Protein analysis of earthworm coelomic fluid IV. Evidence, activity induction and purification of Eisenia fetida andrei lysozyme (Annelidae)

Comp. Biochem. PhysioL Vol. 91B, No. 1, pp. 187-192, 1988 Printed in Great Britain

0305-0491/88 $3.00+ 0.00 © 1988PergamonPress plc

PROTEIN ANALYSIS OF EARTHWORM COELOMIC FLUID--IV. EVIDENCE, ACTIVITY INDUCTION AND PURIFICATION OF E I S E N I A FETIDA A N D R E I LYSOZYME (ANNELIDAE) F. LASSALLE, M. LASSEGUESand PH. ROCH Drpartement de Biologie du Drveloppement, Universit6 de Bordeaux I--UA CNRS 1136, Institut de Biologie Animale, Avenue des Facultrs, 33405 Talence Cedex, France

(Received 21 October 1987) Eiseniafetida andrei. 2. Background activity was 20-fold enhanced by one injection of either Gram + , G r a m - , worm pathogen or non-pathogen bacteria, SRBC. 3. Maximum induced activity occurred 4-5 hr after injection. 4. Purification has been achieved by three steps fast protein liquid chromatography (FPLC): gel filtration; chromatofocusing; and reversed phase chromatography (RPC). Only one active molecule was isolated: MW mol. wt 20,000, pI > 9.0. 5. Comparing numbers of cystein residues, E.f andrei lysozyme is close to vertebrate type c lysozyme, but its amino acid composition seems to be related to Asterias rubens lysozyme (Echinoderm). Abstract--1. Lysozyme activity was found in the coelomic fluid of the earthworm

INTRODUCTION

Invertebrate humoral immunity is mediated by several proteic systems not related to immunoglobulins or complement. Nevertheless, their large spectrum of proteins is able to neutralize invading biological agents. Differing in their structure, mode of action and site of origin, they generally do not show the specificity nor the anamnestic properties of immunoglobulins. Lysins, hemolysins and hemagglutinins have been found in molluscs, annelids, arthropods and echinoderms while naturallyoccurring antimicrobial molecules have been widely reported in insects (Ratcliffe et al., 1985). One of the best known antimicrobial lysin is lysozyme, a Gram+bacteriolytic enzyme specifically directed against the cell wall mucopeptide N-acetyl muramic acid-N-acetyl glucosamine (NAM-NAG). Lysozyme is a basic low mol. wt monomeric protein with intrachain disulfide bonds; its lysis activity is stable when heated in acidic medium and disappears when heated in an alkaline one. Lysozyme is present in the whole living kingdom: in vertebrates, type c has been isolated from hen egg-white and type g from goose egg-white; lysozyme has also been evidenced in the bacteria Bacillus subtilis, the bacteriophage T4 and the plant Papaya latex, the only lysozyme evidenced as a dimeric protein (see Table 2 for references). Lysozyme activity has been reported in many invertebrates including molluscs, insects, echinoderms, and annelids where it has been particularly studied in Nephtys hombergii (P~rin and Joll~s, 1972), Enchytraeus sp., Nereis diversicolor, Tubifex sp., Herpobdella octoculata, Haemopis sanguisuga, Allolobophora ealiginosa, Hirudo medicinalis, Arenieola marina (Schubert and Messner, 1971) and Neoamphitrite (Dales and Dixon, 1980). All these lysozymes differ by their mol.

wt, pI and amino acid composition but their mode of action is always the hydrolysis of the mucopeptide N A M - N A G . In the annelid Lumbricidae Eisenia, we previously reported the presence of hemolysins, hemagglutinins and bacteriostatins, all of them diluted in the coelomic fluid filling the general cavity of the worm body (Valembois et al., 1986). The present report shows a lysozyme-like activity in Eisenia coelomic fluid, its inducibility, purification of active protein and amino acid analysis.

MATERIALS AND METHODS

Animals and coelomic fluid extraction The earthworm Eiseniafetida andrei (E.f andrei, annelid, Oligochaeta, Lumbricidae) lives in farm manure but can be easily grown in artificial composts. All the worms used herein came from our laboratory breedings and had nearly the same age. Worms were washed, dried in soft paper, then excited with 5V stimulation inducing the animals to extrude coelomic fluid through epidermal dorsal pores. After harvesting, pooled fluid was centrifuged at 11,000g and stored at -20°C until use. Lysozyme characterization Standard assay was constituted by Micrococcus luteus (M, luteus also referred to as M. lysodeikticus) cell wall lysis. 700/~1 of 5 mg/ml M. luteus cell wall solution were diluted in 7 ml phosphate buffered agarose (pH 6.4) then spread in Petri dish. When solid, 4 mm diameter wells were sunk and filled with 20 #1 of coelomic fluid or any FPLC fraction to be tested. Lysis diameters were measured after overnight incubation at 30°C. Injection procedure To test possible induction and specificity of lysozyme activity, earthworms were anaesthetized by 4 hr at 4°C followed by 1.5 hr in crushed ice supplemented with 0.3% 187

188

F. LASSALLEet al.

chloroform. Seven groups of 15 times 35 E.f. andrei were each injected with one of the following: (l) ten pl of PBS containing 5 x 103 living Aeromonas hydrophyla ( G r a m - , worm pathogen strain); (2) ten pl of PBS containing 5 x 104 living Bacillus megaterium (Gram + , worm pathogen strain); (3) ten pl of PBS containing 106 living Acinetobacter sp. ( G r a m - , non-pathogen strain); (4) ten pl of PBS containing 105 living Micrococcus luteus (Gram + , non-pathogen strain); (5) ten /~1 of PBS containing 105 living Proteus vulgaris ( G r a m - , non-pathogen strain); (6) ten pl of PBS containing sheep red blood cells (SRBC, 5% final concentration); (7) ten /~1 of PBS. Pooled coelomic fluid from 15 times 35 worms was collected at 15 different times after injection, from 1 hr to 12 days,

Analytical polyacrylamide gel electrophoresis (SDS-PAGE) Electrophoresis analysis was performed on 15% resolution gel topped by a 7% stacking gel, prepared according to the method of Laemmli (1970). The two electrode chambers were filled with the same buffer: 25mM Tris, 192mM glycine, pH 8.5. As we used denaturing conditions, 0.12% SDS was added to the gel and the buffer, when 2% SDS and 5% mercaptoethanol were added to the sample (v/v) heated 5 min at 100°C. Migration ran at 20 mA constant current while voltage evolved from 100 to 400 volts, in a cold room until the bromophenol blue marker reached the bottom of the gel (2.5hr). SDS-PAGE was calibrated with the 6 proteins of the Pharmacia Electrophoresis Calibration Kit LMW, listed in Fig. 6. Protein bands were detected by staining the gel with Coomassie Brilliant Blue A 250 (Weber and Osborn, 1969).

Fast protein liquid chromatography (FPLC) Gel filtration. As the first step of purification, coelomic fluid was fractionated using gel filtration. Two hundred #1 of coelomic fluid (10-fold concentrated by freeze-drying) was applied to a Pharmacia column HR 10/30 containing Superose 12 gel (fractionation range 103 3 x 105 D, exclusion at 2 × 105 D). Equilibration and filtration were made with 0.05 M Tris-Cl, 0.15 M NaC1, pH 8.0, 30ml/hr. The elution profile monitored at 280 nm revealed the presence of 14 fractions. The one containing lysozyme evidenced by M. luteus tysis assay, was submitted to chromatofocusing without desalt!ng. Chromatofocusing. Best separation was obtained as follows: 700 ¢1 of active fraction were applied to a Pharmacia column Mono P HR 5/20 containing PBE 94 gel. Equilibration buffer was 25 mM bis Tris, pH 7.1 and eluent buffer Polybuffer 74, pH 4.0. Other attempts are indicated in Results. Chromatofocusing ran at 20 ml/hr. Both pH and protein (monitored at 280 nm) profiles were recorded simultaneously and revealed 6 major peaks. The one containing lysozyme was submitted to RPC without desalting. Reversed phase chromatography (RPC). The final purification step was by hydrophobic interaction separation created by acetonitrile gradient through gel ProRPC. Three ml of active peak obtained from chromatofocusing were applied to a Pharmacia column HR 5/10. Buffer A was 0.1% TFA in water and buffer B 0.1% TFA in 90% acetonitrile, 10% water. Using only 90% acetonitrile in buffer B enabled us to avoid bubble formation when buffer B mixed with buffer A. RPC was run at 15 ml/hr. Fortunately, lysozyme activity was not suppressed by organic solvent, as we expected, and could be recovered after acetonitrile elimination by freeze-drying. After any FPLC separation, each tube was submitted to M. luteus lytic assay in order to identify the fraction

containing lysozyme. A second control consisted of purity checking by analyzing the fraction containing lysozyme in SDS PAGE. Amino acid analysis. Quantitative amino acid analysis was made with a Kontron Liquimat III Labotron. Samples of purified lysozyme were hydrolyzed at 110~'C for 24 hr in 1 ml 6 N HC1 in a tube sealed under vacuum. After vacuum evaporation of HC1, the amino acids were resuspended in 0.5 ml sodium citrate buffer, pH 2.2 norleucine was added as internal standard. Cystein was determined as cysteic acid and methionine as methionine sulphone.

RESULTS

Lysozyme-like activity M. luteus lytic activity exists in El. andrei coelomic fluid at a c o n c e n t r a t i o n of a b o u t 6 0 - 1 0 0 ~ g / m l equivalent hen egg-white lysozyme, according to individual variation. Table 1 presents p H conditions of lysozyme-like activity stability. After 3 min at 100~C, the activity was just recovered when reconstituted in acidic medium. H e a t i n g in alkaline m e d i u m induced lability. M. luteus lytic assay has been achieved at E~/~ andrei coelomic fluid pH, but at several temperatures, in order to d e m o n s t r a t e the enzymic nature of the lytic protein. Figure 1 shows that reaction velocity was t e m p e r a t u r e - d e p e n d e n t , which is one characteristic of enzymic reaction. Activity induction In worms s u b m i t t e d to a single bacteria strain or S R B C injection, lysozyme activity was 20-fold enhanced. M a x i m u m activity was reached 4--5 hr afterwards, c o r r e s p o n d i n g to 750-2000 # g / m l equivalent according to the injected bacteria strain. N o r m a l titer was recovered 6-8 hr after injection (Fig. 2) and r e m a i n e d c o n s t a n t during at least 12 days.

L ysozyme purification Gelfiltration. Elution with Tris-NaC1 excluded E f andrei respiratory pigment or e r y t h r o c r u o r i n (mol. wt 3.82 x 106 according to Jamieson, 1981) in the void volume while antibacterial yellow pigment (Lassalle a n d Lass~gues, 1986) was eluted at the end o f total c o l u m n volume. So, gel filtration eliminates the two m a j o r pigments a n d also the viscosity associated with mucopolysaccharides. Lysozyme activity has been located in fraction 4 (Fig. 3) but S D S - P A G E revealed the presence of several protein species. Consequently, fraction 4 was submitted to the second F P L C purification step; w i t h o u t desalting. Chromatofocusing. Second-step purification was attempted with 3 different buffer systems. Originally we Table 1. Earthworm lysozyme-likeactivity stability according to pH and heating (determined by M. luteus lysis assay) Activity Samples pH (,ug/ml eq.) Crude coelomic fluid 7.2 60 100 Lyophylized coelomic fluid reconstituted in PBS 7.2 75 Lyophylized coelomic fluid reconstituted in phosphate buffer 4.5 75 Same sample heated at 100"C, 3' 4.5 75 Lyophylized coelomic fluid reconstituted in phosphate buffer 8.5 75 Same sample heated at 100"C, 3' 8.5 0

E.f. andrei l y s o z y m e

E H

!9 ~3

-

•

20*C

~3

2

4

24

M Luteus

48

Lytic

assay

incubation time (hrs)

Fig. 1. Temperature-effect on lysozyme kinetic reaction in E.f andrei crude coelomic fluid. H

2000

--

189

used imidazole 25 mM, pH 7.4 as equilibration buffer and PB 74, pH 4.0 as eluent buffer. But Shinitzky et al. (1966) suggested imidazoles strongly inhibit lysozyme activity. Although contradicted by Powning and Davidson (1976), we chose to use routinely another equilibration buffer: bis-Tris 25 mM, pH 7.1. In this system, lysozyme was eluted before pH gradient was established (Fig. 4) but remained contaminated by other proteins. The third buffer system we assayed, Tris 75mM, pH9.3 as equilibration buffer and PB 96, pH 6.0 as eluent buffer, created a pH gradient from 9.3 to 6.0. Even in this case, lysozyme was eluted before pH gradient formation and was still contaminated by other proteins. So, E.f. andrei lysozyme pI is above 9.0, out of chromatofocusing performance range.

Hydrophobic interaction chromatography (HIC) and reversed phase chromatography (RPC)

I000-

Active chromatofocusing peak was submitted to HIC and RPC without any desalting. Because RPC necessitates organic solvent use to create hydrophobicity gradient and because such solvents prevent enzymic activity, we first chose to separate lysozyme by HIC. Such chromatography uses the same sepaI00 ration criterium as RPC but hydrophobicity gradient is created by phosphate buffer supplemented with I I I I I I / /p 2 3 4 5 6 7 24 ammonium sulfate. Unfortunately, earthworm lysoHours after injection zyme was eluted through phenyl-superose matrix in Fig. 2. Kinetic of lysozyme activity enhancement in coelo- 2 M ammonium sulfate and then necessitates a demic fluid after injection of 5 × 103 Aeromonas hydrophila (0), 5 x 104 Bacillus rnegaterium (©), or 106 Acinetobacter salting step. As E.f. andrei lysozyme activity is not suppressed by organic solvent, lytic enzyme was sp. (~). submitted to acetonitrile gradient elution. In this system, lysozyme was isolated in 35% acetonitrile, 0.1% T F A without other protein contamination I -(Fig. 5). One advantage of this procedure was the use of volatile solvents (acetonitrile, TFA) which are easily eliminated by freeze-drying. Purity checked by S D S - P A G E indicated that E,f. andrei lysozyme has a tool. wt about 20,000 (Fig. 6). 5 0 0 -

Amino acid composition

0,5

I

3

5

7'9

II

13

II

{

l]

I [

I

,;¢ J

Vo

Vr

y/

I

Purified lysozyme was then submitted to amino acid analysis. Table 2 presents the composition of different types of lysozyme: vertebrate type c (hen egg-white) and type g (goose egg-white); insects such as Hyalophora cecropia with the pioneering study of Boman et al. (1986) and Galleria melonella, the closest type c amino acid sequence in invertebrates (Joll+s and Joll6s, 1984); echinoderms such as Asterias rubens; annelids such as Nephtys hombergi; plants such as Papaya latex and bacteriophage T4. E.f. andrei lysozyme presents two common characteristics with the other ones: mol. wt below 25,000 and basic pI above 9.0. Its amino acid composition is remarkable for its important number of proline (14--15) and methionine (10-11) residues.

Etution fractions

2 x 106 MW I03MW

Fig. 3. Gel filtration. Sample: 200/~1 of 10-fold concentrated coelomic fluid. Column Pharmacia HR I0/30, gel Superose 12. Elution conditions: buffer 0.05M Tris~l, pH 8.0, 0.15 M NaCI; flow rate 30 ml/hr. Elution profile measured by absorption at 280nm. Lysozyme activity located in fraction 4 (dark). Erythrocruorin in fraction 1, yellow pigment in fraction 5.

DISCUSSION

(~otuk and Dales (1984) reported the existence of active E. fetida lysozyme in cell extract and resuspended cells but only slightly expressed in coelomic fluid. In the same annelid, Kauschke and Mohrig (1987) also failed to evidence any lysozyme activity in crude coelomic fluid. Using a method based, as the

190

F. LASSALLEet al. 0.5 --8

7

/

I I

I

- - 6 3 = O.

,f

:

;

0.25

5

t !

4

Ekution peaks

Fig. 4. Chromatofocusing. Sample: 700/~1 of active peak eluted from gel filtration. Column Pharmacia Mono P HR 5/20, gel PBE 94. Elution conditions: start buffer 0.025 M bis-Tris, pH 7.1, HCI; eluent buffer Polybuffer 74, pH 4.0, HC1; flow rate 20 ml/hr. Elution profile measured by absorption at 280 nm (solid line) and pH gradient measured in fractions leaving the column (dotted line). Lysozyme in dark peak.

one of (~otuk and Dales, on M. luteus cell wall lysis in agar, we report significant lysozyme-like activity in E.f. andrei coelomic fluid.

E.f. andrei lytic enzyme has been classified as a lysozyme according to its lyric activity against M. luteus (also referred to as M.lysodeikticus) cell wall, heat stability at acidic pH and lability at basic pH (characteristics given by Joll6s and Zuili, 1960). (~otuk and Dales (1984) thought E. fetida lysozyme had a mol. wt of 15,000 because crude coelomic fluid separated by SDS-PAGE presented a prominent band at 15,000 co-migrating with hen egg-white lysozyme. We show herein by SDS--PAGE of purified

lytic enzyme, that El. andrei lysozyme mol. wt is about 20,000. Lysozyme activity can be enhanced by various injections. The induction response is not specific since Gram + , Gram - , pathogen and non-pathogen bacteria, as well as SRBC, provoked its appearance. Moreover, any induction response exhibited the same

kinetic, with a peak at 4--5 hr after injection. As

controls, PBS and sham-injection, as well as anaesthaesia, did not induce any titer modification. E.f. andrei lysozyme activity enhancement represents a rapid and fugitive phenomenon. Disappearance of activity enhancement 6-8 hr after injection can explain the contradictory results of (~otuk and Dales (1984) studying induction only 24 hr post-injection, while normal titer is recovered. Purification has been achieved by fast protein liquid chromatography. While hen egg-white lyso-

zyme can be purified by ion exchange chromatography or crystallization in a one-step method, it is necessary to use a three-step chromatography procedure to isolate E l . andrei lysozyme from other proteic and lipidic components of the coelomic fluid.

Amino acid composition represents a necessary step in comparing E.f. andrei lysozyme to lysozymes

from other sources. It is important to notice that the E.f. andrei one is close to type g according to tool. wt --I00

iI

0.2--

/*

I

I /I

80

iI

I o

iI

1

/ I

6O

iI

m

I

0.1

m --40

iI iI iI

i/I/lll~

-

20

',,_.... 0

/

I L .......

O

ELution peaks

Fig. 5. Reversed Phase Chromatography. Sample: 3 ml of active peak eluted from chromatofocusing. Column Pharmacia HR 5/10, gel ProRPC. Elution conditions: buffer A 0.1% TFA in water; buffer B 0.1% TFA in 90% acetonitrile 10% water; flow rate 15 ml/hr. Elution profile measured by absorption at 280 nm (solid line) and hydrophobicity gradient measured at the top of the column (dotted line). Lysozyme in dark peak.

E.f. andrei lysozyme

191

Table 2. Amino acid composition of lysozymes (residues)

LYS HIS ARG ASX GLX GLY ALA VAL LEU ILE PRO SER THR CYS* METt TYR PHE TRP Total

Eisenia fetida

Hen

Goose

Hyalophora cecropia

7-8 3-4 5--6 13 21 13 8 5-6 9-10 5 14-15 8-9 6-7 7-8 10-11 0 3 N.D. 137-147

6 1 11 21 5 12 12 6 8 6 2 10 7 8 2 3 3 6 129

18 5 10 20 15 21 15 11 7 14 4 10 13 4 3 9 3 3 185

12 3 8 18 8 10 4 6 8 5 2 7 7 8 1 4 5 4 120

.4sterias rubens

Galleria mellonella

Nephtys hombergii

9-10 2-3 6-7 12 9 15 10 9-10 8 5 10-11 9-10 7 12 5 5-6 3 3 139-146

15 2 10 20 10 8 7 6-7 9 5-6 4 6-7 7 8 1 4 4 5 131-134

10 2 6± 1 1-2 2-3 124 __.6

Jollrs & Jollrs (1975) 15.5 + 1 .

Powning & Davidson (1976) --

Prrin & Jollrs (1972) 15

Papaya latex

11 + 1 3-4 9 _+ I 11-12 9-10 14 8 6 4 5 6-7 10

Phage T4

10 3 13 23 11 27 19 7 12 11 18 14 13 8 4 12 11 7 223

13 1 13 22 13 I1 15 9 16 10 3 6 11 2 5 6 5 3 164

Howard & Glazer (1967) 25 10.5

Tsugita

4

*Determined as cysteic acid. "tDetermined as methionine sulphone. Amino acid composition published by MW (kDa) pl

Imoto et al. (1972) 14.6 + 0.2 10.5-11

20 >9.0

Simpson & Morgan (1983) 20.5 .

Engstrfm et al. (1985) 13.8 . .

and close to type c according to the number of cystein residues. Cystein residues pairing enables disulfide bonds formation determining the enzyme tertiary structure. So, positive correlation between E.f. andrei and type c lysozymes is important. Nevertheless, when comparing the number of each amino acid residue, E.f. andrei lysozyme is closest to the Asterias

a

b

c

d

.

(1971) 18 --

rubens one: they present similarities in number of basic amino acids, aromatic ones (except for tyrosine), proline and aspartic residues. Now, Jollrs and Jollrs (1975) defined the latter 'to be the representative of a new type of lysozyme found among the invertebrates'. It will be interesting to determine precisely the degree of homology between these two

e

f

g

h

Fig. 6. S D S - - P A G E of: a, P h a r m a c i a E l e c t r o p h o r e s i s C a l i b r a t i o n Kit ( p h o s p h o r y l a s e b 94 kD, a l b u m i n 67 k D , o v a l b u m i n 43 kD, c a r b o n i c a n h y d r a s e 30 k D , trypsin i n h i b i t o r 20.1 k D , ~t-lactalbumin 14.4 kD); b, n a t i v e c o e l o m i c fluid; c, frozen c o e l o m i c fluid; d, fraction 4 eluted f r o m gel filtration; e, first p e a k eluted from c h r o m a t o f o c u s i n g ; f, lysozyme eluted from R P C ; g, hen egg-white lysozyme; h, c a l i b r a t i o n kit.

F. LASSALLEet al.

192

lysozymes. In this way, complete a m i n o acid sequence a n d cloning in c D N A a n d genornic libraries will be developed.

Acknowledgements--The authors thank the Laboratoire de Biochimie et Technologie des Aliments, Universit6 de Bordeaux I, for amino acid analysis. REFERENCES

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Joll6s P. and Zuili S. (1960) Purification et 6tude compar6e de nouveaux lysozymes extraits du poumon de poule et de Nephtys hombergi. Biochim. biophys. Acta 39, 212 217. Kauschke E. and Mohrig W. (1987) Cytotoxic activity in the coelomic fluid of the annelid Eisenia fetida Sav. J. comp. Physiol. B 157, 77 83. Laemmli U, K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 68(~685. Lassalle F. and Lassegues M. (1986) Humoral defences in earthworms: lysozyme and yellow pigment, lmmunobiology 173, 231. P+rin J. P. and Joll6s P. (1972) The lysozyme from Nephtys hombergii (Annelid). Biochim. biophys. Acta 263, 683 689. Powning R. F. and Davidson W. J. (1976) Studies on insect bacteriolytic enzymes. If--Some physical and enzymatic properties of lysozyme from haemolymph of Galleria mellonella. Comp. Biochem. Physiol. 5fiB, 221 228. Ratcliffe N. A., Rowley A. F., Fitzgerald S. W. and Rhodes C. P. (1985) Invertebrate immunity: basic concepts and recent advances. Int. Rev. Cytol. 97, 183 350. Schubert I. and Messner B. (1971) Untersuchungen uber das vorkommen yon lysozym bei anneliden. Zool. Jb. Physiol. Bd 76, 3f~50. Shinitzky M., Katchalski E., Grisaro V. and Sharon N. (1966) Inhibition of lysozyme by imidazole and indole derivatives. Archs Biochem. Biophys. 116, 332 343. Simpson R. J. and Morgan F. J. (1983) Complete amino acid sequence of Embden goose (Anser anser) egg-white lysozyme, Biochim. biophys. Acta 744, 349 351. Tsugita A. (1971) Phage lysozyme and other lytic enzymes. In The Enzymes, 3rd Edn, pp. 343411. Academic Press, New York. Valembois P., Roch Ph. and Lass6gues M. (1986) Antibacterial molecules in annelids. In lmmuniU' in Invertebrates (Edited by Br6helin M.), pp. 74-93. Springer, Berlin. Weber K. and Osborn M. (1969) The reliability of molecular weight determinations by dodecyl sulfate polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406-4412.