Printed in Sweden Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827/79/040359-06%02.0010
Cell Research 119 (1979) 359-364
OF RECEPTOR-MEDIATED ENZYME
OF A LYSOSOMAL
GLORIA N. SANDO,’ PAULINE TITUS-DILLON; and ELIZAEIETH F. NEUFELD
CLARA W. HALL
Section on Human Biochemical Genetics, National Institute of Arthritis, Metabolism & Digestive Diseases, Nationailnstitutes of Health, Bethesda, MD 20014, USA
SUMMARY Cultured human skin fibroblasts take up o-L-iduronidase by receptor-mediated pinocytosis. Certain lysosomotropic amines such as chloroquine, ammonia and procaine inhibit this process, without affecting the fluid endocytosis of dextran. In contrast to the competitive inhibition by mannose 6-phosphate, the inhibition by amines is non-competitive and is therefore presumed not to affect binding of the enzyme to receptors. The dose response curves are very steep, and equations that best Iit the data use a power of inhibitor concentration (P for procaine, i4 for chloroquine), indicating interaction of several amine molecules at the inhibitory site(s). The inhibition is reversed by removal of the amine from the medium and does not result from accelerated efflux of endocytosed enzyme. We suggest that the amines interfere with delivery of receptor-bound enzyme to lysosomes.
Cultured human diploid tibroblasts take up certain acid hydrolases by a selective and efficient process which involves recognition of a specific marker on the enzyme by receptors on the fibroblast surface [l-lo]. It has been suggested that this process may be important for packaging endogenous hydrolytic enzymes into lysosomes of fibroblasts as well as for the uptake of exogenous enzymes . Although direct chemical proof is not yet available, the evidence points to a phosphorylated sugar as the major determinant of the recognition marker on the enzymes ; mannose 6-phosphate, fructose l-phosphate, some glycoproteins and phosphorylated mannans inhibit the receptormediated uptake competitively, presumably because the steric resemblance of these substances to the recognition marker allows them to bind to the receptors for the
enzyme [5-lo]. The kinetics of uptake and inhibition have been studied in greatest detail for a-L-iduronidase  and /3-glucuronidase , but similar considerations apply to a number of other hydrolytic enzymes [7, lo]. We now report that chloroquine and some other amines also inhibit the receptor-mediated uptake of o-L-iduronidase, but by a mechanism which is noncompetitive and therefore different from that of the phosphorylated compounds. MATERIALS
Fibroblasts from patients deficient in cY-L-iduronidase [ll] were obtained from samples submitted for diagnosis or from the Human Genetic Mutant Cell Rer Present address: Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA. ’ Present address: Department of Medicine, Howard University, Washington, DC 20001, USA. Exp
Res 119 (1979)
Sando et al. I
Fig. 1. Abscissa: inhibitor (M); ordinate: iduronidase uptake (% of control). Inhibitory effect of amines on the uptake of a-~iduronidase. 0, Chloroquine; A, TEMED; 0, procaine; 0, cyclohexylamine; A, ammonium chloride; n , glucosamine were included at the specified concentration in 6 ml of serum-free medium, which also contained 0.7 U of a-L-iduronidase; uptake of the enzyme was measured after 2.5 h . The previously documented effect of mannose 6-phosphate (- --) is shown for the same concentration of enzyme in the medium. Control experiments showed that incubation with the amines did not interfere with the subsequent assay of cr-L-iduronidase activity in the cell homogenates. Except for lo-* M procaine, the drugs appeared to have no visible cytotoxic effects over the 2.5 h incubation.
Fig. 2. Abscissa: I/iduronidase applied (U/ml of medium)-‘; ordinate: l/iduronidase uptake (U/mg cell protein/h)-‘. Double reciprocal plots showing the kinetics of inhibition of a-L-iduronidase uptake by (A) chloroquine; (B) procaine. Chloroquine was used at concentrations of 0, 0; 0, 10; A, 20; & 25 or n , 35 PM, whereas procaine was included at 0, 0; 0, 0.33; A, 1.0; A, 3.3 and n , 10 mM. Conditions were as in fig. 1 except for the variation, as indicated, of enzyme concentration.
were from common commercial sources and of the highest purity available. Analysis of the kinetics of uptake was performed as previously described .
The inhibition of cY-L-iduronidase uptake by chloroquine, N,N,N’,N’-tetramethylethylenediamine (TEMED), procaine, cyclopository (Institute for Medical Research. Camden. N.J.) and cultured at 35°C in Eagle’s Minimal Essen: hexylamine, ammonium chloride and glutial Medium enriched with 10% fetal calf serum (FCS), cosamine are shown in fig. 1. The very non-essential amino acids and antibiotics, in 5% steep dose response curves should be noted co, . Preparation and assay of high uptake cY+iduronid(the more gradual response to mannose 6ase and measurement of its uptake have been dephosphate, previously shown to be a comscribed previously . Briefly, cells are transplanted into 100 mm2 plastic Petri dishes in the usual growth petitive inhibitor, is included for comparimedium; two days later, this is replaced by serum-free medium  for 12 h before the uptake experiment is son). Several amine compounds frequently begun. The enzyme and test substances are Drepared used as buffers in cell culture, Tris, N-Zhyin 1 ml of buffer (0.15 M NaCl, 0.01 M Na phosphate droxyethylpiperazine - N’ - 2 - ethanesulfonic pH 6.0, and 1 mg/ml bovine serum albumin), mixed with 5 ml of serum-free medium, and sterilized by acid (HEPES) and N,N-bis-(Zhydroxyfiltration. The mixtures are applied to the plates which acid (BES), are then incubated at 35°C for 2.5 h. The cells are ethyl)-Zaminoethanesulfonic washed, detached from the dish by trypsinization and showed less inhibition than comparable assaved for a-L-iduronidase activitv 1131. concentrations of glucosamine, whereas [Y?]Chloroquine and [3H]methoxyde&ran were purchased from New England Nuclear Corp. Phenvl-cY- one, piperazine-N,N’-his-(2-ethanesulfonic L-iduronide was provided by Dr B. Weissmann (Uniacid) (PIPES), was as inhibitory as the versity of Illinois, Chicago, Ill.); all other compounds Exp Cell Res 119 fIY79)
procaine and ammonia
Table 1. Uptake of cr-L-iduronidase lowing preincubation
Cont. in preincubation without enzyme
25 PM 0
Fig. 3. Abscissa: lliduronidase applied (U/ml of medium)-‘; ordinate: lliduronidase uptake (U/mg cell
protein/hour)-‘. The data of fig. 2 have been fit to the following equation: “=
VE K uptake(l+inlKis)+E(l+I”/Ki,)r
where v is rate of enzyme uptake; V, maximal velocity of enzyme uptake; K,,,,,, concentration of enzyme in the medium which gives the half-maximal rate of uptake; E and i, concentration of enzyme and inhibitor in the medium, respectively; Ki, and Kit, the dissociation constants for the inhibitor, slope and intercept terms, as used in the notation for steady-state enzyme kinetics . The value of n is 4 for chloroquine (A) and 2 for procaine (B).
amino sugar. The inhibition by relatively high levels of the buffers or of glucosamine may be non-specific effects of hyperosmolarity, since addition of 0.17 M NaCl to the usual culture medium caused uptake to decline to 30% of control. Adenine, adenosine , cytosine, thymidine, and thiamine we’re not inhibitory at 1.4 mM, nor was guanosine at 0.7 mM or arginine at 5 mM; higher concentrations were not examined. Benzidine and chlorpromazine inhibited somewhat at 1 mM and 20 /.LM respectively, the highest concentrations that could be tested without cytotoxicity. The kinetics of inhibition were tested for chloroquine and procaine, and found to be non-competitive [ 141(fig. 2). Analysis of the double reciprocal plots shows that the in-
folof cells with amines
50 /LM 0 3mM 0 9mM 0
Cont. in incubation with enzyme 0
Enzyme uptake (% of control) 92
0 50 /.LM 0 3mM 0 9mM
Ai 5 91 42
The cells were preincubated for 2.5 h at 35°C in serumfree medium in the presence or absence of the amine; the medium was removed, and the cells incubated for an additional 2.5 h in medium containing 0.8 U/6 ml of cY-L-iduronidase, as well as amines at the concentrations indicated. Uptake of the enzyme into the control cells ranged from 0.15 to 0.18 U/mg cell protein.
hibitors reduce the maximal velocity of uptake, without significantly affecting the Kuptake(previously defined as the concentration of enzyme in the medium which corresponds to the half-maximal rate of uptake). However, the data shown in fig. 2 do not follow the equation for linear non-competitive inhibition. Of several simple equations tested, those using a power of the inhibitor concentration, the square for procaine and the fourth power for chloroquine, gave the best fit to the data (fig. 3). This suggests that the cooperation of several inhibitor molecules may be required to decrease the uptake of enzyme, and explains the steep dose response curves seen in fig. 1. The inhibitory effect of the amines on the uptake of a+iduronidase required the simultaneous presence of the enzyme and the amine in the culture medium. Preincubation of cells with chloroquine or ammonia, followed by incubation with the enzyme but without the drug, did not result in Exp Cell Res I19 (1979)
Sando et al.
Table 2. Retention of endocytosed a-~iduronidase during post-incubation with amines
through membranes into all cellular compartments, but become trapped by protonation within lysosomes. However, the trapping is imperfect, since some of the proEnzyme remaining Cow. in nated species can diffuse out of the lysoin cells postsomes, creating a proton leak and a conseAmine incubation (% of control) quent rise in pH . At high concentraChloroquine 25 /.LM 88 tions (e.g., 100 PM chloroquine) the os50 /.LM 78 motic effect of the trapped amine results in NH&I 3mM 97 an influx of water so great as to cause vacu9mM 87 olation easily visible by phase microscopy The cells were incubated for 2.5 h in serum-free medium containing 0.8 U/6 ml of cY-L-iduronidase;the me- [ 181.Fibroblasts treated with lower doses of dium was then removed, and the cells incubated for an- chloroquine have impaired degradation of other 2.5 h with fresh medium without enzyme but with chloroquine or ammonia in the concentrations in- protein  and mucopolysaccharide  dicated. The control plate, which had no added amine as well as morphology reminiscent of mucoduring the post-incubation, showed no loss of enzyme, in expectation wifh the very long retention time of lipidosis II, a multiple enzyme deficiency disease [ 191.The impairment of degradation endocytosed enzyme (tt , 9 days). of endocytosed proteins by chloroquine has frequently been used as evidence for the lysosomal location of such degradation any significant decrease in the uptake of [20-231. a-L-iduronidase (table 1). Yet, it could be Ammonium chloride likewise has been shown by the use of [3H]chloroquine that shown to cause inhibition of degradation of 80 % of the chloroquine taken up during the proteins [22, 24-261 and mucopolysacpreincubation was still cell-associated at the charide  as well as vacuolation  in end of the second incubation without the cultured cells. Although procaine and redrug. Conversely, post-incubation of the cells in medium containing chloroquine or ammonia did not result in any major loss of Table 3. Effect of amines on the uptake of enzyme that had been taken up previously [3H]methoxydextran (table 2). Uptake of The uptake of dextran, believed to be a dextran % of receptor-less process , was not signifi(cpmlmg conCont. protein) trol cantly affected by doses of amines (or of Compound added mannose 6-phosphate) which would have None 310 inhibited the uptake of a+iduronidase al- Chloroquine 50 /.LM 319 103 Procaine 10 mM 277 89 most completely (table 3). NH,Cl IO mM 253 82 Mannose 6-phosphate
DISCUSSION Chloroquine has been extensively studied as a model “lysosomotropic” compound [ 161. Such compounds are weak bases which are lipophilic enough to permeate Exp Cell Res 119 (1979)
[3H]Methoxydextran, 8x lo6 cpm, was added to each culture dish. The uptake of dextran was measured exactly as that of a-L-iduronidase, except that after trypsinization, the cells were dissolved in 0.1 M NaOH-0.4% deoxycholate for counting. The very small amount of dextran taken up (0.004% of the amount applied, in 2.5 h) should be contrasted to the substantial uptake of cY-L-iduronidase(20%. in table 1).
procaine and ammonia
lated local anaesthetics probably exert their V maxby decreasing the pool of available repharmacological action by effects on the ceptors, either by direct inactivation or by cell membrane , they are thought, in ad- causing rapid internalization as an indirect dition, to inhibit protein degradation in cul- consequence of the lysosomotropism and tured fibroblasts by an amine-trapping influx of water. Alternatively, the amines may interfere with the delivery of surfacemechanism similar to that of chloroquine and ammonia . The inhibition of de- bound enzyme to the lysosomes. This degradation of macromolecules is probably livery (of which there are as yet no experidue in all these instances to inhibition of mental studies) probably involves a comacid hydrolases within lysosomes by the plex sequence of events including clusprotonation of the amines and the resulting tering, invagination, and vesicle formation, followed by inte,rnalization, fusion, and rise in pH. release of the enzyme from its surface reThe inhibition of the uptake of a-~iduronidase described in this paper appears ceptor. In this connection the reported disto depend on the continued presence of the organizing effects of local anaesthetics on amines in the medium; a change of medium the cytoskeleton and on receptor mobility released the inhibition by chloroquine, even may be of interest [29, 301. But whatever though much of the chloroquine remained the step affected, it appears specific to the uptake of lysosomal enzyme; the uptake of associated with the cells. It is unlikely, dextran by fluid endocytosis (above, and therefore, that the inhibition of enzyme uptake is a direct effect of the amine already ), as well as the specific receptor-bindwithin the lysosome compartment. How- ing and uptake of epidermal growth factor ever, the inhibition of uptake and the intra- , low density lipoprotein  and translysosomal rise in pH could both be a con- cobalamin II  are not affected. Insequence of interference with proton tran- terestingly the transport (but not the bindsport by the amines . Preliminary ex- ing) of diphtheria toxin is also inhibited by periments have shown that the ionophore ammonia [32, 331. Wiesmann et al. [31, 341 have reported nigericin also inhibits the uptake of (Y-Liduronidase (complete inhibition at 1 PM that chloroquine at concentrations below 5 PM inhibited the uptake and accelerated nigericin; Karson, Myerowitz & Neufeld, the efflux of another acid hydrolase, aryl unpublished experiments). Direct interference of binding of the en- sulfatase A. The effect persisted after rezyme to the cell receptors appears excluded moval of chloroquine from the medium and by the kinetic studies; inhibition of uptake was thought to occur by interference with by chloroquine and procaine was shown to receptor binding. Those findings are so difbe non-competitive, affecting V,,, but not ferent from ours that we must presume a K uptalte,whereas inhibition of binding should different mechanism for the internalization result in competitive kinetics (i.e., affecting of the two enzymes or a marked effect of K uptakebut not V,,,) as previously shown the different experimental conditions. The drug effect may prove to be a valufor mannose 6-phosphate . Acceleration of enzyme efflux has also been ruled out by able tool in sorting out the mechanisms of receptor-mediated endocytosis of hydrothe experimental results. Several hypotheses are compatible with lytic enzymes and the role of the uptake the existing data. The amines may decrease process in the normal function of lysoExp Cell Res 119 (1979)
somes. However, the inhibitory effect of ammonium ions may create difficulties in studies of lysosomal enzyme uptake or exchange in cell culture, since the concentration of ammonium ions which gives halfmaximal inhibition (3 mM) can be readily generated by decomposition of glutamine. Cyclohexylammonium salts of phosphate esters should also be avoided in uptake studies because of the inhibitory effects of cyclohexylamine. P. T.-D. was a recipient of a MARC fellowship from the National Institute of General Medical Sciences.
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