TRANSFERRIN COUPLED AZANTHRAQUINONE ENHANCES THE KILLING EFFECT ON TRYPANOSOMES.
THE ROLE OF LYSOSOMAL MANNOSIDASE
NOK AJ.* & NOCK I.H.**
S u m m a r y :
Partially purified azanthraquinone (AQ) extract from Mitracarpus scaber was coupled to bovine transferrin (Tf) using azidophenyl glyoxal (APG|. The AQ-APG-Tf conjugate was found to possess an enhanced in vitro trypanocidal activity against Trypanosoma congolense and T. brucei brucei. At low concenttations of 0 . 3 9 - 9 0 mg/ml, the conjugate diminished the growth of T. congolense and T. b. brucei dose dependency at the logarithmic phase. Both parasites were more sensitive to AQ-APGTf than to the free (AQ) extract. Growth inhibition on the parasites by the free extract was observed at 20-200 mg/ml. The total activity of the lysosomal enzyme a-mannosidase was reduced in the T. congolense cells treated with AQAPGTf in a dose related pattern. However, the activity of the mannosidase in the T. b. brucei treated cells is less affected. The AQ-APG-Tf is more effective on a mannosidase than free AQ, eight and four fold for T. congolense and T. b. brucei respectively. The results ate discussed as regards the potency of using transferrin as suitable drug carrier in the chemotherapy of Human sleeping sickness.
KEY W O R D S : azanthraquinone, transferrin, Trypanosoma congolense.
Resume : AUGMENTATION DE L'EFFET TRYI'ANOCIDE DE L'AZANTHRAQUINONE ASSOCIÉE À UNE TRANSFERRINE. RÔLE D'UNE MANNOSIDASE LYSOSOMIALE
L'azanthraquinone (AQ) partiellement purifiée, extraite de Mitracarpus scaber, a été associée à une tranferrine (Tf) d'origine bovine à l'aide d'azidophenyl glyoxal (APC). Le conjugué AQ- APG-Tf est connu pour accroître in vitro l'activité trypanocide de l'AQ envers Trypanosoma congolense et T . brucei brucei. A basse concentration ( 0 , 3 0 - 9 0 μg/ml), le conjugué diminue la croissance de T . congolense ef de T . b. brucei de façon logarithmique dose dépendante. Les deux parasites sont plus sensibles à l'association AQ-APG-Tf qu'à l'extrait libre d'AQ. L'inhibition de la croissance des parasites par l'extrait libre est observée entre 20 et
200 μg/ml. L'activité de l'enzyme lysosomiale α-mannosidase a été réduite de façon dose dépendante dans les cellules de T. congolense soumises à AQ-APG-Tf. Cette activité est moindre chez T . b. brucei. L'association AQ-APG-Tf est plus effective sur l'α-mannosldase que l'extrait libre d'AQ, respectivement huit et quatre fois plus pour T . congolense ef T . b. brucei. Les résultats sont discutés au regard de la possibilité d'associer la transferrine à la chimiothérapie de la maladie du sommeil.
MOTS CLÉS : azanthraquinone, transferrine, Trypanosoma congolense.
A
frican t r y p a n o s o m e s are p r o t o z o a n parasites that c a u s e s l e e p i n g s i c k n e s s in h u m a n s and o t h e r related d i s e a s e s in cattle. African trypan o s o m i a s i s is c o n s i d e r e d to b e o n e o f the most serious d i s e a s e s affecting m a n a n d his d o m e s t i c animals and has b e e n the b a n e against animal p r o d u c t i o n in s u b humid T r o p i c a l Africa ( T D R , 1 9 8 4 ) .
T h e c h e m o t h e r a p y o f t r y p a n o s o m i a s i s is b e s e t with p r o b l e m s including a limited repertoire o f c o m p o u n d s , resistance to drugs, drug toxicity and protracted treat
ment p r o t o c o l . M o r e o v e r , there is n o available trypa
n o c i d a l drug that c a n b e u s e d routinely for the pre
vention o f the d i s e a s e during b l o o d transfusion ( N o k
Abbreviations: AQ, azanthraquinone; Tf. transferrin; APG, azido
phenyl glyoxal.
* Departments o f Biochemistry and ** Biological sciences, Ahmadu Bello University, Zaria. Nigeria.
Correspondence: Andrew ) . Nok.
E-mail: jandrew@skannet.com
et al., 1 9 9 3 ) . Also fewer drugs are available for t h e t r e a t m e n t o f c h r o n i c t r y p a n o s o m i a s i s ( G u t t e r i d g e , 1 9 8 5 ) .
T r y p a n o s o m e s require transferrin for growth ( S c h e l l et al., 1996a, 1 9 9 6 b ) and require it in a receptor-mediated fashion ( C o p p e n s et al, 1987; W e b s t e r & G r a b , 1 9 8 8 ) . In contrast to t h e m a m m a l i a n cells, T f and its r e c e p t o r is d e l i v e r e d into African t r y p a n o s o m e s a n d m a y n o t b e r e c y c l e d ( G r a b et al, 1 9 9 2 , 1 9 9 3 ) . O n a c c o u n t o f the foregoing, it implies that with p r e c i s e Tf/drug c o m b i n a t i o n s , it is p o s s i b l e to s e l e c t i v e l y kill the para
site b y directing t h e drug to the t r y p a n o s o m e lyso- s o m e , living the m a m m a l i a n cell unaffected. T h e i n h e rent difficulty at raising v a c c i n e s against t r y p a n o s o m e s , d u e to the p h e n o m e n o n o f variable surface g l y c o protein ( V S G ) has m a d e t h e glycosyl p h o s p h a t i d y l inositol ( G P I ) a n c h o r a m o r e a p p e a l i n g target in drug d e v e l o p m e n t .
In the p r e s e n t paper, the e n h a n c e d trypanocidal effect o f A Q - A P G - T f a n d the p o s s i b l e interference with the synthesis o f GPI synthesis o f the t r y p a n o s o m e para
site is reported.
Parasite. 2002, 9. 375-379
Note de recherche 375
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/2002094375
N O K A . J . & N O C K I . H .
MATERIALS AND METHODS
B
ovine transferrin, azidophenyl glyoxal ( A P G ) , Sephadex G 2 5 , para-nitrophenyl-f3-D-mannopy- ranoside (pNP-man) w e r e purchased from Sigma Chemical Company St-Louis, USA. General laboratory and inorganic chemicals were obtained from W a k o , Japan.TRYPANOSOMES
A stabilate o f Trypanosoma congolense stb 2 1 2 and T. brucei brncei stb 4 2 1 w e r e supplied by the Depart
ment o f Veterinary Parasitology, Ahmadu B e l l o Uni
versity, Zaria, Nigeria.
EXTRACTION OF ACTIVE COMPONENTS
OF MLTRACARPUS SCABP.RMitracarpus scaber leaves were collected fresh from the Botanical garden o f Ahmadu B e l l o University, Zaria, Nigeria. T h e leaves ( 8 0 0 g ) w a s h o m o g e n i z e d in 4 L o f chilled EtOH, filtered and c o n d e n s e d by v a c u u m evaporation.
T h e c o n c e n t r a t e w a s partitioned b e t w e e n EtOAc and H20 a n d the EtOAc fractions that exhibited trypano
cidal activity w a s further partitioned b e t w e e n 9 0 % a q u e o u s M e O H and n - h e x a n e . T h e final m e t h a n o l i c extract, which was h o m o g e n o u s on TLC, w a s r e d u c e d by evaporation yielding about 12 g o f the active residue.
ISOLATION OF ACTIVE COMPONENT
About 10 g o f the active residue w a s further c h r o m a - tographed o n silica gel column. Elution o f the fractions w a s c o n d u c t e d using: n - h e x a n e : EtOAc ( 1 0 : 1-0.2) gra
dient system and EtOAc: M e O H (4: 1). Fractions o f 100 ml w e r e collected. T h e y w e r e pooled according to TLC analysis. T h e EtOAc: M e O H fractions ( 1 0 : 1 and 10: 0 . 5 ) w e r e c o m b i n e d a n d r e - c h r o m a t o g r a p h e d with the s a m e solvent system. T h e extract had the s a m e Rf with standard AQ a n d a p p e a r e d as dark spots after e x p o sure to UV radiation.
PREPARATION OF A Q - A P G - T F
Bovine a p o T f ( 1 0 0 m g ) was loaded with F e3 + as des
cribed by Klausner et al. ( 1 9 8 3 ) . T h e d i F e3 +- T f was passed through S e p h a d e x G 2 5 to separate free F e3 +. T h e d i F e3 +- T f was reserved for coupling. T h e AQ extract from the preceding section was then added to the diFe3 +-Tf.
Cross linking o f the extract and d i F e3 +- T f was d o n e by dispensing about 100 mg o f the AQ in 0.5 ml 100 mM N a H C 03 pH 8.3 to 0.2 ml APG (1 mg/ml DMSO). T h e mixture was gently stirred in a volumetric flask covered with aluminum foil at 2 5 " C . This w a s followed by the addition o f 1 ml o f d i F e3 +- T f ( 5 0 m g / m l ) in a buffer.
T h e transferrin w a s non-specifically linked with A Q - APG c o m p l e x b y e x p o s u r e to the sun for ten minutes.
T h e mixture w a s then centrifuged at 5,000 g for ten minutes and the r e c o v e r e d supernatant l o a d e d o n t o S e p h a d e x G 2 5 to r e m o v e the u n r e a c t e d AQ-APG frac
tion. T h e estimated AQ-APG-Transferin c o m p l e x w a s 15 mg A Q / m g protein.
IN VITRO SENSITIVITY ON BLOOD STREAM
TRYPANOSOME CONGOLENSE AND T. B. BRUCEIT. congolense a n d T. b. brucei w e r e cultured in a 2 4 well culture plates at varying levels o f AQ-APG-Tf ( 0 . 0 4 - 1 0 0 p g / m l ) or AQ ( 5 - 2 0 0 p g / m l ) . T h e parasites w e r e s e e d e d at 4 x 1 05/ m l in D u l b e c c o m e d i u m ( G I B C O BRL) s u p p l e m e n t e d with 100 uM h y p o x a n - thine, 3 0 pM thymidine, 4 0 pM adenosine, 1 pM sodium pyruvate, 50 pM L-glutamine, 100 pM 2 - m e r c a p t o e - thanol and 2 0 % FCS, and incubated at 3 7 " C u n d e r an a t m o s p h e r e o f 5 % C O , a n d 9 5 % air. T h e control culture w a s incubated without AQ and AQ-APG-Tf.
Cell growth w a s s c r e e n e d b a s e d o n motility and c o l o r c h a n g e as described b y Hirumi et al. ( 1 9 9 3 ) . In the system, live t r y p a n o s o m e s that g r o w well to stationary p h a s e acidify the m e d i u m and c h a n g e the c o l o r o f the indicator dye p h e n o l red from pink to y e l l o w ( p H less than 6 ) . T h e m e d i u m remains pinkish (pH 7 - 8 ) after three-five days incubation w h e n the parasites are killed.
ASSAY FOR OC -1,2 MANNOSIDASE ACTIVITY
Both T. congolense and T. b. brucei that w e r e treated with AQ-APG-Tf, AQ for o n e h o u r and the control (i.e untreated parasites) w e r e separated from the m e d i u m by passing through DEAE cellulose c o l u m n . T h e flow through fractions containing a b o u t 4 x 1 0s c e l l s / m l w e r e s p a n n e d d o w n at 5,000 g x 5 min. T h e parasites in e a c h case were lysed in 50-mM acetate buffer pH 5.0.
About 100 pg o f the crude lysate was incubated with 50 mM o f pNP-mannoside in 50 mM acetate buffer pH 5.0.
At the end o f o n e hour, the reaction was stopped using 0.1 ml o f 1 M NaOH. T h e released para-nitro p h e n o - late ( p N P ) ion hydrolyzed b y the e n z y m e w a s deter
m i n e d at 5 4 0 nm using a s p e c t r o p h o t o m e t e r . O n e unit o f oc-mannosidase activity w a s defined as that a m o u n t o f e n z y m e that catalysed the release o f 1 u m o l e o f m a n n o s e from p N P - m a n n o s e p e r min p e r mg protein.
Total protein o f the parasite lysate w a s quantified as d e s c r i b e d b y Lowry et al. ( 1 9 5 1 ) .
RESULTS
EFFECT OF AQ EXTRACT ON THE IN VITRO GROWTH
OF T. CONGOLENSE AND T. B. BRUCEIA
s observed in Table I, the AQ extract at 2 5 - 200 ug/ml inhibited the growth o f both T. congolense and T. brucei in a d o s e d e p e n d e n t pat-
3 7 6 Note de recherche Parasite, 2002, 9, 375-379
TRANSFF.RRINE COUPLED AZANTHRAQUINONE EFFECT ON TRYPANOSOMEN
Cell g r o w t h ( c e l l s / m l )
AQ ( u g / m l ) T. congolense T. b. bnicei
200 0 0
100 0 0
50 0 Ki ± 50
25 0 lo1 ± 1 03
12.5 10" ± 10-' 106 ± 10-
6.25 10" ± 103 10" ± 103
Table I. - Trypanocidal activity of azanthraquinone extract (AQ) on T. congolense and T. b. brucei.
The results are expressed as averages of the number of parasites in three experiments. The parasites were treated for 48 h with the res
pective level of the AQ. Only motile parasites were counted. At '0' the parasites were completely lysed.
tern. T h e minimum growth inhibitory level o f the AQ extract was 2 5 pg/ml for T. congolense and. T. b. bnicei respectively. B e l o w t h e s e levels o f AQ for the res
pective parasites, there w a s n o c o m p l e t e inhibition o f growth on both parasites. However, they exhibited diminished motility as o b s e r v e d under phase contrast microscopy. Furthermore, w h e n the parasites at 1 0 ' cells/ml w e r e sub-inoculated into healthy mice, they failed to elicit any parasitemia after 3 0 days after infec
tion.
EFFECT
OF
A Q - A P G - T FON BLOOD STREAM TRYPANOSOMES
T h e c o u p l e d AQ-APG-Tf w a s incubated with either T. congolense or T. b. brucei. Both parasites w e r e found to b e highly sensitive to the growth inhibitory effects o f the conjugate. As seen in Table II, at 0.39-100 pg/ml, the conjugate inhibited the growth o f T. congolense dose dependently within two days post incubation. T h e flagellic motility at less than 0 . 7 8 p g / m l o f AQ-APG-
AQ-APG-Tf ( u g / m l )
°/o g r o w t h
AQ-APG-Tf ( u g / m l ) T. congolense T. b. bmcei
100 0 0
50 10 5
25 20 10
12.5 40 15
6.25 50 20
3.12 60 50
1.56 75 60
0.78 80 65
0.39 100 70
0.19 100 85
0.09 100 100
Table II. - Trypanocidal activity of transferrin coupled methanolic extract of azanthraquinone (AQ-APG-Tf) on T. congolense and
7'. b. bnicei.
The results are expressed as averages of three experiments. Cell growth was measured after 48 h of incubation with the various amounts of AQ-APG-Tf.
T f was very slow and the parasites appeared clustered.
Also they w e r e easily subdued w h e n sub-inoculated into healthy mice. Similarly, the growth o f T. b. bnicei cells w a s inhibited by the AQ-APG-Tf conjugate. T h e minimal amount o f AQ-APG-Tf required to inhibit
T. b. brucei growth w a s 0 . 1 9 pg/ml, i.e. about two to four folds m o r e than that o f T. congolense.
EFFECTS OF A Q AND AQ-APG-TF ON
α-MANNOSIDASE OF T. CONGOLENSE AND T. B. BRUCEI The activity of acid a-mannosidase from T. congolense and 77. b. brucei were assayed after treatment with AQ and AQ-APG-Tf. A summary of the results is presented in Tables III & IV. The level of a-mannosidase acti
vity in the control parasites (i.e. untreated) was 230 and 260 pmole/min/mg for T. congolense and T. b. bnicei respectively. The level of a-mannosidase activity dimi
nished significantly for T. congolense when AQ-APG- Tf was used as the growth inhibitor (Table III).
The inhibition was clearly dose dependent with about 90 % inhibition when 100 pg/ml of AQ-APG-Tf was used to inhibit growth. At 12.5 pg/ml, about 50 % of the activity was inhibited. The inhibition was not rever
sible even after dialysis suggesting strong covalent interaction between the conjugate AQ-APG-Tf and the enzyme. In contrast AQ-APG-Tf had a lower inhibitory effect on the activity of a-mannosidase from 77. b. bni
cei. The enzyme activity on both 7'. congolense and
OC-mannosidase activity ( u m o l e / m i n / m g )
AQ-APG-Tf ( u g / m l ) T. congolense T. b. bmcei
100 18 ± 8 180 ± 25
50 30 ± 8 195 ± 20
25 43 ± 15 212 ± 40
12.5 110 ± 24 201 ± 35
6.25 180 ± 20 230 ± 25
3.12 200 ± 20 235 ± 40
0 230 ± 30 260 ± 30
Table IV. - Effect of azanthraquinone extract (AQ) on T. congolense and T. b. brucei.
The results are expressed as averages of three experiments.
Parasite, 2002, 9, 375-379
Note de recherche 377
Table III. - a-mannosidase activity in extracts of AQ-APG-Tf treated parasites. The results are expressed as averages of three experiments.
AQ ( u g / m l )
a - m a n n o s i d a s e activity ( u m o l e / m i n / m g )
AQ ( u g / m l ) T. congolense T. b. bnicei
200 85 ± 15 210 ± 25
100 105 ± 22 210 ± 30
50 180 ± 40 250 ± 35
25 210 ± 4 0 260 ± 40
0 218 + 30 260 ± 30
NOK A.J. & NOCK I.H.
T. b. brucei w e r e u n c h a n g e d as c o m p a r e d with the control w h e n the free AQ was used to inhibit growth ( T a b l e I V ) .
DISCUSSION
H
ere, w e report for the first time the trypanocidal activity o f a carrier c o m p l e x AQ-APG-Tf c o n j u g a t e . In t h e p r e s e n t w o r k w e h a v e demonstrated the feasibility o f binding the AQ extract with transferrin using APG. T h e conjugate had a higher trypanocidal activity than free AQ. This observation suggests that the AQ-APG-Tf mediated trypanocidal effect is synergistic and construe the involvement o f m o r e than a target. This is reinforced by the fact that though the free AQ was growth inhibitory to both para
sites, only the AQ-APG-Tf had a greater effect o n the parasites' a - m a n n o s i d a s e .
Furthermore, the effect o n the e n z y m e was e x c l u s i v e with the m o r e susceptible T. congolense loosing about 50 % o f the a-mannosidase activity in the p r e s e n c e o f 12.5 ug/ml o f AQ-APG-Tf. T h e T. b. brucei a - m a n n o sidase was less r e s p o n s i v e to the p r e s e n c e o f the conjugate. T h e e n h a n c e d susceptibility thus suggests that the conjugate is delivered through a special system.
Previous reports have s h o w n that unlike the m a m m a lian cell, in the acidic environment o f t r y p a n o s o m e e n d o s ó m e system, serum transferrin along with its r e c e p t o r is d e l i v e r e d to l y s o s o m e s ( M b a w a et al., 1992). This essentially suggests that trypanosom.es have evolved a modified m e c h a n i s m for iron uptake. ( G r a b et al, 1992, 1 9 9 3 ) previously p r o p o s e d that since trans
ferrin is delivered to lysosomes in African trypanosome.
it might b e possible to covalently bind drugs with plant or bacterial toxins to transferrin for use as c h e m o t h e - rapeutic agent.
Also. Shin et al. ( 1 9 9 5 ) reported that transferrin can b e used as targeting moiety for molecules across the blood brain barrier presumably through and interaction with T f receptors in brain endothelial cells. Since T f and low- density lipoprotein (LDL) particles enter the cell through receptor mediated endocytosis, a receptor-associated transport o f AQ-APG-Tf conjugate to the lysosomal compartment o f the parasite is contemplated.
Both T. congolense and T. b. brucei were sensitive albeit to a different extent to the conjugate. This could imply that the m e c h a n i s m by which the parasites handle the A Q - A P G - T f in b o t h s y s t e m s c o u l d b e d i f f e r e n t . Moreover, the sensitivity o f the T. congolense a - m a n - nosidases and the attendant comparative insensitivity by the T. b. brucei e n z y m e supports this observation. T h e decreased a-mannosidase activity in the treated cells could b e due to irreversible inhibition or repression in the synthesis o f the enzyme. In support o f the foregoing, Gerrits et al. ( 1 9 9 6 ) reported that T. b. brucei has mul
tiple expression sites encoding for different mammalian T f receptors, which allow trypanosomes to efficiently, take up T f from a wide range o f host. T h e T. congo
lense stb 2 1 2 used in the present study w a s maintained in a culture media with bovine serum presupposing that the receptors for Tf-bovine are expressed. O n the other hand, the T. b. brucei was grown in B a l b - c mice sug
gesting that the parasites could have adjusted to the expression o f m o u s e specific T f receptors.
T h e e n z y m e a - m a n n o s i d a s e is a lysosomal e n z y m e especially with its pH optimum. It has b e e n d e s c r i b e d in 7'. cruzi and T. rangelli (Alison et al., 1 9 9 8 ; Nok et al, 2 0 0 0 ) . O u r report is the first to s h o w the p r e s e n c e o f the e n z y m e in the African t r y p a n o s o m e s : T. congo
lense and T. b. brucei. T h e i m p o r t a n c e o f a - m a n n o s i d a s e in the p r o c e s s i n g system o f g l y c o p r o t e i n s in higher cells is well k n o w n (Alison et al, 1998). In mammalian cells, a - l , 2 m a n n o s i d a s e play an essential role in early steps o f A'-linked o l i g o s a c c h a r i d e matu
ration. It c o u l d also serve the parasites in reprocessing derived oligosaccharides portion o f glycoprotein to b e mobilized in the synthesis o f the G P I a n c h o r .
From the observed results, A Q treated parasites had there enzymes unaffected by the plant extract. However, the tranferrin-coupled extract had a clear suppression on the activity o f a-mannosidase by at least 3 0 %. Since the a-mannosidase is a lysosomal enzyme, its fall in acti
vity implies a successful delivery o f the extract to the lysosome. T h e c o m p r o m i s e d activity will have a far-rea
ching effect in the process o f mobilizing the mannosyl residues b y the parasite. Apart from preventing the association o f the parasite with the host cells, the action o f AQ-APG-Tf could induce the susceptible parasite to produce defective oligosaccharides, rich in m a n n o s e . Comparatively, there was n o effect o n the 7'. brucei enzyme which could b e rationalized on the possible pre
s e n c e o f other forms o f receptors in T. brucei.
T h e summation o f the observation s h o w s that the drug delivery system with T f is host specific. O u r work clearly s h o w e d that transferrin has high poten
tial for a p p l i c a t i o n in drug d e l i v e r y s y s t e m with e n h a n c e d trypanocidal effect. Furthermore, that at least an e n z y m e system involved in reprocessing g l y c o p r o teins is affected by the daig-conjugate strongly implies the possibility o f targeting GPI synthesis. Results from this w o r k should stimulate interest in the design o f try- panocide-host specific T f carrier systems for the assess
ment o f different forms o f trypanocide and their effect o n the e n z y m e s involved in G P I synthesis.
ACKNOWLEDGEMENTS
A
ndrew J . Nok whishes to acknowledge the contribution o f the Alexander von Humboldt Founda
tion, G e r m a n y .
378 Note de recherche Parasite. 2002, 9, 375-379
TRANSFERRINE COUPLED AZANTHRAQUINONE EFFECT ON TRYPANOSOMES
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Reçu le 7 mars 2002 Accepté le 4 juillet 2002
Note de recherche -379
Parasite. 2002. 9, 375-379
