cells. In homogenates of fibroblasts grown in normal medium three distinct zones of P-glucuronidase activity ((1, h. c.1 are formed. the strongest of which is c’. The isozyme h was absent or very weak in homogenates of 2deoxyglucose-grown cells. Finally. there was no change in the position of the bands corresponding to hexosaminidases A and B in the 2-deoxyglucose-grown cells. No band of activity corresponding to hexosaminidase C could be detected, either in control or in 2-deoxyglucose-grown cells.
Fig. 5. Ahscissu: amount ConA (ml); ordinure: activity of cells grown in the absence Binding of the activity (B) a-galactosidase; and ConA coupled to Sepharose
Sepharose 4B-coupled (% of that in homogenate of 2-deoxyglucose). of (A) P-hexosaminidase: (C) acid phosphatase to 4B in homogenates of cells
cultured with I-deoxyglucose (0-O) and ceils cultured without 2-deoxyglucose (O-G). The cells were grown to confluence and then grown further for 7 days in either the absence or -presence of 5 mM 2-deoxyglucose. One ml Sepharose 4B coupled ConA contained 10 mg ConA.
In the experiment of fig. 5, homogenates of cells grown in the presence or absence of 2-deoxyglucose were incubated with different amounts of Sepharose 4B to which the lectin ConA was bound. As a control, homogenates were incubated with Sepharose 4B only. In the control cells, about 95% of the a-galactosidase and P-hexosaminidase activities were removed by treatment with immobilized ConA, leaving about
5% in the supernatant. In the I-deoxyglucase-treated cells, incubation with immobilized ConA lowered the residual activities to about the same level as in control cells. Similar results were obtained with the acid phosphatase activity, except that the amount remaining in the supernatant after ConA treatment was much higher (about 35%’ of the total activity present in control cells) than in the case of the other acid hydrolases. It is of importance to note that the activity of acid phosphatase was measured with /?-glycerophosphate. which is generally considered to be a specific substrate for lysosomal acid phosphatase [57, 581. Furthermore, both activities, i.e., that able to bind to ConA and that unable to bind to the lectin, are inhibited by 1 mM sodium-potassium tartrate, an inhibitor of lysosomal acid phosphatase [57. 581. In cultured human skin fibroblasts, some lysosomal enzymes are found in the culture medium [7]. In the case of P-hexosaminidase, 2-deoxyglucose had no effect on the amount of extracellular enzyme (fig. 61. On the other hand, extracellular P-glucuronidase was lower in 2-deoxyglucose-grown cells than in control cells (fig. 6). The amount of lactate dehydrogenase found in
Fig.
of
6. Abscissu: time after confluence (days); ordinate: activity (mu/ml medium). Effect of 5 mM 2-deoxyglucose on P-hexosaminidase (0, 0) and /3-glucuronidase (A, A) in culture medium of fibroblasts. After confluency, the cells were incubated further in the presence or absence of 2deoxyglucose. Every day the activity was measured in control cells (0, A) and in the cells cultured in the presence of 2-deoxyglucose (0, A).
EJfect of 2-deosyglucose the medium was not influenced by growing the cells in 2-deoxyglucose (not shown). DISCUSSION The glycosylation of glycoproteins can be inhibited by a number of different compounds including the antibiotic tunicamycin [20] and sugar analogues like glucosamine [22], 2-deoxyglucose [44], 2-deoxy-2-fluoroglucose and 2-deoxy-2-fluoromannose [45]. It has been shown that inhibitors of glycosylation can inhibit the synthesis of glycoprotein-containing viruses such as influenza virus [46], Sindbis virus [47] and vesicular stomatitis virus [47]. Inhibition of glycosylation can also lead to impairment of secretion of a protein; this is the case with IgA and IgE secretion in plasma cells [48]. In other cases, inhibition of glycosylation leads to formation of an unstable product. Olden et al. [49] have shown by means of pulse-labelling experiments that when tunicamycin is added to cultured chick embryo fibroblasts, the major cell-surface protein appears to be synthesized in a form that breaks down more rapidly than the fully glycosylated protein. In cultured chick tendon fibroblasts or chick bone cultures, addition of tunicamycin prevents the conversion of procollagen, which apparently accumulates in a non-glycosylated form, to collagen [50, 5 I]. We have investigated the possible role of the oligosaccharide moiety in the packaging of lysosomal enzymes by employing 2deoxyglucose as an inhibitor of glycosylation [44]. Datema & Schwarz [21] have shown that inhibition by 2-deoxyglucose of glycosylation is caused by the formation of 2-deoxyglucose-containing lipid-linked oligosaccharides, which are not metabolized further. In confluent cultures of human skin fibro-
on lysosomal
enzymes
213
blasts, there is a gradual increase in the activity of lysosomal enzymes [23-261. We have shown that when 2-deoxyglucose is present in the culture medium, this increase in activity is prevented, at least in the case of the acid hydrolase activities measured. A similar effect is found with the plasma membrane glycoprotein phosphodiesterase I. On the other hand, there is no effect of 2-deoxyglucose on succinate dehydrogenase and lactate dehydrogenase, on the protein content of the cells, on the gross morphology of the cells. or on their proliferating capacity when the cells are transferred to flasks containing 2-deoxyglucose-free medium (not shown). The following mechanisms may be brought forward to explain the effect of 2deoxyglucose on the activity of the lysosomal enzymes: (1) 2-Deoxyglucose inhibits glycosylation of the nascent lysosomal enzyme polypeptides, thus interfering with their routing to the lysosomes and causing feedback inhibition of synthesis of the polypeptides. (2) The non-glycosylated lysosomal enzymes are rapidly broken down in the lysosomes. (3) The non-glycosylated enzymes are less active than the normal enzymes or even inactive. (4) Addition of 2-deoxyglucose may lead to an accumulation of phosphorylated sugars and a depletion of ATP [52], thus interfering with ATP-dependent processes in the cell, such as protein synthesis or the functioning of the contractile elements. The following observations are in agreement with the first two mechanisms and not with the others: (I) The 2-deoxyglucose effect appears to be specific for glycoproteins; the acid hydrolases and phosphodiesterase I are affected, and not lactate dehydrogenase or succinate dehydrogen-
ase. (2) No evidence could be obtained for the synthesis in the presence of 2-deoxyglucose of forms of acid hydrolases with altered enzymological or physico-chemical properties: the K,,, values of the enzymes and their electrophoretic mobility were unaffected and no increase was detected in the activity unable to bind to the carbohydrate lectin ConA. (3) Fructose which. like 2-deoxyglucose. can lead to a decrease in the ATP content of cells (see [5?]l has no effect on the level of acid hydrolases in the cultured fibroblasts (results not shown). At present we can not distinguish between mechanisms 1 and 2. If an unstable non-glycosylated form is. indeed. produced, it must be broken down so rapidly that it can not be detected. Mechanism 2 implies that the oligosaccharide moiety protects the acid hydrolases from being degraded by lysosomal cathepsins. Brown et al. [54] have suggested that treatment of yeast invertase with endo-N-acetyl$-hexosaminidase to remove oligosaccharides leads to some decrease in the half-life of the enzyme after it is endocytosed by rat yolk sacs. However, close examination of their data suggest that heating of the deglycosylated enzyme converts it to a form with about half the specific activity of the native enzyme. Other studies have indicated that non-glycosylated glycoproteins may be less stable than the native proteins (for a discussion see [55]). If non-glycosylated peptides were formed which were completely devoid of enzymic activity, it would not be possible to distinguish between mechanisms I. 2 and 3. However. von Figura et al. [I41 have brought forward evidence that after treatment of a lysosomal enzyme with endoglycosidase H. some activity at least remains, so that one might have expected to be able to detect enzymically active. non-glycosylated forms
of the acid hydrolases. Studie\ on the intracellular distribution and turnover of Iys~omal enzymes in the presence and absence of 2-deouvglucose are in progress. i\t first sight. the effect of 2-deosyglucase resembles that of cytochalasin B. which. as shown by von Figura & Kresse [56]. leads to a decrease in the intracellular activity of lysosomal hydrolases. However. in the case of cytochalasin B. there is a concomitant increase in the extracellular activity of at least one of these enzymes. phexosaminidase. This increase is not seen with 2-deoxyglucose (fig. 61. which suggests that 2-deoxyglucose and cytochalasin B exert different effects. The response of the acid phosphatase activity (measured with P-glycerophosphate as substrate) to 2-deoxyglucose is exactly analogous to that of the other acid hydrolases in that only the activity able to bind to ConA is decreased in the cells grown in the presence of the sugar analogue. The activity that does not bind to the lectin is unaffected. It is of interest that both activities were measured with /3glycerophosphate as substrate and that both were inhibited to about 95?? by I mM sodium-potassium tartrate. These criteria are usually employed to distinguish lysosomal acid phosphatase from other phosphatases [57, 58]. The relationship between the two activities that can be distinguished on the basis of the ability to bind to Con4 is at present unclear. After this study was completed an abstract by Vladutiu & Rattazzi [59] appeared in which they reported that 2-deoxyglucose reduces the excretion of /3-hexosaminidase by cultured skin fibroblasts from patients with mucolipidosis II (I-cell disease). In contrast. it had no effect on the intracellular level of the enzyme in normal fibroblasts. The apparent lack of effect in normal fibro-
blasts was probably due to the fact that the cells were cultured with 2-deoxyglucose for 24 h only (cf fig. 1). The authors are grateful to all the members of the Rotterdam-Leiden-Amsterdam ‘Lvsosomal Enzvmes’ group for stimulating discussions and to Mr-Nice Leschot for orovidine a line of fetal human skin fibroblasts. This study ways supported by a grant from the Netherlands Organization for the Advancement of Pure Scientific Research (Z.W.O.) under the auspices of the Netherlands Foundation for Fundamental Medical Research tF.U.N.G.O.1.
23. 24. 25. 26. 27, 28. 29.
30. 31.
REFERENCES I. Bouma. J M W. Enzyme therapy in lysosomal storage diseases (ed J M Tager. G J M Hoogwinkel & W Th Daems) p. 197. North-Holland Publ. Co.. Amsterdam t 1974). 2. Fiddler. M B. Ben-Yoseph. Y & Nadler. H L. Biochem j 177 (1979) 175. 3. Dale, G L. Villacorte, D G & Beutler, E, Biochem bionhvs res commun 71 (1976) 1048. 4. Dean.‘R T & Barrett. A J. Essays in biochemistry I2 (1976) I. 5 Novikoff. A B, Essmer E & Quintana. N. Fed proc 23 ( 1964) 1010. 6. De Duve. C & Wattiaux R. Ann rev physiol 28 (1966) 435. 7. Hickman, S & Neufeld E F, Biochem biophys res commun 49 t 1972) 992. 8. Lloyd, J B. Biochem j 164 (1977) 281. 9. Hickman. S. Shapiro, L J & Neufeld. E F. Biochem biophys res commun 57 t 1974) 55. IO. Kaplan, A. Achord, D T & Sly, W S. Proc natl acad sci US ?4 (1977) 2026. Il. Kaplan, A. Fisher. D, Achord. D T & Sly. W S, J clin invest 60 t 1977) 1088. I?. Sando G N &‘Neufeld E F, Cell I2 (1977) 619. 13. Ullrich, K, Mersmann G, Weber. E & von Figura. K. Biochem j 170 (1978) 643. 14. von Figura. K & Klein, U. Eurj biochem 94 t 1979) 347. 15. Neufeld, E F. Sando. G N. Garvin A J & Rome L H. J supramol struct 6 (1977) 95. 16. Sando, G N, Titus-Dillon, P, Hall, C W & Neufeld. E F, Exp cell res I I9 (1979) 359. 17. von Figura. K & Weber. E. Biochem j 176 (1978) 943. 18. Sly, W S. Gonzales-Noriega, A. Natowitz. M. Fischer, H D & Chambers. J P. Fed oroc 38 t 1979) 467. 19. Novikoff, A B, Lysosomes and storage diseases ted H G Hers & F van Hoof) p. 2. Academic Press, New York (1973). 20. Takatsuki, .4 & Tamura. G. J antibiot (Tokyo) 24 (1971) 224. 21. Datema. R & Schwarz, R T. Eur j biochem 90 (1978) 505. 22. Koch. H U. Schwarz. R T & Scholtissek, C. Eur j biochem 94 (1979) 515.
32. 33. 34.
35.
36. 37. 38. 39. 40.
41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52.
Heukels-Dully. M J & Niermeijer. M F. Exp cell res 97 t 19761 304. Horvat, A & Acs. G, J cell physiol 83 f 1973) 59. Lockwood. T D & Shier, W T. Nature 267 (1977) 252. Kaplan. J. Arch biochem biophys 187 t 1978) 376. Aooelmans. F & De Duve. C. Biochem j 59 t 1955) 4%. Schram. A W. De Groot. P G. Hamers. M N. Brouwer-Kelder, B. Donker-Koopman. W E & Tager J M, Biochim biophys acta 525 t 1978) 410. Morre. D J. Methods in enzymology (ed W B Press. New Jakoby) vol. 22. p. 130. .4cademic York (1971). Bergmeyer. H U & Bemt. E, Methoden der enzymatischen Analyse ted H U Bergmeyer) vol. I. p. 533. Verlag Chemie. Weinheim ( 1970). Razzell. W E. Methods in enzymology ted S P Colowick & N 0 Kaplan) vol. 6. p. 236. Academic Press, New York (1963). Lowry. 0 H, Rosebrough, N J. Farr. A L & Randall, R J. J biol them 193 (1951) 265. Wijnen, L M M. Grzeschik. K-H. Pearson. P L & Meera Khan. P, Human genet 37 t 1977) 271. Grzeschik, K-H, Grzeschik, A M, Banhof. S. Romeo, G. Siniscalco. M. van Someren. H, Meera Khan. P. Westerveld. A & Bootsma. D. Nature new biol 240 t 1972) 48. van Someren, H. Beijershergen van Henegouwen. H. Los. W. Wurzer-Fiaurelli. E. Doonert. B. Vervloet, M & Meer Khan. P. Human genet 75 (1974) 189. Grzeschik. K H. Cytogenet cell genet I6 (1976) 142. Davis. K A & Hatefi. Y. Biochemistry IO t 1971) 2509. Reitzer, L J. Wice. B M & Kennell. D. J biol them 254 (1979) 2669. Evans, W H, Hood, D 0 & Gurd. J W. Biochem j 135 (1973) 819. Eylar. E H & Hagopian, A, Methods in enzvmoloav ted W B Jacobv) vol. 22. P. 123. Academic Press. New York (1971). Schram. A W. Hamers. M N & Tager. J M. Biochim biophys acta 482 I 1977) 138. Dean. K J. Sung, S S J & Sweeley. C C. Biochem biophys res common 77 (1977) I41 I. Groot. P G de. Westerveld. A, Meera Khan, P & Tager, J M, Human genet 44 (1978) 305. Scholtissek, C, Current topics microbial immunol 70(1975) 101. Schmidt. M F G. Schwarz, R T & Ludwig. H. J viral I8 (1976) 819. Kilbourne. E D. Nature 183 ( 1959) 271. Leavitt, R. Schlesineer. S & Komfeld. S. J biol them 252 (1977) 901s. Hickman. S. Kulczycki Jr, A. Lynch. R G & Kornfeld. S, J hiol them 252 ( 1977) 4402. Olden, K, Pratt. R M & Yamada, K M. Cell I3 (1978) 461. Duksin. D & Bornstein, P. J biol them 252 (1977) 955. Tanzer. M L. Rowland, F N. Murray. L W & Kaplan. J. Biochim biophys acta 500 t 1977) 187. Kletzien. R F & Perdue. .I F. J biol them 249
53. 54. 55. 56. 57.
58.
Woods. H F. Eggleston. L V & Kreb\. H .A, Biochemj 119~19701501. Brown. J A, Seaal. H L. Males. F. Trimble. R B & Chu. F. J biol &em 354 r 1575) 3685. Gibson. R. Schlesinger, S Sr Kornfeld. S. J biol them 254 (1575) 3600. van Figura. K & Kresse. H. Eur j biochem J8 f 157-l) 357. Beaufay. H. Lysosomes, a laboratory handbook 1st edn fed J T Dinglet p. 33. North-Holland Publ. Co.. Amsterdam (19721. Barrett. A J & Heath. M F. Lysosomes. a labord-
59. ho.
tory handbook. 2nd rdn led J I Dtnglet p. IY. North-Holland Publ. Co.. .~msterdaw I lY771. Vladutiu. G D d Rattazri. M C. Fed proc 3X I IY~YI 637. Committee on human gene nomen~l,ttut~e. Human gene mapping file: Proceeding\ Vth intrrnational workshop on human gene mapping birth defects original article series. March of Dimes National Foundation. New York I 19791. In me\\.
Received Accepted
August October
30. 1979 16. I979
OF 2-DEOXYGLUCOSE CULTURED PH.
G. de
GROOT,’ A.
ON
HUMAN A.
WESTERVELD.:’
SKIN
STRLJLAND.’ M.
R. N.
LYSOSOMAL
IN
FIBROBLASTS
KALSBEEK.’
HAMERS’
ENZYMES
and
P. MEERA J. M.
KHAN.’
TAGER’
SUMMARY Addition of 2-deoxyglucose, an inhibitor of glycosylation of proteins. to the medium of contluent cultures of human skin fibroblasts prevents the increase in specific activity of lysosomal enzymes that normally occurs after confluence. Maximal inhibition is obtained at a concentration of about I mM 2-deoxyglucose. The inhibition by I-deoxyglucose is reversible. The K,,, . pH dependence and electrophoretic mobility of the acid hydrolases tested was the same in cells cultured with or without Z-deoxyglucose. In homogenates of cultured human skin fibroblasts, about 95 “r of the /3hexosaminidase and a-galactosidase activity and about 65% of the acid phosphatase activity with fi-glycerolphosphate as substrate binds to concanavalin A (ConA); 2-deoxyglucose affects only the activity able to bind to ConA. In cells cultured in the presence of ?-deoxyglucose. the specific activity of alkaline phosphodiesterase I. a plasma membrane glycoprotein is lowered. ?-Deoxyglucase has no effect on the specific activity of succinate dehydrogenase. lactate dehydrogenase or total cellular protein.
All lysosomal enzymes investigated so far, including glucocerebrosidase, a membranebound enzyme, are glycoproteins [l-3]. It is believed that the carbohydrate moiety plays an important role in the intracellular transport of the enzymes and their packaging into lysosomes [4]. Indeed, in all current hypotheses about the mechanisms involved in the packaging of lysosomal enzymes [S81, an important role is assigned to the oligosaccharide moiety as a recognition marker for the enzymes. As demonstrated initially by Neufeld and co-workers [9]. the rapid endocytosis of extracellular lysosomal enzymes by cultured human skin fibroblasts is dependent on the presence in the enzymes of a specific oligosaccharide structure, which was subsequently identified by Kaplan et al. [ 10, 1l] as mannoIA-7YIXIX
sylphosphate (see also [ 12-141). According to Neufeld and co-workers the packaging of lysosomal enzymes involves transport of the enzymes to the cell surface followed by their endocytosis via a specific receptor [7] which recognises mannosylphosphate groups [ 15. 161. An alternative hypothesis, according to which lysosomal enzymes are cycled via the plasma membrane without being released into the extracellular space has been advanced by Lloyd [8] and Von Figura & Weber [ 171. Sly et al. [ 181 too, propose that intracellular transport only is involved in the packaging of lysosomal enzymes, and that secretion and recapture seen in cultured fibroblasts is a minor pathway. Novikoff [19] postulated that the glycosylation of lysosomal enzymes occurs
'08
Groat et 111.
The substrates were the methylumbelliferylglyco~ides. final volume 0.5 ml. The reaction mixture 100 mM sodium acetate at the pH indicated. for the substrate (column 2) was measured indicated
contained The K,,, at the pH
Enzyme
R,r, (mM)
Substrate cont. (mM)
PH
/3-Hexosaminidase a-Galactosidase P-Glucuronidase a-Glucosidase Arylsulphatase P-Glucosidase
0.3’ 3.4 OS6 0.44 I.2 0.78
I.5 3.6 1.7 0.44 3.5 1.8
5.0 4.6 3.5 4.6 5.0 5.0
during passage through the smooth endoplasmatic reticulum and the Golgi apparatus, where packaging occurs. Since glycosylation can be inhibited by a number of compounds, including tunicamycin [20], 2deoxyglucose [2 I] and glucosamine [22], it should be possible to investigate the role of the oligosaccharide moiety in the packaging of lysosomal enzymes by making use of these inhibitors. In this paper we show that the addition of 2-deoxyglucose to confluent cultures of human skin fibroblasts prevents the increase in lysosomal enzyme activity that normally occurs after confluence [see refs 23-261.
MATERIALS Cell
AND
carried out on confluent cultures. In t~dt’tto hat-\r\r the cells. they were first washed three time> with Hanks’ balanced sa1t solution IFI~M L.ab\~ and then detached from the flash by incubation with 0.25 c; trvpsin (Flow Labs) in Hank<’ balanced salt \oIution ioll-5 min. depending on the age of the tr\p\in \alution. When the cells were detached. 5 ml of medium containing FCS were added. and the cell> \\el-e centrifuged. The washing was repeated once with a phorphate-free medium containing 130 mM NaC‘I. 2.7 mM KCI, 14.3 mM NaHCO,, and 10 mM glucose. The cells were subsequently lysed hl addmg 0.5 ml of a solution containing 0.05 ci Triton X-100 and 2 mM EDTA. The solutions of 2-deoxyglucose (Sigma) and lactate (Sigma) were sterilized by passing through a Millipore filter (0.X pm) before adding to the medium.
All the acid hydrolases except acid pho\phatase were measured using the methylumbelliferyl glycosides as substrates (Koch-Light). The reaction mixture (final volume 0.5 ml) contained fibroblast homogenate or medium, 100 mM sodium acetate buffer at the pH shown in table 1, and substrate at a concentration at least I .5 times the K,,, value (see table I ). except in the case of a-glucosidase. since methylumbelliferyl-cglucoside is poorly soluble. The reaction was carried out for 0.5-3.0 h at 37°C and stopped by the addition of I.5 ml 300 mM glycine-NaOH (pH 10.6). The fluorescence of the liberated 4-methylumbelliferone wa>
Table 2. .EfTect qf’ 2-deolc?‘gllrc.o.sll otz enzyme actilqities und totrd protein in cultured humnn skin ,fibroblosts Fibroblasts were grown to confluence as described in Materials and Methods and then grown further either in the absence or in the presence of 5 mM ?-deoxyglucose. The cells were harvested 7 days after confluence and the enzyme activities were measured in the homogenates. Values are means + S.D., with the number of observations given in parentheses. or values from individual observations
METHODS Parameter
Activity or amount after culture with 7-deoxyglucose (ci of control)
Protein Succinate dehydrogenase Lactate dehydrogenase P-Hexosaminidase Acid phosphatase cY-Galactosidase Arylsulphatase /3-Glucuronidase cu-Glucosidase Phosphodiesterase I
99?lO (6) 91. 109(Z) 103. I IO II) SO+ IO (6) 78+ll (61 37t Ii (61 4ot 614) 26+ 614) 50,64(2t 43. 36 (2)
crrlt1we
Human skin fibroblasts were grown in Ham’s F-IO medium (Flow Laboratories), supplemented with 15 % heat-inactivated fetal calf serum (FCS) (Flow Labs), I6 mM NaHCO,, penicillin (250 U/ml) and streptomycin (250 pg/ml). If the medium was older than one week, extra glutamine (5 mM) was added. All the experiments described in the tables and figures were carried out with a line of fetal human skin fibroblasts, kindly provided by Mr Nice Leschot (Dept of Anthropogenetics, University of Amsterdam). The cells were grown in 25 cm2 Falcon plastic flasks containing 5 ml medium. The medium was refreshed every 3 days. Unless otherwise stated, all the experiments were
qf 2-deoxyglucose
Effect
on lysosomal
enzymes
209
2. Abscissa: 2-deoxyglucose cont. (mM) in medium; ordinate: activity (5% of that in cells grown in the absence of 2-deoxyglucose). Effect of adding different concentrations of 2-deoxyglucose to the medium of a confluent cell culture on the activity after 7 days of O-O, /3-glucuronidase; A-A. cY-galactosidase; U--O, /3-hexosaminidase; O-O, acid phosphatase; W--m, succinate dehydrogenase; n--n, on total cell protein after 7 days. Fig.
I
I
1
I
I
I
,
I
I
o
2
4
6
0
2
4
6
8
I. Abscissa: time after confluence (days); ordifrufe: (A-E) activity (mU/mg protein); (F) protein (mg/flask). Effect of 5 mM 2-deoxyglucose, added to the medium of a confluent cell culture, on total cellular protein (F) and on the activity of /3-hexosaminidase (A), arylsulphatase (B), o-glucosidase (C), N-acetylru-galactosaminidase (D). and P-glucosidase (E). Culture in O-O, absence; O-O, presence of 2-deoxyglucose. For conditions see text. Fig.
measured in an Eppendorf fluorimeter with a 3 13-366 nm filter for excitation and a 430-3 000 nm secondary filter. Acid phosphatase was measured with /3-glycerophosphate as substrate [27]. N-acetyl-o-galactosaminidase with p-nitrophenyl-a-galactosaminide as substrate [28]. succinate dehydrogenase according to Morre [29], lactate dehydrogenase following Bergmeyer & Bernt [30]. and phosphodiesterase I as described by Razzell [31]. The rates of the enzymic reactions were linear for the time intervals used in the assays. Triton X-100 at the concentration used in the assays had no effect on enzyme activities when tested with a homogenate made by freezing and thawing of the cells. Protein was estimated according to Lowry [32]. All enzyme activities are expressed as nmol product formed per min per mg total protein in the cell extract. The abbreviations for the enzymes are according to the recommendations of the Committee on Human Gene Nomenclature [60]. The electrophoretic characterization of acid phosphatase (ACP). P-hexosaminidase (HEX), /3-glucuronidase (GUSB). and cY-galactosidase (GALA), was carried out on cellogel (Chemetron) as described elsewhere [33-361.
RESULTS The specific activity of acid hydrolases in cultured human skin fibroblasts changes dramatically during growth of the cells [2326]. In the log phase, the specific activity first declines and then increases, and this increase continues for several days after the cells become confluent. The effect of adding 2-deoxyglucose at a concentration of 5 mM to the medium of cultures of confluent fibroblasts is shown in fig. 1. Whereas 2-deoxyglucose had no effect on the protein content of the cells, it prevented the increase in the activity of acid hydrolases that is characteristic of cells cultivated without 2-deoxyglucose. The concentration dependence of the inhibitory effect of 2-deoxyglucose is shown in fig. 2. In this experiment, a half-maximal effect on acid hydrolases was observed at about 0.5 mM 2-deoxyglucose. On the other hand, 2-deoxyglucose had relatively little effect on the total cellular protein, or on the mitochondrial enzyme succinate dehydrogenase, which is apparently not a glycoprotein [37]; the activity of succinate de-
2 I0
(;l~oot
1’1 t/l.
Enryme Il..37 II. I6
a-Galactosidase
Normal +2-deox\gluco~e
1.11 -1.1)
Acid
Not-mal +2-deoxyglucose
4.6 4.6
3 I’)
L.1 II x
Nor-ma1 t 2-deoxyglucose
s.s > .\
7’ 3x
0.3: II..36
phosphatahe
/3Hexosaminidase
hydrogenase varied between 8&l IOf of the control value (see also table 7). In order to eliminate the possibility that the effect of I-deoxyglucose may have been caused by depletion of respiratory substrate, due to inhibition of glucose transport into the cells. 5 mM lactate was routinely added to the culture medium. Furthermore. the glutamine present in the medium may have been the main respiratory substrate [38]. These considerations. together with
F/,6,. 3. r\b.\ci.v.\cr: time after beeding the cells tdayhl: ,~~/i~rtc,: activity (8 of that at confluence (day 71 in cells without 2-deoxyglucose). Reversibility of the effect of 5 mM 2.deoxyglucobe on b-glucuronidase. For incubation conditions. see text. Ceils cultured (S--O. continuously without ?deoxyglucose: O-U. continuously in the presence of 2-deoxyglucose; m-B, for 7 days in the presence of 5 mM 7-deoxyglucose after which the medium VW, changed to one without 2-deoxyglucose.
2.s -.‘-I
the fact that culture of the cell5 in the presence of 2-deoxyglucose had no effect on the morphology of the cells. total cellular protein. the activity of succinate dehydrogenase and of the cytosolic enzyme lactate dehydrogenase (table 21. suggest that 1deosyglucose had no gross effect on thr energy metabolism of the cells. Table 2 summarizes the result5 of different experiments in which cells were grown for 7 days in the presence of 5 mM Z-deosyglucose. The magnitude of the influence of 2-deoxyglucose on the acid hydrolaseb was dependent on the enzyme:, measured: ,!!Lglucuronidase and u-galactosidasr were intluenced most strongly. uhereaa acid phosphatase and n-glucosidaae showed the lowest response. In addition, in cells cultured with 2-deoxyglucose there uas a marked decrease in the activity of alkaline phosphodiesterase I. a glycoprotein [39] located in the plasma membrane [30]. On the other hand. 2-deoxyglucose had no detectable effect on lactate dehydrogenasr. succinate dehydrogenaar (see also fig. 2) or total cellular protein ccf figs I. 2). In the experiment of fig. 3 cell5 Mere cultured for 7 dab5 in the pt-esence or absence of 5 mM 2-deoxyglucose. The control cells uere cultured in the absence of 2-deoxyglucose for ;I further I I days. The
F;q. 4. .Acid phosphatase IACPI, a-galactosidase (GALA), P-glucuronidase (CiUSBI and P-helosaminidase t HEX) electropherograms on Cellogel. Origin at bottom and migration anodal. Channels I and 2 carry the extracts of human fetal skin fibroblasts cultured in the growth medium with and without 2.deosyglucose. respectively. The letter< refer to different iso7ymes (see text).
cells grown in the presence of 2-deoxyglucose were divided into two portions. One half was cultured for I I days in control medium, the other half in a medium containing 2-deoxyglucose. On day 7. the activity of P-glucuronidase in cells grown in the presence of 2-deoxyglucose was about 30 Q? of that in control cells. The activity in cells grown for a further I I days in the presence of 2-deoxyglucose remained at about the same level. When the cells were transferred to control medium on day 7, there was an
almost immediate rise in the activity of pglucuronidase. the extent of which was similar to that seen in cells grown continuously in the control medium. All acid hydrolases tested responded in an analogous manner. Thus the effect of 2-deoxyglucose on acid hydrolases is reversible. In table 3. the characteristics of the acid hydrolase activities of cells grown in the presence and absence of 2-deoxyglucose are compared. 3-Deoxyglucose had no effect on the pH optimum but caused a decrease in V without altering the K,,, for the substrates. The effect of 2-deoxyglucose on the electrophoretic mobility of several acid hydrolases in cellulose acetate gels is shown in fig. 4. The major isozyme of acid phosphatase (fig. 4~) did not exhibit a change in the rate of migration. while the minor isozyme (fig. 417) was not seen in homogenates of cells grown in the presence of 2-deoxyglucose. Regions A and B of (Ygalactosidase activity correspond to (Ygalactosidase A and B, respectively. (The latter activity is identical with N-acetyl-agalactosaminidase [41-43].) The activity band corresponding to a-galactosidase A could not be detected in 2-deoxyglucosegrown cells. The intensity of the band corresponding to a-galactosidase B was reduced in 2-deoxyglucose-grown cells. In homogenates of fibroblasts grown in normal medium three distinct zones of P-glucuronidase activity ((1, h. c.1 are formed. the strongest of which is c’. The isozyme h was absent or very weak in homogenates of 2deoxyglucose-grown cells. Finally. there was no change in the position of the bands corresponding to hexosaminidases A and B in the 2-deoxyglucose-grown cells. No band of activity corresponding to hexosaminidase C could be detected, either in control or in 2-deoxyglucose-grown cells.
Fig. 5. Ahscissu: amount ConA (ml); ordinure: activity of cells grown in the absence Binding of the activity (B) a-galactosidase; and ConA coupled to Sepharose
Sepharose 4B-coupled (% of that in homogenate of 2-deoxyglucose). of (A) P-hexosaminidase: (C) acid phosphatase to 4B in homogenates of cells
cultured with I-deoxyglucose (0-O) and ceils cultured without 2-deoxyglucose (O-G). The cells were grown to confluence and then grown further for 7 days in either the absence or -presence of 5 mM 2-deoxyglucose. One ml Sepharose 4B coupled ConA contained 10 mg ConA.
In the experiment of fig. 5, homogenates of cells grown in the presence or absence of 2-deoxyglucose were incubated with different amounts of Sepharose 4B to which the lectin ConA was bound. As a control, homogenates were incubated with Sepharose 4B only. In the control cells, about 95% of the a-galactosidase and P-hexosaminidase activities were removed by treatment with immobilized ConA, leaving about
5% in the supernatant. In the I-deoxyglucase-treated cells, incubation with immobilized ConA lowered the residual activities to about the same level as in control cells. Similar results were obtained with the acid phosphatase activity, except that the amount remaining in the supernatant after ConA treatment was much higher (about 35%’ of the total activity present in control cells) than in the case of the other acid hydrolases. It is of importance to note that the activity of acid phosphatase was measured with /?-glycerophosphate. which is generally considered to be a specific substrate for lysosomal acid phosphatase [57, 581. Furthermore, both activities, i.e., that able to bind to ConA and that unable to bind to the lectin, are inhibited by 1 mM sodium-potassium tartrate, an inhibitor of lysosomal acid phosphatase [57. 581. In cultured human skin fibroblasts, some lysosomal enzymes are found in the culture medium [7]. In the case of P-hexosaminidase, 2-deoxyglucose had no effect on the amount of extracellular enzyme (fig. 61. On the other hand, extracellular P-glucuronidase was lower in 2-deoxyglucose-grown cells than in control cells (fig. 6). The amount of lactate dehydrogenase found in
Fig.
of
6. Abscissu: time after confluence (days); ordinate: activity (mu/ml medium). Effect of 5 mM 2-deoxyglucose on P-hexosaminidase (0, 0) and /3-glucuronidase (A, A) in culture medium of fibroblasts. After confluency, the cells were incubated further in the presence or absence of 2deoxyglucose. Every day the activity was measured in control cells (0, A) and in the cells cultured in the presence of 2-deoxyglucose (0, A).
EJfect of 2-deosyglucose the medium was not influenced by growing the cells in 2-deoxyglucose (not shown). DISCUSSION The glycosylation of glycoproteins can be inhibited by a number of different compounds including the antibiotic tunicamycin [20] and sugar analogues like glucosamine [22], 2-deoxyglucose [44], 2-deoxy-2-fluoroglucose and 2-deoxy-2-fluoromannose [45]. It has been shown that inhibitors of glycosylation can inhibit the synthesis of glycoprotein-containing viruses such as influenza virus [46], Sindbis virus [47] and vesicular stomatitis virus [47]. Inhibition of glycosylation can also lead to impairment of secretion of a protein; this is the case with IgA and IgE secretion in plasma cells [48]. In other cases, inhibition of glycosylation leads to formation of an unstable product. Olden et al. [49] have shown by means of pulse-labelling experiments that when tunicamycin is added to cultured chick embryo fibroblasts, the major cell-surface protein appears to be synthesized in a form that breaks down more rapidly than the fully glycosylated protein. In cultured chick tendon fibroblasts or chick bone cultures, addition of tunicamycin prevents the conversion of procollagen, which apparently accumulates in a non-glycosylated form, to collagen [50, 5 I]. We have investigated the possible role of the oligosaccharide moiety in the packaging of lysosomal enzymes by employing 2deoxyglucose as an inhibitor of glycosylation [44]. Datema & Schwarz [21] have shown that inhibition by 2-deoxyglucose of glycosylation is caused by the formation of 2-deoxyglucose-containing lipid-linked oligosaccharides, which are not metabolized further. In confluent cultures of human skin fibro-
on lysosomal
enzymes
213
blasts, there is a gradual increase in the activity of lysosomal enzymes [23-261. We have shown that when 2-deoxyglucose is present in the culture medium, this increase in activity is prevented, at least in the case of the acid hydrolase activities measured. A similar effect is found with the plasma membrane glycoprotein phosphodiesterase I. On the other hand, there is no effect of 2-deoxyglucose on succinate dehydrogenase and lactate dehydrogenase, on the protein content of the cells, on the gross morphology of the cells. or on their proliferating capacity when the cells are transferred to flasks containing 2-deoxyglucose-free medium (not shown). The following mechanisms may be brought forward to explain the effect of 2deoxyglucose on the activity of the lysosomal enzymes: (1) 2-Deoxyglucose inhibits glycosylation of the nascent lysosomal enzyme polypeptides, thus interfering with their routing to the lysosomes and causing feedback inhibition of synthesis of the polypeptides. (2) The non-glycosylated lysosomal enzymes are rapidly broken down in the lysosomes. (3) The non-glycosylated enzymes are less active than the normal enzymes or even inactive. (4) Addition of 2-deoxyglucose may lead to an accumulation of phosphorylated sugars and a depletion of ATP [52], thus interfering with ATP-dependent processes in the cell, such as protein synthesis or the functioning of the contractile elements. The following observations are in agreement with the first two mechanisms and not with the others: (I) The 2-deoxyglucose effect appears to be specific for glycoproteins; the acid hydrolases and phosphodiesterase I are affected, and not lactate dehydrogenase or succinate dehydrogen-
ase. (2) No evidence could be obtained for the synthesis in the presence of 2-deoxyglucose of forms of acid hydrolases with altered enzymological or physico-chemical properties: the K,,, values of the enzymes and their electrophoretic mobility were unaffected and no increase was detected in the activity unable to bind to the carbohydrate lectin ConA. (3) Fructose which. like 2-deoxyglucose. can lead to a decrease in the ATP content of cells (see [5?]l has no effect on the level of acid hydrolases in the cultured fibroblasts (results not shown). At present we can not distinguish between mechanisms 1 and 2. If an unstable non-glycosylated form is. indeed. produced, it must be broken down so rapidly that it can not be detected. Mechanism 2 implies that the oligosaccharide moiety protects the acid hydrolases from being degraded by lysosomal cathepsins. Brown et al. [54] have suggested that treatment of yeast invertase with endo-N-acetyl$-hexosaminidase to remove oligosaccharides leads to some decrease in the half-life of the enzyme after it is endocytosed by rat yolk sacs. However, close examination of their data suggest that heating of the deglycosylated enzyme converts it to a form with about half the specific activity of the native enzyme. Other studies have indicated that non-glycosylated glycoproteins may be less stable than the native proteins (for a discussion see [55]). If non-glycosylated peptides were formed which were completely devoid of enzymic activity, it would not be possible to distinguish between mechanisms I. 2 and 3. However. von Figura et al. [I41 have brought forward evidence that after treatment of a lysosomal enzyme with endoglycosidase H. some activity at least remains, so that one might have expected to be able to detect enzymically active. non-glycosylated forms
of the acid hydrolases. Studie\ on the intracellular distribution and turnover of Iys~omal enzymes in the presence and absence of 2-deouvglucose are in progress. i\t first sight. the effect of 2-deosyglucase resembles that of cytochalasin B. which. as shown by von Figura & Kresse [56]. leads to a decrease in the intracellular activity of lysosomal hydrolases. However. in the case of cytochalasin B. there is a concomitant increase in the extracellular activity of at least one of these enzymes. phexosaminidase. This increase is not seen with 2-deoxyglucose (fig. 61. which suggests that 2-deoxyglucose and cytochalasin B exert different effects. The response of the acid phosphatase activity (measured with P-glycerophosphate as substrate) to 2-deoxyglucose is exactly analogous to that of the other acid hydrolases in that only the activity able to bind to ConA is decreased in the cells grown in the presence of the sugar analogue. The activity that does not bind to the lectin is unaffected. It is of interest that both activities were measured with /3glycerophosphate as substrate and that both were inhibited to about 95?? by I mM sodium-potassium tartrate. These criteria are usually employed to distinguish lysosomal acid phosphatase from other phosphatases [57, 58]. The relationship between the two activities that can be distinguished on the basis of the ability to bind to Con4 is at present unclear. After this study was completed an abstract by Vladutiu & Rattazzi [59] appeared in which they reported that 2-deoxyglucose reduces the excretion of /3-hexosaminidase by cultured skin fibroblasts from patients with mucolipidosis II (I-cell disease). In contrast. it had no effect on the intracellular level of the enzyme in normal fibroblasts. The apparent lack of effect in normal fibro-
blasts was probably due to the fact that the cells were cultured with 2-deoxyglucose for 24 h only (cf fig. 1). The authors are grateful to all the members of the Rotterdam-Leiden-Amsterdam ‘Lvsosomal Enzvmes’ group for stimulating discussions and to Mr-Nice Leschot for orovidine a line of fetal human skin fibroblasts. This study ways supported by a grant from the Netherlands Organization for the Advancement of Pure Scientific Research (Z.W.O.) under the auspices of the Netherlands Foundation for Fundamental Medical Research tF.U.N.G.O.1.
23. 24. 25. 26. 27, 28. 29.
30. 31.
REFERENCES I. Bouma. J M W. Enzyme therapy in lysosomal storage diseases (ed J M Tager. G J M Hoogwinkel & W Th Daems) p. 197. North-Holland Publ. Co.. Amsterdam t 1974). 2. Fiddler. M B. Ben-Yoseph. Y & Nadler. H L. Biochem j 177 (1979) 175. 3. Dale, G L. Villacorte, D G & Beutler, E, Biochem bionhvs res commun 71 (1976) 1048. 4. Dean.‘R T & Barrett. A J. Essays in biochemistry I2 (1976) I. 5 Novikoff. A B, Essmer E & Quintana. N. Fed proc 23 ( 1964) 1010. 6. De Duve. C & Wattiaux R. Ann rev physiol 28 (1966) 435. 7. Hickman, S & Neufeld E F, Biochem biophys res commun 49 t 1972) 992. 8. Lloyd, J B. Biochem j 164 (1977) 281. 9. Hickman. S. Shapiro, L J & Neufeld. E F. Biochem biophys res commun 57 t 1974) 55. IO. Kaplan, A. Achord, D T & Sly, W S. Proc natl acad sci US ?4 (1977) 2026. Il. Kaplan, A. Fisher. D, Achord. D T & Sly. W S, J clin invest 60 t 1977) 1088. I?. Sando G N &‘Neufeld E F, Cell I2 (1977) 619. 13. Ullrich, K, Mersmann G, Weber. E & von Figura. K. Biochem j 170 (1978) 643. 14. von Figura. K & Klein, U. Eurj biochem 94 t 1979) 347. 15. Neufeld, E F. Sando. G N. Garvin A J & Rome L H. J supramol struct 6 (1977) 95. 16. Sando, G N, Titus-Dillon, P, Hall, C W & Neufeld. E F, Exp cell res I I9 (1979) 359. 17. von Figura. K & Weber. E. Biochem j 176 (1978) 943. 18. Sly, W S. Gonzales-Noriega, A. Natowitz. M. Fischer, H D & Chambers. J P. Fed oroc 38 t 1979) 467. 19. Novikoff, A B, Lysosomes and storage diseases ted H G Hers & F van Hoof) p. 2. Academic Press, New York (1973). 20. Takatsuki, .4 & Tamura. G. J antibiot (Tokyo) 24 (1971) 224. 21. Datema. R & Schwarz, R T. Eur j biochem 90 (1978) 505. 22. Koch. H U. Schwarz. R T & Scholtissek, C. Eur j biochem 94 (1979) 515.
32. 33. 34.
35.
36. 37. 38. 39. 40.
41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52.
Heukels-Dully. M J & Niermeijer. M F. Exp cell res 97 t 19761 304. Horvat, A & Acs. G, J cell physiol 83 f 1973) 59. Lockwood. T D & Shier, W T. Nature 267 (1977) 252. Kaplan. J. Arch biochem biophys 187 t 1978) 376. Aooelmans. F & De Duve. C. Biochem j 59 t 1955) 4%. Schram. A W. De Groot. P G. Hamers. M N. Brouwer-Kelder, B. Donker-Koopman. W E & Tager J M, Biochim biophys acta 525 t 1978) 410. Morre. D J. Methods in enzymology (ed W B Press. New Jakoby) vol. 22. p. 130. .4cademic York (1971). Bergmeyer. H U & Bemt. E, Methoden der enzymatischen Analyse ted H U Bergmeyer) vol. I. p. 533. Verlag Chemie. Weinheim ( 1970). Razzell. W E. Methods in enzymology ted S P Colowick & N 0 Kaplan) vol. 6. p. 236. Academic Press, New York (1963). Lowry. 0 H, Rosebrough, N J. Farr. A L & Randall, R J. J biol them 193 (1951) 265. Wijnen, L M M. Grzeschik. K-H. Pearson. P L & Meera Khan. P, Human genet 37 t 1977) 271. Grzeschik, K-H, Grzeschik, A M, Banhof. S. Romeo, G. Siniscalco. M. van Someren. H, Meera Khan. P. Westerveld. A & Bootsma. D. Nature new biol 240 t 1972) 48. van Someren, H. Beijershergen van Henegouwen. H. Los. W. Wurzer-Fiaurelli. E. Doonert. B. Vervloet, M & Meer Khan. P. Human genet 75 (1974) 189. Grzeschik. K H. Cytogenet cell genet I6 (1976) 142. Davis. K A & Hatefi. Y. Biochemistry IO t 1971) 2509. Reitzer, L J. Wice. B M & Kennell. D. J biol them 254 (1979) 2669. Evans, W H, Hood, D 0 & Gurd. J W. Biochem j 135 (1973) 819. Eylar. E H & Hagopian, A, Methods in enzvmoloav ted W B Jacobv) vol. 22. P. 123. Academic Press. New York (1971). Schram. A W. Hamers. M N & Tager. J M. Biochim biophys acta 482 I 1977) 138. Dean. K J. Sung, S S J & Sweeley. C C. Biochem biophys res common 77 (1977) I41 I. Groot. P G de. Westerveld. A, Meera Khan, P & Tager, J M, Human genet 44 (1978) 305. Scholtissek, C, Current topics microbial immunol 70(1975) 101. Schmidt. M F G. Schwarz, R T & Ludwig. H. J viral I8 (1976) 819. Kilbourne. E D. Nature 183 ( 1959) 271. Leavitt, R. Schlesineer. S & Komfeld. S. J biol them 252 (1977) 901s. Hickman. S. Kulczycki Jr, A. Lynch. R G & Kornfeld. S, J hiol them 252 ( 1977) 4402. Olden, K, Pratt. R M & Yamada, K M. Cell I3 (1978) 461. Duksin. D & Bornstein, P. J biol them 252 (1977) 955. Tanzer. M L. Rowland, F N. Murray. L W & Kaplan. J. Biochim biophys acta 500 t 1977) 187. Kletzien. R F & Perdue. .I F. J biol them 249
53. 54. 55. 56. 57.
58.
Woods. H F. Eggleston. L V & Kreb\. H .A, Biochemj 119~19701501. Brown. J A, Seaal. H L. Males. F. Trimble. R B & Chu. F. J biol &em 354 r 1575) 3685. Gibson. R. Schlesinger, S Sr Kornfeld. S. J biol them 254 (1575) 3600. van Figura. K & Kresse. H. Eur j biochem J8 f 157-l) 357. Beaufay. H. Lysosomes, a laboratory handbook 1st edn fed J T Dinglet p. 33. North-Holland Publ. Co.. Amsterdam (19721. Barrett. A J & Heath. M F. Lysosomes. a labord-
59. ho.
tory handbook. 2nd rdn led J I Dtnglet p. IY. North-Holland Publ. Co.. .~msterdaw I lY771. Vladutiu. G D d Rattazri. M C. Fed proc 3X I IY~YI 637. Committee on human gene nomen~l,ttut~e. Human gene mapping file: Proceeding\ Vth intrrnational workshop on human gene mapping birth defects original article series. March of Dimes National Foundation. New York I 19791. In me\\.
Received Accepted
August October
30. 1979 16. I979