An Electrophoretic Analysis of Schistosoma mansoni Soluble Egg Antigen Preparation

Author(s): Clint E. Carter and Daniel G. Colley

Source: The Journal of Parasitology, Vol. 64, No. 3 (Jun., 1978), pp. 385-390
Published by: Allen Press on behalf of The American Society of Parasitologists
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 THE JOURNAL OF PARASITOLOGY
VOLUME 64 JUNE 1978 NUMBER 3

J. Parasitol., 64(3), 1978, pp. 385-390

? American Society of Parasitologists 1978

AN ELECTROPHORETIC ANALYSIS OF
SCHISTOSOMA MANSONI SOLUBLE EGG ANTIGEN PREPARATION*

Clint E. Carter and Daniel G. Colley

The Departments of General Biology and Microbiology, Vanderbilt University,
and the Veterans Administration Hospital, Nashville, Tennessee 37235

ABSTRACT: Schistosoma mansoni soluble egg antigen (SEA) has been examined electrophoretically.
Sodium dodecyl sulfate (SDS) electrophoresis of SEA reveals an extremely heterogeneous protein
composition. At least 18-20 distinct bands stain with Coomassie blue and at least 6 bands stain
with periodic acid Schiff (PAS). Four of the PAS-positive bands stain only faintly with Coomassie
blue. The estimated molecular weight range for these proteins is between 16,000 and 200,000 dal-
tons. An acid soluble fraction was isolated from SEA which contained 5 of the 6 glycoproteins. An
immunoelectrophoretic analysis of SEA reveals at least 5 distinct precipitin arcs when developed
with serum from mice infected with S. mansoni for 16 weeks.

Schistosoma mansoni egg-induced granulo-polysaccharides. The qualitative and quantita-

matous lesions, which are a major cause of
tive aspects of these substances are understood
pathology in murine S. mansoni infection, can only partially (Boros et al., 1976; Pelley et al.,
be induced artificially and elicited by a hetero-
1976). The current study was initiated to
geneous preparation from eggs termed soluble provide an analytical basis for the continued
egg antigens (SEA) (Boros and Warren, elucidation of the chemical composition of
1970). A wide variety of immune responses
SEA using electrophoretic techniques and to
against SEA have been described during the
course of S. mansoni infections in various hosts lead to preparative procedures for the produc-
tion of workable quantities of these moieties.
(Boros et al., 1975; Colley, 1975; Colley
et al., 1977). The host responses observed
MATERIALS AND METHODS
include the production of: (1) several classes
of antibody, and (2) different lymphokines,Preparation of soluble egg antigen
including lymphocyte transformation. Many of
Soluble egg antigen was prepared according to
these host reactions to SEA wax and wane, Boros and Warren (1970) with only slight modi-
depending upon the duration of infection.fications in the procedure. Seven and one-half to
Due to the heterogeneity of SEA, usually it is 8 weeks after infection of mice with S. mansoni
cercariae, eggs were harvested from mouse livers
difficult to form conclusions regarding the
by differential centrifugation. These eggs were di-
relevance of such changes in host anti-SEA luted to -80,000/ml with phosphate buffer (pH
responsiveness in relationship to the concomi-7.4 at 0.01 M) and homogenized in a Potter-
tant immunopathologic events. Previous anal- Elvehjem tissue homogenizer at 4 C. The homog-
enate was centrifuged at 4 C for 90 min, at
yses have shown SEA to be a complex mixture100,000 g. After centrifugation, the supernatant
of proteins, glycoproteins, lipoproteins, and
fraction was concentrated by ultrafiltration using
Amicon ultrafiltration cells equipped with a diaflo
ultrafiltration PM-10 membrane.
Received for publication 30 September 1977.
*This study was supported primarily by the Preparation of the acid glycoprotein fraction
United States-Japan Cooperative Medical Sci-
ence Program through the NIAID, NIH, USPHS This procedure essentially follows that described
Grant AI 12996 and in part by USPHS Grant by Reznick and Winzler (1975). SEA prepared
AI 11289 and the Veterans Administration. as described above was adjusted to a final con-
385

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386 THE JOURNAL OF PARASITOLOGY, VOL. 64, NO. 3, JUNE 1978

centration of 0.6 M perchloric acid using 10 M Holes were 4.0 mm in diameter with centers 9.5
perchloric acid stock. The resultant precipitate mm apart. The plates were incubated for 24 hr
was removed by centrifugation at 10,000 g for at 23 C. Excess protein was removed by diffusion
10 min, and the supernatant fraction was dialyzed into 10 mm sodium phosphate buffer, pH 7.4,
against distilled water and lyophilized. with 0.15 N NaCl and subsequently stained with
Coomassie blue.
Chemical components of SEA
Immunoprecipitation
Protein was measured by the method of Lowry
et al. (1951) using bovine serum albumin as a One hundred /Jiter of serum, pooled from mice
standard. DNA was assayed using a modification infected with S. mansoni for 14 weeks, was over-
of the Burton diphenylamine technique as de- laid with 100 Aliter SEA at a concentration of 2.5
scribed by Giles and Myers (1965) with salmon mg/ml protein in a 1.0-ml conical tube. This
sperm DNA as a standard. Pentose sugar content material was allowed to diffuse for 24-48 hr at
was determined by the method of Dische et al. 4 C. The resultant precipitate was collected by
(1955). Total hexose was measured using the centrifugation at 2,000 g for 10 min. The super-
phenol sulfuric assay as described by Dubois et al. natant fraction was discarded and the pellet was
(1956). washed 3 X using 0.01 M P04 pH 7.2 plus 0.15
M NaCl. The immunoprecipitates were disso-
Disc gel electrophoresis
ciated in 0.01 M sodium phosphate, pH 7.2, 0.1%
Analytical disc gel electrophoresis was carried2-mercaptoethanol. This material was then elec-
out on 11-cm 7.0% acrylamide gels (pH 8.9 sys-trophoresed on SDS gels as described above.
tem) according to the procedures of Gabriel
(1971). Protein bands were stained with Coomas- RESULTS
sie Brilliant blue. Carbohydrates were stained
with the periodic acid Schiff (PAS) procedure of Chemical composition of SEA
Zacharius et al. (1969). Sodium dodecyl sulfate
At a concentration of -80,000 eggs/ml
(SDS) electrophoresis was performed by the pro-
cedure of Weber et al. (1969) using 7.5% acryl- the 100,000 g supernatant fraction contained
amide gels in the presence of mercaptoethanol. between 600 and 800 /jg/ml Lowry reactive
A calibration curve of log molecular weight against protein. This same material contained 200-
relative mobility was prepared, using the following 250 jug/ml total hexose. DNA concentrations
proteins as standard markers: rabbit myosin
(220,000), bovine serum albumin (136,000 and were quite low at 3-5 ytg/ml (from 5 assays)
68,000), ovalbumin (43,000), immunoglobulin G while the pentose sugar reaction often used
(50,000 and 23,500), and cytochrome C (11,700). for RNA indicated between 60-80 u/g/ml.
Protein and carbohydrate staining in SDS gels Coomassie blue staining patterns of SEA,
follow above references except those gels used for
separated using 7.0% polyacrylamide gels, in-
carbohydrate staining were incubated in a sat-
urated solution of 2,4-dinitrophenylhydrazine for dicates a rather heterogeneous protein com-
4 hr, rinsed with distilled water repeatedly until position with 8-15 bands depending on the
excess phenylhydrazine was removed before incu- amount of protein loaded on the gel (Fig.
bation in periodic acid. 1A). The major Coomassie blue staining
bands have electrophoretic mobilities of 0.09,
Immunoelectrophoresis
0.13, 0.25, 0.34, and 0.49 with a relatively
Immunoelectrophoresis was carried out accord- high background throughout most of the gel.
ing to the method of Axelsen et al. (1975).
PAS staining of identical gels electrophoresed
Agarose (1% w/v) plates were prepared using a
barbital buffer (pH 8.6 ionic strength 0.1) con- in parallel with those stained with Coomassie
taining 0.1%o sodium azide (w/v). Electrophoresis blue show only three PAS-positive bands with
of SEA (20 /uliter added to wells at 1.0 mg/ml) mobilities of 0.01, 0.13, and 0.26 (Fig. 1B).
was run at 95 v (10 v/cm) for 1 hr at 15 C. Whereas two of these PAS-positive bands are
Antigen diffusion patterns were developed by the
use of serum pools collected from CF1 mice coincident with major Coomassie blue staining
(Charles Rivers Lab., Wilmington, Mass.) 16 bands, a third PAS-positive band at the origin,
weeks after infection with 30-60 S. mansoni cer- which reacts intensely using PAS, stains only
cariae.
faintly with Coomassie blue. SEA electro-
Ouchterlony double diffusion
phoresed on PAGE gels, which are first
stained with PAS and then counterstained with
Ouchterlony double diffusion analyses were per-
formed on 84 X 94 mm plates coated with 1.0%
Coomassie blue, displayed the same pattern.
Agarose in 10 mM sodium phosphate buffer,The pH major PAS-positive band (relative mo-
bility 0.1) indicates a skewing to the light side
7.4, with 0.15 N NaCl and 0.1% sodium azide.

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 CARTER AND COLLEY-SCHISTOSOMA MANSONI SOLUBLE EGG ANTIGEN PREPARATION 387

LlJ \
0

C)
m
<: I B

i 9111^W
A B C 0 E F

0.25 0.5
FIGURE 2. SDS-polyacryla
MOBILITY
phoresis comparing protei
from SEA and the acid soluble fraction of SEA.
FIGURE 1. Analytical disc gel electrophoresisDetails of procedure as described in text. A, 150
of S. mansoni SEA run on 7.0% acrylamide ug of SEA protein
gels electrophoresed and stained
(pH 8.9 system) as described in text. with Coomassie
A, Absor- blue; B, E, 150 Lg of SEA protein
bance scan (550 nm) of Coomassie blue-stained electrophoresed gel and stained with PAS; C, 80 /g
after electrophoresis of 150 ,ug SEA of protein from B,
protein; the acid soluble fraction of SEA
absorbance scan (565 nm) of PAS stained gel stained with Coomassie blue; D, F, 80 jug of pro-
co-electrophoresed with gel A using 150 ,tg SEA tein from the acid soluble fraction of SEA stained
protein. with PAS. These gels are typical patterns from 20
different preparations.

which may represent the presence of another

species that is not well resolved. (Holden et al., 1971; Kobylka and Carraway,
To determine the number and relative size 1972; Carraway et al., 1971).
of the constituent polypeptide chains of SEA, Boros et al. (1976) and Pelley et al. (1976)
the material was subjected to SDS-acrylamide reported that the major antigenic fractions of
gel electrophoresis. The results indicate an SEA appear to be glycoprotein. In view of
extremely heterogeneous protein composition. the fact that several glycoproteins are soluble
Coomassie blue staining patterns of SEA run in dilute perchloric acid (PCA), we have
on SDS gels are shown in Figure 2A. At least subjected SEA to PCA fractionation. The
18-20 bands can be observed. The estimated carbohydrate (total hexose)/protein ratio of
molecular weights for these proteins fell in SEA
the is 0.28 while the carbohydrate to protein
range of 16,000-200,000 daltons. SDS gelsratio
of of the PCA soluble fraction from SEA
SEA run in parallel with those used for Coo-is 0.73. This enrichment in carbohydrate
massie blue staining were stained for carbo-moieties also is reflected in the electrophoretic
patterns on SDS gels (Fig. 2C, D). The total
hydrate. At least six PAS-positive bands were
observed (Fig. 2B). As can be seen from Fig-protein profiles of the two preparations using
ure 2A, B, at least four of these PAS-positive
Coomassie blue staining on SDS gels are quite
different (Fig. 2A, C). However, five of the
bands stain only slightly with Coomassie blue.
A lack of Coomassie blue staining previouslysix PAS-positive glycoprotein bands present in
has been reported for several glycoproteins SEA identified by SDS gel electrophoresis

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388 THE JOURNAL OF PARASITOLOGY, VOL. 64, NO. 3, JUNE 1978

*i

LJ

A, 150 t~g SEA protein; B

FIGURE 3. Ouchterlony double immunodiffu-

sion (A) and immunoelectrophoresis (B) of SEA
and serum from mice infected for 16 weeks with
S. mansoni. A, Center well contains 20 Mliter of 0 0.5 0pr
MOBILITY
SEA at 2.5 mg proteins/ml. Outer wells contain
20 Aliter of 16-week-infected mouse serum. B, FIGURE 4. Absorbance scans of SDS electro-
Wells contain 20 tliter SEA at 2.5 mg protein/mlphoresis
phoresis gelsgels
stained with Coomassie
stained blue. Con- blue. Con-
with Coomassie
and trough was filled with serum from the same ditions as described in "Materials and Methods."
serum pool used in the double diffusion assays. A, 150 oug SEA protein; B, immunoprecipitate
Conditions were as described in "Materials and from SEA and 16-week-infected mouse serum; C,
Methods." 80 tg protein from the acid soluble fraction of
SEA.

were observed in the PCA soluble fraction

nine bands on SDS-polyacrylamide gels stained
(Fig. 2E, F). In studies to be reported else-
with Coomassie blue (Fig. 4B). Seven of
where, both SEA and the enriched glycopro-
these bands are distinct from the polypeptides
tein fraction were observed to induce a strong
of mouse immunoglobulins. The relative mo-
blastogenic response using lymphocytes from
bilities for mouse y-globulin heavy and light
mice infected with S. mansoni for 8 weeks.
chains are 0.48 and 0.79, respectively. The
Ouchterlony immunodiffusion analysis ofelectrophoretic pattern of this solubilized im-
SEA developed with serum from mice infectedmunoprecipitate is compared to the electro-
with S. mansoni for 16 weeks indicates three
phoretic pattern of SEA and the glycoprotein
to four precipitin bands (Fig. 3B) which de-
enriched fractions of SEA in Figure 4. PAS-
veloped within 24 hr. An immunoelectropho-staining of solubilized immunoprecipitates on
retic analysis of the same SEA preparation and
SDS-gels run in parallel with those used for
developed with the same serum pool displays Coomassie blue staining indicate at least six
at least five distinct precipitin arcs (Fig. 3A).PAS-positive bands (Fig. 5). Five of these
Solubilization of the antigen-antibody pre-PAS-positive bands are distinct from the heavy
cipitates produced by mixtures of SEA and chain of mouse y-globulin (relative mobility
serum from mice infected for 16 weeks yielded of 0.49). For comparative purposes a tracing

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 CARTER AND COLLEY-SCHISTOSOMA MANSONI SOLUBLE EGG ANTIGEN PREPARATION 389

of the PAS-positive profile of SEA on SDS

gels is also presented in Figure 5.

DISCUSSION

As previously described (Boros and Warren,

1970; Pelley et al., 1976), an electrophoretic
analysis of SEA on polyacrylamide gels re-
veals a complex pattern of proteins and glyco- LLJ

proteins. As indicated by the electrophoretic

m
patterns of SEA on SDS-acrylamide gels, even
regular polyacrylamide gels represent a simpli-
n

fication of the complexity of the heterogeneous

SEA preparation. Similarly, an analysis of the
antigenic components of SEA using Ouchter-
lony double diffusion assays often reveals
three precipitin bands whereas immunoelectro-
phoresis indicates at least five distinct pre-
cipitin bands. Using Ouchterlony immunodif-
fusion, Pelley et al. (1976) reported one
precipitin line which developed after 24 hr at 0 0.5
MOB I LITY
1.0

room temperature while two additional pre-

cipitin lines developed after incubation at 4FIGURE
C 5. Absorbance scans of SDS electro-
for another 36 hr. phoresis gels stained with PAS. Conditions as de-
scribed in "Materials and Methods." A, 150 ,ug
From electrophoretic patterns of solubilized
SEA protein; B, immunoprecipitate from SEA and
immunoprecipitates stained with Coomassie
16-week-infected serum.
blue (Fig. 4B), it is possible to identify several
components (polypeptides) which clearly par-
ticipate in immunoprecipitation reactions with are highly reproducible, and provide the base-
sera from 16-week infected mice. From a line data against which we currently are com-
comparison of the electrophoretic patterns paring of the results of various separative and
solubilized immunoprecipitates stained with
preparative methodologies that are being ap-
Coomassie blue (Fig. 4B) and PAS (Fig. plied
5B) to SEA. It is hoped that preparative
with complete SEA (Fig. 2A, B) using the separations of SEA into its immunogenic and
same stains, it is unlikely that all the major
functional components will be analyzed more
precipitable antigens are glycoproteins. The
completely by use of a system based on these
glycoprotein components of SEA appear to be
analytical data for a reference foundation.
slow migrating with electrophoretic mobilities
not exceeding 0.5. Even though the polypep- ACKNOWLEDGMENTS
tides observed in SDS-gels of solubilized im-
The expert technical assistance of Ms. Sara
munoprecipitates stained with Coomassie blue
Murphy and Mr. Richard Gillerman is grate-
are slow migrating with mobilities not greater
fully acknowledged.
than 0.37, there is no good correlation between
PAS staining bands from solubilized immuno-
LITERATURE CITED
precipitates and PAS staining bands from com-
plete SEA. AXELSEN, N. H., H. KROLL, AND B. WEEKE. 1975.
Quantitative Immunoelectrophoresis. Methods
While all the proteins observed as a result
and Applications. Universitiesforlaget, Oslo.
of solubilizing immunoprecipitates appear to 1975, p. 15-36.
be larger than 50,000 daltons, this may be BoRos, D. L., R. P. PELLEY, AND K. S. WARREN.
only a reflection of the difficulty of obtaining 1975. Spontaneous modulation of granulo-
matous hypersensitivity in Schistosomiasis
accurate molecular weights of glycoproteins
mansoni. J Immunol 114: 1437-1441.
using SDS-polyacrylamide gel electrophoresis. , R. TOMFORD, AND K. S. WARREN. 1976.
The electrophoretic patterns herein reported Induction of granulomatous and elicitation

This content downloaded from 179.210.183.214 on Tue, 28 Sep 2021 14:23:36 UTC
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390 THE JOURNAL OF PARASITOLOGY, VOL. 64, NO. 3, JUNE 1978

of cutaneous sensitivity by partially purified GILES, K. W., AND A. MEYERS. 1965. An im-
SEA of Schistosoma mansoni. J Immunol proved method for the estimation of deoxy-
118: 373-376. ribonucleic acid. Nature 206: 93.
, AND K. S. WARREN. 1970. DelayedHOLDEN,
hy- K. G., N. C. F. YIM, L. J. GRIGGS, AND
persensitivity-type granuloma formation andJ. A. WEISBACH. 1971. Gel electrophoresis
dermal reaction induced and elicited by aof mucous glycoproteins. I. Effect of gel
soluble factor isolated from Schistosoma man-
porosity. Biochemistry 10: 3105-3109.
soni eggs. J Exp Med 132: 448-507. KOBYLKA, D., AND K. L. CARRAWAY. 1972. Pro-
CARRAWAY, K. L., D. KOBYLKA, AND R. B. TRIP- teins and glycoproteins of the milk fat globule
LETT. 1971. Surface proteins of erythrocyte membrane. Biochim Biophys Acta 288: 282-
membranes. Biochim Biophys Acta 241: 295.
934-940. LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, AND
COLLEY, D. G. 1975. Immune responses to a R. J. RANDALL. 1951. Protein measurements
soluble schistosomal egg antigen preparation with the Folin phenol reagent. J Biol Chem
during chronic primary infection with Schisto- 193: 265-275.
soma mansoni. J Immunol 115: 150-156. PELLEY, R. P., R. J. PELLEY, J. HAMBURGER, P. A.
, J. A. COOK, G. K. FREEMAN, JR., R. K. PETERS, AND K. S. WARREN. 1976. Schisto-
BARTHOLOMEW, AND P. JORDAN. 1977. Im- soma mansoni soluble egg antigens I. Iden-
mune responses during human schistosomiasis tification and purification of three major
mansoni. I. In vitro lymphocyte blastogenic antigens, and the employment of radioim-
responses to heterogeneous antigenic prepara- munoassay for their further characterization.
tions from schistosome eggs, worms, and J Immunol 117: 1553-1560.
cercariae. Int Arch Allergy Appl Immunol REZNICK, A. Z., AND R. J. WINZLER. 1975. Gly-
53: 420-433. coproteins from ascitic fluid of Ehrlich ascites
DISCHE, Z. 1955. Color reactions of nucleic tumor.acid Isolation and characterization. Bio-
components. In Chargaff and J. N. Davidson chim Biophys Acta 404: 268-273.
(eds.), The Nucleic Acids. Vol. I, Academic
WEBER, K., AND M. OSBORN. 1969. The reli-
Press, New York, p. 285-305. ability of molecular weight determination by
DUBOIS, M., K. A. GILLES, J. K. HAMILTON, P. dodecyl
A. sulfate-polyacrylamide gel electro-
REBERS, AND F. SMITH. 1956. Colorimetric phoresis. J Biol Chem 244: 4406-4412.
method for determination of sugars ZACHARIUS,
and re- R. M., T. E. ZELL, J. H. MORRISON,
lated substances. Anal Chem 28: 350-356. AND J. J. WOODLOCK. 1969. Glycoprotein
GABRIEL, 0. 1971. Analytical disc gel electro- staining following electrophoresis on acrylam-
phoresis. Methods Enzymol 22: 565-578. ide gels. Anal Biochem 30: 148-152.

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