Presence of proteolytic enzymes in Dicrocoelium dendriticum eggs

lnrernrrrronal Journal/or Prrnred ,n Grror Brrtorn

Parosrmlog~

Vol.

23. No.

5. pp.

681-684.

1993 ‘f

1993

0020-7519:93 $6.00 + 0.00 Pergamon Pre.rs Lid socier.v/or Pazsiml0g.b

,4usrra/run

RESEARCH NOTE PRESENCE

OF PROTEOLYTIC ENZYMES IN DZCROCOELIUA4 DENDRITICUM EGGS

CRISTINADE ARMAS-SERRA, CONSUELO GIMENEZ-PARDO and FILOMENA RODRIGUEZ-CAABEIRO Laboratory of Parasitology, Faculty of Pharmacy, University of Alcall de Henares, 28871 Alcala de Henares (Madrid), Spain (Received 15 December

1992; accepted 6 April 1993)

Abstract-ARMAS-SERRA C. DE, GIMENEZ-PARDO C. and RODRIGUEZ-CAABEIRO F. 1993. Presence of proteolytic enzymes in Dicrocoelium dendriticum eggs. International Journal for Parasitology 23: 68 I-684. The proteolytic activity of eggs from Dicrocoelium dendriticum was examined using azocoll as protein substrate. Enzymes from the egg were capable of hydrolyzing the protein in a wide range of pH (2, 3 and 4), with maximal activity in acid ranges (pH 34). These enzymes were sensitive to serine, cystein, aspartyl and metallo protease inhibitors. Subsequent analysis by modified SDS-PAGE in gels containing gelatine demonstrate the presence of three bands with proteolytic activity. INDEX

KEY WORDS:

Dicrocoelium dendriticum; egg; proteases.

PROTEASES have been found to play a critical role in biological cycles of many parasites (McKerrow, 1989). In the case of digenetic Trematoda several authors have discovered their presence in different parasite stages and they have been implicated in vital processes of the parasite. Thus, in Fasciola hepatica there are proteases involved in the nutrition of the parasite (Rupova & Keilova, 1979; Simpkin, Chapman & Coles, 1980; Coles & Rubano, 1988; Herrera, Lopez & Dresden, 1989), Rege, Paragonimus sp. uses a hemoglobinase in its nutrition and other proteases for the invasion of host tissues (Yamakami, 1986), Schistosoma sp. presents several types of proteases that are involved in the nutrition of the parasite, invasion of host tissue and evasion of host immune response (Auriault, Quaissi, Torpier, Eisin & Capron, 1981; McKerrow, Jones, Sage & Pino-Heiss, 1985; Chapel1 & Dresden, 1986) and Clonorchis sinensis also uses proteolytic enzymes in several vital processes (Yong Song, Dresden & Rege, 1990). However to our knowledge no investigation of the proteases of digenetic Trematoda eggs has been reported up to now. One of the generally accepted hypotheses is that the miracidium releases a hatching enzyme which loosens bonds holding the operculum in place, and permits the miracidium to escape (Rowan, 1956; 1957). On the other hand, the apical gland and paired cephalic glands of Trematoda larvae are known to have histolytic secretions that participate in the host penetration process. For all these reasons the present

paper studies the presence of proteolytic enzymes in eggs of D. dendriticum, and also determines the optimal conditions for proteolytic activity using ranges of pH and temperature similar to those found in the snail. In addition, the proteases have been characterized by inhibition assays and by gelatin SDS-PAGE electrophoresis. Eggs of Dicrocoelium dendriticum were obtained from adult parasites. The trematodes were collected in the biliary vesicule and ducts of sheep from the municipal abattoir of Alcala de Henares (Spain). The helminths were washed with 0.2 M sterile phosphate saline buffer (PBS, pH= 7.2) supplemented with penicillin (1 mg ml-‘), streptomycin sulfate (2 mg ml-’ and 5-fluorocytosine (0.1 mg ml-‘). These trematodes were treated with a digestive solution (1: 10,000 pepsin 5 g, NaCl 9 g and HCl 7 ml in 1 liter distilled water) for 10 min to obtain the eggs, which were washed 10 times by sedimentation with sterile 10 mM-PBS supplemented with the antibiotics and antifungal agent. Next, eggs were homogenized in a glass-Teflon homogenizer in 10 mM-PBS (pH=7.2) and centrifuged at 100,000 g for 30 min. The supernatant was recovered and recentrifuged. The supernatant protein content was determined according to Lowry, Rosebrough, Farr & Randall (195I) and protein concentration in the crude extract was adjusted to 2 mg ml-’ by appropriate dilution with homogenization buffer. Crude extract was used immediately or frozen at - 80°C. Proteinase assays used calorimetric measurement 681

682

C. ~)f: ARMAS-SERRA~~~

2 ._ .t 5

7060-

2

50-

'2

40

3

30 20 10 II I 2345678

I

PB

FIG.

1. pH

hydrolysis of The point of maximal activity was taken as 100%. Points are means for 4 Dicrocoefium

dependence

for

azocoll

dendriticum egg proteases.

100

f

90 g

-.

80

t+ 70 .z .E 60 5

50

;

40

2 '2

20 30 I 10 LL

I/

I

I 4'

20 Temperature

37 (“C)

I

4s

FIG. 2. Temperature dependence for azocoll hydrolysis of Dicrocoelium dendriticum egg proteases. The point of

maximal activity was taken as 100%. Points are means for 4 experiments.

by hydrolysis of azo-dye-coupled hide powder (azocoll) as described by Monroy, Cayzer, Adams & Dobson (1989). Incubation time was 24 h in assays at several temperatures (4, 20, 37 and 45°C). The following buffers were used: 0. I M-citric acid/sodium citrate, pH 2, 3, 4 and 5; 0.1 M-PBS pH 6, 7, 8 and glycine-OH pH 9. Proteolytic activity was also measured in the presence of several inhibitors at pH inhibitors were: 5 rnM3. The used phenylmethylsulfonyl fluoride (PMSF), 5 mM-

ethylenediaminetetraacetic acid (EDTA), 500 ,ug ml-’ chymostatin, 50 pg ml-’ soybean trypsin inhibitor (STI), 10 mM-MgCIZ, 100 PM-leupeptin, 2 mM-No-~-tosyl-1-lysine ~hloromethyl ketone (TLCK) and I rn~-pepstatin A. Electrophoretic analysis was performed at 4°C in polyacrylamide gel electrophoresis (SDS-PAGE) using Miniprotean II BioRad equipment. The minigels contained 10% (w/v) acrylamide and 0.15% (w/v) gelatin as substrate (Criado-Fornelio, ArmasSerra, Gimenez-Pardo, Casado-Escribano, JimenezGonzalez & Rodriguez-Caabeiro, 1992) in the presence of 10% sodium dodecyl sulphate (SDS gelatin-PAGE). The samples were diluted 3:l in sample buffer and electrophoresed at 70 V per gel. After electrophoresis gels were washed in 2.5% Triton X-100 for 1 h to remove SDS, incubated in 0.1 M-citrate buffer (pH 5) and 2 mM-CaCl 2for 8 h at 37”C, and stained with Coomassie blue. After destaining, proteases were located as clear bands in a blue gel. Azocoll, gelatin, proteinase inhibitors and eiectrophoresis calibration kits were purchased from SIGMA (St Louis, MO). All other chemicals were of analytical grade. Proteolytic activity was detected in the samples studied. Spectrophotometric measurements showed that proteases from D. dendrikwm eggs have azocollytic activity over a relatively broad pH range (Fig. I). This result may reflect the presence of multiple proteases, each with its own optimum pH. However, this activity does have optimal conditions (pH and temperature) that coincide with those found by the parasite within the host. Thus. the highest proteolytic activity for the trematode eggs was at acid ranges of pH (pH 2, 3 and 4) like those found in the digestive tract of the mollusc, where the miracidia hatch from the eggs. Also, we must mention that at these pH, the proteases from crude extracts showed a strong azocollytic activity, degrading some 60% 01 the azocoll present in the medium after 24 h 01 incubation. In respect to the optimal temperature. we determined that proteolytic activity was thermosensitive, and the enzyme was inactive at 4°C (Fig. 2). It is interesting to note that all this is related to the distribution of the parasite in nature and may well reflect that the proteases function as lytic enzymes used to hatch the egg and penetrate the digestive tract and tissues of the snail. Electrophoretic analysis demonstrated the presence of 3 bands with proteolytic activity in crude extracts (Fig. 3). These experiments probably reflect the strong proteolytic activity present in n. de~dr~fi~~~7 eggs. Thus, the incubation in the presence of n. ~e~~r~Iie~m egg proteinase enzymes

Research TABLE I-----EFFE~ OF PROTEASEINHIBITORSON Dicrocoelium dendriticun? EGG PROTEINASE ENZYMES Inhibitors’

Inhibitors

types

% Inhibition

+ s.a

Note

683

3B

3A

-

MW kDa

-

(Concentration) Leupeptin

Serine/cystein proteases

2.12+0.05

Serine proteases Cysteinyl proteases Aspartyl proteases

(100 PM) Chymostatin (50 pg ml-‘) PMSF (5mM) ST1 (50 pg ml-‘) MgCl~(iOm~) Pepstatin A

10.84+0.05 10.84 I- 0.05 4.14+0.05 65.54+0.10

Metallo-proteases

(l mM) TLCK (2 mM) EDTA (5 mM)

41.18fO.06 17.93 to.04

11.63+ 0.05

Abbreviutions: PMSF, phenylmethylsulphonylfluoride; STI, soybean trypsin inhibitor; TLCK, Na-p-tosylI- lysine chloromethyl ketone; EDTA, ethylenediaminetetraacetic

acid.

(protein

concentration

degrade

the geiatin

2 mg ml-‘)

took only 8 h to In contrast, for helminths such as T. ~~~r~I~s (even when used at a protein concentration of 6 mg ml-‘), 24 h were needed to detect the presence of proteolytic activity (Criado-Fornelio et al., 1992). Studies on inhibition of azocollytic activity were carried out at optimum pH (pH = 3). We determined partial inhibition with all inhibitors assayed (Table 1). These data reflected the presence of serine/ cysteine, serine, metallo and aspartyl proteases in eggs of D. dendriticum The inhibition of aspartyl proteases by Pepstatin A and TLCK was the most significant. The presence of cysteine and serine proteases seems to be a very common feature in parasitic trematodes. Since these proteases are also present in I). dendriti~um eggs, it seems reasonable that they would play a similar role in this parasite. On the other hand, to our knowledge, this is the first time that aspartyl and metallo-proteases have been found in digenetic Trematoda, but it is possible that this may not have been studied previously. We suspect that aspartyl and metallo-proteases are also involved in parasite nutrition and penetration through the mollusc tissues to the hepatopancreas. However it is possible that these enzymes may also play an important role in the hatching mechanism, one of the enigmas of these helminths. Additional studies on D. dendr~tie~m proteases involving enzyme purification and structural studies are underway to ascertain their origin and principal role in this digenetic parasite. protein

substrate.

-

180 * 116~-

1

84 * 58 w 48.5

*

36.5 26.6

) m-

FIG. 3. Electrophoretic analysis of eggs proteases from Dicroroelium dendriticum. (A) 10% SDS-PAGE copolymerized with 0.15% gelatin as substrate. Gels were incubated in citrate buffer (pH= 5) for 8 h at 37°C. (B) Schematic representation of SDS-gelatin-PAGE.

Acknowledgements-We would like to thank C. F. Warren of the I.C.E. of the U.A.H. for her editorial assistance.

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Pumsitolog~

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K. G..

Fasciola

hepaticu:

a

C. R. & Coots G. C. 1980. proteolytic digestive enzyme.

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e~iI/

YAMAKAMI

K.

thiolprotcase c‘omparuiiw SONG

1986.

from

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Purification and propertIes lung fluke adult Puru~onbwus

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