Molecular and Biochemical Parasitology, 18 (1986) 17-24 17 Elsevier

MBP 00610

T U R N O V E R O F T H E S U R F A C E P R O T E I N S O F A D U L T A N D T H I R D A N D

F O U R T H S T A G E L A R V A L B R U G I A P A H A N G I

ELIZABETH MARSHALL and ROBERT E. HOWELLS

Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, U.K.

(Received 16 May 1985; accepted 5 August 1985)

The turnover of surface proteins in adults, fourth-stage and third-stage larvae of Brugia pahangi was measured using [1251]iodosulfanilic acid. Groups of worms (n = 10 adult, 20 L4, 50 L3) were labelled and surgically implanted into the peritoneal cavity of naive jirds. The amount of radioactivity remaining on worms recovered over a 7-8 day period was determined. Adult females showed no significant loss of label during a 7 day period. The recovery of fourth-stage larvae was low but the counts per minute remaining on each group of larvae recovered over an 8 day period, encompassing the major part of the instar, did not fall below the limits of the standard deviation of the time 0 groups, indicating that no significant loss of surface label had occurred. Third-stage larvae showed a significant loss of ~25I-labelled proteins prior to the third moult, although it was not confirmed that these proteins occur on the worm surface. Electrophoresis and autoradiography of labelled homogenates of adult, fourth and third stage larvae suggested that [12~I]iodo- sulfanilic acid labels polypeptides of different molecular weights on each life cycle stage of B. pahangi.

Key words: Brugia pahangi; Filaria; Cuticle; Surface proteins; [~251]iodosulfanilic acid

INTRODUCTION

S t u d i e s o n Trichinella spiralis [1] a n d Toxocara canis [2] u s e d r a d i o i o d i n a t i o n

t e c h n i q u e s to d e m o n s t r a t e t he loss o f l a b e l l e d p r o t e i n s f r o m the su r f ace o f t he se

w o r m s . T h e r e su l t s s u g g e s t e d t h a t in t h e s e n e m a t o d e spec ies , t he cu t ic le is a d y n a m i c

s t r u c t u r e in w h i c h p r o t e i n s a re c o n t i n u a l l y b e i n g s h e d a n d r e p l a c e d . H o w e v e r , m o r -

p h o l o g i c a l s t u d i e s o n t h e f o u r t h - s t a g e l a r v a e o f t he f i l a r i id n e m a t o d e Brugia pahangi

s u g g e s t e d t h a t t h e cu t i c l e o f t h i s spec ies is a r e l a t i ve ly s t a b l e s t r u c t u r e w i t h a l i m i t e d

c a p a c i t y f o r t u r n o v e r o f s u r f a c e c o m p o n e n t s [3]. T h e a i m o f th i s s t u d y was to

d e t e r m i n e w h e t h e r o r n o t s u r f a c e p r o t e i n s were los t f r o m d i f f e r e n t life cycle s t ages o f B.

pahangi. S u r f a c e p r o t e i n s we re l a b e l l e d u s i n g the h i g h l y s u r f a c e - s p e c i f i c r e a g e n t

[125I] iodosul fani l ic a c id (125ISA) [4].

Abbreviations: cpm, counts per minute; 12~ISA, [t25I]iodosulfanilic acid; SDS, sodium dodecyl sulfate.

0166-6851/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

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MATERIALS AND METHODS

Maintenance of parasites. Mosquitoes (Aedes aegypti SS) were infected by feeding on a suspension of microfilariae in stock rabbit blood, using the membrane feeding method of Wade [5]. Thirteen days post infection the mosquitoes were lightly crushed and the infective third-stage larvae recovered [6]. Jirds (Meriones unguiculatus) were infected by the intraperitoneal injection of 100 infective larvae. Fourth-stage larvae were recovered 8 days post infection. Patent adult worms were recovered at least 3 months post infection.

lodosulfanilic acid labelling. Iodosulfanilic acid (NEN) was diazotised according to the manufacturer's instructions. Following diazotisation 0.2 ml of phosphate-buffered saline (PBS) containing 0.1 mCi of the reagent was dispensed into centrifuge tubes and groups of 10 adult worms were added to each tube. In the case of the larvae the concentration was adjusted so that groups of 200 fourth-stage larvae were iodinated using 0.5 mCi of 125I or 103 third-stage larvae using 0.5 mCi in 0.2 ml of PBS. The reaction was performed on ice for 15 min and was stopped by the addition of 0.8 ml of Hanks' Balanced Salt Solution (HBSS) containing bovine serum albumin (1 mg ml-~). The worms were washed once in 10 ml of this solution for 5 min and then given a further 3 washes in 15 ml of prewarmed (30-37°C) HBSS.

Labelling with Bolton and Hunter reagent. Adult female worms were labelled using ~25I-labelled Bolton and Hunter reagent as described by Marshall and Howells [4].

Measurement of radioactivity. Adult worms were counted individually using a Packard multi prias gamma counter. Fourth-stage larvae were counted in groups of 20 and third-stage larvae in groups of 50. Radioactivity was expressed in counts per minute (cpm) and corrected for the decay of ~25I.

Electrophoresis and autoradiography. Groups of 10 labelled worms were homogeniz- ed on ice in 1-ml glass-glass homogenizers (Jencons Ltd.) in 0.1 ml of 10 mM Tris-HCl buffer containing 10 lal of 5% sodium deoxycholate, 5 lal of a 100 mM solution of the protease inhibitor phenyl methyl sulphonyl fluoride (Sigma Ltd.) in isopropanol, and the protease inhibitors e-l-tosylamide-2-phenylethyl-chloromethyl ketone (TPCK; Sigma Ltd.) (50 lag ml -~) and N<t-p-tosyl-L-lysine chloromethyl ketone (TLCK; Sigma Ltd.) (60 lag ml -~) [7]. The homogenates were centrifuged for 2 min at 13 400 )< g using a MSE microcentaur centrifuge and the supernatants were retained. These were boiled for 5 min in an equal volume of 10 mM Tris buffer containing 1% sodium dodecyl sulfate (SDS; BDH), 0.5% mercaptoethanol, 2 mM phenyl methyl sulphonyl fluoride, 1 mM ethylenediaminetetraacetic acid (EDTA; BDH) and 10% glycerol [7]. The samples were electrophoresed using a discontinuous Tris glycine buffer system on 10% SDS-polyacrylamide gels with 5% stacking gels [8]. The gels

19

were dried and autoradiographed using Kodak X-Omat AR film in the presence of a Dupont Cromex Xtra Life Hi Speed intensifying screen. The films were exposed overnight at -70°C [9] and developed according to the manufacturer 's instructions.

RESULTS

Loss of label from adult female B. pahangi. Groups of 10 adult female B. pahangi previously labelled using 0.1 mCi of 125ISA and implanted in the peritoneal cavity of

naive jirds, were recovered after intervals of 0, 1, 3, 5 and 7 days. Autoradiographs prepared from histological sections of 125ISA-labelled worms confirmed that the label was restricted to the worms' surface [4]. The amount of radioactivity associated with

the individual worms in each group was measured immediately after labelling (time 0) and again at the end of each incubation. The results were corrected to the mean time 0 value and also corected to compensate for the decay of 125I (Fig. 1).

The results showed that there was no significant loss of label over 7 days (0 .05<P<0 .01 ) . Autoradiography of SDS-polyacrylamide gels prepared from worms homogenised at the end of each incubation showed no apparent loss of the most heavily labelled surface protein (mol. mass 30 kDa, Fig. 2). In contrast, when adult female worms were labelled using 125I-labelled Bolton and Hunter reagent the 30 kDa protein was not heavily labelled, but there was significant loss of labelled proteins with a wide range of molecular masses (Fig. 3).

Loss of label from fourth-stage larvae. Groups of fourth-stage larvae were labelled and incubated in vivo for 0, 1, 3 and 8 days. At the end of each incubation the worms were recovered and the radioactivity remaining on 20 worms from each group was

250

% x

0

10C 1 3 5

DAYS

Fig. 1. The amount of t25I (cpm) remaining on individual worms following in vivo incubation for 0, 1,3, 5, 7

days after labelling with '2~ISA. Each point is the mean of at least 10 worms 5: S.D.

20

30

7 5 3 1 0 DAYS

7 ! 4 21 2'8

I

10

8 ¸

~ 0 6

× I

qO

4 J

2

DAYS

Fig. 2. An autoradiograph of a SDS-PAGE gel prepared from adult female B. pahangi homogenised after incubation for up to 7 days following labelling with ~2~ISA.

Fig. 3. A graph showing the loss of Bolton and Hunter reagent-labelled proteins during incubation in vivo for 0-28 days. Each line represents an area cut from a gel containing proteins of approximate molecular masses (w) 14 kDa, (o) 110 kDa, (.) 97 kDa, (A) 43 kDa, (e) 54 kDa, (n) 67 kDa, (z~) 43 kDa, (e) 20 kDa. The radioactivity is expressed as cpm X 104.

determined. Recovery of labelled fourth-stage larvae was low, however in three similar

experiments the radioactivi ty remaining on the larvae I, 3 and 8 days after labelling

was within the s tandard deviat ion of the time 0 value, indicat ing that there was no

significant tu rnover of surface polypeptides, i.e. in one experiment at time 0 groups of

20 fourth-stage larvae had a mean cpm of 1.4 × 106 9- 0.6 × 106, at 24 h 1.6 X 106 cpm,

at 3 days 1.0 X 106 cpm, and at 8 days 1.2 X 106 cpm.

Fol lowing moul t ing the larvae had a background level of radioactivi ty (420 cpm),

indicat ing that all the radioactivi ty had been shed with the cuticle on moult ing.

The ability of labelled worms to complete the moul t indicates that the presence of

125ISA on their surface does not adversely affect their normal development . The low

recovery rate may be a result of damage by handl ing dur ing labelling rather than a

direct result of the label.

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Autoradiographs of electrophoresed preparations of homogenised worms showed

that in contrast to the adult, in which only one protein of molecular mass 30 kDa was labelled, in the fourth-stage larvae the most heavily labelled polypeptide had a molecu-

lar mass of 14 kDa. Proteins with molecular masses of 30, 43, 54, 67, 94, 100, 140 and 180 kDa were also labelled, though to a lesser extent (Fig. 4). This may indicate that the surface of fourth-stage larvae is more complex than that of the adult.

Loss of label from third-stage larvae. In contrast to the results seen in L4 and adults the third-stage larvae showed a significant loss of label over the 8 day period of incubation and by day 8 less than 10% of the original label remained (Fig. 5). Ap- proximately two thirds of the label was lost during the first 5 days in vivo and all the

6 7

•o 10

X

t,2

t

0 0 1 3 5 8 A g o A , s

Fig. 4. An autoradiograph of a SDS-polyacrylamide gel showing the proteins on L 4 (A) and adult B. pahangi (B) which are labelled using 125ISA.

Fig. 5. The amount of ~25I (cpm) remaining on groups of 50 L3 following incubation for 0, 1, 3, 5, 8 days after labelling with 125ISA.

22

9 4

6 7 ~

3 0

14

A B Fig. 6. An autoradiograph of an SDS-polyacrylamide gel electrophoresis showing the proteins on infective larvae (A) and adult (B) B. pahangi which are labelled using ~25ISA.

label was lost at the third larval moult (day 7-8). Electrophoresis and autoradiography

of labelled worms showed that the 30 kDa polypeptide was not labelled in infective

larvae. The labelled proteins had molecular masses of 67 and 94 kDa and a significant

amount of labelled material remained at the starting slot of the gel (Fig. 6). A greater

number of polypeptides was labelled in the infective larvae than in the adult and this

may be an indication that ~25ISA has penetrated the cuticle and labelled underlying

somatic tissue. However, the possibility that surface turnover is occurring in infective

larvae cannot be ruled out.

DISCUSSION

125Iodine has been used to demonstrate the loss of labelled proteins from the surface

of the nematode T. spiralis [1]. In that study it was found that 10% of the proteins

which had been labelled at time 0 had been lost following the culture of larvae in vitro for 24 h, and that 25% of the label was shed from adult worms during the same time

period. There is no such evidence for protein turnover on the surface of ill'trial

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nematodes. Two antigenic proteins resembling proteins from the adult and microfil- arial stages of B. malayi were found in the excretory-secretory products of adult B. malayi following in vitro cultivation, but it was not confirmed that these antigens had originated from the surface of the adult worms [10]. The presence of microfilarial antigens in excretory-secretory products is consistent with the results of studies on Litomosoides carinii [ 11] and O. volvulus [ 12]. The results of the present study demon- strated that there was no significant turnover of labelled protein from adult or fourth- stage larvae of B. pahangi over 7 and 8 day periods of observation, respectively, using the highly surface-specific reagent 125ISA. These results contrasted with the observa- tioff that in Bolton and Hunter reagent-labelled adult female worms a significant loss of labelled proteins was demonstrated over a 28 day period of incubation. As Bolton and Hunter reagent penetrates the cuticle and labels the underlying somatic tissue the labelled proteins were presumably being lost as a result of normal metabolic activity. As the somatic proteins were showing normal turnover, and the worms were active and producing normal microfilariae it is unlikely that they were adversely affected either by the labelling procedure or by the presence of 1251 in their tissues. The results of the present study showed that the 30 kDa protein differs from the somatic proteins by not showing significant turnover. In previous studies, autoradiography of sections through 125ISA-labelled adult worms, showed that the label was distributed over the whole area of the cuticle. These studies also demonstrated that the 30 kDa polypeptide was labelled using a variety of other 125I-labelling techniques, suggesting that it is a major surface protein [4]. Failure to demonstrate turnover of this material suggests that there is no surface turnover in adult B. pahangi.

The studies on fourth-stage larvae, although incomplete, suggest that there is no turnover of surface proteins in this life cycle stage ofB. pahangi. During the period of study there was a 2-3 fold increase in the length of the larvae, consistent with their normal development. As this period encompassed almost the entire larval instar, it seems unlikely that intermoult growth was accomplished by the shedding and replace- ment of surface proteins but is consistent with the mechanism proposed by Howells and Blainey [3]. Those authors studied the morphological development of fourth-stage larvae and demonstrated that, during the instar, increase in length occurred without a significant increase in surface area; the larval growth being accommodated by a gradual expansion of the cuticular folds which are tightly compressed immediately after the moult.

The results obtained for the third-stage larvae showed a constant and significant loss of label over the period studied. It is possible that the thinner cuticle of the infective larvae might allow passage of I25ISA to label underlying somatic structures and that the labelled polypeptides being shed were of somatic rather than cuticular origin. A similar pattern of loss of labelled material was seen in adult worms labelled with Bolton and Hunter reagent [14], which passes freely across the cuticle [4]. However, these results do not preclude the possibility that infective larvae show significant surface turnover.

This study shows that 125ISA labels polypeptides of different molecular weights on

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the sur face o f d i f fe ren t s tages o f the life cycle o f B . pahangi, a n d d e m o n s t r a t e s tha t in

adu l t and poss ib ly also f o u r t h - s t a g e l a rvae the m a j o r su r face p o l y p e p t i d e s are re la t ive-

ly s table , s h o w i n g no s ign i f i can t t u r n o v e r d u r i n g a 7 -8 p e r i o d o f o b s e r v a t i o n . O t h e r

s tudies have s h o w n tha t the 30 k D a p r o t e i n on the sur face o f adu l t B. pahangi is a m a j o r

sur face an t igen [ 15] and m a y p lay an i m p o r t a n t ro le in the h o s t - p a r a s i t e r e l a t ionsh ip .

ACKNOWLEDGEMENTS

This w o r k was s u p p o r t e d by a g r an t f r o m the U N D P / W o r l d B a n k / W H O Specia l

P r o g r a m m e for Resea rch a n d T r a i n i n g in T r o p i c a l Diseases and by Med ica l Resea rch

C o u n c i l P ro jec t G r a n t No . G 8 2 2 0 4 9 9 / T . E .M. was the rec ip ien t o f a U n i v e r s i t y o f

L i v e r p o o l R e s e a r c h S tuden t sh ip .

REFERENCES

1 Philipp, M., Parkhouse, R.M.E. and Ogilvie, B.M. (1980) Changing proteins on the surface of a parasitic nematode. Nature 287, 538-540.

2 Maizels, R.M., De Savigny, D. and Ogilvie, B.M. (1984) Characterisation of surface and excretory secretory antigens of Toxocara canis infective larvae. Parasite Immunol. 6, 23-37.

3 Howells, R.E. and Blainley, L.J. (1983) The moulting process and the phenomenon of intermoult growth in the filarial nematode Brugia pahangi. Parasitology 87, 493-505.

4 Marshall, E. and Howells, R.E. (1985) An evaluation of different methods for labelling the surface of the filarial nematode Brugia pahangi with ~:5Iodine. Mol. Biochem. Parasitol. 15, 295-304.

5 Wade, J.O. (1976) A new design of membrane feeder incorporating an electrically powered blood stirring device. Ann. Trop. Med. Parasitol. 70, 113-120.

6 Ash, L.R. (1974) Rodent models for the study of lymphatic dwelling filarial worms. WHO Cyclostyled Report WHO/FIL/74.121.

7 Parkhouse, R.M.E., Philipp, M. and Ogilvie, B.M. (1981) Characterisation of surface antigens of T. spiralis infective larvae. Parasite Immunol. 3, 339-352.

8 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacterio- phage T4. Nature 227, 680-685.

9 Laskey, R.A. and Mills, A.D. (1977) Enhanced autoradiographic detection of 32p and ~2~I using intensifying screens and hypersensitised film. FEBS Lett. 82, 314-316.

10 Kaushal, N.A., Hussain, R., Nash, T.E. and Ottesen, E.A. (1982) Identification and characterisation of E-S products of Brugia malayi adult filarial parasites. J. Immunol. 29, 338-343.

11 Ishii, A. (1970) Antigenicity of excretory and secretory products of the cotton rat filariae Litomosoides carinii. Jpn. J. Exp. Med. 40, 39-45.

12 Des Moutis, I., Ouassi, A., Grzych, J.M., Yarzabel, L., Haque, A. and Capron, A. (1983) Onchocerca volvulus: detection of circulating antigen by monoclonal antibodies in human onchocerciasis. Am. J. Trop. Med. Hyg. 32, 533-542.

13 Bolton, A.E. and Hunter, W.M. (1973) The labelling of proteins to high specific radioactivities by conjugation to a ~251odine containing acylating agent: application to the radioimmunoassay. Biochem. J. 133, 529-538.

14 Marshall, E. and Howells, R.E. (1984) Surface turnover in the cuticle of Brugiapahangi. Parasitology 89, Xlii.

15 Sutanto, I., Maizels, R.M. and Denham, D.A. (1985) Surface antigens of a filarial nematode: analysis of adult Brugia pahangi surface components and their use in monoclonal antibody production. Mol. Biochem. Parasitol. 15, 203-214.