ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 221, No. 2, March, pp. 593-597, 1983

COMMUNICATION

Castanospetmine, a Tetrahydroxylated that Inhibits &Glucosidase and p-Glucocerebrosidase

RICK SAUL,* JAMES P. CHAMBERS,? RUSSELL J. MOLYNEUX,$ AND ALAN D. ELBEIN*

*Depa&nent of Biochemistry, University of T- Health science Center, San Antonio, T- 78,284 ~Department of Pediatrics, University of T- Health solace Center, How&an, T- 77025; and $Natural Proo!ucb Chemistry Research Unit, Western Rep&d Research Center, Agricultural Research Seruice, USDA, Berhdey, Califonia 94710 Received December 7. 1982

Castanospermine (1,6,7&tetrahydroxyoctahydroindolizine) was tested against a va- riety of commercially available glycosidases and found to be a potent inhibitor of almond emulsin P-glucosidase, and also to inhibit fungal &xylosidase. This alkaloid was inactive on yeast a-glucosidase, (Y- or @galactosidase, a-mannosidase, B-N-ace- tylhexosaminidase, @glucuronidase, cY+fucosidase. Fifty-percent inhibition of &glu- cosidase required about 10 pg/ml of castanospermine. The amount of inhibition was uniform throughout the time course, and the inhibition with regard to substrate con- centration (pnitrophenyl-B-D-glucopyranoside) appeared to be of the mixed type. Cas- tanospermine was also a potent inhibitor of 8-glucocerebrosidase when assayed with fibroblast extracts using either a fluorimetric or a radioactive assay. Interestingly enough, castanospermine also inhibited the lysosomal a-glucosidase, and this inhibition required comparable levels of alkaloid to that required for inhibition of /I-glucocere- brosidase. However, a number of other lysosomal glycosidases were not sensitive to castanospermine (i.e., (Y- or /3-galactosidase, (Y- or &mannosidase, (Y- or /3-L-fucosidase, 8-N-acetylhexosaminidase, @glucuronidase).

Castanospermine (1,6,7,8-tetrahydroxyoctahydro- GlcNAq protein, but had no effect on the a-1,2-man- ) is an alkaloid of the class, nosidases (6). first isolated from the seeds of the Australian le- Because of the structural similarity between caa- gume, Casta noqw-mum austrak (1). The structure of tanospermine and swainsonine, we tested castano- this compound is shown in Fig. 1. The seeds of this spermine on a number of commercially available gly- plant are toxic and cause gastrointestinal irritation cosidases. This alkaloid only inhibited almond emul- when eaten by horses and cattle. Castanospermine sin /3-glucosidase, but was without effect on yeast bears a structural relationship to another indolizi- a-glucosidase, jack bean a-mannosidase, liver @-glue- dine alkaloid, swainsonine, which is also toxic to an- uronidase, liver &galactosidase, coffee bean a-galac- imals and gives rise to symptoms analogous to those tosidase, kidney p-N-acetylhexosaminidase, or epi- of human a-mannosidosis (2). Swainsonine was shown didymis a-L-fucosidase. The alkaloid did show some to be a potent inhibitor of lysosomal and jack bean inhibitory activity toward AspergiUus @-xylosidase. a-mannosidase (3). and was also found to inhibit gly- In addition, castanospermine was a potent inhibitor coprotein processing by preventing the formation of of fibroblast and white blood cell @glucosidase and the normal, complex types of oligosaccharides (4,5). fi-glucocerebrosidase. This latter activity might ex- In these processing reactions, swainsonine specifi- plain the toxicity of this compound, since a genetic cally inhibited mannosidase II that removes a-1,3- insufficiency of the latter enzyme is characteristic of and a-1,6-linked mannoses from the GlcNAc-Man,- Gauche& disease. It will be of interest to determine

593 0003-9861/83/040593-05$03.00/O Copyright Q 1989 by Academic press, Inc. All rights of reproduction in any form reserved SAUL ET AL.

orometer with excitation and emission settings of 365 and 449 nm, respectively. @Glucocerebrosidase was also assayed by the radioactive method using fl- [8HMlucocerebroside (8). White blood cells were iso- lated from 10 ml of freshly drawn blood and were CASTANOSPERMINE suspended in 1 ml of deionized water and ruptured by sonication. Protein was measured as described by Lowry et al. (9). FIG. 1. The structure of castanospermine.

RESULTS AND DISCUSSION whether this alkaloid can produce symptoms of Castanospermine was tested against a number of Gaucher’s disease in experimental animals. Inter- commercially available glycosidases to determine estingly enough, castanospermine also proved to be whether it was an inhibitor of any of these enzymes. a powerful inhibitor of lysosomal a-glucosidase in In each case, enzyme and alkaloid were mixed in buffer fibroblast extracts, but it did not inhibit any of the and the reaction was started by the addition of the other lysosomal glycosidases. This latter activity could appropriate pnitrophenyl glycoside. After an incu- explain why castanospermine also inhibits glycopro- bation of 30 min, the liberated pnitrophenol was tein processing in cultured cells. measured at 410 nm. Table I presents the resdts of this experiment. The data indicate that castanosper- EXPERIMENTAL METHODS mine was a fairly specific inhibitor of almond emulsin p-glucosidase, but did not inhibit yeast a-glucosidase, The following glycosidases were purchased from liver &galactosidaae, coffee bean a-galactosidase, jack Sigma Chemical Company: almond emulsin j3-glu- bean a-mannosidase, kidney B-N-acetylhexosamini- cosidase, yeast a-glucosidase, bovine liver @galac- dase, liver &glucuronidase, or epididymis a-L-fucos- tosidase, coffee bean a-galactosidase, bovine liver @- idase, even at levels of 250 pg/ml. However, castano- glucuronidase, bovine kidney @-N-acetylhexosamin- spermine did inhibit @xylosidase, although in this idase, jack bean a-mannosidase, bovine epididymis case, 50% inhibition required about 100 pg/ml. On a-L-fucosidase, and Aspet@h n&r &xylosidase. the other hand, j3-glucosidase was almost completely These enzymes were assayed with the appropriate p inhibited in this experiment at 50 pg/ml of alkaloid. nitrophenyl glycoside substrates which were also ob- In order to determine the optimum amount of cas- tained from Sigma. The reaction mixtures for assay tanospermine required for inhibition of the almond of these enzymes generally contained the following emulsin fl-glucosidase, a curve of alkaloid concentra- components in a final volume of 0.5 ml: 25 mM sodium tion versus activity was done as shown in Fig. 2. In- buffer, pH 5.0; 5 rnM pnitrophenyl glycoside; creasing amounts of castanospermine, from 109 ng/ various amounts of castanospermine; and enzyme ml to 100 pg/ml, caused increasing amounts of in- (usually enough enzyme to give an optical density hibition of the enzyme with 50% inhibition being ob- change of 1.0 to 2.0 at 410 nm during an incubation served at about 10 pg/ml. However, it should be of 30 min). Generally, enzyme and alkaloid were mixed pointed out that the optimum concentration of al- in buffer and the reaction was started by the addition kaloid depended to some extent on the amount of of the pnitrophenyl glycoside. After an incubation enzyme used. Similar results were previously shown of 30 min, 2.5 ml of 0.4 Y glycine buffer, pH 10.4 were with swainsonine, i.e., the amount of this alkaloid added, and the liberated pnitrophenol was measured required to inhibit jack bean a-mannosidase in- at 410 nm (7). creased with increasing amounts of protein (3). How- Castanospermine was isolated in 0.3% yield from ever, in contrast to swainsonine, preincubation of en- mature seeds of C. australe by extraction with water zyme and castanospermine was not necessary for op- and purification by ion-exchange chromatography on timum activity. Dowex 5OW-X8 (1, 2). The formation of pnitrophenol from pnitro- Glycosidases were also measured in extracts of hu- phenyl-p-n-glucopyranoside by the &glucosidase was man fibroblasta using the appropriate 4-methylum- fairly linear with time of incubation as shown in Fig. belliferyl glycosides. Confluent fibroblasts were har- 3, and also with the amount of protein (data not vested by trypsinization, washed, and ruptured by shown). Castanospermine inhibited the reaction at sonication. These extracts were used to assay for fl- all times, and the percent of inhibition at a given glucocerebrosidase (8), a-glucosidase, a- and @-ga- alkaloid concentration remained the same through- lactosidase, a- and fi-mannosidase, a- and @-L-fucos- out the time course (Fig. 3). The effect of concentra- idase, B-N-acetylhexosaminidase, @-glucuronidase, tion of substrate (pnitrophenyl-/3-n-glucoside) on the and fl-xylosidase using the appropriate I-methylum- inhibition by caztanospermine was measured as shown belliferyl glycosides. The release of I-methylumbel- in Fig. 4. The velocity was proportional to substrate liferone was measured using an Aminco spectroflu- concentration to about 5-8 mrd, and the K,,, for this CASTANOSPERMINE AS INHIBITOR 595

TABLE I

EFFECT OF CASTANOSPERMINE ON VARIOUS GLYCOSIDASES

Amount of Activity castanospermine Enzyme activity remaining Enzyme tested &z/ml) (O.D. 410 nm) (S)

fl-Glucosidase 0 1.58 (almond emulsin) 50 0.29 18 100 0.15 10 250 0.09 6 cY-Glucosidase 0 2.47 - (yeast) 50 2.57 104 100 2.61 106 250 2.63 107 fl-Galactosidase 0 1.00 - (bovine liver) 100 1.02 102 250 1.07 107 cu-Galactosidase 0 2.39 - (coffee bean) 50 2.48 103 100 2.46 104 250 2.43 102 @Glucuronidase 0 2.10 - (bovine liver) 50 2.11 100 100 2.09 99 250 2.07 98 B-N-Acetylhexosaminidase 0 0.85 - (bovine kidney) 50 0.85 100 100 0.92 108 250 0.84 98 a-Mannosidase 0 0.86 - (jack bean) 50 0.84 97 100 0.78 91 250 0.84 98 a-L-Fucosidase 0 1.10 - (bovine epididymis) 100 0.94 85 250 0.98 89 /3-Xylosidase 0 1.13 - (AspergiUus) 50 0.87 72 100 0.66 58 250 0.46 40

Note. Assay mixtures were as described in the text and contained the indicated amounts of castanospermine. After an incubation of 30 min, the liberated pnitrophenol was measured at 410 nm. fi-glucoside was calculated to be about 4 mrd. These appear to be competitive, the binding of castanosper- substrate concentration curves were also determined mine to the enzyme was reversible. Thus, when en- in the presence of various amounts of castanosper- zyme was mixed with an amount of alkaloid that mine and the data were plotted by the method of almost completely inhibited the activity and the mix- Lineweaver and Burk (Fig. 4). These studies indicated ture serially diluted with buffer, most of the enzy- that the inhibition was of the mixed type since both matic activity could be recovered. Similar results were the K,,, and the V, were altered by increasing previously shown with the swainsonine inhibition of amounts of the inhibitor. Although inhibition did not jack bean a-mannosidase (3). 596 SAUL ET AL.

8-

6-

4-

I I I I 2- 25 50 75 100 / CASTANOSPERMINE (pg/,,,l) / 4 FIG. 2. Effect of concentration of eaatanospermine _I - NoCASTAN. on the almond emulsin b-glucosidaae activity. Incu- 0- bations were as described under Experimental Meth- ods, except that various amounts of castanospermine were mixed with the enzyme as shown. Reactions FIG. 4. Effect of substrate (pnitrophenyl-~-glu- were initiated by the addition of pnitrophenyl-8-D- coside) concentration on the castanospermine inhi- glucopyranoside, and following an incubation of 30 bition. Incubations were az described in the text ex- min, the amount of pnitrophenol was measured at cept that various amounts of substrate were added 410 nm. Each incubation contained 0.33 mg of fi-glu- as indicated. The data were plotted according to the cosidase. method of Lineweaver and Burk. Castanospermine inhibition of j3-glucosidase was also examined using several b-linked glucose disac- charides, such as cellobiose and laminaribiose. In general, @-glucosidase hydrolysis of these substrates was inhibited by the same concentrations of castano- spermine as were required to inhibit the hydrolysis of the pnitrophenyl-@-glucoside. Since castanospermine was such a specific inhibi- tor of fi-glucosidase, we also tested this compound on mammalian cell homogenates to determine whether / _’ it would inhibit @-glucosidase and/or fl-glucocere- brosidase. These activities were assayed in white blood cell and in fibroblast extracts. The enzymes were as- sayed using 4-methylumbelliferyl-Bglucoside as sub- strate, and fl-glucocerebrosidase was also specifically assayed using the PHIglucose-labeled glucocerebro- side. Figure 5 shows a concentration curve of cas- tanospermine with both white blood cell and fibro- blast extracts. It can be seen from this data that both activities were inhibited by 50% at about 7 to 10 pg8/ ml of alkaloid. These data suggest that both mam- malian @-glucosidase and /3-glucocerebrosidase are TIME (mi.) inhibited to the same extent by this alkaloid. Similar FIG. 3. Time course of fl-glucosidase and caatano- results were obtained when @-glucocerebrosidase was spermine inhibition. Incubations were as described specifically assayed by the radioactive method. in the text with pnitrophenyl-@-glucoside and al- A number of other lysosomal glycosidases were as- mond emulsin &glucosidase. Various amounts of cas- sayed with the fibroblast extracts using the 4-meth- tanospermine were added and mixtures were incu- ylumbelliferyl glucosides as substrates. In this ex- bated for the times shown. Released pnitrophenol periment, castanospermine was also found to be a was measured at 410 nm. Each incubation contained potent inhibitor of the lysosomal a-glucosidase, 0.33 mg of @glucosidase. showing approximately the same degree of inhibition CASTANOSPERMINE AS INHIBITOR 597

ferent stereochemistry will be valuable inhibitors of other glycosidases. These kinds of compounds should be important tools for studying glycoprotein pro- cessing or lysosomal function.

ACKNOWLEDGMENTS This work was supported by a grant from the Rob- ert A. Welch Foundation and NIH Grant AM 21800 to ADE, and NIH Grant GM28949 to JPC. We thank James Bauml of the Huntington Botanical Gardens, San Marino, California, for providing the seeds of

CASTANOSPERMINE CONCENTRATION hlml) Castonospcrmum australe. We are also grateful to Dr. Irving Goldstein of the University of Michigan for FIG. 5. Effect of castanospermine concentration on providing some of the glucose disaccharides. fl-glucocerebrosidase (and j3-glucosidase) activity. Extracts of white blood cells or fibroblasts were in- cubated with 4-methylumbelliferyl-@glucoside in the REFERENCES 1. HOHENSCHUTZ,L. D., BELL, E. A., JEWESS, P. J., The liberated 4-methylumbelliferone was measured LEWORTHY, D. P., PRYCE, R. J., ARNOLD, E., in a spectrofluorimeter as described under Experi- AND CLARDY,J. (1981) Phytochist?y 20,811- mental Methods. 814. 2. COLEGATE, S. M.. DORLING, P. R., AND HUXTABLE, C. R. (1979) Au&. J. Chxm. 32, 2257-2264. on this enzyme as on the B-glucosidase. However, it 3. DORLING, P. R., HUXTABLE, C. R., AND COLEGATE, still did not inhibit the yeast o-glucosidase. Why cas- S. M. (1986) Biochem J. 191,649-651. tanospermine inhibits lysosomal a-glucosidase but 4. ELBEIN, A. D., SOLF, R., DORLING, P. R., ANDVOS- not the yeast a-glucosidase is not clear, but it will BECK, K. (1981) Proc Nat Ad sci. USA 78, be of interest to test this alkaloid on other glucosi- 7393-7397. dases. In that regard, it is of interest to point out 5. ELBEIN, A. D., DORLING, P. R., VOSBECK, K., AND that this alkaloid has also been found to be an in- HORISBERGER, M. (1982) J. Bid Ckem 257, hibitor of glycoprotein processing, and to cause al- 1573-15’76. terations in the oligosaccharide portion of the influ- 6. TULZXANI, D. R. P., HARRIS, T. M., AND TOUSTER, enza viral hemagglutinin (10). However, we also tested 0. (1982) J. Bid Chem 257,7936-7939. castanospermine on a number of other lysosomal gly- 7. RUDICK, M. J., AND ELBEIN, A. D. (1973) J. Bid cosidases in the fibroblast extracts and found it to Chem 248, 6506-6513. be without effect on a- and @-galactosidase, a- and 8. PETERS, S. P., LEE, R. E., AND GLEW, R. H. (1975) p-mannosidase, a- and B-L-fucosidase, 6-N-acetyl- Clin Chim Acta 60,391-396. hexosaminidase or @-glucuronidase. In these ex- 9. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, S. L., tracts, it also inhibited the &xylosidase. AND RANDALL, R. J. (1951) J. Bid Chem 193, The finding that castanoepermine inhibits gluco- 265-275. sidase (especially 6 but also some a), while swain- 10. PAN, Y. T., SAUL, R., SANFORD,B. A., MOLYNEUX, sonine inhibits a-mannosidase strongly suggests that R. J., AND ELBEIN, A. D., manuscript in prep- other hydroxylated indolizidine with dif- aration.