Molecular and Biochemical Parasitology, 14 (1985) 127-139 127 Elsevier

MBP 00505

B I O S Y N T H E S I S O F T W O S T A G E - S P E C I F I C . M E M B R A N E P R O T E I N S

D U R I N G T R A N S F O R M A T I O N O F P L A S M O D I U M G A L L I N A C E U M Z Y G O T E S

I N T O O O K I N E T E S

NIRBHAY KUMAR and RICHARD CARTER

Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20205, U.S.A.

(Received 3 July 1984; accepted 19 September 1984)

We have studied the synthesis and expression of surface proteins in zygotes ofPlasmodium gallinaceum during their transformation to mature ookinetes. The cells were biosynthetically labelled in vitro using [JSS]methionine and proteins were immunoprecipitated with rabbit anti-ookinete serum or monoclonal antibodies. Early zygotes (approx. 2 h post-gametogenesis and fertilization) synthesized and expressed on their surface a protein of M r 26 000 as observed under reducing conditions on polyacrylamide gel electro- phoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) (31000 under non-reducing conditions) and continued to do so for 8-10 h; thereafter synthesis of the Mr 26000 protein declined and little or none was synthesized in the mature ookinetes (>20 h post-gametogenesis). Between 3-5 h post-gametogenesis, zygotes also began to synthesize a protein of Mr 28 000 (34 000 under non-reducing conditions). Synthesis and expression of this surface protein continued throughout development; and the Mr 28 000 protein was the predominant surface protein synthesized by the mature ookinete. Mr 26 000 and Mr 28 000 proteins have been designated earlier as PgO-I and PgO-2 respectively (Carter and Kaushal, Mol. Biochem. Parasitol. (1984) 13, 235-241). Neither protein was synthesized in the gametocytes prior to gametogenesis. Both proteins could be labelled with [JH]glucosamine or [~H]mannose. When zygotes were incubated with [3H]palmitic acid both PgO-1 and PgO-2 bound fatty acids in covalent linkage. The two proteins do not otherwise appear to be structurally related. They were differentially immunoprecipitated by different monoclonal antibodies and gave rise to distinct patterns of peptides following digestion with proteases such as Staphylococcus aureus V-8, trypsin and chymotrypsin.

Key words: Plasmodium gallinaceum; Zygotes; Ookinetes; Biosynthesis; Transmission-blocking immunity; Glycosylation; Acylation

INTRODUCTION

T r a n s m i s s i o n o f m a l a r i a p a r a s i t e s f r o m the v e r t e b r a t e h o s t to t he m o s q u i t o v e c t o r is

Abbreviations: BOB, bromophenol blue; MAb, monoclonal antibody; NRS, normal rabbit serum. SDS- PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TSG, 10 mM Tris, 145 mM NaCI, 10 mM glucose, pH 7.3.

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

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mediated by the gametocytes of the parasite. The sexual stages arise as intracellular parasites of the host red blood cells and become extracellular gametes in the midgut of a mosquito during a blood meal. The gametes then fertilize in the mosquito midgut lumen, a process largely completed within 30 min of ingestion of the blood, and the zygotes undergo transformation over the following 20-24 h to form motile stages, the ookinetes, which transverse the midgut wall to establish the infection in the vector.

Antibodies against the mosquito midgut stages of malarial parasites are capable of preventing the infection in the vector [1]. The antibodies may be directed against the malarial gametes preventing fertilization [2] or against the zygote to prevent subse-

quent development [3]. The targets of these antibodies represent stage specific parasite surface proteins. In the chicken parasite Plasmodium gallinaceum, the anti-gamete antibodies immuno-precipitate three proteins o f M r 240 000, 56 000 and 54 000 present on male and female gametes and newly fertilized zygotes [4]. As zygotes undergo transformation into ookinetes these proteins are subsequently lost from the parasite

surface [5]. In addition, two new proteins of apparent M r under reducing conditions of 26 000 (PgO- 1) and 28 000 (PgO-2) (31 000 and 34 000 respectively under non-reducing conditions) appear on the zygote surface [5] of which the M r 26 000 protein is the target of a monoclonal antibody which blocks the post fertilization development of the parasites in the mosquito [3]. In the present paper we have studied the biosynthesis and expression on the surface of PgO- 1 and PgO-2 proteins* during transformation of P. gallinaceum zygotes to ookinetes.

MATERIALS AND METHODS

Parasites. Newly fertilized zygotes (approximately 2 h post-fertilization) of P. galli-

naceum were prepared as described [6] and further incubated under conditions [3] supporting the formation of mature and infectious ookinetes. Typically 10 7 zygotes are incubated for 24 h at 26°C in 4.0 ml medium-199 supplemented with 1 mM L-glutamine, 125 lag m1-1 gentamycin, 100 units ml -L of penicillin and 100 lag m1-1 streptomycin.

Antibodies. Polyspecific hyperimmune rabbit serum was prepared against mature ookinetes as previously described for zygotes [6]. Monoclonal antibodies (Mab) (IID2B3B3 and IID2C5) used in this study were derived from the fusion of myeloma cells with spleen cells of mice immunized with ookinetes. Their production, character- ization and effects on infection are described elsewhere [3].

*Since we found that PgO-1 and PgO-2 are more clearly resolved under non-reducing conditions, we have mostly carried out SDS-PAGE under non-reducing conditions.

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Radioiodination and biosynthetic labelling of parasites. Intact ookinetes were labelled with ~5I by the iactoperoxidase method as described previously for zygotes [6].

Newly fertilized zygotes were labelled with [35S]methionine [approx. 1200 Ci mmol -~] according to two different protocols. In the first, zygotes at 2.5 X 10 7

ml -~ of methionine-free medium RPMI-1640 were incubated for 60 min at 24°C with [35S]methionine (100 laCi ml-~). The cells (without further washing) were then diluted to 2.5 X 106 ml -~ and incubated under the conditions supporting transformation of zygotes into ookinetes (as described above). At the end of incubation the cells were washed and extracted with Triton X-100 for immunoprecipitation.

In the second protocol purified zygotes were incubated under conditions support- ing ookinete formation. Cells were harvested at different time points during transfor- mation, washed three times with methionine-free RPMI- 1640 and given a 10 min pulse of [35S]methionine at room temperature under the same conditions used in the first protocol, e-Methionine (2 mM final concentration) was added, the cells washed twice (300 X g, I min using Dade bench top clinical immufuge) and incubated for 60 min with 2 mM e-methionine in medium-199 for maximum expression of labelled proteins on cell surface (data not shown).

Zygotes during transformation were labelled with [3H]glucosamine (30.3 Ci mmo1-1 Amersham, Arlington Heights, IL) or [3H]mannose (15-23 Ci mmol -Z, New England Nuclear, Boston, MA), for 24 h in the presence of either radioactive sugar (approx. 10 laCi ml-L). Zygotes were labelled in a similar way with [3H]palmitic acid (25-50 laCi ml-l). Toluene was removed from 9,10-[3H]palmitic acid (NEN, 15.2 Ci mmo1-1) by passing N2 gas and the fatty acid then redissolved in a minimum volume of dimethyl sulfoxide and added to the medium. Labelled cells were washed and extract- ed with Triton X-100 as described below.

Immunoprecipitation and extraction of labelled proteins from the surface of intact cells and extracts. Zygotes, labelled as described above, were washed three times with 10 mM Tris, 145 mM NaC1, 10 mM glucose pH 7.3 (TSG)and incubated with antibodies (heat inactivated 30 min at 56°C) for 20 min at room temperature. The patterns of immunoprecipitation were similar both quantitatively and qualitatively, when the intact cells were reacted with antibodies at 4°C or at room temperature (data not shown). Cells were washed three times with TSG buffer (300 X g, 1 min in immufuge) and lysed (20 min at 4°C) with 1% Triton X-100 in TSG containing 2 mM phenylme- thylsulfonyl fluoride (Sigma, St. Louis, MO), 0.2 mM L- l-tosylamide-2-phenylmethyl chloromethyl ketone (Sigma), 0.2 mM p-tosyl-L-lysine chloromethyl ketone (Sigma), 10 mM EDTA, 0.25 lag m1-1 leupeptin (Boehringer-Mannheim, Indianapolis, IN) and 10 lag m1-1 pepstatin (Sigma). Lysed cells were centrifuged in a Beckman Airfuge for 15 min at 10 000 × g and supernatants were immediately incubated for 60 min at room temperature with Protein A-Sepharose (Pharmacia Fine Chemicals, Piscataway, N J). The beads were then washed twice with NETTS (10 mM Tris, 5 mM EDTA, 0.5 M NaC1, 0.5% Triton X- 100) twice with NETT (10 mM Tris, 5 mM EDTA, 0.15 M NaCI,

130

0.5% Triton X-100, pH 7.6) and twice with NET (10 mM Tris, 5 mM EDTA, 0.15 M NaCI, pH 7.6). Immune-complexes were extracted by boiling the beads for 3-5 min

in 50 lal of SDS-sample buffer (62.5 mM Tris, pH 6.8, containing 5% SDS, 10% glycerol, and 0.005% bromophenol blue [B~B]) for analysis by SDS-PAGE.

For immunoprecipitation from the whole cell lysates, the cells were lysed with 1%

Triton X-100 with protease inhibitors (as above) and the supernatants reacted with antibodies for 2-3 h at 4°C. Immune-complexes were collected by Protein A-Sepha- rose beads, washed and extracted for SDS-PAGE.

Biosynthetic labelling of parasitized blood before or after induction of gametogenesis. To label parasites before gametogenesis, 25 ml of parasitized blood was washed in

250 ml of TSG at 30°C, resuspended in 200 ml TSG at 35°C containing 2 mCi [35S]methionine and incubated for 45 min at room temperature. The labelled blood

was washed twice in TSG, induced to undergo gametogenesis and zygotes prepared as previously described [6]. Purified zygotes were extracted in Triton X-100.

Parasites were also labelled soon after inducing gametogenesis as previously describ- ed [6]. 15 min following induction, the parasitized blood was labelled with [35S]me-

thionine for 30 min and zygotes purified and extracted with Triton X-100.

SDS-PAGE, autoradiography andfluorography. Samples were analyzed either on a 5-15% gradient or 10%, 12.5% or 15% acrylamide gels according to the procedure of Laemmli [7]. Gels were stained with Coomassie Blue and destained with 10% acetic acid and 25% methanol, dried under vacuum and exposed to Kodak X-omat XAR 5 films at -80°C. For autoradiography of 3H- or 35S-labelled samples, gels, after

destaining, were soaked in Autofluor (National Diagnostics, Somerville, N J) for 1-2 h

before drying.

Comparison of peptide maps of M r 28 000 and 26 000 proteins. Ookinetes labelled with [35S]methionine during transformation as described in the first protocol (above) were extracted in Triton X-100 and immunoprecipitated with MAbs II D2B3B3 (specific for M r 28 000 protein) or with II D2C5 (specific for M r 26 000 protein) and the immunoprecipitated proteins were separated by SDS-PAGE (10% acrylamide). Gels were treated for 30 min with 25% methanol and 10% acetic acid, dried and exposed to X-ray film to locate the respective labelled protein bands. Regions of gels correspond- ing to M r 28 000 and 26 000 proteins were excised and soaked in 50 mM Tris, pH 6.8, with four to five changes over a period of 90-120 min. The washed gel pieces were

digested with proteases as described by Cleveland et al. [8]. Briefly, gel pieces were placed in the sample wells in the stacking gels containing 30 ~1

of SDS-buffer (62.5 mM Tris, pH 6.8, 5% SDS, 0.005% B~B and 20% glycerol). Freshly prepared solutions of Staphylococcus aureus V-8 (Miles Laboratories, Eck- hart, IN), Trypsin-TPCK (Sigma) and L-chymotrypsin-TLCK (Sigma) were diluted to 0.1 mg ml -~ in the SDS-buffer containing only 10% glycerol and 20 ~tl added to gel

131

pieces in the wells. When the BOB dye reached the interphase of stacking gel and separating gel, the current was switched off for 30 min. The digested products were separated by SDS-PAGE using a 10-30% acrylamide gradient.

Hydrolysis of [3Hlpalmitic acid labelled proteins and analysis of the released lipids. To determine the nature of linkage of [3H]palmitic acid in the proteins, gels

immediately after electrophoresis were fixed in 25% methanol and 10% acetic acid, washed with distilled water and treated with 0.1 M KOH in 20% methanol at room temperature for 90 min. They were then processed for fluorography as described above.

Lipids released by methanol alkali hydrolysis were analyzed according to the method of Schmidt et al. [9]. Briefly, the gel pieces containing 3H-labelled proteins were cut out and incubated with 2 ml 0.1 M KOH in 20% methanol for 60 min at room temperature. The mixture was acidified with HCI; CHCI 3 and H20 were added to yield a ratio of CHCI3/CH3OH/H~O of 8:4:3. The lower phase was washed three times with 1 ml CHC13/CH3OH/HEO (1:10:10) and analyzed by thin layer chromatography on Whatman LK5D Linear-K silica gel coated plates (Pierce, Rockford, IL) using C H C l J C H 3 O H / H 2 0 (65:25:4). The plates divided into 1 cm strips were scraped off and counted in a liquid scintillation counter. The position of standard methyl palmi- tate was located by charring the plate in 50% H2SO4.

RESULTS AND DISCUSSION

Stage specificity and kinetics of synthesis and expression of M r 26000 and 28000 proteins in transforming zygotes. Newly fertilized zygotes of P. gallinaceum were labelled continuously with [35S]methionine for 24 h during transformation into ooki- netes. Two proteins of apparent M r of 31 000 (PgO-l) and 34000 (PgO-2) under non-reducing conditions were labelled and immunoprecipitated with rabbit anti- ookinete serum (Fig. 1, lane 2). These corresponded in electrophoretic mobility on SDS-PAGE with labelled proteins immunoprecipitated by rabbit anti-ookinete serum from extracts of ookinetes surface radioiodinated with 1251 (Fig. 1, lane 3). Fig. 1, lanes

4 and 5, also show that the apparent M r of these proteins shifts from 31 000 to 26 000 and 34 000 to 28 000 if samples are analyzed under reducing conditions. The biosynthe- tically labelled proteins identified by immunoprecipitation with anti-ookinete serum thus represent the ookinete stage specific surface proteins previously designated as PgO-I and PgO-2 [5] and are synthesized de novo during the transformation of zygotes into ookinetes.

These proteins were not synthesized bygametocytes. Neither PgO-I nor PgO-2 were immuno-precipitated by anti-ookinete serum from extracts of zygotes prepared from gametocytes labelled with [ 'S]methionine (Fig. 2, lane 1). However, when [35S]me- thionine was added to the cell suspensions after inducing gametogenesis, biosyntheti- cally labelled PgO-1 (M r 31 000 under non-reducing conditions) was immunoprecipi-

132

M r x l O - 3

T o p -

9 7 . 4 -

6 6 . 2 -

4 5 . 0 -

P g O - 2 , , ~ 3 1 . 0 - , , ~ P g O - 1

2 1 . 5 -

1 4 . 4 -

1

® 2 3 5 4

I I

P g O - 2

P g O - 1

Bd~B -

Fig. 1. Autoradiogram of immunoprecipitates from ookinetes labelled by surface radio-iodination or with [3sS]methionine during transformation. Newly fertilized zygotes were biosynthetically labelled with [3SS]methionine during transformation into ookinetes as described in Materials and Methods, and mature ookinetes surface labelled with 1251 as previously described [6]. Labelled cells were extracted in Triton X-100 for immunoprecipitation and analyzed on a 12.5% acrylamide SDS-PAGE. Lanes 1 and 2 are immunopre- cipitates from extracts of [35S]methionine-labelled cells; lanes 3 and 4 are immunoprecipitates from extracts of t25I-surface-labelled cells and lane 5 shows total labelled proteins extracted from [~SS]methionine-labelled cells. Immunoprecipitations were with normal rabbit serum (NRS)(lane 1) and rabbit anti-ookinete serum (lanes 2-4). Low molecular weight standards (Bin Rad, Richmond, CA) were used to calibrate the gels. Samples were analyzed under non-reducing (lanes 1-3) and reducing (lanes 4--5) conditions during SDS-PAGE. Positions of the two ookinete stage-specific proteins PgO-1 and PgO-2 are marked by arrows (right side for reduced samples and left side for non-reduced samples).

t a t ed f r o m the ex t rac t s o f pu r i f i ed zygotes (Fig. 2, lane 2). These resul ts ind ica te tha t

PgO-1 is syn thes i zed by the sexua l s tages o f P. gallinaceum on ly a f te r in i t i a t ion o f

g a m e t o g e n e s i s .

T h e k ine t ics o f syn thes i s o f P g O - I a n d P g O - 2 were fu r t he r s tud ied d u r i n g zygo te

t r a n s f o r m a t i o n . A t d i f fe ren t t imes a f te r g a m e t o g e n e s i s (2, 5, 10 and 22 h), zygo tes

were i n c u b a t e d fo r l0 m i n at r o o m t e m p e r a t u r e in the p resence o f [35S]methionine. A t

each t ime p o i n t l abe l led p ro t e in s were i m m u n o p r e c i p i t a t e d wi th r abb i t a n t i - o o k i n e t e

s e r u m o r wi th e i the r o f two M a b s IID2C5 a n d IID2B3B3, specif ic fo r P g O - I a n d

133

Fig. 2. Biosynthetic labelling of surface proteins before orafter induction ofgametogenesis. Parasitized blood containing gametocytes of Plasmodium gallinaceum was biosynthetically labelled with [35S]methionine either before or after inducing gametogenesis as described in Materials and Methods. After labelling by either method, zygotes were purified, extracted with Triton X-100. Extracts containing equal numbers of trichloroacetic acid-precipitable counts were immunoprecipitated with rabbit anti-ookinete serum and run on a 12.5% SDS-PAGE under non-reducing conditions. Lane 1, zygotes from blood labelled before inducing gametogenesis; lane 2 zygotes from blood labelled after inducing gametogenesis.

PgO-2, respectively. I m m u n o p r e c i p i t a t l o n s were done either (i) f rom to ta l cell Tr i ton

X-100 extracts p repa red immedia te ly af ter label l ing (Fig. 3A) or (ii) by reac t ion

of an t ibod ies with intact cells af ter a 60 min chase with unlabel led meth ionine dur ing

which t~me m a x i m u m expression of label led prote ins occur red on the cell surface (da ta

not shown) fo l lowed by ext rac t ion in Tr i ton X-100 (Fig. 3B).

Up to 2 h pos t -gametogenes is , only PgO-1 was synthesized and expressed on the

zygote surface. By 5 h pos t -gametogenes is the zygotes also began synthesizing PgO-2

in add i t i on to PgO-1 and at 10 h pos t -gametogenes is their rates of synthesis and

expression on the cell surface were similar . Af te r 22 h (mature ookinetes) PgO-2 was

the p r e d o m i n a n t p ro te in synthesized by the paras i tes and expressed on the cell surface

and there was no detect ible synthesis o f PgO-1.

134

M r x 10 -3

TOP

9 7 . 4 6 6 . 2 - -

4 5 . 0 - -

3 1 . 0 - -

2 1 . 5 - -

1 4 . 4

B B--

M r x 1 0 - 3

TOP

9 7 . 4 - -

6 6 . 2 - -

4 5 . 0 - -

31 .0 - -

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

2 h r s . ] 5 h r s . 1 1 0 h r s . I 2 2 h r s .

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 ( B )

S u r f a c e

. . /PgO +2

PgO - 1

2 1 . 5 - -

14 .4 - -

B+B - -

Fig. 3 (A,B). Synthesis and expression of ookinete proteins during transformation of zygotes. Zygotes (ap- prox. 2 h post-gametogenesis) were allowed to develop under conditions supporting transformation into ookinetes. At various times (as indicated) during development, cells were pulse-labelled with [~SS]methio- nine for 10 min. The cells were either extracted immediately for immunoprecipitation (A) or washed and chased with 2 mM L-methionine in medium 199. Following 60 min of chase the cells were reacted intact with antibodies, extracted in Triton X-100 and processed as in Materials and Methods (B). All samples were analyzed under non-reducing conditions on SDS-PAGE (12.5% acrylamide). Immunoprecipitations were with NRS (lane 1), rabbit anti-ookinete serum (lane 2), MAb IID2B3B3 (lane 3) and IID2Cs (lane 4).

135

Further biochemical characterization of ookinete surface proteins. Zygotes were bio- synthetically labelled with [3H]glucosamine or [3H]mannose for 24 h during transfor- mation into ookinetes. Mature ookinetes were reacted intact with rabbit anti-ookinete serum or with Mab IID2B3B3 prior to extraction with Triton X-100. Both PgO-I and PgO-2 were labelled with [3H]glucosamine and [3H]mannose (Fig. 4). Other proteins in the cells were not labelled with these sugars (data not shown). Labelling of PgO-1 and PgO-2 was, therefore, probably due to their direct glycosylation and not to incorporation of tritiated moieties into amino acids.

Both PgO-1 and PgO-2 were also labelled when zygotes were incubated in the presence of [3H]palmitic acid during transformation (Fig. 5). Similar results (using rabbit anti-ookinetic serum or Mab IID2B3B3) were obtained by immunoprecipitation from intact cells or from Triton X-100 extracts.

The [3H]palmitic acid appeared to be tightly associated with the proteins. First the label remained associated with the proteins during extraction with Triton X-100 and electrophoresis in the presence of sodium dodecyl sulphate and was not removed by

PgO-2 PgO-1

Fig. 4. Incorporation of [3H]glucosamine (A) or [3H]mannose (B) into proteins during transformation of

zygotes into ookinetes. Zygotes were labelled with radioactive sugars under conditions supporting their

t ransformation into ookinetes. After 24 h cells were reacted intact with various antibodies and processed as

in Materials and Methods. Immunoprecipitat ions were done with rabbit anti-ookinete serum (lane 1) or MAb IID2B3B3 (lane 2).

136

Mr x 10- 3 [3HI PALMITATE

Top,,__ 97.4 - - 66.2/ -

45.0--

31.0--

21.5--

14.4--

[35S] METHIONINE 1251

i

~i~i!ili~iiiii~i~i~!~ i~ii ~i ~

B~B-- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

~ / P g O - 2

"ql-"x p g O_ 1

B A B A B A

Fig. 5. [3H]palmitate labelling of ookinete proteins: fl uorograms and autoradiogram of immunoprecipita- ted proteins before (B) and after (A) methanolic-KOH hydrolysis. Zygotes during transformation were labelled with radioactive precursors and extracted in Triton X- 100. Labelled proteins were immunoprecipi- tared with rabbit anti-ookinete serum (lanes 1,3, 7, 11, 13, 15), monoclonal antibody IID2B~B 3 (lanes 2, 4, 6, 10, 14, 16), monoclonal antibody IID2C 5 (lanes 5, 9) and normal rabbit serum (lanes 8, 12). Methanolic- KOH hydrolysis was carried out as described under Materials and Methods in situ on gels. Arrows show the positions of ookinete-proteins.

repeated extraction of immunoprecipitated proteins with chloroform and methanol. Second, the label was released, however, by mild methanolic-alkali hydrolysis (Fig. 5, lanes 1-4). Most of the label released co-migrated with methyl-palmitate in thin layer chromatography on silica gels (Fig. 6). Fig. 5 also shows that similar methanolic-alkali hydrolysis of the proteins labelled with [aSS]methionine (lanes 5-12) or 125I (lanes 13-16) did not remove or reduce these labels.

Covalent binding of fatty acids in an ester (serine, -OH group) or thioester (cysteine, -SH group) linkage has been shown to be a post-translational modification of many bacterial, viral and mammalian glycoproteins ([9,10, and 1 1] for review). This is the first observation of acylation of membrane proteins in a malarial system. Recently, myristylation of the membrane form of a Trypanosoma brucei variant surface glyco- protein, probably not directly linked to amino acid groups [12] and presence of glycerol and fatty acids in the C-terminal end of a variant surface glycoprotein from T. equiperdum have also been reported [ 13]. Recently we have also observed that both PgO- 1 and PgO-2 are quantitatively heavily labelled by [3H]myristic acid as compared to [3H]palmitic acid (N. Kumar, unpublished observations).

The functional significance of protein acylation in any biological system is not known. It has been proposed that acylation may help anchor the proteins into the lipid bilayer, or function in intracellular transport of membrane proteins and in membrane

137

8 C

~60

~,40 z

20

~Origin

, L , L C - C -

2

I I I I I [ (AI

f IB) n t

q / _ _ • : _~ - ~ i

4 6 8 10 12 14 Distance (cm)

Fig. 6. Identity ofmethanolic-KOH hydrolysis product from ookinete proteins with methylpalmitate. Details of procedures are described under Materials and Methods. (A) Standard methylpalmitate marker and (B) distribution of [3HI counts released from immunoprecipitated ookinete proteins by methanolic-KOH hydrolysis.

fusion events (review [11]). Both PgO-I and PgO-2 show strong interaction with the detergent Triton X-114 [14] and are highly hydrophobic proteins (N. Kumar and R. Carter, 1983, J. Cell Biochem. 7A, 43A). It is possible that fatty acids covalently linked to proteins increase the hydrophobicity of the proteins and facilitate their interaction with the lipid bilayer. The fatty acids bound to proteins could also faciliate the interaction of ookinetes with mosquito tissue, during their passage through the mosquito midgut wall.

In spite of the shared chemical characteristics of glycosylation and acylation recorded above, PgO-I and PgO-2 are otherwise structurally dissimilar. Following immunoprecipitation from Triton X-100 extracts of [35S]methionine labelled cells with Mabs IID2B3B3 or IID2C5 and separation on SDS-PAGE, gel pieces containing the respective labelled proteins were digested with proteases (Staphylococcus aureus V-8, trypsin-TPCK or tt-chymotrypsin-TLCK) and the digested peptides separated on a 10-30% acrylamide gradient SDS-PAGE. The patterns of labelled peptide frag- ments were different for each protein (Fig. 7). Further evidence of the structural dissimilarity of PgO-1 and PgO-2 is provided by the very fact of their differential recognition by different Mabs (IID2B3B3 and IID2C5) and also by certain immune rabbit sera raised against early zygotes and female gametes of P. gallinaceum (C. French and D. Wirth, personal communication). The proteins have also been shown to be products of two different mRNAs whose in vitro translation products show differential sensitivity to trypsin digestion (C. French and D. Wirth, personal commu- nication).

Results in this paper clearly demonstrate that P. gallinaceum zygotes during trans- formation into ookinetes synthesize two new major membrane proteins (PgO-1 and PgO-2) whose synthesis is developmentally regulated. In addition to glycosylation

138

1 2 1 2 1 2

S.a.V-8 Trypsin Chymotrypsin Fig. 7. Peptide maps of PgO- 1 (M 26 000) and PgO-2 (M r 28 000) proteins. Immunoprecipitates of Triton X- 100 extracts of [35S]methionine labelled cells with MAb IID2B3B 3 (specific for M r 28 000 protein) or MAb IID2C5 (specific for Mr 26000 protein) were run on 10% SDS-PAGE. Gel pieces containing the protein bands were cut out and digested with various proteases according to Cleveland et al. [8] as described in Materials and Methods. The digested products were separated on a 10-30% acrylamide SDS-PAGE. Lane 1 shows the peptide maps of PgO-2 (Mr 28 000) protein, and lane 2 shows the peptide maps of PgO-I (Mr 26 000) protein.

these pro te ins also share ano the r i m p o r t a n t feature and tha t is, bo th con ta in covalen t -

ly l inked fa t ty acids a n d thus add ing mala r ia l ant igens to the list o f fast g rowing

l i te ra ture on this in teres t ing modi f i ca t ion o f m e m b r a n e prote ins . Despi te all these

c o m m o n features , they appea r to be s t ruc tura l ly different and are cer ta in ly func t iona l -

ly dis t inct as only one o f them (PgO- 1) is the target ant igen for monoc lona l an t ibod ies

b lock ing pos t - fe r t i l i za t ion deve lopmen t of the paras i tes in the mosqu i to midgu t

lumen.

ACKNOWLEDGEMENTS

We are grateful to Dr. L .H. Mil ler for encouragemen t and suppor t . The au thors

t hank Mrs. C. G r o t e n d o r s t and Mrs. R. H e a r n for technical ass is tance and Mrs.

W i l m a Davis and Mrs. Brenda Mar t in for p r epa ra t i on of this manuscr ip t . These

studies received a par t ia l f inancia l suppor t o f the U N D P / W o r l d B a n k / W H O Special

P r o g r a m m e for Research and Tra in ing in Trop ica l Diseases and Senior Fe l lowsh ip

f rom the Bur roughs Wel lcome F o u n d a t i o n to N i rbh a y Kumar .

139

REFERENCES

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