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Schistosoma mansoni: Pattern of release of secretory products
EXPERIMENTAL
48, 92-99 ( 1979)
PARASITOLOGY
Schistosoma
mansoni:
Pattern
of Release of Secretory
Products
THEODORE E. NASH Laboratory
of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National lnstitutes of Health, Bethesda, Maryland 20205, U.S.A. (Accepted for publication
7 April 1979)
T. E. 1979. Schistosoma munsoni: Pattern of release of secretory products. Experi48, 92-99. Adult Schistosoma mansoni were maintained in vitro for 1 hr with radioactively labeled precursors of protein, glycoprotein, and polysaccharides. The worms were then washed extensively and the supernates analyzed. The precursors N-acetylglucosamine, N-acetylgalactosamine, glucosamine, galactosamine, glucose, leucine, and fucose were incorporated into the worms and both large and small molecular weight products accumulated in the supernatant. For all the precursors except fucose, there was an initial rapid and then slower phase of release for both the large and small molecular weight materials. The amount of label retained by the worms as well as the proportion excreted as large molecular weight material was characteristic for the precursor used. In contrast, the products of fucose were released within 4 to 6 hr and therefore only exhibited the early secretory phase. There was no retention of fucose by the worms. Hydrolysis of large molecular weight products revealed that the N-acetylglucosamine-derived material was incorporated as amino sugars and fucose was incorporated as fucose. Therefore, Nacetylglucosamine and fucose precursors can specifically label secretory glycoproteins of schistosomes in a manner similar to that in mammalian systems. INDEX DESCRIPTORS: Schktosoma mansoni; Trematode; Blood fluke; Maintenance, in vitro; Secretion; Glycoprotein; Radioactive label. NASH,
mental
Parasitology
The understanding of these processes becomes important because of the recent emphasis placed on the measurement of schistosome E-S products in the host in the hope that the amount of antigen found would reflect the worm burden of the host. The factors modifying production and release of E-S products from the adult worm are largely unstudied (Wilson and Barnes 1974). It is known for instance that the weight of adult schistosomes in acutely infected mice varies inversely with the total worm burden (Radke et al. 1957; Lennox and Schiller 1972). This probably affects total antigen production and, therefore, the amount of antigen in the circulation would
INTRODUCTION
Since adult Schistosoma munsoni are intravascuIar parasites all the parasitic products released from the parasite enter the bloodstream of the host. However, the nature of these materials broadly defined as excretory-secretory (E-S) material with few exceptions have not been characterized. The manner of synthesis and release of materials as well as the fate of these materials in the host has received little attention (Kusel 1972; Kusel et al. 1975; Kusel and Mackenzie 1975). The amount of released material in the criculation must be related to the amount produced as well as the amount of E-S products removed. 92 0014-4894/79/040092-08$02.00/O Copyright All rights
@I 1979 by Academic of reproduction in any
Press. Inc. form reserved.
Schistosoma munsoni: SECRETION PATTERN
not vary linearly with the worm burden. Although most of the worm E-S substances are not defined (Murrell et al. 1974)) it is clear that in some cases the host responds to secretory material (Nash et al. 1974; Nash 1978; Nash et al. 1978). In recent studies, antibody response to an antigen released from the schistosome gut depended on the duration of infection (Nash et al. 1978). Therefore, there are many factors which can possibly modify the release of adult worm material and the host response; these factors would be expected to alter the relationship between worm burden and the amount of E-S products in circulation. To study the biosynthesis, secretion, and nature of released E-S products of adult schistosomes, adult worms were cultured in oitro in defined media containing labeled small molecular weight precursor substances. Since in mammalian systems nglucosamine and L-fucose are incorporated into large molecular weight material as amino sugars or L-fucose, respectively ( Kraemer 1972), these sugars as well as other possible precursors of glycoproteins, proteins, or polysaccharides were added to cultures of adults maintained in proteinfree media for short periods of time. The ES products were then harvested in the super-r-ratesand analyzed
93
units/ml penicillin, and 2 mM glutamine, transferred to a large petri dish containing 20 ml of the above protein-supplemented medium, and cultured overnight (14 to 18 hr) at 37 C in 5% CO,-95% air. After rinsing in protein-free medium, 25 mature male and 25 mature female worms were placed in 2 ml of fresh Medium 199 without FCS. In order to facilitate transfer and rinsing of worms, they were held in silastic holders with monofilament nylon screening that fit into small 35 x lo-mm tissue culture dishes (Costar, Cambridge, Mass., U.S.A.). In typical experiments 10 to 100 &i of label was added to duplicate, triplicate, or quadruplicate culture dishes; the worms were then incubated for 1 hr at 37C in 5% C02-95% air as before. After extensive washing the worms were placed in 2 ml of protein-containing medium. Samples of medium in duplicate were taken from each culture dish at this time to check completeness of washing. At specific timed periods, usually at 4, 7, 24, and 29 hr after addition of label, the supernates were harvested and the worms washed and placed in a new culture dish containing 2 ml of fresh protein medium. Cultures for bacteria were done in the first few experiments and were always sterile. In a parallel experiment done under the same conditions and with the same batch of worms, release of label from the adult MATERIALS AND METHODS schistosomes was studied. In a separate culture dish containing 25 males and 25 NIH Swiss mice were infected with 500 female schistosomes, 2 male and 2 female cercariae of a Puerto Rican strain of worms were picked at random, washed, Schistosoma mans& subcutaneously; and saved for counting. After addition of worms were harvested at 7 weeks. The label for 1 hr, the worms were washed and schistosomes were collected aseptically two male and two female worms saved for with sterile normal saline as the perfusate counting. Worms were similarily harvested (Duvall and Dewitt 1967). They were after washing, usually at 4, 7, 24, and 29 hr then gathered on monofilament nylon cloth, Nitex (Tobler, Ernst, and Traber, Inc., after the addition of label. Samples were New York, N.Y., U.S.A.) rinsed with solubilized in NCS solubilizer (Amersham/ Medium 199 in Earle’s balanced salts Searle, Arlington Heights, Ill., U.S.A.), (Microbiological Association, Walkersville, Two separate four-worm counts were done Md., U.S.A.) containing 10% fetal calf per time period and experiments were reserum (FCS), 100 pg/ml streptomycin, 100 peated at least twice. Results are expressed
94
THEODORE
E. NASH
as percentage ‘of label retained by Schisto- n- [ I-14C] glucosamine (223 &i/mg ), Nmansoni following a 1-hr pulse of acetyl-n- [ 1J4C] galactosamine (215 &i/ label. mg), n-[l-3H]glucosamine (14.8 mCi/mg), Uptake of label over time was studied in D- [ 1J4C] galactosamine hydrochloride (221 another series of experiments. About 100 to ,uCi/mg), L-[6-3H]fucose (96-104 mCi/ 200 worms were incubated in label for up mg), n-[1-14C]glucose (15.8 &i/mg), L[ lJ4C]leucine (444 &i/mg ) , L- [ 4, ~JH] to 48 hr. Four male and four female schistosomes were washed as bef,ore and leucine (370 mCi/mg ), and Na 35S04 (51.0 solubilized in 1 N NaOH followed by to 52.7 mCi/mmole). Initially the salt-free neutralization with 1 N NCl. A known compounds were lyophilized and reconvolume was counted and another portion stituted with Hanks’ balanced salt s&rwas used for protein determination by the tion but later it was found that the commethod of Lowry et al. ( 1951). mercially obtained compounds could be and reconstituKnown volumes were taken from the used without lyophilization tion. Samples were counted in a Beckman total supernates for counts. The supernates 9000 scintillation counter. Disintegrations were then lyophilized, dissolved in water, per minute were determined for 3H and and separated into large and small molec14C. 35S04 was counted as counts per minular weight components on Bio-Rad (Richute only with the same window as that for mond, Calif., U.S.A.) disposable glass 1%. columns (30 x 0.7 cm) containing BioTo obtain enough labeled large molecGel P-4, 100 to 200 mesh, (Bio-Rad) in ular weight E-S products for analysis water. Two peaks were usually discernible. supernates were harvested after 24 hr The first peak corresponded to the exclufrom 200 to 500 worms cultured in 20 ml sion volume and the second peak which included the indicator present in the me- medium containing the labeled precursor. The supernatant was lyophilized and sepadium contained the small molecular weight rated into large and small molecular commaterial. The amount of large and small ponents by chromatography in disposable molecular weight E-S product was taken glass Bio-Rad columns (1.5 x 30 cm) conas the disintergrations per minute in tubes taining Bio-Gel P-4 or P-2 in water, PBS, containing large or small molecular weight or 1.0 M NaCl. The large molecular weight material, fractions were at different times dialyzed In later experiments large and small against water or rechromatographed as molecular weight products were separated above. DEAE chromatography was perby precipitation in cold 86% ethanol. In these experiments 200 pl of supernate was formed on this material (Nash et al. 1977); the fractions were pooled, dialyzed, and added to 1.5 ml Eppendorf ( Bio-Rad) centrifuge tubes held on ice. Then, 1.3 ml subjected to analysis and hydrolysis. Hydrolysis of large molecular weight of iced ethanol was added followed by vortexing and centrifuging at 12,800 x G. material from cultures labeled with fucose was performed after the addition of 2 mg Samples were washed three times with 100% ethanol. The precipitate was dis- of cold fucose to the radioactive material. Hydrolysis was performed in 0.1 M H2S04 solved in water and counted; the initial supernate and washes were pooled and and 2 M H2S04 for 10 min 1 hr, respectively, at 100 C under nitrogen. After passcounted. Samples were done in duplicate. ing through AG 1-x 8 ( 100 to 200 mesh, The radioactive compounds were purchased from Amersham/Searle. These inacetate form) resin ( Bio-Rad), the samples cluded N-acetyl-n- [ 1-3H] glucosamine ( spe- were lyophilized and chromatographed on Whatman No 1 (Whatman Ltd., Springcific activity 12.6 to 23 mCi/mg), N-acetylSOW
Schistosoma mansoni: SECmTION PATTERN TABLE Schistosoma mansoni:
Percentage large molecular weight (mean f SE)b
I
Percentage Excretory-Secretory Large Molecular Weight Material
gluNacG
19.6 f
1.2
Leucine
4.6 f
1.0
95
Fucose
11.5 f
0.7
Products
glcNHz
14.4 f 0.8
Released
as
galNHz
galNac
9.0 f
1.6
22.7
Glucose
1.6 f
0.4
0 Abbreviations : gluNac = N-acetylglucosamine ; glcNHz = glucosamine; galNac = N-acetylgalactosamine; galNHz = galactosamine. b Results represent the percentage of supernates released as large molecular weight material as determined by column chromatography. With the exception of fucose and galactosamine, all values were significantly different from each other at the p < 0.05 level. GalNac results represent the mean of two experiments; significance was not calculated.
field Mill, Maidstone, England) paper in pyridine : ethyl acetate : HZ0 : acetic acid (5:5:3:1) and compared to authentic Lfucose treated in a similar manner. Onecentimeter strips were cut out and counted. Samples derived from labeled N-acetylglucosamine were hydrolized in 4 N HCl for 5 hr, lyophilized, and chromatographed in the same system as above for 24 hr. Standards included labeled glucosamine and galactosamine mixed with cold authentic standards. Total secreted radioactivity in each dish was calculated from samples taken from total supernates before lyophilization and subsequent chromatography. The amount of large or small molecular weight products was calculated by multiplying the percentage of large or small molecular weight material derived from columns by the total supernate counts and dividing by the speTABLE Percentage
of Label Retained
gluNac= Percentage of label remaining in S. mansoni (mean f SE)
38.2 f
2.3b
cific activity. The specific activity was calculated by adding the amount of label to the precursor already present in the media, if any. Results are expressed as the amount of label released during a certain time period per 50 worms. RESULTS
All the compounds used with the exception of sulfate were incorporated into the adult Schistosoma man.soni and subsequently released as both large and small molecular weight material. The percentage of large or small molecular weight material depended on the precursor used as well as on the method used to separate the secretory materials. The percentage large molecular weight material found in the supernate after 28 hr in label-free media is shown in Table I. The N-acetyl derivatives of the amino II by Schistosoma mansoni
Leucine
Fucose
glcNHz
galNHz
galNac
Glucose
50.4 f 4.9
0.7 3~ 0.4
47.7 zk 0.4
60.1*
28.5*
22%
glcNHz = glucosamine; galNac = N-acetylgalacto0 Abbreviations : gluNac = N-acetylglucosamine; samine; galNHz = galactosamine. b Results represent the percentage of label retained by adult S. mansoni following a I-hr pulse of labeled precursor. Values were obtained 24 to 29 hr after addition of label. Leucine and glucosamine were not significantly different at the p <0.05 level; all the rest were significantly different at p < 0.05. * Mean of two experiments.
96
THEODORE
sugars glucosamine and galactosamine excreted 19.6 and 22.7% of the total material released as large molecular weight substances compared to 14.4% for glucosamine, 9.0% for galactosamine, 4.6% for leucine, 11.5% for fucose, and 1.6% for glucose. By using column-separated large and small molecular weight N-acetylglucosamine-derived supernates, 84% of the large molecular weight material was precipitated with 86% cold ethanol and about 1.8% of the small molecular weight fraction contaminated the precipitate compared to 0.7% for authentic N-acetylglucosamine. The percentage of large molecular weight material released into the supernates was 3.49 * 0.17% compared to 19.6% for column-separated material (Table I). The percentage of label retained by the worms following 28 hr in label-free media was also dependent on the precursor used (Table II). Leucine, glucosamine, and galactosamine were retained by the worms to a greater degree than the other precur-
6
5
4 v)
:
IV
I
I
I
I
I
I
5
10
15
20
25
30
hrs.
FIG. 1. Cumulative small and large molecular weight excretory-secretory products derived from N-acetylglucosamine-labeled Schistosoma mansoni. Separation was performed by column chromatography and done in quadruplicate. Points and bars represent mean k SD. Similar E-S patterns were seen in all the other precursors used except fucose.
E. NASH 400
loot
i* i
I.-5
10
15
20
25
hrs.
FIG. 2. Large molecular weight excretory-secretory pattern derived from N-acetylglucosaminelabeled Schktosoma mansoni. Separation was performed using 867% ethanol precipitation and represent mean percentage SD of three different experiments run in triplicate. Results are corrected for 1.8% supemate contamination of precipitate and 84% precipitation of large molecular weight material.
sors. Interestingly, the N-acetyl derivatives of the amino sugars tended to be released to a greater degree than their corresponding amino sugars and they tended to be released as higher molecular weight material. This suggests different handling of the free amino sugar compared to their Nacetyl counterparts. The pattern of secretion of large and small molecular weight secretory material was similar for most of the precursors studied. As demonstrated with N-acetylglucosamine-labeled worms, there was a rapid early phase of release followed by a later slower linear phase (Fig. 1). There tended to be day to day variability for each precursor when comparing the total amount found per time period, but the cumulative curves always revealed this pattern. Curves derived by sampling earlier time periods and by employing ethanol precipitation showed a pattern similar to that derived from column-separated supernates (Fig. 2). Loss of label from adult worms showed a similar pattern ‘of early rapid phase followed by a slower phase (Fig. 3). The pattern of release of fucose-derived products was unique in that practically all the label was released by the worms into
Schistosoma mansoni: SECRETION PATTERN
97
+...a -Large
I
40
Small
5
10
15
m.w:5Q
Worms
mw/50
worms
20
25
30
tlE.. I
5
,
10
15 20 hrs.
,
I
25
30
FIG. 3. Regression of percentage of initial label retained by Schistosoma mansoni following pulselabeling of schistosomes with N-acetylglucosamine and culturing in label-free media. Points are derived from three experiments. Y = A + B (log x), T = 0.95.
the supernant after 4 hr (Fig. 4). That large molecular weight materials were indeed found in the supernate is based on isolation of material using 1.0 M NaCl columns, isolation of a single salt-eluted peak after DEAE-column chromatography, multiple bands following SDS-acrylamide electrophoresis, and autoradiography, and release of fucose following hydrolysis. The loss of fucose from adult worms was practically complete by 4 to 6 hr ( Fig. 5), confirming the pattern of release into the supernate. The same pattern of release was noted whether the worms were incubated in label for 1 or 24 hr. N-Acetylglucosamine was incorporated into large molecular weight products as amino sugars; fucose was primarily incorporated as fucose. Large molecular weight E-S products for both fucose and N-acetylglucosamine were prepared by repeated chromatography in water or saline followed by DEAE chromatography in some experiments (Nash et al 1977). Large molecular weight fucose-labeled E-S products were found in a single salt-eluted peak after chromatography on DEAE. Mild hydrolysis of this peak followed by paper chromatography resulted in the release of
FIG. 4. Cumulative release of large and small excretory-secretory products following pulselabeling of Schistosoma mansoni with fucose. Separation was performed by column chromatography. Points represent mean of duplicate samples.
a small amount of fucose with most material remaining at the origin, After hydrolysis in 2 M H2S04 for 1 hr, most of the material migrated similarly to authentic fucose. Total large molecular weight Nacetylglucosamine secretory products following hydrolysis yielded a product which migrated as glucosamine. A slight asymmetric early portion suggested a minor galactosamine peak, a finding previously suggested by hydrolysis of DEAE-purified peaks (unpublished data). However, the vast majority of the material migrated as glucosamine.
100 80
t;rn m 0 : 40 20
5
10
15 hrs.
20
25
FIG. 5. Regression of percentage of initial label retained by Schistosoma mansoni following pulselabeling of schistosomes with fucose and culturing in label-free media. Points are derived from two expermients. y = A x*, r = 0.99.
9s
THEODORE
DISCUSSION Adult Schistosoma munsoni incorporated and then released both large and small molecular weight products when incubated with labeled precursors including N-acetylglucosamine, N-acetylgalactosamine, glucosamine, galactosamine, glucose, fucose, and leucine but not sodium sulfate. As in mammalian system, addition of N-acetylglucosamine and fucose in short-term cultures resulted in incorporation primarily into amino sugars and fucose, respectively, and not noticeably into the glycolytic pathway. However, some differences were seen in the amount of material retained by the worms as well as in the proportion of label released as small or large molecular weight substance. The pattern of excretion-secretion in schistosomes was characterized by a rapid phase for all precursors studied followed by a slower linear phase which was present in all precursors except fucose. Although the reasons for the biphasic loss of material from schistosomes is unknown, the findings that fucose release differs from the other precursors suggest that there are at least two different methods of release. Although schistosomes release materials from the tegument (Kusel and Mackenzie 1975; Wilson and Barnes 1974) and gut (Nash et al. 1974), the relative contribution of each in the E-S process has not been studied. Kusel and Mackenzie (1975) using a double-label ratio technique with Lleucine as the precursor found different relative turnover rates of schistosome tegumental membrane, soluble secreted materials, and immune precipitated supernatant material, suggesting faster and slower release. Comparison of adult Schistosoma mansoni experiments to mammalian experiments is difficult because of different culture systems and methodologies (Hubbard and Cohn 1968; Warren and Glick 1968; Kaplan and Moskowitz 1975; Yurchenco and Atkinson 1977; Hudson and Johnson 1977; Baumann and Doyle 1978). When studied in detail differences have
E. NASH
been noted in the rates of release of specifically labeled constituents (Hubbard and Cohn 1968; Kaplan and Moskowitz 1975; Yurchenco and Atkinson 1977; Baumann and Doyle 1978; MacDermott et al. 1974) which along with other factors accounted f or the biphasic release pattern noted. The methodology of the separation of large and small molecular weight secretory components is a problem not addressed by most investigators. Commonly, a standard protein-precipitating method is used without regard to whether some or all of the large molecular weight material is precipitated (Kusel et al. 1975). For instance, at least two schistosome secretory antigens are trichloroacetic acid (TCA) soluble and would therefore not precipitate if TCA were used (Nash et al. 1974; Deelder et al. 1976). The use of 86% ethanol in precipitating large molecuar weight material operationally worked well. However, problems included different solubilities of the small molecular weight products in 86% ethanol compared to those of authentic Nacetyglucosamine and possible different solubilities of the released material each collection period
during
The reason for the discrepancy between the percentage of large molecular weight material released as obtained by column chromatography compared to 86% ethanol precipitation is not known. However, comparisons of the different labeled E-S products were made from data obtained from the same technique. Furthermore, although the results differed quantitatively, they were similar qualitatively. The system described can be useful in several ways. First, small numbers of Schistosoma mansoni worms can yield enough secretory material to analyze products by standards counting or visualization techniques. This bypasses the need to use thousands of worms, cultured over long periods of time with the risk of death and disintegration of worms and release of nonsecretory products. Second, the analysis of substances is nonimmunological so that
Schistosoma mansoni: SECRETION PATTERN substances that are not immunogenic can be defined. Materials can be defined by molecular weight and by types of precursors incorporated. Third, the system can be used to study the process of secretion itself. Fourth, labeled secretory products can be used to study metabolism of secretions in the host. Last, the biosynthesis of secretory products of schistosomes can be studied. ACKNOWLEDGMENTS The author thanks Dr. Allen W. Cheever his advice and guidance and James Merritt his technical assistance.
for for
REFERENCES BAUMANN, H., AND DOYLE, D. 1978. Turnover of plasma membrane glycoproteins and glycolipids of hepatoma tissue culture cells. Journal of Biological Chemistry 253, 4408-4418. DEELDER, A. M., KLAPPE, H. T. M., VAN DEN AARDWEG, G. J. J. G., AND VAN MEERBEKE, E. H. E. M. 1976. Schistosoma mansoni: Demonstration of two circulating antigens in infected hamsters. Experimental Parasitology 40, 189-197. DWALL, R. H., AND DEWITT, W. B. 1967. An improved perfusion technique for recovering adult schistosomes from laboratory animals. American Journal of Tropical Medicine and Hygiene 16,
483-486. HUBBARD, A. L., AND COHN, Z. A. 1968. Externally disposed plasma membrane proteins. II. Metabolic fate of iodinated polypeptides of mouse L cells. .lournul of Cell Biology 37, 729-746. HUDSON, J. E., AND JOHNSON, T. C. 1977. The degradation and turnover of fucosylated glycoproteins in the plasma membrane of a neuroblastoma cell line. Biochemistry Journal 166,
217-223. KAPLAN, J., AND MOSKOWITZ, M. 1975. Studies on the turnover of plasma membranes in cultured mammalian cells. II. Demonstration of heterogeneous rates of turnover for plasma membrane proteins and glycoproteins. Biochimica et Biophysica Acta 389, 306-313. KRAEMER, P. M. 1972. Complex carbohydrates of mammalian cells in culture. In “Growth, Nutrition and Metabolism of Cells in Culture” (G. H. Rothblatt and V. J. Cristafalo, eds.), pp. 371426. Academic Press, New York. KUSEL, J. R. 1972. Protein composition and protein synthesis Bn the surface membranes of Schistosoma munsoni. Parasitology 65, 55-69. KUSEL, J. R., AND MACKENZIE, P. E. 1975. The measurement of the relative turnover rates of proteins of the surface membranes and other
99
fractions of Schistosoma mansoni in culture. Parasitology 71, 261-273. KUSEL, J. R., MACKENZIE, P. E., AND MCLAREN, D. J. 1975. The release of membrane antigens into culture by adult Schistosoma mansoni. Parasitology 71, 247-259. LENNOX, R. W. AND SCHILLER, E. L. 1972. Changes in dry weight and glycogen content criteria for measuring the postcercarial growth and development of Schistosoma mansoni. Journal of Parasitology 58, 489494. LOWRY, 0. H., ROSEBROUGH, N. R., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurement with the Folin-phenol reagent. Journal of Biological Chemistry 193, 265-275. MACDERMOTT, R. P., DONALDSON, R. M., AND TRIER, J. S. 1974. Glycoprotein synthesis and secretion by mucosal biopsies of rabbit colon and human rectum. Journal of Clinical Investigation 54, 545-554. MURRELL, K. D., VANNIER, W. E., AND AHMED, A. 1974. Schistosoma mansoni: Antigenic heterogenicity of excretions and secretions. Experimental Parasitology 36, 316-330. NASH, T. E. 1978. Antibody response to a polysaccharide antigen in schistosomiasis. I. Sensitivity and specificity. American Journal of Tropical Medicine and Hygiene 27, 939-943. NASH, T. E., NASIR-UD-DIN, AND JEANLEZ, R. W. 1977. Further purification and characterization of a circulating antigen in schistosomiasis. Journal of Immunology 119, 1627-1633. NASH, T. E., OTTESEN, E. A., AND CHEEVER, A. W. 1978. Antibody response to a polysaccharide antigen present in schistosome gut. II. Modulation of antibody response. American Journal of Tropical Medicine and Hygiene 27, 944-950. NASH, T. E., PRESCOTT, B., AND NEVA, F. A. 1974. The characteristics of a circulating antigen in schistosomiasis. Journal of Immunology 112,
1500-1507. FIADKE, M. G., SCHNEIDER, M. D., AND HOUGHTALING, D. G. 1957. Dry weight, nitrogen and phosphorus content of Schistosoma mansoni. Experimental Parasitology 6, 202-207. WARREN, L., AND GLICK, M. C. 1968. Membranes of animal cells. II. The metabolism and tumover of the surface membrane. Journal of Cell Biology 37, 729-746. WILSON, R. A., AND BARNES, P. E. 1974. An in vitro investigation of dynamic processes occurring in the schistosome tegument using compounds known to disrupt secretary processes. Parasitology 68, 259-270. YURCHENCO, P. D., AND ATKINSON, P. H. 1977. Equilibration of fucosyl glycoprotein pools in HeLa cells. Biochemistry 16, 944.
48, 92-99 ( 1979)
PARASITOLOGY
Schistosoma
mansoni:
Pattern
of Release of Secretory
Products
THEODORE E. NASH Laboratory
of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National lnstitutes of Health, Bethesda, Maryland 20205, U.S.A. (Accepted for publication
7 April 1979)
T. E. 1979. Schistosoma munsoni: Pattern of release of secretory products. Experi48, 92-99. Adult Schistosoma mansoni were maintained in vitro for 1 hr with radioactively labeled precursors of protein, glycoprotein, and polysaccharides. The worms were then washed extensively and the supernates analyzed. The precursors N-acetylglucosamine, N-acetylgalactosamine, glucosamine, galactosamine, glucose, leucine, and fucose were incorporated into the worms and both large and small molecular weight products accumulated in the supernatant. For all the precursors except fucose, there was an initial rapid and then slower phase of release for both the large and small molecular weight materials. The amount of label retained by the worms as well as the proportion excreted as large molecular weight material was characteristic for the precursor used. In contrast, the products of fucose were released within 4 to 6 hr and therefore only exhibited the early secretory phase. There was no retention of fucose by the worms. Hydrolysis of large molecular weight products revealed that the N-acetylglucosamine-derived material was incorporated as amino sugars and fucose was incorporated as fucose. Therefore, Nacetylglucosamine and fucose precursors can specifically label secretory glycoproteins of schistosomes in a manner similar to that in mammalian systems. INDEX DESCRIPTORS: Schktosoma mansoni; Trematode; Blood fluke; Maintenance, in vitro; Secretion; Glycoprotein; Radioactive label. NASH,
mental
Parasitology
The understanding of these processes becomes important because of the recent emphasis placed on the measurement of schistosome E-S products in the host in the hope that the amount of antigen found would reflect the worm burden of the host. The factors modifying production and release of E-S products from the adult worm are largely unstudied (Wilson and Barnes 1974). It is known for instance that the weight of adult schistosomes in acutely infected mice varies inversely with the total worm burden (Radke et al. 1957; Lennox and Schiller 1972). This probably affects total antigen production and, therefore, the amount of antigen in the circulation would
INTRODUCTION
Since adult Schistosoma munsoni are intravascuIar parasites all the parasitic products released from the parasite enter the bloodstream of the host. However, the nature of these materials broadly defined as excretory-secretory (E-S) material with few exceptions have not been characterized. The manner of synthesis and release of materials as well as the fate of these materials in the host has received little attention (Kusel 1972; Kusel et al. 1975; Kusel and Mackenzie 1975). The amount of released material in the criculation must be related to the amount produced as well as the amount of E-S products removed. 92 0014-4894/79/040092-08$02.00/O Copyright All rights
@I 1979 by Academic of reproduction in any
Press. Inc. form reserved.
Schistosoma munsoni: SECRETION PATTERN
not vary linearly with the worm burden. Although most of the worm E-S substances are not defined (Murrell et al. 1974)) it is clear that in some cases the host responds to secretory material (Nash et al. 1974; Nash 1978; Nash et al. 1978). In recent studies, antibody response to an antigen released from the schistosome gut depended on the duration of infection (Nash et al. 1978). Therefore, there are many factors which can possibly modify the release of adult worm material and the host response; these factors would be expected to alter the relationship between worm burden and the amount of E-S products in circulation. To study the biosynthesis, secretion, and nature of released E-S products of adult schistosomes, adult worms were cultured in oitro in defined media containing labeled small molecular weight precursor substances. Since in mammalian systems nglucosamine and L-fucose are incorporated into large molecular weight material as amino sugars or L-fucose, respectively ( Kraemer 1972), these sugars as well as other possible precursors of glycoproteins, proteins, or polysaccharides were added to cultures of adults maintained in proteinfree media for short periods of time. The ES products were then harvested in the super-r-ratesand analyzed
93
units/ml penicillin, and 2 mM glutamine, transferred to a large petri dish containing 20 ml of the above protein-supplemented medium, and cultured overnight (14 to 18 hr) at 37 C in 5% CO,-95% air. After rinsing in protein-free medium, 25 mature male and 25 mature female worms were placed in 2 ml of fresh Medium 199 without FCS. In order to facilitate transfer and rinsing of worms, they were held in silastic holders with monofilament nylon screening that fit into small 35 x lo-mm tissue culture dishes (Costar, Cambridge, Mass., U.S.A.). In typical experiments 10 to 100 &i of label was added to duplicate, triplicate, or quadruplicate culture dishes; the worms were then incubated for 1 hr at 37C in 5% C02-95% air as before. After extensive washing the worms were placed in 2 ml of protein-containing medium. Samples of medium in duplicate were taken from each culture dish at this time to check completeness of washing. At specific timed periods, usually at 4, 7, 24, and 29 hr after addition of label, the supernates were harvested and the worms washed and placed in a new culture dish containing 2 ml of fresh protein medium. Cultures for bacteria were done in the first few experiments and were always sterile. In a parallel experiment done under the same conditions and with the same batch of worms, release of label from the adult MATERIALS AND METHODS schistosomes was studied. In a separate culture dish containing 25 males and 25 NIH Swiss mice were infected with 500 female schistosomes, 2 male and 2 female cercariae of a Puerto Rican strain of worms were picked at random, washed, Schistosoma mans& subcutaneously; and saved for counting. After addition of worms were harvested at 7 weeks. The label for 1 hr, the worms were washed and schistosomes were collected aseptically two male and two female worms saved for with sterile normal saline as the perfusate counting. Worms were similarily harvested (Duvall and Dewitt 1967). They were after washing, usually at 4, 7, 24, and 29 hr then gathered on monofilament nylon cloth, Nitex (Tobler, Ernst, and Traber, Inc., after the addition of label. Samples were New York, N.Y., U.S.A.) rinsed with solubilized in NCS solubilizer (Amersham/ Medium 199 in Earle’s balanced salts Searle, Arlington Heights, Ill., U.S.A.), (Microbiological Association, Walkersville, Two separate four-worm counts were done Md., U.S.A.) containing 10% fetal calf per time period and experiments were reserum (FCS), 100 pg/ml streptomycin, 100 peated at least twice. Results are expressed
94
THEODORE
E. NASH
as percentage ‘of label retained by Schisto- n- [ I-14C] glucosamine (223 &i/mg ), Nmansoni following a 1-hr pulse of acetyl-n- [ 1J4C] galactosamine (215 &i/ label. mg), n-[l-3H]glucosamine (14.8 mCi/mg), Uptake of label over time was studied in D- [ 1J4C] galactosamine hydrochloride (221 another series of experiments. About 100 to ,uCi/mg), L-[6-3H]fucose (96-104 mCi/ 200 worms were incubated in label for up mg), n-[1-14C]glucose (15.8 &i/mg), L[ lJ4C]leucine (444 &i/mg ) , L- [ 4, ~JH] to 48 hr. Four male and four female schistosomes were washed as bef,ore and leucine (370 mCi/mg ), and Na 35S04 (51.0 solubilized in 1 N NaOH followed by to 52.7 mCi/mmole). Initially the salt-free neutralization with 1 N NCl. A known compounds were lyophilized and reconvolume was counted and another portion stituted with Hanks’ balanced salt s&rwas used for protein determination by the tion but later it was found that the commethod of Lowry et al. ( 1951). mercially obtained compounds could be and reconstituKnown volumes were taken from the used without lyophilization tion. Samples were counted in a Beckman total supernates for counts. The supernates 9000 scintillation counter. Disintegrations were then lyophilized, dissolved in water, per minute were determined for 3H and and separated into large and small molec14C. 35S04 was counted as counts per minular weight components on Bio-Rad (Richute only with the same window as that for mond, Calif., U.S.A.) disposable glass 1%. columns (30 x 0.7 cm) containing BioTo obtain enough labeled large molecGel P-4, 100 to 200 mesh, (Bio-Rad) in ular weight E-S products for analysis water. Two peaks were usually discernible. supernates were harvested after 24 hr The first peak corresponded to the exclufrom 200 to 500 worms cultured in 20 ml sion volume and the second peak which included the indicator present in the me- medium containing the labeled precursor. The supernatant was lyophilized and sepadium contained the small molecular weight rated into large and small molecular commaterial. The amount of large and small ponents by chromatography in disposable molecular weight E-S product was taken glass Bio-Rad columns (1.5 x 30 cm) conas the disintergrations per minute in tubes taining Bio-Gel P-4 or P-2 in water, PBS, containing large or small molecular weight or 1.0 M NaCl. The large molecular weight material, fractions were at different times dialyzed In later experiments large and small against water or rechromatographed as molecular weight products were separated above. DEAE chromatography was perby precipitation in cold 86% ethanol. In these experiments 200 pl of supernate was formed on this material (Nash et al. 1977); the fractions were pooled, dialyzed, and added to 1.5 ml Eppendorf ( Bio-Rad) centrifuge tubes held on ice. Then, 1.3 ml subjected to analysis and hydrolysis. Hydrolysis of large molecular weight of iced ethanol was added followed by vortexing and centrifuging at 12,800 x G. material from cultures labeled with fucose was performed after the addition of 2 mg Samples were washed three times with 100% ethanol. The precipitate was dis- of cold fucose to the radioactive material. Hydrolysis was performed in 0.1 M H2S04 solved in water and counted; the initial supernate and washes were pooled and and 2 M H2S04 for 10 min 1 hr, respectively, at 100 C under nitrogen. After passcounted. Samples were done in duplicate. ing through AG 1-x 8 ( 100 to 200 mesh, The radioactive compounds were purchased from Amersham/Searle. These inacetate form) resin ( Bio-Rad), the samples cluded N-acetyl-n- [ 1-3H] glucosamine ( spe- were lyophilized and chromatographed on Whatman No 1 (Whatman Ltd., Springcific activity 12.6 to 23 mCi/mg), N-acetylSOW
Schistosoma mansoni: SECmTION PATTERN TABLE Schistosoma mansoni:
Percentage large molecular weight (mean f SE)b
I
Percentage Excretory-Secretory Large Molecular Weight Material
gluNacG
19.6 f
1.2
Leucine
4.6 f
1.0
95
Fucose
11.5 f
0.7
Products
glcNHz
14.4 f 0.8
Released
as
galNHz
galNac
9.0 f
1.6
22.7
Glucose
1.6 f
0.4
0 Abbreviations : gluNac = N-acetylglucosamine ; glcNHz = glucosamine; galNac = N-acetylgalactosamine; galNHz = galactosamine. b Results represent the percentage of supernates released as large molecular weight material as determined by column chromatography. With the exception of fucose and galactosamine, all values were significantly different from each other at the p < 0.05 level. GalNac results represent the mean of two experiments; significance was not calculated.
field Mill, Maidstone, England) paper in pyridine : ethyl acetate : HZ0 : acetic acid (5:5:3:1) and compared to authentic Lfucose treated in a similar manner. Onecentimeter strips were cut out and counted. Samples derived from labeled N-acetylglucosamine were hydrolized in 4 N HCl for 5 hr, lyophilized, and chromatographed in the same system as above for 24 hr. Standards included labeled glucosamine and galactosamine mixed with cold authentic standards. Total secreted radioactivity in each dish was calculated from samples taken from total supernates before lyophilization and subsequent chromatography. The amount of large or small molecular weight products was calculated by multiplying the percentage of large or small molecular weight material derived from columns by the total supernate counts and dividing by the speTABLE Percentage
of Label Retained
gluNac= Percentage of label remaining in S. mansoni (mean f SE)
38.2 f
2.3b
cific activity. The specific activity was calculated by adding the amount of label to the precursor already present in the media, if any. Results are expressed as the amount of label released during a certain time period per 50 worms. RESULTS
All the compounds used with the exception of sulfate were incorporated into the adult Schistosoma man.soni and subsequently released as both large and small molecular weight material. The percentage of large or small molecular weight material depended on the precursor used as well as on the method used to separate the secretory materials. The percentage large molecular weight material found in the supernate after 28 hr in label-free media is shown in Table I. The N-acetyl derivatives of the amino II by Schistosoma mansoni
Leucine
Fucose
glcNHz
galNHz
galNac
Glucose
50.4 f 4.9
0.7 3~ 0.4
47.7 zk 0.4
60.1*
28.5*
22%
glcNHz = glucosamine; galNac = N-acetylgalacto0 Abbreviations : gluNac = N-acetylglucosamine; samine; galNHz = galactosamine. b Results represent the percentage of label retained by adult S. mansoni following a I-hr pulse of labeled precursor. Values were obtained 24 to 29 hr after addition of label. Leucine and glucosamine were not significantly different at the p <0.05 level; all the rest were significantly different at p < 0.05. * Mean of two experiments.
96
THEODORE
sugars glucosamine and galactosamine excreted 19.6 and 22.7% of the total material released as large molecular weight substances compared to 14.4% for glucosamine, 9.0% for galactosamine, 4.6% for leucine, 11.5% for fucose, and 1.6% for glucose. By using column-separated large and small molecular weight N-acetylglucosamine-derived supernates, 84% of the large molecular weight material was precipitated with 86% cold ethanol and about 1.8% of the small molecular weight fraction contaminated the precipitate compared to 0.7% for authentic N-acetylglucosamine. The percentage of large molecular weight material released into the supernates was 3.49 * 0.17% compared to 19.6% for column-separated material (Table I). The percentage of label retained by the worms following 28 hr in label-free media was also dependent on the precursor used (Table II). Leucine, glucosamine, and galactosamine were retained by the worms to a greater degree than the other precur-
6
5
4 v)
:
IV
I
I
I
I
I
I
5
10
15
20
25
30
hrs.
FIG. 1. Cumulative small and large molecular weight excretory-secretory products derived from N-acetylglucosamine-labeled Schistosoma mansoni. Separation was performed by column chromatography and done in quadruplicate. Points and bars represent mean k SD. Similar E-S patterns were seen in all the other precursors used except fucose.
E. NASH 400
loot
i* i
I.-5
10
15
20
25
hrs.
FIG. 2. Large molecular weight excretory-secretory pattern derived from N-acetylglucosaminelabeled Schktosoma mansoni. Separation was performed using 867% ethanol precipitation and represent mean percentage SD of three different experiments run in triplicate. Results are corrected for 1.8% supemate contamination of precipitate and 84% precipitation of large molecular weight material.
sors. Interestingly, the N-acetyl derivatives of the amino sugars tended to be released to a greater degree than their corresponding amino sugars and they tended to be released as higher molecular weight material. This suggests different handling of the free amino sugar compared to their Nacetyl counterparts. The pattern of secretion of large and small molecular weight secretory material was similar for most of the precursors studied. As demonstrated with N-acetylglucosamine-labeled worms, there was a rapid early phase of release followed by a later slower linear phase (Fig. 1). There tended to be day to day variability for each precursor when comparing the total amount found per time period, but the cumulative curves always revealed this pattern. Curves derived by sampling earlier time periods and by employing ethanol precipitation showed a pattern similar to that derived from column-separated supernates (Fig. 2). Loss of label from adult worms showed a similar pattern ‘of early rapid phase followed by a slower phase (Fig. 3). The pattern of release of fucose-derived products was unique in that practically all the label was released by the worms into
Schistosoma mansoni: SECRETION PATTERN
97
+...a -Large
I
40
Small
5
10
15
m.w:5Q
Worms
mw/50
worms
20
25
30
tlE.. I
5
,
10
15 20 hrs.
,
I
25
30
FIG. 3. Regression of percentage of initial label retained by Schistosoma mansoni following pulselabeling of schistosomes with N-acetylglucosamine and culturing in label-free media. Points are derived from three experiments. Y = A + B (log x), T = 0.95.
the supernant after 4 hr (Fig. 4). That large molecular weight materials were indeed found in the supernate is based on isolation of material using 1.0 M NaCl columns, isolation of a single salt-eluted peak after DEAE-column chromatography, multiple bands following SDS-acrylamide electrophoresis, and autoradiography, and release of fucose following hydrolysis. The loss of fucose from adult worms was practically complete by 4 to 6 hr ( Fig. 5), confirming the pattern of release into the supernate. The same pattern of release was noted whether the worms were incubated in label for 1 or 24 hr. N-Acetylglucosamine was incorporated into large molecular weight products as amino sugars; fucose was primarily incorporated as fucose. Large molecular weight E-S products for both fucose and N-acetylglucosamine were prepared by repeated chromatography in water or saline followed by DEAE chromatography in some experiments (Nash et al 1977). Large molecular weight fucose-labeled E-S products were found in a single salt-eluted peak after chromatography on DEAE. Mild hydrolysis of this peak followed by paper chromatography resulted in the release of
FIG. 4. Cumulative release of large and small excretory-secretory products following pulselabeling of Schistosoma mansoni with fucose. Separation was performed by column chromatography. Points represent mean of duplicate samples.
a small amount of fucose with most material remaining at the origin, After hydrolysis in 2 M H2S04 for 1 hr, most of the material migrated similarly to authentic fucose. Total large molecular weight Nacetylglucosamine secretory products following hydrolysis yielded a product which migrated as glucosamine. A slight asymmetric early portion suggested a minor galactosamine peak, a finding previously suggested by hydrolysis of DEAE-purified peaks (unpublished data). However, the vast majority of the material migrated as glucosamine.
100 80
t;rn m 0 : 40 20
5
10
15 hrs.
20
25
FIG. 5. Regression of percentage of initial label retained by Schistosoma mansoni following pulselabeling of schistosomes with fucose and culturing in label-free media. Points are derived from two expermients. y = A x*, r = 0.99.
9s
THEODORE
DISCUSSION Adult Schistosoma munsoni incorporated and then released both large and small molecular weight products when incubated with labeled precursors including N-acetylglucosamine, N-acetylgalactosamine, glucosamine, galactosamine, glucose, fucose, and leucine but not sodium sulfate. As in mammalian system, addition of N-acetylglucosamine and fucose in short-term cultures resulted in incorporation primarily into amino sugars and fucose, respectively, and not noticeably into the glycolytic pathway. However, some differences were seen in the amount of material retained by the worms as well as in the proportion of label released as small or large molecular weight substance. The pattern of excretion-secretion in schistosomes was characterized by a rapid phase for all precursors studied followed by a slower linear phase which was present in all precursors except fucose. Although the reasons for the biphasic loss of material from schistosomes is unknown, the findings that fucose release differs from the other precursors suggest that there are at least two different methods of release. Although schistosomes release materials from the tegument (Kusel and Mackenzie 1975; Wilson and Barnes 1974) and gut (Nash et al. 1974), the relative contribution of each in the E-S process has not been studied. Kusel and Mackenzie (1975) using a double-label ratio technique with Lleucine as the precursor found different relative turnover rates of schistosome tegumental membrane, soluble secreted materials, and immune precipitated supernatant material, suggesting faster and slower release. Comparison of adult Schistosoma mansoni experiments to mammalian experiments is difficult because of different culture systems and methodologies (Hubbard and Cohn 1968; Warren and Glick 1968; Kaplan and Moskowitz 1975; Yurchenco and Atkinson 1977; Hudson and Johnson 1977; Baumann and Doyle 1978). When studied in detail differences have
E. NASH
been noted in the rates of release of specifically labeled constituents (Hubbard and Cohn 1968; Kaplan and Moskowitz 1975; Yurchenco and Atkinson 1977; Baumann and Doyle 1978; MacDermott et al. 1974) which along with other factors accounted f or the biphasic release pattern noted. The methodology of the separation of large and small molecular weight secretory components is a problem not addressed by most investigators. Commonly, a standard protein-precipitating method is used without regard to whether some or all of the large molecular weight material is precipitated (Kusel et al. 1975). For instance, at least two schistosome secretory antigens are trichloroacetic acid (TCA) soluble and would therefore not precipitate if TCA were used (Nash et al. 1974; Deelder et al. 1976). The use of 86% ethanol in precipitating large molecuar weight material operationally worked well. However, problems included different solubilities of the small molecular weight products in 86% ethanol compared to those of authentic Nacetyglucosamine and possible different solubilities of the released material each collection period
during
The reason for the discrepancy between the percentage of large molecular weight material released as obtained by column chromatography compared to 86% ethanol precipitation is not known. However, comparisons of the different labeled E-S products were made from data obtained from the same technique. Furthermore, although the results differed quantitatively, they were similar qualitatively. The system described can be useful in several ways. First, small numbers of Schistosoma mansoni worms can yield enough secretory material to analyze products by standards counting or visualization techniques. This bypasses the need to use thousands of worms, cultured over long periods of time with the risk of death and disintegration of worms and release of nonsecretory products. Second, the analysis of substances is nonimmunological so that
Schistosoma mansoni: SECRETION PATTERN substances that are not immunogenic can be defined. Materials can be defined by molecular weight and by types of precursors incorporated. Third, the system can be used to study the process of secretion itself. Fourth, labeled secretory products can be used to study metabolism of secretions in the host. Last, the biosynthesis of secretory products of schistosomes can be studied. ACKNOWLEDGMENTS The author thanks Dr. Allen W. Cheever his advice and guidance and James Merritt his technical assistance.
for for
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fractions of Schistosoma mansoni in culture. Parasitology 71, 261-273. KUSEL, J. R., MACKENZIE, P. E., AND MCLAREN, D. J. 1975. The release of membrane antigens into culture by adult Schistosoma mansoni. Parasitology 71, 247-259. LENNOX, R. W. AND SCHILLER, E. L. 1972. Changes in dry weight and glycogen content criteria for measuring the postcercarial growth and development of Schistosoma mansoni. Journal of Parasitology 58, 489494. LOWRY, 0. H., ROSEBROUGH, N. R., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurement with the Folin-phenol reagent. Journal of Biological Chemistry 193, 265-275. MACDERMOTT, R. P., DONALDSON, R. M., AND TRIER, J. S. 1974. Glycoprotein synthesis and secretion by mucosal biopsies of rabbit colon and human rectum. Journal of Clinical Investigation 54, 545-554. MURRELL, K. D., VANNIER, W. E., AND AHMED, A. 1974. Schistosoma mansoni: Antigenic heterogenicity of excretions and secretions. Experimental Parasitology 36, 316-330. NASH, T. E. 1978. Antibody response to a polysaccharide antigen in schistosomiasis. I. Sensitivity and specificity. American Journal of Tropical Medicine and Hygiene 27, 939-943. NASH, T. E., NASIR-UD-DIN, AND JEANLEZ, R. W. 1977. Further purification and characterization of a circulating antigen in schistosomiasis. Journal of Immunology 119, 1627-1633. NASH, T. E., OTTESEN, E. A., AND CHEEVER, A. W. 1978. Antibody response to a polysaccharide antigen present in schistosome gut. II. Modulation of antibody response. American Journal of Tropical Medicine and Hygiene 27, 944-950. NASH, T. E., PRESCOTT, B., AND NEVA, F. A. 1974. The characteristics of a circulating antigen in schistosomiasis. Journal of Immunology 112,
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