PRELIMINARY ANALYSIS OF THE PROTEOLYTIC ENZYMES IN THE EXCRETORY-SECRETORY PRODUCTS OF THE ADULT STAGES

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PRELIMINARY ANALYSIS OF THE PROTEOLYTIC ENZYMES IN THE EXCRETORY-SECRETORY PRODUCTS OF THE ADULT STAGES

OF THE DOG HOOKWORM UNCINARIA STENOCEPHALA

KOTOMSKI G.* & WEDRYCHOWICZ H*

S u m m a r y :

The paper describes an introductory characterisation of proteinases present in the excretory-secretory products (ESP) of adult Uncinaria slenocephala. In S D S - P A G E gelatine substrate gels E S P resolved as a s i x bands of proteolytic activity, with a molecular weight of 1 8 2 , 1 5 9 , 9 8 , 5 0 , 3 9 and 2 6 kDa. The 9 8 and 3 9 kDa components were serine proteinases. The 5 0 kDa band w a s sensitive to a metalloproteinase inhibitor. The 2 6 kDa component w a s highly sensitive to cysteine proteinase inhibitors and w a s also partially inhibited in the presence of E D T A . The bands of 1 8 2 and 1 5 9 kDa were sensitive to a Z n -

metalloproteinase inhibitor.

The enzymes present in E S P showed the highest proteolytic activity at p H 8 - 9 . Quantitative analysis revealed maximum proteolytic activity of the polypeptides of 1 5 9 and 1 8 2 kDa at p H 7 ; 9 8 and 2 6 kDa at p H 8 while the 5 0 kDa and 3 9 kDa components showed the highest activity at p H 9 .

KEY WORDS : hookworms, excretory-secretory products, proteinases, Uncinaria sienocephala.

Résumé :

ÉTUDE PRÉLIMINAIRE DES CARACTÉRISTIQUES DES PROTÉASES PRÉSENTES DANS LES PRODUITS D'EXCRÉTION-SÉCRÉTION D'UNCINARIA STENOCEPHALA ADULTE

Uncinaria stenocephala est un parasite des canidés appartenant à la famille des ankylostomes. On ignore toujours la composition et la fonction des produits d'excrétion-sécrétion (ESP). Cet essai démontre les caractéristiques préliminaires des proteases présentes dans les ESP d'U. stenocephala adulte.

En utilisant la méthode de l'électrophorèse SDS-PAGE, on a découvert six protéines présentant des activités protéolytiques différenciées, de masse moléculaire : 182. 159, 98, 50, 39 et 26 kDa dans le gel-substrat, les épreuves avec les inhibiteurs spécifiques des proteases ont démontré que les composants de masse 98 et 39 kDa appartiennent aux proteases à serine, ta bande 50 kDa a des propriétés de métalloprotéase. la bande 26 kDa démontre des caractères de protease à cysteine. Les composants 182 et 159 semblent être des métalloprotéases à zinc. Les enzymes découvertes dans les ESP montrent une plus grande activité à pti 8-9.

MOTS CLÉS : ankyiostome, produits d'excrétion-sécrétion, protéases, Uncinaria stenocephala.

INTRODUCTION

N

e m a t o d e s release a variety o f proteins with e n z y m a t i c p r o p e r t i e s w h i c h , in vivo, a r e believed to play important roles in the host- parasite relationship. Among the specific functions attri- b u t e d to the e x c r e t o r y - s e c r e t o r y products ( E S P ) of n e m a t o d e s are invasion and migration through host tissues, facilitation o f feeding and evasion o f host immune responses (Tort et al, 1999), S o m e proteinases secreted by nematodes have b e e n shown to b e inhibited by host antibodies. This is particularly important b e c a u s e it indicates that proteinases o f parasite origin constitute targets for the host's immune responses and suggests a w a y through which immunotherapeutic intervention m a y protect the host from disease (Tort et al, 1999).

Uncinaria stenocepbala is a c a n i n e h o o k w o r m w h i c h is prevalent in dogs all over the world (Arene, 1984;

* Department of Parasitology, Faculty of Veterinary Medicine, Warsaw Agricultural University, Grochowska 272, 03-849 Warsaw, Poland.

Correspondence: H. Wedrychowicz.

T e l / F a x : +48 22 810 18 51.

E-mail: Wedrychowicz@alpha.SGGW.waw.pl

B l a k e & Overend, 1982; Gorski et al, 1996; Vanparijs et al, 1991). Attempts to understand a molecular patho- biology o f h o o k w o r m infections have largely c o n c e n - trated o n Ancylostoma caninum in dogs and o n the human parasites ( B r o w n et al, 1995; Hotez et al, 1995;

Furmidge et al, 1995; Pritchard et al, 1990). In contrast, U. stenocephala has received scarce attention and very little is k n o w n about molecular interactions in this host- parasite relationship, including enzymes secreted by the parasite. In this report w e describe the range o f pro- teolytic activities present in the ESP o f adult U. steno- cepbala and w e provide a preliminary characterization o f the principal e n z y m e s involved.

MATERIAL AND METHODS

COLLECTION OF THE EXCRETORY-SECRETORY PRODUCTS (ESP)

A

dult nematodes w e r e isolated from the intestines of experimentally infected dogs, washed exten- sively in 0.15 M sodium chloride containing 100 i.u/ml penicillin and 100 ug/ml streptomycin and

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KOTOMSKI G. & WEDRYCHOWICZ H.

then left over a period o f 3 h at 37° C in RPMI 1 6 4 0 containing 100 i.u./ml penicillin and 100 pg/ml strep

tomycin in order to permit loss o f any remaining host tissues from the worms' intestines ( B r o w n ET AL, 1995).

Following this "cleaning" period adult worms w e r e cul

tured in sterile RPMI 1640, enriched with antibiotics (as above), for a further 24 h at 37° C. As assessed by moni

toring worm motility, the parasites remained viable in culture. T h e culture fluid containing ESP was dialysed against distilled water pH 7.0, freeze dried and recons

tituted in distilled water prior to use (Brown ET AL, 1995).

MEASUREMENT OF PROTEINASE ACTIVITY

Proteinases w e r e detected following their e l e c t r o p h o - retic separation using gelatin-SDS PAGE assay system (Hotez et al, 1990; Hawdon et al, 1995) which consisted o f 12 % w / v acrylamide gels containing 0.2 % w / v gelatin. TRIS-HC1 buffer pH 8.8 and 0.1 % ( w / v ) SDS.

Typically 10 pg o f total protein was separated per lane o f gels. Following electrophoresis gels w e r e incubated at 25° C in 25 % v / w Triton X - 1 0 0 for 1 h and then in glycine buffer ( 0 . 0 5 M, pH 6 - 8 ) with or without pro

teinase inhibitors at 37° C.

CHARACTERIZATION OF PROTEINASES USING PROTEINASE INHIBITORS

T h e proteinase inhibitors w e r e obtained from Sigma- Aldrich Germany and applied at concentrations reported by Kumar & Pritchard ( 1 9 9 2 ) , Hawdon et al. ( 1 9 9 5 ) and Cordova et al. ( 1 9 9 7 ) . T h e inhibitors used w e r e as fol

lows: 1,10 phenanhtroline (2 mM; Zn-metalloproteinase inhibitor); ethylendiaminetetracetic acid (EDTA; 10 mM;

metalloproteinase inhibitor); phenylmethanesulphonyl fluoride (PMSF; 10 mM & 1 mM, serine proteinase inh- bibitor); pepstatin ( 7 0 pM, aspartic proteinase inhibitor),

Nethylmaleimide (NEM; 10 or 5 mM, cysteine protei

nase inhibitor); iodoacetamide (IA; 5 mM, cysteine pro

teinase inhibitor). T h e assays w e r e conducted at pH 7.0.

ESTIMATION OF THE PH OPTIMA FOR PROTEINASE ACTIVITY

Assays w e r e c o n d u c t e d with a pH range from 3 to 11.

After electrophoresis, gels w e r e immersed in different buffers prepared as described by D o w d et al. ( 1 9 9 4 ) and incubated at 37° C. After the incubation, the gels w e r e s c a n n e d then area o f proteolysis w e r e measured and c o m p a r e d using a c o m p u t e r program "Diversi- t i O n e ™ version 1.3 (pdi). T h e p r o g r a m m e calculates the proteolytic activity index by measuring the area and intensity o f substrate degradation.

RESULTS

S

ix bands o f proteolysis at 182, 159, 9 8 , 50, 39 and 26 kDa were observed in substrate gels incubated without inhibitors (Fig. 1, lane A). Several proteinase inhibitors affected the proteolysis associated with com

ponents of differing molecular weight (Fig. 1B-G, Table I).

T h e 182, 159, 5 0 and 2 6 k D a bands w e r e absent from gels incubated in the p r e s e n c e o f EDTA (metallopro-

Proteolytic activity

P r o t e i n a s e 182 159 98 50 39 26

inhibitor kDa kDa kDa kdA kDa kDa

No inhibitor + + + + + +

EDTA

10 mM - - ⁺ - ⁺ ^{+ / -}

1,10 phenathroline

2 mM - - ⁺ ⁺ ⁺ ⁺

PMSF

10 mM _{1 mM} -₊ -₊ -_- -+ --

-

^I

Pepstatin

70 mM + + + + + +

N-ethylmaleimide

10 mM + + / - +/— + -

5 mM + + + +

-

⁺

Iodoacetamide

5 mM + + + + +

Table I. - Sensitivity of proteinases secreted by adult (7. stenocephala to inhibitors.

Fig. 1. - Characterization of gelatinolytic proteinases in the excre

tory-secretory products (ESP) of adult Uncinaria stenocephala using proteinase inhibitors.

Lanes: M - molecular weight markers (kDa); A - the SDS-PAGE pro

file of gelatinolytic proteinases in U. stenocephala ESP (no inhibitor);

B - metalloproteinase inhibitor (EDTA); C - Zn-metalloproteinase inhi

bitor (1,10-phenanthroline); D - serine proteinase inhibitor (phe

nylmethanesulphonyl fluoride); E - aspartic proteinase inhibitor (pepstatin); F - cysteine proteinase inhibitor (N-ethylmaleimide);

G - cysteine proteinase inhibitor (iodoacetamide).

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teinase inhibitor, F i g . I B ) while Zn metalloproteinase inhibitor ( 1 , 1 0 phenanthroline) c a u s e d deprivation o f the proteolytic activity o f 182 and 159 kDa components (Fig. 1C). In the presence o f 10 mM PMSF only 182 and 159 bands exhibited a weak activity (Fig. I D ) . However, w h e n the incubation buffer contained 1 mM PMSF only 9 8 and 3 9 kDa bands w e r e inhibited (Table I ) . N o n e o f the ESP proteinases w e r e affected b y the aspartic proteinase inhibitor (Fig. I E ) . 10 mM NEM b l o c k e d the enzymatic activity o f the 2 6 kDa and atte

nuated 159 k D a and 9 8 k D a bands (Fig. I F ) . W h e n l o w e r concentration o f the inhibitor was used only the proteolytic activity o f 26 kDa declined (Fig. 1G, Table I ) . T h e optimal pH conditions for proteolysis by ESP were evaluated and quantitative analysis revealed maximum proteolytic activity o f the polypepetides o f 182 and 159 kDa at pH 7.0; those o f 9 8 and 26 kDa at pH 8 while the 50 kDa and 39 kDa components s h o w e d higher acti

vity at pH 9- No proteolytic activity was observed at pH 10 and 11. T h e results are shown in Figure 2.

DISCUSSION

O

ur e x p e r i m e n t s demonstrated that excretory- secretory products o f adult U. stenocephalia include a heterogeneous mixture o f proteolytic

activities. T o our knowledge, there have b e e n n o reports o n proteinases o f U. stenocephala. Reports c o n c e r n i n g proteolytic activities found in the ESP o f other h o o k w o r m s are limited and a m o n g those w h i c h have b e e n described so far, differences in structure and function have b e e n reported ( D o w d et al, 1994; Hotez et al, 1985; Kumar & Pritchard, 1992; Pritchard et al, 1 9 9 0 ; Tort et al, 1 9 9 9 ) .

Results obtained using various proteinase inhibitors strongly suggested lack o f aspartic proteinases in the ESP o f adult U. stenocephala. T h e 26, 50, 159 and 182 kDa c o m p o n e n t s lost their proteolytic activities in the pre

s e n c e o f EDTA (metalloproteinase inhibitor), while 1,10 phenathroline which is a strong Zn atoms chelator, inhibited only 159 and 182 k D a bands. B e c a u s e EDTA is much less effective against Zn and more effective in chelating Ca, the results s e e m to suggest that the 159 and 182 k D a bands contain Zn metalloproteina- ses, w h e r e a s the 5 0 and 2 6 kDa c o m p o n e n t s include enzymes utilising Ca or another cofactor ( B o n d & Butler, 1 9 8 7 ) . An activity o f m e t a l l o p r o t e i n a s e s has b e e n detected in ES products o f other h o o k w o r m species. For instance, H a w d o n et al. ( 1 9 9 5 ) found metalloprotei

nases o f 5 0 and 9 0 kDa in ES products o f A. caninum.

Diverse results w e r e o b t a i n e d in the present experi

ment using serine proteinase inhibitor (PMSF). W e ini

tially used the 10 mM concentration o f PMSF since

Fig. 2. - Results of computer analysis of effects of pH on proteolytic activity of enzymes present in excretory-secretory products of adult U. stenocephala. PA = proteolytic activity.

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EXCRETORY-SECRETORY PRODUCTS OF UNCINARIA STENOCEPHALA

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K O T O M S K I G . & W E D R Y C H O W I C Z H .

Kumar & Pritchard ( 1 9 9 2 ) reported that only 10 mM but not 1 mM o r 5 mM PMSF inhibited ESP proteinases o f Necator americanus. However, t h e 1 0 mM PMSF b l o c k e d entirely the proteolytic activity o f ESP o f U. ste

nocephalia. T h e r e f o r e w e repeated e x p e r i m e n t s using 1 mM PMSF and found that proteolytic activity o f only 39 a n d 9 8 kDa c o m p o n e n t s w a s inhibited. T h e c o m p l e t e inhibition o f p r o t e o l y t i c activity o f t h e E S P observed in p r e s e n c e o f 10 mM PMSF resulted possibly from a non-specific inhibition ( B o n d & Butler, 1 9 8 7 ) . Similarly, a non-specific inhibition h a s probably also o c c u r r e d in test using 10 mM NEM. T h e 10 mM NEM affected proteolytic activity o f three c o m p o n e n t s ( 1 5 9 , 9 8 and 2 6 k D a ) while 5 mM concentration o f the inhi

bitor b l o c k e d activity only o f the 26 k D a c o m p o n e n t . The 2 6 k D a b a n d was also sensitive to 1A a n d EDTA inhibition. O n the strength o f the fact that both IA and NEM, w h i c h are cysteine proteinase inhibitors, c a u s e d similar effect and that EDTA is a strong metal chelator, w e c a n tentatively c o n c l u d e that t h e 26 k D a c o m p o nent b e l o n g s t o cysteine class o f proteinases and pos

s e s s e s a metal cofactor. T h e metal ions are often required for t h e activity o f other than metalloprotei- nases classes ( B o n d & Butler, 1 9 8 7 ) . T h e proteolytic activity o f the 26 k D a c o m p o n e n t was most p r o n o u n ced at pH 8 although a clear activity was also observed at pH 7 a n d 9. Similar p H values have b e e n reported by D o w d et al., ( 1 9 9 4 ) a n d B r o w n et al. ( 1 9 9 5 ) for cysteine proteinases secreted b y adult A. caninum.

The results o f o u r preliminary analysis o f the ESP o f adult U. stenocephala suggest that serine a n d metallo- proteinases a c c o u n t for the most p r o n o u n c e d proteo

lytic activity in the ESP o f the n e m a t o d e . However, fur

ther investigations o f t h e p r o t e i n a s e s r e l e a s e d b y U. stenocephala are n e e d e d in order t o characterise their role in p a t h o g e n e s i s a n d i m m u n o b i o l o g y o f t h e n e m a t o d e infection.

ACKNOWLEDGEMENTS

This w o r k w a s supported b y Polish Committee for Scientific R e s e a r c h (Grant K B N Nr 5 P O 6 K 0 2 1 1 3 ) .

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Reçu le 1 0 avril 2 0 0 0 Accepté le 2 1 septembre 2 0 0 0

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