Studies of a Glycoprotein in the Oocysts of Eimeria tenelk (Received f’or publication, August 29, 197.5)

RONALD L. STOTISH, CHING C. WANG, MARTIN HICHENS, WILLIAM J. A. VANDENHEUVEL, AND PAUL GALE

From the Merck Institute for Therapeutic Research, Rahway, New Jersey 07065

A glycoprotein unique to the cytoplasm of the unsporulated oocyst of’ Eimeria tenella has been purified

and partially characterized. The protein has a molecular weight of 30,000, of which approximately 40% is

carbohydrate. The carbohydrate portion of the molecule consists of glucose, galactose, mannose, xylose,

glucosamine, and galactosamine, with no detectable sialic acid. The protein portion contains approxi-

mately 141 residues, being rich in hydrophilic amino acids with very few aromatic amino acids and no

cystine.

The protein comprises 14% of the total soluble protein of the unsporulated oocyst but has not been

identified in the cytoplasm of any other developmental stage of the organism. Using polyacrylamide gel

electrophoresis and a radioimmunoassay specific for the protein, it has been shown to disappear from the

cytoplasm between the 15th and 20th hour of the 20-hour sporulation process. Subsequent immunofluo-

rescence experiments have shown a reactive material as a component of the sporozoite membrane. These

results indicate that the glycoprotein is a structural protein of the sporozoite membrane, apparently

synthesized by the unsporulated oocyst and incorporated into the sporozoite membrane as one of the last

steps involved in the sporulation process.

Eimeria tenella (Merck #18) is a parasitic protozoon infect-

ing the caecal epithelial cells of chickens (1). The parasite has a

rather complex life cycle, most of which is intracellular.

Toward the end of its development within the host, the asexual

merozoites differentiate to macrogametocytes and mi-

crogametocytes. Sexual reproduction then occurs forming

zygotes and then unsporulated oocysts. Upon maturation, the

unsporulated oocysts of E. tenella accumulate in the caeca and

are gradually excreted in the feces (2). They can be harvested

in large numbers and easily purified under anaerobic and

sterile conditions (3). Exposure to oxygen initiates the process

of sporulation which can be controlled and manipulated in

uitro. This segment of the life cycle of E. tenella, which

involves one meiosis, two mitoses, and the formation of

sporocysts and sporozoites within the oocyst (4), thus provides

a good opportunity for biochemical studies of development of

the parasite.

Much investigation of E. tenella sporulation has already

been carried out. Degradation of the storage polysaccharide (5)

and respiration during sporulation (6, 7) have been studied.

The authors have also noted the decrease of many enzyme

activities such as amylopectin phosphorylase (8) and dihy-

drofolate reductase (9) as well as the changes of properties of

leucine aminopeptidase (10) during sporulation. There is also

limited synthesis of RNA and protein in the early phase of

sporulation (ll), although the total content of protein in the

unsporulated and sporulated oocysts remains equal. There is,

however, a shift of 13 to 14% of the total protein from

cytoplasm to the membrane fraction after sporulation (11).

This observation corresponds quantitatively to a major protein

band found in disc electrophoresis of the soluble cytoplasm of

unsporulated oocysts. The present report deals with the

isolation and identification of this protein and elucidation of its

possible biological function.

EXPERIMENTAL PROCEDURE

Preparation of Crude Extracts of Eimeria tenella Oocysts-A pure strain of E. tenella (Merck #18) was maintained in the laboratory by monthly serial passage in chickens. The unsporulated oocysts of the parasite were harvested, purified, and stored under anaerobic condi- tions as previously described (7). Sporulated oocysts of E. tenella were obtained by a 24.hour aerobic incubation of the unsporulated oocysts at 30” (7). The process of sporulation was synchronous and resulted in over 9010 sporulated oocysts.

Crude extracts of the oocysts were made from suspensions of 1.5 x lo7 oocysts/ml in 0.05 M Tris-Cl, pH 7.2. The suspension, kept at O-4”, was sonicated in a Branson W185 sonifier at 60 watts for 8 min in 60-s bursts. Cell debris was removed by centrifugation at 105,000 x g for 1 hour.

Purification of Glycoprotein-The crude extract of unsporulated oocysts of E. tenella (109 ml, 2.7 mg of protein/ml) was brought to 25% saturation with ammonium sulfate by the gradual addition of 36.3 ml of saturated ammonium sulfate solution with stirring over a 30.min period. After stirring 1 hour at 4’ the precipitate was removed by centrifugation at 10,000 x g for 15 min. The supernatant fraction was brought to 60% saturation by addition of 127.2 ml of saturated ammonium sulfate by the same procedure. This precipitate was resuspended in 10 ml of 0.05 M Tris-Cl, pH 7.2, and dialyzed against the same buffer with four changes in 24 hours. The dialysate was mixed with an equal volume of a thick slurry of DEAE-cellulose (Cellex-D, Bio-Rad) previously equilibrated with 0.05 M Tris-Cl, pH 7.2, stirred gently for 15 min, and the resin collected by a brief centrifugation. It was then eluted batchwise with 50.ml aliquots of 0.1 M NaCl in 0.05 M

T&Cl, pH 7.2, twice and 0.2 M NaCl in 0.05 M Tris-Cl, pH 7.2, twice. The 0.2 M NaCl eluates were then combined and concentrated to 5 ml in an Amicon ultrafilter with a PM-10 membrane under pressure of 40 p.s.i. It was next passed through a column (2.5 x 70 cm) of Sephadex G-200 (Pharmacia) and eluted with 0.05 M Tris-Cl, pH 7.2. The effluent was collected in 1.0.ml fractions. The entire procedure was carried out at O-4”.

302

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Glycoprotein of Eimeria tenellu Oocysts 303

Disc Electrophoresis and Ultracentrifugation-Disc electrophoresis in 7.5% polyacrylamide gel was carried out in Tris-borate buffer, pH 9.0 (12). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was done according to the procedure of Weber and Osborn (13) with the modification of Segrest and Jackson (14) for glycoproteins. The gels were stained with Coomassie brilliant blue and destained by diffusion in 7Y0 acetic acid. The glycoprotein in gels was identified by periodic acid-Schiff staining (15). Protein concentrations were determined by the method of Lowry et al. (16). Sedimentation coefficient of the glycoprotein was measured by the sedimentation velocity analysis described by Chervenka (17) in a Beckman analytical ultracentrifuge; the data was analyzed by plotting log r versus t.

Amino Acid and Sugar Analysis-Samples for amino acid analysis were hydrolyzed in 6 N HCl in uacuo at 110” for 16 hours and analyzed with a Beckman 120C amino acid analyzer.

Acid hydrolysates from in uacuo heating in 2 N HCl at 110’ for 5 hours were used for neutral sugar analysis. HCl was removed by evaporation under vacuum at 4O, and the neutral sugars reduced by sodium borohydride and acetylated by acetic anhydride according to Abdel-Akher et al. (18). The alditol acetate derivatives were separated by gas-liquid chromatography (19) using a 3% ECNSS.M on Gas- Chrom Q column (Regis Chemicals) and analyzed by a mass spectrome- ter connected with the chromatographic column. Identification of the neutral sugar derivatives was made from the retention times in gas-liquid chromatography and comparison of mass spectra with authentic samples of alditol acetates.

Total content of hexosamine was estimated by the method of Berman and Gatt (20). Identification and quantitative estimation of glucosamine and galastosamine in the acid hydrolysates were carried out in the amino acid analyzer using a Beckman AA-15 custom research resin column at 65O. The column was eluted with 0.2 M sodium citrate, pH 3.25, followed by 0.2 M sodium citrate, pH 4.51, + 5% methylcellosolve. Authentic samples of glucosamine and galactosa- mine (Calbiochem) were utilized for comparison. Hydrolysates of the glycoprotein from a 90-min heating at 80” in 1 N H,SO, were assayed for sialic acid by the thiobarbituric acid method of Warren (21). @ elimination was carried out according to the procedure of Spiro (22).

Preparation of Antiglycoprotein Serum-Male New Zealand white rabbits of 3 to 4 kg were each injected subcutaneously and intrader- mally with 0.5 mg of the purified glycoprotein in 1.0 ml of an emulsion containing 50% (v/v) Freund’s complete adjuvant (Difco). The injec- tion was repeated with incomplete adjuvant on days 40 and 76 accompanied by an additional intravenous dose of 0.25 mg of glycopro- tein for each rabbit on Day 76. All rabbits developed antibody against the glycoprotein detectably by immunodiffusion by Day 58.

Double Diffusion Test-Antiglycoprotein serum from Day 97 was placed in the center wells of Ouchterlony immunodiffusion plates (Hyland) against samples of serial dilutions in the outer wells. Precipitin lines were read at 24 and 48 hours of incubation. Two-tenths micrograms or more of the purified glycoprotein formed a visible precipitin band, but the test could not detect 0.15 pg or less of the glycoprotein.

Radioimmunoassay-Fifty microliters of 0.25 M phosphate buffer, pH 7.5, containing 20 pg of the purified glycoprotein and 1 mCi of lzsI (added as NaI (New England Nuclear) in 5 pl of 0.1 N NaOH) were mixed with 10 ~1 of 0.05 M phosphate buffer, pH 7.5, containing 20 rg of chloramine-T. After 20 s, 50 ~1 of sodium metabisulfite (2.5 mgiml) and 100 ~1 of potassium iodide (10 mg/ml) were added. The mixture was fractionated on a lo-ml Bio-Gel P-60 (Bio-Rad) column in 0.05 M phosphate buffer, pH 7.5. The peak fraction of the glycoprotein, with an estimated specific activity of 15 wCi/pg, was used for radioim- munoassay.

The assay was carried out by the general procedure of Hichens et al. (23). Samples (0.10 ml) of serial dilutions were mixed with 0.10 ml of ‘*9+glycoprotein (4.0 rig/ml) and 0.20 ml of 1:5000 diluted antiglyco- protein serum collected on Day 97. All dilutions were made with 0.01 M

phosphate-buffered saline, pH 7.6, containing 1 mg/ml of bovine serum albumin. After overnight incubation at 4”, 1 ml of a dextran-charcoal suspension containing 0.1% dextran T-80 (Pharmacia) and 0.5% Norit A (Amend Drug & Chemical Co.) in phosphate-buffered saline was added and the mixture allowed to stand at 0’ for 20 min. Following centrifugation at 1200 x g for 15 min the antibody-bound label remained in the supernatant while the free glycoprotein was sedi- mented with the charcoal. The radioactivity in both supernatant and charcoal was counted in a Beckman Gamma 300 counter. A standard curve was constructed between 0.5 and 50 ng of the purified glycopro- tein.

Immunofluorescence Staining-Sporocysts of E. tenella were re- leased from the sporulated oocysts by grinding in a tissue homogenizer for 10 min and purified through a glass bead column (2 x 10 cm) (Super Brite 100, 3M Co.) (24). Sporozoites of E. tenella were obtained from the sporocysts by in vitro excystation as previously described (25) and purified similarly by filtering through glass beads. Samples of oocysts, sporocysts, and sporozoites were air-dried on microscopic slides and incubated with the 113th day rabbit antiglycoprotein serum samples of a-fold serial dilutions at room temperature for 30 min (26). Normal rabbit serum was included as controls. The slides were washed in phosphate-buffered saline and incubated with fluorescein-conju- gated anti-rabbit IgG serum (Cappel Laboratories) at room tempera- ture for another 30 min. The goat anti-rabbit IgG was also used in 2X serial dilutions in a cross-hatch-type pattern. Following one more wash in phosphate-buffered saline, the slides were air-dried, mounted, and examined under a Leitz Dialux microscope equipped with a mercury lamp and a Bausch and Lomb 5-58 exciter filter with maximum transmittance at 400 nm.

Immunization of Chickens against E. tenella Infection-Two-week- old female White Leghorn chickens isolated in sterile boxes were repeatedly infected with increasing oral doses of sporulated oocysts of E. tenella at a-week intervals. Beginning at 500 oocysts/bird, the inocula were gradually increased to 5,000, 50,000, and, finally, 100,000. The infection was monitored by examining the output of oocysts from the chickens 1 week following inoculation. Total immunity was achieved by the 13th week. Sera samples were collected from the chickens by bleeding from the wing vein in the 13th and 15th week.

RESULTS

Identification and Purification of Glycoprotein from Crude Extracts of Unsporuluted Oocysts of Eimeria tenella-Poly- acrylamide gel electrophoresis of the crude extracts of un- sporulated oocysts of E. tenella revealed a major protein band

with an RF value of approximately 0.5 by Coomassie blue stain

(Fig. 1). The major protein was also the only band in the gel

reacting positively to periodic acid-Schiff staining. When crude

extracts of the sporulated oocysts of E. tenella were also

examined by electrophoresis (Fig. l), the glycoprotein was

FIG. 1. Comparison of unsporulated and sporulated soluble protein by polyacrylamide gel electrophoresis. Polyacrylamide gels (7.5%) stained with Coomassie brilliant blue (A and C) and by the periodic acid-Schiff technique (B and D). A and B contain 100 pg each of unsporulated oocyst-soluble protein; C and D contain 100 pg each of soluble protein from sporulated oocysts.

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304 Glycoprotein of Eimeria tenella Oocysts

absent by both methods of staining. Further studies of the time samples of E. tenella oocysts during sporulation by radioim- munoassay indicated rapid disappearance of the glycoprotein between 15 and 20 hours into the process of sporulation (Table I). At this late phase of sporulation, it was observed microscop- ically that sporocysts and sporozoites were being formed inside the oocysts (4).

The glycoprotein was purified from the crude extracts of unsporulated oocysts according to the methods described under “Experimental Procedure.” Gel electrophoresis was originally utilized to monitor the purification. At the gel filtration step, the glycoprotein was eluted from Sephadex G-200 column in a single, sharp peak (Fig. 2). When 25 pg of this peak sample were examined by sodium dodecyl sulfate gel electrophoresis (Fig. 3), only a single, dense protein band was visible, indicating the homogeneity of the purified glycopro- tein. Three barely visible bands were also observed but accounted for less than 1% of the total material by gel scanning. The purified sample was then used to immunize rabbits as well as to prepare the Y-glycoprotein. Radioim- munoassays were then used as a quantitative assay of the subsequent repetitions of the same purification procedure. The results summarized in Table II indicate an overall yield of approximately 50% of the original glycoprotein in crude ex- tracts.

Studies of Molecular Weight and Composition of Glyco- protein-Sedimentation velocity analysis of the purified glyco- protein gave a sedimentation coefficient of 3.1 S, corresponding to a molecular weight of approximately 30,000 daltons assum- ing a spherical molecule (Fig. 4). Sodium dodecyl sulfate gel electrophoresis, shown in Fig. 3, agreed with the data from ultracentrifugation and yielded a molecular weight of 30,000 f 3,000 for the glycoprotein. It must be stated that both of these methods yield only approximations of the true molecular weight. The sodium dodecyl sulfate gel electrophoresis (14), as performed, is known to give values approaching the true molecular weight for glycoproteins. Mole composition data for amino acids and carbohydrates based on these approximations (Table III) total to approximately 34,000. This agreement seems to support the validity of the molecular weight estima- tions.

The glycoprotein appeared to be hydrophilic because it remained in solution at 100” for at least 5 min (27). Amino acid analysis of the glycoprotein (Table III) showed high levels of hydrophilic amino acids like glutamate, alanine, threonine, aspartate, arginine, serine, low levels of aromatic amino acids, and no cysteine. Based on the estimated molecular weight, there are about 141 amino acid residues in each molecule, accounting for 18,488 daltons or approximately two-thirds of the molecular weight of the glycoprotein.

TABLE I

Radioimmunoassay analysis of glycoprotein content of sporulating oocysts

Sample pg of glycoprotein/ mg of total protein

Unsporulated oocysts 144

At 2 hours sporulation 118

At 6 hours sporulation 137

At 10 hours sporulation 120

At 14.5 hours sporulation 121

At 21.5 hours sporulation 23.0

94% Sporulated oocysts 8.9

Extract of sporozoites and sporocysts 10.0

Identification and quantitative estimation of the neutral sugars in glycoprotein by gas-liquid chromatography are shown in Fig. 5. The identification of each neutral sugar was corroborated by mass spectrometry. The results indicate each glycoprotein molecule contains 60 glucose, 18 xylose, 5 man- nose, and 3 galactose residues (Table III). The Berman-Gatt method (20) gave a value of 7.5 c(g of hexosamine/mg of the purified glycoprotein or approximately 1.2 mol of hexosamine/ mol of glycoprotein. Further studies with an amino acid analyzer indicated the presence of 0.6 glucosamine and 0.4 galactosamine residues in a glycoprotein molecule (Table III). The total sugar content is thus estimated to be more than one-third of the mass of the glycoprotein molecule. No sialic acid was detected in purified samples of the glycoprotein by the thiobarbituric acid method of Warren (21).

Studies on Glycoprotein during Sporulation of E. tenella-With the availability of rabbit antiglycoprotein se- rum, crude extracts of unsporulated and sporulated oocysts, sporocysts, and sporozoites of E. tenella were examined in the

SEPHADEX G-200 ELUTION

FIG. 2. Elution profile of a column (2.5 x 90 cm) of Sephadex G-200. Elution was performed at 4’ with 0.05 M Tris-Cl, pH 7.2, and l-ml fractions collected.

FIG. 3. Sodium dodecyl sulfate gels of purified glycoprotein from Eimeria tenella. A is 7.5% acrylamide gel; B is 10% acrylamide; and C is 12.5% acrylamide. Each gel contains 25 pg of protein.

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Glycoprotein of Eimeria tenella Oocysts 305

TABLE II

Radioimmunoassay of glycoprotein purification and characterization

Pg

Sample Total glyco-

protein protem/

mg of proten?

-

Puri- fica- tion

Yield

Purification Unsporulated 0.25 saturated (NH,),SO,

precipitate 0.25-0.60 saturated

(NH,),SO, precipitate 0.2 M NaCl wash of DEAE-

cellulose Sephadex G-200 peak

Characterization Pronase digestion products

of 1 mg of purified glyco- protein

@ Elimination products of 1 mg of pure glycoprotein

a-Amylase digestion products of 1 mg of purified glycopro- tein

&Amylase digestion products of 1 mg of purified glycopro- tern

w

294 144 10.7 100

133 310

34 665

20 1000

0.0

0.0

0.4

0.0

- fold 9%

1.0 2.8 sz 0.3

2.2 97.0 * 3.0

4.6 53.3 * 5.0

7.0 47.5

TABLE III

Amino acid and sugar analysis of glycoprotein from Eimeria tenella unsporulated oocysts

Ammo acid Mole/mole Of Dl’Oteln

Sugar MOle/~Ol~ of protem

Lysine 5.8 Glucosamine 0.6

Histidine 1.2 Galactosamine 0.4

Arginine 11.6 Glucose 60.0

Aspartic acid 11.6 Galactose 3.0

Threonine 12.2 Xylose 18.0

Serine 9.2 Mannose 5.0

Glutamic acid 24.1

Proline 7.1 Glycine 6.9 Alanine 15.3 Valine 8.6 Methionine 7.3

Isoleucine 2.7 Leucine 8.1

Tyrosine 3.6 Phenylalanine 2.9

Tryptophan 2.9

I GLC ANALYSIS OF NEUTRAL SUGARS

I I I I I I 0 16 5 21 5 245 ‘276 34

P’ / / 1000 2000 3000

TIME (SECONDS) ----w

FIG. 4. Sedimentation coefficient determination for purified glyco- protein of Eimeria tenella. Run performed in a Beckman mode1 E analytical ultracentrifuge using ultraviolet absorption optics.

double diffusion tests shown in Fig. 7. The glycoprotein was present only in extracts of unsporulated oocysts; all other samples (up to 0.5 mg total protein) failed to form a visible precipitin band with the antiglycoprotein serum. The concen- tration of the glycoprotein in extracts of E. tenella un- sporulated oocysts, estimated by radioimmunoassay (Table II), turned out to be 16 f 3% of the total soluble protein or 32 + 6 pg/lOs unsporulated oocysts. The glycoprotein with its carbohydrate moiety removed by p elimination showed no detectable reactivity in either the radioimmuno- or immunodif- fusion assay (Fig. 6 and Table II).

The unsporulated, sporulated oocysts, sporocysts, and sporo- zoites of E. tenella were also examined by immunofluorescence staining. The sporozoites were the only cells exhibiting fluores- cence (Fig. 7).

Studies of Possible Role of Glycoprotein in Developing Immunity to Coccidiosis in Chickens-Two-week-old White

TIME WINSI -

FIG. 5. Gas-liquid chromatography (GLC) elution profile of alditol acetate derivatives of neutral sugars of the Eimeria tenella glycopro- tein. Chromatography performed on 3% ECNSS or Gas-chrom Q at 190” constant temperature using a flame ionization detector system in a Perkin Elmer mode1 900 gas chromatograph.

Rock chickens, each orally inoculated with 5 x 10’ E. tenella

sporulated oocysts, were injected intravenously with 1.0 ml of rabbit antiglycoprotein sera at time 0, 1 day and 2 days after inoculation. The results indicated no detectable protective activity from the infected sera against the parasitic infection. On the other hand, sera samples from chickens immunized against E. tenella infection were tested against the purified glycoprotein on Ouchterlony immunodiffusion plates. There was no demonstrable antibody to the glycoprotein in the immune chicken’s sera.

Unsporulated oocysts of Eimeria acervulina, an intestinal species of coccidia infecting the duodenum of chickens, were harvested and purified by a procedure similar to that for E. tenella. Crude extracts of E. acervulina unsporulated oocysts were prepared by the same method as previously described. Polyacrylamide gel electrophoresis of the extracts showed neither a major protein band nor glycoprotein band. Double diffusion test and radioimmunoassay, however, revealed some material capable of cross-reacting with the antiglycoprotein serum (Fig. 6).

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306 Glycoprotein of Eimeria tenella Oocysts

FIG. 6. Kddml lmmunodlffuwm of samples agamst rdbblt anti- glycoprotein serum. All sample wells contam 15 pg of protein; center wells contam 800 pg of the rabbit antiserum. a, Elmerza tenella unsporulated oocyst-soluble protein; b, E. tenella sporulated (94%) oocyst-soluble protein; c, E. tenella sporocyst and sporozoite-soluble protein; d, soluble protein from Ezmerm aceruulina unsporulated oocysts (note spur formation); e, purified glycoprotein; f, same as a; g, @ elimination products of purified glycoprotem; h, same as e; 1, Pronase digestion products of purified glycoprotein; j, same as b.

DISCUSSION

The discovery of a glycoprotein comprising 16 5 3% of the soluble protein of the unsporulated oocysts of E. tenella agrees quantitatively with two of our previous observations. The sporulated oocysts of E. tenella have been shown to contain 13 to 14% less soluble protein as compared with the unsporulated oocysts (11). This decrease of soluble protein, however, is offset by an equal increase of the membrane protein of the sporulated oocysts, the total protein per cell thus remaining constant. The gross arithmetic agreement between the protein distribution changes and glycoprotein concentration, coupled with the immunofluorescence staining of the sporozoite membranes by antiglycoprotein serum, have led us to tentatively conclude that the glycoprotein is a protein component of the sporozoite membrane. The failure to detect this particular protein in all other developmental stages examined implies its specific syn- thesis by the unsporulated oocyst for use in the next step of differentiation. This should not be surprising when one reasons that the unsporulated oocyst is only a brief, transient period in the normal life cycle of the organism, the survival of which depends upon the successful completion of the sporulation process. The obligatory differentiation from unsporulated to sporulated oocyst allows one to infer that the unsporulated oocyst is only a preparatory stage, during which the organism must synthesize materials necessary for sporulation and hence survival.

Perhaps a more definitive proof of the protein’s incorpora- tion into the membranes of sporozoites would be to actually disrupt the membranes and recover the material intact. We have attempted this using the lithium 3,5-diiodosalicylate method of Marchesi and Andrews (28) and by sodium dodecyl sulfate-urea extraction. In neither case did we recover any material which could be identified by either polyacryl- amide gel electrophoresis, immunodiffusion, or radioimmuno- assay as being glycoprotein. In fact very little demon- strably solubilized protein was recovered by either technique (<2%). Since the structural organization of the sporozoite membrane is not understood, the failure to recover authentic glycoprotein may be a result of an incorrect choice of tech- nique. A second possibility could be that during the membrane extraction process fragments of the glycoprotein are released but are unidentifiable by our techniques. In fact we have not established that the 30,000 molecular weight protein is indeed incorporated intact into the membrane. Our studies with

with antiglycoprotein serum (rabbit) and then with fluorescen-conju- gated goat antl-rabbit serum. Illummated with Bausch and Lomb mercury arc lamp usmg 5-58 (B & L) exciter filter. Fluorescence observed at a 1:16 dilution of rabbit antisera and a 1:8 dilution of goat anti-rabbit serum (X 400 magnification).

intact protein have shown that deglycosidation and/or Pronase treatment can either reduce or eliminate our capability to identify the protein, although the immunofluorescence data suggests that the protein is incorporated largely intact.

Unsuccessful efforts have been devoted to examining any possible additional enzymic function, such as phosphatase, dehydrogenase, and protease, of the glycoprotein. The compo- sition of the glycoprotein appears unusual because of the high content of hydrophilic amino acids and extraordinarily high number of glucose and xylose residues in the molecule.

Interestingly, another glycoprotein of similar composition, but with a 3 times higher molecular weight, was found as the only prominent protein in the cyst fluid of the larvae of the tapeworm Cysticercus tenuicollis (29). The authors did not pursue the fate of the glycoprotein in subsequent larval development but suggested that it may function as an osmoti- cally active macromolecule maintaining the turgidity of the cyst. It is not known whether the E. tenella glycoprotein has a similar secondary function in unsporulated occysts.

The high glucose content of the glycoprotein prompted speculation as to whether the protein was in any way involved with the amylopectin granules (8) found in the cytoplasm of both unsporulated and sporulated oocysts. The amylopectin granules account for approximately 235 pmol of maltose (4.7 x 10-l pmol of glucose)/106 unsporulated oocysts (8) and are consumed as an energy source during sporulation (sporulated oocysts contain only 93 pmo1/106 cells). Two lines of evidence, however, suggest that the consumption of amylopectin gran- ules and disappearance of the glycoprotein from the cyto- plasm are unrelated. Chronologically during sporulation, the amylopectin is consumed at a linear rate during the first lo- to 12-hour period, at which time the value reaches a constant level (30). The glycoprotein, however, is present at a fixed concentration (Table I) up to the final stages of sporulation (12th to 20th hour), at which time it disappears completely. Secondly, as stated previously, the ability to detect material on the sporozoite membrane by antiglycoprotein serum would seem to indicate that it has been incorporated at least antigenically reactive. There thus would appear no obvious correlation between amylopectin synthesis and degradation and that of the changes associated with the glycoprotein.

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Glycoprotein of Eimeria tenella Oocysts 307

The total amino sugar content of the glycoprotein deter- mined by both Berman-Gatt hexosamine and amino acid analysis is less than 1% by weight. Conditions of hydrolysis employed are known to give less than 15% destruction of amino sugar. Varying the time of hydrolysis from between 4 to 8 hours had no apparent effect on the levels of freed amino sugar. It is our interpretation that the presence of 0.4 mol of galactosa- mine and 0.6 mol of glucosamine/mol of glycoprotein (Table III) reflect a degree of microheterogeneity of the glycoprotein. The distribution of molecules containing glucosamine as op- posed to those containing galactosamine is approximately equal. Such microheterogeneity is well established for many, if not all, glycoproteins (31). The presence of xylose and absence of sialic acid is consistent with the data available on glyco- proteins from invertebrate sources (32).

Since the glycoprotein is apparently a surface antigen of the sporozoite, it was hoped that antibody could be demonstrated in immune chickens. Likewise the possibility of passive immu- nization against E. tenella infection using rabbit antiglyco- protein serum was tested. Neither approach was successful, although this result is neither surprising nor conclusive. It has not been shown that there is a protective humoral antibody response to E. tenellu infection. Also, it is thought that host immunity development occurs during schizogony (33), at which time it is improbable that the glycoprotein would be an exposed antigen, regardless of the type of response generated.

The presence of a material in the unsporulated oocyst of another species of coccidia, E. acervulinn, as shown by

radial immunodiffusion and radioimmunoassay (Table I and Fig. 7) indicates that the observations performed with E. tenella may possibily be expandable to include at least some other species of Eimeria.

Acknourledgments-The authors wish to thank Dr. Carl

Bennett, Dr. Yashwant Karkhanis, and Mr. Carl Homnick for their technical assistance in performing amino acid analysis and hexosamine determinations. We also wish to express our

gratitude to Mr. Jordan Hirschfield for performing the sedi-

mentation coefficient determinations.

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R L Stotish, C C Wang, M Hichens, W J VandenHeuvel and P GaleStudies of a glycoprotein in the oocysts of Eimeria tenella.

1976, 251:302-307.J. Biol. Chem. 

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