ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 221, No. 2, March, pp. 593-597, 1983 COMMUNICATION Castanospetmine, a Tetrahydroxylated Alkaloid 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- indolizine) is an alkaloid of the indolizidine 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- acetate 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.