Serine proteases in coelomic fluids of annelids Eisenia foetida and Lumbricus terrestris

Comp. Biochem.PhysioLVol. 105B,Nos 3/4, pp. 637~41, 1993 Printed in Great Britain

0305-0491/93 $6.00+ 0.00 © 1993PergamonPress Ltd

SERINE PROTEASES IN COELOMIC FLUIDS OF ANNELIDS EISENIA FOETIDA A N D LUMBRICUS TERRESTRIS C. LEIPNER,* L. TU(~KOVA.,t J. REJNEK~f and J, LANGNER* *Department of Immunology, Medical Faculty, Martin Luther University, D-4010 Halle, Germany; and fDepartment of Immunology, Institute of Microbiology, Czech Academy of Science, CS 142 20, Prague, Czech Republic

(Received 11 December 1992; accepted 15 January 1993) Almtraet--1. The pH optimum of the proteolytic enzymes in the coelomic fluids of Lumbricus terrestris and Eisenia foetida earthworms lies in the alkaline pH range of 8.0--10.0. 2. According to inhibition experiments, they belong to serine proteases. 3. The mean proteolytic activity was found to be higher in Eiseniafoetida than in Lumbricus terrestris coelomic fluids and individual variations were more pronounced in Lumbricus terrestris. 4. The comparison of the heterogeneity of enzymes by SDS-PAGE also showed individual variations in both species. 5. No changes in specific proteolytic activity after primary and secondary stimulation of earthworms with protein antigens were observed.

INTRODUCTION In the defence mechanisms of annelids against microorganisms and foreign materials natural haemolytic, agglutinating and bacteriostatic proteins of coelomic fluid play an important role (Kauschke and Mrhrig, 1987; Vaillier et al., 1985; Roch et al., 1986; Roch et al., 1991; Stein et al., 1982; Wojdani et aL, 1982). Furthermore, a proteolytic activity that very successfully digests vertebrate proteins but not proteins of its own coelomic fluids, or those of a relative species, was detected in Lumbricus terrestris and Eiseniafoetida (Tu~kov~i et al., 1986a). This activity may not be restricted to food digestion only, but may also participate in defence mechanisms. The aim of this report was to study the heterogenity of proteases of coelomic fluids Lumbricus terrestris and Eisenia foetida earthworms before and after parenteral stimulation with foreign proteins.

MATERIALS AND METHODS

Animals and their stimulation Adult Lumbricus terrestris earthworms kept at 15°C in soil, and Eisenia foetida kept at room temperature in mould, were stimulated by injection of 10 #g arsanilic acid (Hopkins and Williams, Essex, U.K.) coupled to human serum albumin (HSA, Institute of Sera and Vaccines, Prague) (ARS-HSA) into the coelomic cavity. The A R S - H S A complex was embedded in 3% agar gel to prevent its expulsion through dorsal pores. The same dose of A R S - H S A was used for a secondary challenge and administered on day 19 after the primary stimulation. Coelomic fluids from individual earthworms were obtained by

puncturing the coelomic cavity with a Pasteur pipette. The cell-free coelomic fluid was separated by centrifugation (1200 rpm, 10 min and 10,000 rpm, 20 min) and the samples were kept at - 2 0 ° C until use.

Preparation of tissue homogenates and cell lysates Earthworm tissues were prepared in PBS using an MSE homogenizer, centrifuged at 10,000rpm for 20 min at 0°C, and the supernatant collected and immediately frozen at - 6 0 ° C . Separated coelomocytes (106 cells per sample) were lysed by 0.75 M Tris-HC1 buffer (pH 8.2) containing 1% Nonidet-40 and 2 mM EDTA for 30 min on ice. Samples were centrifuged (10,000 rpm, 20 min, 0°C) and supernatants kept at -60°C.

Proteolytic activity Protease activities were assayed with azocasein (0.5%) as substrate in 0.2M borate buffer with 0.1 M NaCI, pH 9.5 (Langner et al., 1973), with and without protease inhibitors. For measurements of the pH dependence of the enzymic activity, BrittonRobinsons buffer, with 0.1 M NaCI (pH 5.2-10.8), was used. The specific activity was expressed in terms of units/mg protein using HSA as standard (Langner et al., 1971).

Demonstration of heterogeneity of proteolytic activity of individual coelom& fluids in SDS gels S D S - P A G E was performed using the LKB 2050 slab-gel Midget electrophoresis unit (Pharmacia LKB Biotechnology, Uppsala, Sweden) according to the method of Laemmli (Laemmli and Favre, 1979). Acrylamide slab gels (7.5 and 10%) (0.75 mm thick) were prepared in the usual manner to examine 637

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the azocaseinolytic activity except that azocasein was incorporated in the running gel (1 mg/ml). The samples (0.25-0.5 units) were not boiled prior to analysis and the gel was cooled during electrophoresis to 0°C. After electrophoresis, the gels were washed in ice-cold 0.2 M borate buffer, 0.1 M NaC1, 1% Triton X-100 (v/v), pH 9.5, for 13 min then the gels were rinsed briefly (3 min, 4°C) in the same buffer without Triton X-100 and were then incubated again in 0.2 M borate buffer, 0.1 M NaC1 1% Triton X-100 (v/v), pH 9.5, for 2 0 m i n at 37°C. Following incubation, the gels were stained with 0.5% Coomassie Brilliant Blue R-250 in 53% ethanol, 10% acetic acid (v/v) overnight and destained in 40% methanol and 10% acetic acid (v/v). Zones of enzymic activity were indicated by negative staining.

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fluids The pH dependence of coelomic fluids of Lumbricus terrestris and Eisenia foetida showed a parallel increase of proteolytic activities in both earthworm species with a maximum in the alkaline pH range from 8.0 to 10.0 (Fig.l). The bulk of the proteolytic activity at p H 9.5 can be characterized as serine protease activity since more than 90% of the proteolytic activity can be inhibited with 0 . 5 m M P M S F as a typical serine protease inhibitor or 4 g M soybean trypsin inhibitor. E D T A (10 mM) and o-phenanthrolin (1 mM) were without an inhibition effect.

Specific proteolytic activity in individual samples The specific proteolytic activity was tested in individual cell-free coelomic fluids of Lumbricus terrestris and Eisenia foetida before and after primary or secondary stimulation with A R S - H S A . No significant increase in specific proteolytic activity was seen during either primary or secondary stimulation (Table 1) but, in each group, individual variations of proteolytic activity were found (Fig. 2). More individuals with low, or without any, dectable proteolytic activity under the experimental

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RESULTS

Characterization of proteolytic activity of coelomic

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Fig. l. pH activity curves for proteases. (A E---difference in extinction measured at 366 nm at time 0 and after 30 min incubation at 37°C. conditions used were seen in Lumbricus terrestris than in Eiseniafoetida. The mean specific proteolytic activity of individual coelomic fluids in nonstimulated Eisenia foetida (1651.5 ___227.7, N = 10) was significantly ( P < 0 . 0 5 ) higher than that in Lumbricus terrestris (632.0 + 190.1; N = 10).

Demonstration of heterogenity of proteolytic enzymes by S D S - P A G E The coelomic fluids of Lumbricus terrestrisand Eisenia foetida non-stimulated individuals showed different protease patterns at pH9.5. In Eisenia foetida, up to six azocaseinolytic proteins in a molecular weight range of 30-70 kDa were found (Fig. 2a). In contrast, the bulk of proteolytically active proteins of Lumbricus terrestris was greater than 67 kDa (Fig. 2b). U p to eight different azocaseinolytic proteins could be seen in some of the coelomic fluids. However, in some cases individuals that showed no proteolytically active proteins in S D S - P A G E were seen (Fig. 2b). These findings

Table 1. Specificproteolytic activity in coelomic fluids of earthworms L. terrestrisand E. foetida collected after primary and secondarystimulation CF collection Specific after stimulation Numberof enzymaticactivity Annelid species Antigen Dose (hr) experiments (U/mg)* ARS-HSA I 24 5 1 104.0_+254.2 48 5 2 032.6 + 94.9 Eiseniafoetida 96 5 1 157.6_ 190.9 II 168 10 1 286.6+ 290.4 0 0 0 10 1 651.5 _+227.7 ARS-HSA I 24 10 432.7 ___144.5 72 5 350.1 _ 222.7 Lumbricus terrestris 96 5 491.1 + 270.1 II 168 10 580.3 _'!-237.8 0 0 0 10 632.0 + 190.1 *Values are expressed as means (five to ten experiments)+ SD.

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Fig. 2. Heterogeneity of proteases in individual samples of Eiseniafoetida (a) and Lumbricus terrestris (b). The first three lines (from left to right) represent SDS-PAGE analyses of enzymic activities in coelomic fluid samples of non-stimulated and, the other three lines, ARS-HSA-stimulated earthworms. correlate with the above-mentioned results showing no, or low, specific proteolytic activities in some of these samples that were more frequent in Lumbricus terrestris. However, differences among individual samples in specific proteolytic activity or number of proteolytically active proteins, demonstrable by S D S - P A G E of Eisenia foetida or Lumbricus terrestris coeiomic fluids, seem not to be influenced by the stimulation with A R S - H S A (Fig. 2a,b). Incubation of coeiomic fluids with 2-mercaptoethanol (2 ME) before SDS--PAGE results in an increase in the number of proteolytic enzymes (Fig. 3). The addition of 1.0 mM P M S F caused complete inhibition of proteolysis. Some of the proteolytically active proteins are heat-labile at 56°C. The heating of Eisenia foetida

coelomic fluids at 56°C for 30 min resulted in the disappearance of most proteolytic zones in S D S - P A G E (Fig. 4). The proteolytic activities and their heterogeneity were observed not only in Eisenia foetida coelomic fluids but also in tissue homogenates and lysates of coelomocytes. The incubation of tissue homogenates with 2 ME also caused an increase in heterogeneity of proteolytic enzymes to the level observed in Eisenia foetida coelomic fluids (Fig. 3b). DISCUSSION The presence of pronounced proteolytic activities in coelomic fluids of Lumbricus terrestris and Eisenia foetida that cleave foreign, but not own proteins, has

Fig. 3. SDS-PAGE analyses of azocaseinolytic proteins before and after addition of 2 ME to coelomic fluids of Lumbricus terrestris. (a) Lines 1,2 and 3: without; 6 and 7 (same samples as in lines 1,2): with 2 ME; 4: mol. wt standard; 5: control coelomic fluid. (b) Line I: Eiseniafoetida tissue homogenate; 2: coelomocyte lysate; 3: coelomic fluid; 4: tissue homogenate after addition of 2 ME (lines numbered from left to right).

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Fig. 4. The effect of temperature (56°C; 30 min) on proteases in coelomic fluids of Eisenia foetida. Line 1: mol. wt standard; 2: normal coelomic fluid; 3: heated coelomic fluid, as followed by SDS-PAGE.

already been published (Tu6kovfi et al., 1986a). We suggest that proteolytie activities may be involved not only in nutritive functions but also in defence mechanisms of annelids. Furthermore, antigenic stimulation of earthworms led to an increase of coelomic fluid total protein concentration (Wojdani et al., 1982; Tu6kovfi et aL, 1988) and one would expect that the increase also includes the concentration of enzymes. The experiments described above showed that the specific proteolytic activity (units activity/mg protein) was not significantly influenced either by primary or secondary stimulation (Table 1). However, when the enzymic activity, estimated independently of protein concentration, was followed it was shown that the presence of antigen led to an increase of proteolysis. We have demonstrated that coelomocytes, cultivated in vitro in the presence or absence of antigen, release proteolytic enzymes into the culture medium, and that the increased release (and probably also formation) of proteolytie enzymes is induced by antigenie stimulation (Bilej et al., 1992). In in vivo experiments it has been shown that 4 hr after administration no intact antigen is detected in Eisenia foetida and Lumbricus terrestris coelomic fluids, which is much earlier than the first formation of antigen-binding protein (ABP). This, and the results of our preliminary experiments, indicates that proteolytie enzymes in the coelomic fluid and coelomocytes play a role in antigen-processing and subsequent ABP formation that represents part of the defence system in earthworms (Tu6kovfi et al., 1991 a, b; Tu~kov~ et al., 1993). The earthworms used in the experiments were collected in different regions so their possible previous stimulation with naturally occurring antigens cannot be excluded. This fact, together with the

increase in the production of proteolytic enzymes, and their release from coelomocytes by stimulation as discussed above, can explain variations in proteolytic activities and S D S - P A G E patterns in individual earthworms (Table 1, Figs 2 and 3). The more frequent occurrence of coelomic fluids with undetectable or low proteolytic activities, observed in the Lumbricus terrestris individuals tested, can be explained by our previous findings showing that L u m bricus terrestris coelomic fluids contain protease inhibitors that, after separation, are able to inhibit the digestion of vertebrate serum proteins by Eisenia foetida coelomic fluids (Tu~kowi et al., 1986b). Coelomic fluids of Lumbricus terrestris also contain inhibitors of haemolytic activity (M6hrig et al., 1993). Interesting is the effect of 2 ME on coelomic fluid and cell lysates, which increases the number of proteolytically active proteins in SDS-PAGE. One would expect that addition of 2 ME results in the reduction of interchain disulphide bonds and leads to separation of polypeptide chains and thus increases the number of proteolytic zones. However, it is also possible that 2 ME acts as a stimulator of proteolysis and causes dissociation of enzymeinhibitor complexes. REFERENCES

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