Molecular and Biochemical Parasitology, 15 (1985) 295-304 Elsevier

MBP 00546

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AN EVALUATION O F D I F F E R E N T M E T H O D S F O R LABELLING TH E

SURFACE OF THE FILARIAL N E M A T O D E BRUGIA P A H A N G I W I T H

1251ODINE

ELIZABETH MARSHALL and ROBERT E. HOWELLS

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

(Received 30 November 1984; accepted 21 January 1985)

The specificity of a range of t25I labelling techniques (Chloramine T, lodogen, Bolton and Hunter reagent, lactoperoxidase and iodosulfanilic acid) to the surface of the filarial nematode Brugia pahangi was evaluated by autoradiography of sections of labelled worms and of dried SDS-polyacrylamide gels following electrophoresis of homogenised worm extracts. It was concluded that Bolton and Hunter reagent was not surface specific but labelled proteins throughout the body of the worm. At the light microscope level autoradiography of worms labelled using Chloramine T, iodogen, lactoperoxidase and iodosulfanilic acid demonstrated that the J251 labelling was restricted to the worm surface. Electrophoresis and autoradio- graphy showed that each method produced a different pattern of labelled polypeptide. A polypeptide of molecular weight 30 kDa was labelled using each method except Bolton and Hunter reagent, and appears to be a major surface component.

Key words: lodination; SDS-polyacrylamide gel electrophoresis; Autoradiography; Filarial nematode; Surface proteins

INTRODUCTION

Recent studies on the surface ant igens of parasitic nematodes , par t icular ly Trichi-

nella spiralis, have used 125I to label accessible surface proteins (see reviews by Maizels

et al. [1], Philipp and Rumjaneck [2]). Those studies indicated that the use of different

iod ina t ion techniques may lead to differences in the proteins labelled in individual

parasite species. Differences may result from the differing specificities of the reagents

used; Chloramine T, Iodogen and lactoperoxidase mediate the b ind ing of ~25I princi-

pally to tyrosine residues [3], Bolton and Hun te r reagent b inds to amino acids with

epsilon amino groups such as lysine [4] and iodosulfanil ic acid binds to lysine, tyrosine

and histidine [5]. The physical characteristics of the reagents may also determine

which proteins are labelled. The large size of the lactoperoxidase molecule and the

insolubil i ty of Iodogen in aqueous solut ions may prevent labelling of less accessible

surface proteins, while the strong negative charge of iodosulfanil ic acid will limit its

b ind ing to basic proteins. In labelling the surface of nematodes the permeabil i ty of the

0166-6851/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

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cuticle to particular reagents may be an important factor in determining the surface specificity of a labelling technique. In this study a range of ~25I labelling techniques has been used to label the filarial nematode Brugia pahangi. Following each labelling procedure, the intensity and surface specificity of labelling, and the effect of iodina- tion on worm mobility were determined.

MATERIALS AND METHODS

The parasites. Adult B. pahangi were recovered from the peritoneal cavity of jirds (Meriones unguiculatus) 3 months or more after the intraperitoneal injection of 100 infective larvae. Adult female worms were used throughout this study.

Iodine labelling. Two categories of iodination technique were used: direct labelling using catalysts such as Chloramine T and conjugation labelling using ~5I-labelled reagents such as Bolton and Hunter reagent and iodosuifanilic acid.

(1) Chloramine T [3]. Groups of 10 adult female B. pahangi were incubated at room temperature in 0.2 ml of phosphate buffered saline (PBS) pH 7.0 and 0.1 mCi of carrier-free radioactive iodine (~25I, Radiochemical Centre, Amersham) was added.

The reaction was initiated by the addition of 10 lal of Chloramine T solution (1 mg m1-1) and a further 10 lal was added 2 min later [6]. The reaction was stopped after 4 min by the addition of 0.8 ml of Hanks Balanced Salt Solution (HBSS) containing bovine serum albumin (BSA, I mg ml -~) and potassium iodide (20 mg ml-l). The

worms were removed and washed for 5 min in 5 ml of this solution, and then given a further three washes in 15 ml of warm (30-37°C) HBSS. This washing protocol was followed in all the iodination experiments reported here.

(2) Lactoperoxidase-glucose oxidase [7]. Groups of 10 worms were incubated at room temperature in 0.2 ml of PBS containing 20 mM o-glucose, and 0.1 lag of 125I was

added. The reaction was initiated by the addition of 7 lag of lactoperoxidase (Calbio- chem Ltd.) followed by 7 lag of glucose oxidase (Sigma Ltd.). The reaction continued for 15 min and was then stopped as described previously.

(3) Iodogen [8,9]. Iodogen (Pearce Chemical Company) was dissolved in chloroform giving a final concentration of 1 mg ml -~, and 0.1 ml amounts were dispensed into 15 ml acid washed conical bot tomed centrifuge tubes. The solvent was evaporated under nitrogen and the coated tubes were either used immediately or stored in a desiccator. Before use the tubes were rinsed with PBS and groups of 10 worms were added to 0.2 ml of PBS in each tube. The reaction commenced on addition of 0.1 mCi of ~25I and continued for I0 rain at room temperature when it was stopped as described previous-

ly.

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(4) Bolton and Hunter reagent [4]. Bolton and Hunter reagent (N-succinimydil 3-(4-hydroxy 5-[~25I]iodophenyl) propionate, Radiochemicai Centre, Amersham)is supplied in benzene and prior to use this was evaporated under nitrogen. The label was redissolved in 0.2 ml of HBSS, giving a concentration of 500 laCi ml -~. Groups of 10 worms were incubated on ice in 0.2 ml (100 laCi) of this solution for 10 min. The reaction was stopped by the addition of 0.2 M glycine in HBSS, and the worms were washed as described previously.

(5) Iodosulfanilic acid. Iodosulfanilic acid (NEN) was diazotised according to the manufacturer's instructions. Following diazotisation 0.2 ml of PBS containing 0.1 mCi of the reagent was dispensed into centrifuge tubes and groups of 10 worms were added to each tube. The reaction was performed on ice for 15 min and was stopped by the addition of 0.8 ml of HBSS containing bovine serum albumin (1 mg ml-~). The worms were washed as described previously.

Determination of radioactivity. The worms were counted individually using a Packard multi prias gamma counter. Radioactivity was expressed as counts per minute (cpm) and corrected for the decay of 125I.

Electrophoresis and autoradiography. Groups of 10 labelled worms were homoge- nised in 5% sodium deoxycholate solution in 10 mM Tris-HCl buffer containing the protease inhibitors L-l-tosylamide-2-phenyl ethyl chloromethyl ketone (TPCK, Sigma (50 lag ml-~)), N-p-tosyl-l-lysine chloromethyl ketone (TLCK, Sigma (50 lag ml-L)) and phenyl methyl sulphonyl fluoride (PMSF, Sigma; 0.1 M in isopropanol) [6]. The homogenates were centrifuged for 2 min at approximately 13 000 rpm using an 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 PMSF, 1 mM ethylenediaminetet- ra-acetic acid (EDTA; BDH) and 10% glycerol [6]. The samples were electropho- resed using a discontinuous Tris-glycine buffer system [10] on 10% SDS-polyacrylam- ide gels with 5% stacking gels. The gels were dried and autoradiographed using Kodak X-Omat AR film in the presence of a Dupont Cronex Xtra-life Hi Speed intensifying screen. The films were exposed overnight a t -70°C [ 11] and developed according to the manufacturer's instructions.

Autoradiography of sections through labelled worms. Autoradiography was perform- ed on 1 lam sections by the stripping film technique using Ilford K5 nuclear emulsion. The film was exposed at 4°C and developed according to the manufacturer's instructions.

In vitro incubation. Groups of 10 labelled worms were incubated in I0 ml of RPM11640 plus 20 mM Hepes (Flow Ltd.) containing 20 mM glutamine and 100 units

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of s t reptomycin and benzyl penicillin, supplemented with 10% fetal bovine serum

(FBS).

RESULTS

The mean cpm obtained using each method is shown in Table I. Bolton and Hunte r

reagent was incorporated in the greatest quant i ty (the mean cpm per g roup of 10

worms was 6278766 5: 311416) and iodosulfanilic acid in the smallest quanti ty

(228 325 5:51 493). Of the direct labelling methods Iodogen mediated the incorpora- tion o f the greatest amoun t o f 125I (3 378 060 ± 689 704) followed by Chloramine T

(1 751 323 5 :575 579) and lactoperoxidase-glucose oxidase (1 579 599 5 :387 314).

The viability o f worms labelled using Bolton and Hunte r reagent, iodosulfanilic

acid and lactoperoxidase was 100% following 24 h in vitro incubation. At this time the

worms were active and appeared normal in compar i son with control groups. Worms

labelled using Iodogen and Chioramine T were mor ibund immediately after labelling

and there was a 40% mortal i ty following 24 h incubat ion in vitro.

The differentiation o f ~25I-labelled polypeptides on SDS-polyacrylamide gels was limited by the resolution of the au torad iographs (Fig. 1). In au torad iographs o f gels

prepared f rom Bolton and Hunter reagent labelled worms at least 24 bands were

identified. At least 12 polypeptide bands were labelled following Chloramine T and

Iodogen and 7 were clearly differentiated following lactoperoxidase. The majori ty o f

preparat ions o f iodosulfanilic labelled worms yielded a single polypeptide band of 30

kDa (Fig. 1) but in occasional preparat ions a 94 kDa polypeptide was also labelled.

The reasons for this variability remain unclear. It is unlikely that insolubility o f the 94

kDa polypeptide in sodium deoxycholate is responsible for the inconsistency since

TABLE I

The mean radioactivity (cpm) on groups of 10 labelled adult female B. pahangi using the Bolton and Hunter reagent, lactoperoxidase-glucose oxidase, Iodogen, chloramine T and iodosulfanilic acid methods

Label cpm 4- SD Worm survival (%)

Bolton and Hunter reagent 6278 766 + 311 316 100 Lactoperoxidase-glucose oxidase 1579599 + 387314 100 Iodogen 3 378 060 4- 689 704 60 Chloramine T 1 751323 4- 575 579 60 lodosulfanilic acid 228 325 4- 54492 100

Standard deviations (SD) are also shown. The worm survival was assessed following 24 h in vitro incubation with the exception of iodosulfanilic acid labelled worms which were incubated in vivo. These results are representative of at least 3 experiments.

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Fig. 1. An autoradiograph of an SDS-polyacrylamide gel prepared from worms labelled using different ~2SI-labelling techniques. The worms were labelled using the following methods: (a) lactoperoxidase-glucose oxidase, (b) Iodogen, (c) iodosulfanilic acid, (d) Chloramine T, (e) Bolton and Hunter reagent. The approximate molecular weights are shown on the left hand side of the figure.

s imi lar results were ob ta ined fo l lowing ex t rac t ion o f worms with mercap toe thano l .

The 94 k D a po lypep t ide was ident i f ied also in deoxycho la te extracts of worms label led

using all the o the r methods . It is poss ible that the 94 k D a po lypep t ide lies near the

surface of the worms and was avai lab le to react with iodosulfani l ic acid only under

cer tain undef ined cond i t ions or fo l lowing pe r tu rba t ion of the worm surface. Light

microscope a u t o r a d i o g r a p h y o f sect ions th rough labelled worms d e m o n s t r a t e d that

Bol ton and Hun te r reagent had label led pro te ins t h roughou t the somat ic tissues of the

worms including the reproduc t ive tissues and the gut (Fig. 2). In worms label led using

iodosulfani l ic acid (Fig. 3), Iodogen (Fig. 4), Ch lo ramine T and l ac toperox idase

(results not shown) the ~25I was res t r ic ted to the cuticle and there was no label l ing o f

the somat ic tissues. A l though the worms label led using Iodogen were more heavi ly

label led than iodosul fani l ic ac id- label led worms, the 12sI d id not a p p e a r to pene t ra te

be low the cuticle and there was no label l ing of the somat ic tissues.

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Fig. 4. An autoradiograph of a transverse section through an adult female B. pahangi labelled using lodogen.

DISCUSSION

The Bolton and Hunter reagent has been used to label parasitic nematodes in several previous studies [6,12,13,14]. This method has been of particular value in immunological studies where the use of harsher methods such as Chloramine T may interfere with the antigenicity of the proteins [15]. In the intestinal nematode T. spiralis, labelling with Bolton and Hunter reagent was surface specific and the pattern of labelled proteins similar to that seen following labelling with Chloramine T and lactoperoxidase [6]. However, when the intestinal nematode Nippostrongylus brasi- liensis was iodinated using Bolton and Hunter reagent the pattern of labelled proteins

Fig. 2. An autoradiograph of a transverse section through an adult female B. pahangi labelled using Bolton

and Hunter reagent.

Fig. 3. An autoradiograph of a transverse section through an adult female B. pahangi labelled using iodosulfanilic acid.

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was different to that obtained using Chloramine T [16]. The present study has shown that Bolton and Hunter reagent was not specific for the surface of the filarial nematode B. pahangi, but penetrated and labelled the underlying somatic tissues. The labelling of the somatic tissues ofB. pahangi using Bolton and Hunter reagent may be explained by the greater permeability of the cuticle of this nematode compared with T. spiralis. The transcuticular uptake of low molecular weight peptides and sugars has been demonstrated in & pahangi [17,18].

In this study Iodogen labelled at least 7 polypeptides and autoradiography of sections through labelled worms demonstrated that the label was restricted to the surface. Iodogen has been used to mediate highly surface specific labelling of Oncho- cerca gibsoni [13], Toxocara canis [20], adult Dipetalonema viteae [21] and Litomo- soides carinii [ 14]. The only parasite in which Iodogen has been shown to label somatic tissues was the microfilariae of D. viteae and in this case the worms died during labelling [21].

Chloramine T gave similar results to Iodogen in this study. At least 7 polypeptides were labelled and the label was localised on the cuticle, although the labelling was less intense than when Iodogen was used. This method has frequently been used to label T. spiralis and autoradiography has demonstrated that it is surface specific [6]. Autora- diography has shown that Chloramine T mediates surface specific labelling of the filarial nematode L. carinii [14] though not of the filarial nematode D. viteae [21].

The iactoperoxidase method labelled a smaller number of polypeptides on adult female B. pahangi than Chloramine T or Iodogen, but the appearance following light microscope autoradiography was the same as when these two reagents were used. As discussed in the introduction the large size of the lactoperoxidase molecule may prevent its labelling less accessible surface proteins. Lactoperoxidase also mediated weak surface specific binding to the filarial nematode D. viteae [21] and to T. spiralis [6].

There are no reports of the use of iodosulfanilic acid to label parasitic nematodes, although it has been used to label the outer tegumental membrane of Schistosoma mansoni [22]. In the present study only one polypeptide of molecular weight 30 kDa was labelled, although occasionally a heavier protein (94 kDa) was detected. This selective labelling does not imply that there is only one major surface protein in B. pahangi as is the case in L. carinii [14], but may be a result of the physical properties of the reagent. The 30 kDa polypeptide was heavily labelled using all the methods except Bolton and Hunter reagent and autoradiography of sections through labelled worms confirmed that the label was evenly distributed over the worm surface.

These results demonstrate that different patterns of labelling are obtained using different iodinatioh techniques to label a single parasite species and emphasise the need to critically evaluate the available techniques to find appropriate methods for labelling particular parasite species.

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ACKNOWLEDGEMENTS

T h i s w o r k was s u p p o r t e d b y a g r a n t f r o m the U N D P / W o r l d B a n k / W H O Spec ia l

P r o g r a m m e f o r R e s e a r c h a n d T r a i n i n g in T r o p i c a l D i s e a s e s a n d b y M e d i c a l R e s e a r c h

C o u n c i l P r o j e c t G r a n t N o . G 8 2 2 0 4 9 9 / T . E . M . was t he r e c i p i e n 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 t u d e n t s h i p .

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