Separation and partial purification of agglutinins from coelomic fluid of the earthworm, Lumbricus terrestris

Comp. Biochem. PhysioL Vol. 97B, No. 4, pp. 701-705, 1990 Printed in Great Britain

0305-0491/90$3.00+ 0.00 © 1990PergamonPress pie

SEPARATION A N D PARTIAL PURIFICATION OF AGGLUTININS FROM COELOMIC FLUID OF THE EARTHWORM, LUMBRICUS TERRESTRIS ELIZABETH A. STEIN, SOHEIL YOUNAI and EDWIN L. COOP~ Department of Anatomy and Cell Biology, School of Medicine University of California, Los Angeles, CA 90024, USA (Received 2 May 1990)

Abstraet--l. Agglutinins from coelomic fluid of the earthworm, Lumbricus terrestris were separated by gel chromatography. Fluid from erythrocyte-injected (induced) worms contained five agglutinin peaks with molecular wts ranging from 4000 to 400,000, whereas fluid from untreated (naturally occurring) worms contained only one agglutinin peak of mol. wt 30,000. 2. Gel chromatography-separated agglutinin from naturally occurring fluid was unaffected by heating at either 60 or 100°C. Three of the agglutinins from induced fluid were unaffected by heating whereas two were reduced in titer by 36.5 to 88%. 3. Affinity chromatography of induced coelomic fluid using bovine submaxillary mucin (BSM)conjugated Affigel 15, eluted with potassium thiocyanate (KSCN), resulted in a purification factor of 105-fold, while bovine submaxillary mucin-Sepharose, eluted with 1 M NaCI, purified agglutinins(s) 93-fold.

INTRODUCTION Earthworm coelomic fluid contains agglutinins against vertebrate erythrocytes and bacteria (Stein et al., 1982, 1986; Stein and Cooper, 1988; Wojdani et al., 1982). Naturally occurring coelomic fluid exhibits low levels of agglutinating activity; however, if worms are injected with either bacteria or certain erythrocytes such as those from rabbits, agglutinin levels increase significantly within 24 hr (Stein et al., 1982). Physiochemical data, absorption analyses and electrophoresis of coelomic fluid indicate the presence of only one or two agglutinins in naturally occurring fluid, but at least three and possibly more in induced fluid (Stein et al., 1982; Wojdani et aL, 1982). To determine the molecular size and properties of these agglutinins, we have separated and partially purified the different agglutinins by gel filtration and affinity chromatography from both naturally occurring and induced coelomic fluid. Gel filtration using Sephadex G-200 resulted in one agglutinin containing peak from naturally occurring fluid and four additional peaks from induced fluid. Purification of induced fluid by affinity chromatography utilized Sepharose 4B conjugated to bovine submaxillary mucin (BSM), Affigel-15 conjugated to BSM, and erythrocyte stroma. BSM-conjugated Affigel-15 was the most efficient purifying agent, which resulted in a purification factor of 105 when eluted with potassium thiocyanate. With respect to defense mechanisms, it is significant that immunized worms had an increased n u m b e r of peaks when compared to non-immunized worlns. MATERIALS AND METHODS Earthworms Adult Lumbricus terrestris were purchased from Golden West Cricket Company (Paramount, CA) and kept at 15°C 701

in Buss bedding (Buss Manufacturing Company, Lanark, IL) with supplemental feedings of dry baby cereal. Coelomic fluid Fluid was collected by inserting a sharpened Pasteur pipette into the coelomic cavity posterior to the clitellum which allowed intra-co¢lomic pressure to force fluid into the pipette. Cells and other particulate matter were removed by centrifugation at 1000g for 10rain, and the supernatant either used fresh or stored at -20°C. For most procedures, fluid was pooled from 40-80 worms. Agglutinin induction Induction was performed as described previously, using rabbit erythrocytes (RRBC) as the inducing antigen (Stein et al., 1982). Briefly, worms were injected with a 5% suspension (v/v) of RRBC in 0.85% NaCI (normal saline). Twenty-four hours after RRBC injection, coelomic fluid was collected and processed as described above. Agglutination assay Coelomic fluid (50 #1) was serially diluted in microtiter "U" plates, using 0.15 M phosphate buffered saline (PBS) as diluent. Twenty-five microliters of a 2% (v/v) suspension of washed RRBC in PBS were added to each well, the plate shaken gently and allowed to stand at room temperature for 1 hr. Titers were read and recorded as the reciprocal of the last well showing agglutination. Gel chromatography Codomic fluid was concentrated 3- to 5-fold by negative pressure ultra-filtration to 1.5 ml and added to a 1.5 x 70 cm column of Sephadex G-200 (Pharmacia, Uppsala). Three milliliter fractions were eluted with a buffer of 4 mm CaCI2, 1 mM MgC12, 20mM Tris HC1 and 100raM NaCI (CMSTB), adjusted to a pH of 7.2 and each fraction analyzed for titer and protein concentration. Both naturally occurring and induced ccelomic fluid were chromatographed, running three separate samples of pooled fluid (40-50 worms each pool) for each type. Standard proteins were also chromatographed (ferritin, aldolase, bovine serum albumin and ribonucleas¢) along with Dextran Blue. A standard plot

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Both eluates were dialyzed for 24 hr in CMSTB, reconstituted to the original volume and analyzed for agglutination titer and protein concentration (Hartree, 1972). (b) Bovine submaxillary mucin conjugated to Sepharose 4B. BSM-Sepharose was prepared according to the procedure recommended by Pharmacia (Pharmacia, 1979), coupling 10mg BSM/gm of CNBr-Sepharose 4B. The slurry was poured into a 10-ml column and washed with 30ml CMSTB. Two milliliters of induced coelomic fluid, diluted 1: 1 with CMSTB, was added slowly. The column was allowed to stand at 4°C for 1 hr, then washed with CMSTB to remove non-absorbed coelomic fluid. Agglutinins were eluted with 10 ml of 1 M NaCI in pH 7.3 Tris buffer. Eluant was dialyzed for 24 hr at 4°C in CMSTB, concentrated at ml then analyzed for hemagglutination titers and protein concentration. (c) Affigel-15 coupled to BSM. Affigel-15, 12ml, was coupled to 100 mg BSM according to the manufacturer's directions. The gel was poured into a 20-ml column, washed, 12 ml of induced coelomic fluid was added and the gel was then allowed to stand for 1 hr. The gel was washed with 80ml CMSTB, eluted with 12ml of 1M NaCI, 10 mM EDTA and 20 mM Tris-HC1, pH 7.2. The column was next eluted with 23 ml of 1 M potassium thiocyanate (KSCN), 10mM EDTA and 20mM Tris-HC1, pH7.2. Eluates were dialyzed for 48 hr in CMSTB and then concentrated with a Minicon LS-15 chamber (Amicon, Danvers, MA) to 1 ml. Titers and protein concentrations were then measured.

with logarithms of mol. wts as the abscissa and the distribution coefficients,/Ca, as the ordinate, was then constructed to determine molecular sizes of the column eluates. K~ values were calculated as: K~ = [(elution volume of protein) - (void volume)] + [(total column volume) - (void volume)]. Those eluted samples showing highest agglutinating activity were heated at 60 and 100°C to determine heat sensitivity of these fractions.

Affinity media Three types of affinity media were used: RRBC stroma, and bovine submaxillary mucin (BSM) (Sigma Chemical Company, St Louis, MO), conjugated either to Sepharose CNBR (Pharmacia) or Affigel-15 (BioRad, Richmond, CA). Each procedure was repeated in triplicate. (a) Erythrocyte stroma. RRBC stroma were prepared by the method of Kabat and Mayer (1961). Briefly, RRBC were washed twice in normal saline then hemolyzed at 37°C for 25 min in normal saline containing 10% glycerol. The lysed RRBC were washed twice in normal saline containing 30 mM sodium citrate, once in hypotonic (10% N) PBS and, finally, in CMSTB. One milliliter of induced coelomic fluid, diluted 1: 1 with CMSTB, was added to 1.0 ml of packed RRBC stroma. The suspension was shaken gently for 1.5 hr at 4°C, centrifuged at 10,000g for 20min and the supernatant assayed for agglutination titer and protein concentration. The stromal pellet was then washed three times in cold CMSTB and agglutinins eluted by two different procedures. In the first, stroma were eluted for 30 min at 24°C in 1.0 M NaC1 buffered at pH 7.3 with Tris-HC1. For the second, stroma were mixed with glycine HCI (10raM, pH3.0) for 15min at 24°C, centrifuged, and supernatant immediately neutralized with 0.1 N NaOH.

Thermal sensitivity of agglutinins Eluate fractions from Sephadex G-200 chromatography of induced and naturally occurring coelomic fluid that

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Lumbricus agglutinins contained agglutinin activity were concentrated 10-fold and heated for 30 rain at 60 and 100°C. Titers of heated fractions from each of the three sets of chromatographed coelomic fluid samples were compared to titers of corresponding unheated fractions, and expressed as the per cent decrease in activity, i.e. % decrease = [(titer unheated fluid)- (titer heated fluid) - (titer unheated fluid)] x 100. RESULTS

Gel filtration Sephadex G-200 chromatography of naturally occurring coelomic fluid resulted in only one agglutinin containing peak at a molecular weight of approximately 3 x 10~ daltons (Fig. 1). Chromatography of induced fluid showed four primary agglutinin peaks at mol. wts of approximately 4 x 105, 6.5 x 104, 1.5 x 104, and 4 x 103 and a minor peak at 3 x 104 (Fig. 2). Agglutinin molecular sizes were determined by the calibration curve constructed from standard protein markers (Fig. 3).

Thermal sensitivity of agglutinins Agglutinin-containing fractions from Sephadex G-200 chromatography showed the following effects of heating: for induced fluid, peak number 1, no change in titer at either 60 or 100°C; peak 2, 25% reduction at 60°C, 62.5% reduction of titer at 100°C; peak 3a, no change at either temperature; peak 3b, 68.4% reduction at 60°C, 73.8% reduction at 100°C; peak 4, 25% reduction at 60°C, 31.3% at 100°C

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(Table 1). The agglutinin-containing fraction from naturally occurring fluid was not affected by heating at either 60 or 100°C.

Affinity purification (a) Erythrocyte stroma. RRBC stroma removed all measurable agglutinins from induced coelomic fluid, but did not serve to purify agglutinins above the level of the original coelomic fluid (Table 2). Both the 1 N NaC1 and pH 3 glycine buffer eluates possessed agglutinin activity but also contained sufficient protein (possibly from stromal proteins) so that specific activity was no higher than that of the original coelomic fluid. (b) BSM-Sehparose 4B affinity chromatography. BSM-Sepharose, eluted with 1 M NaC1, purified agglutinins by a factor of 93.0 (Table 2). (c) BSM-Affigel-15 affinity chromatography. 1 N NaC1, with added EDTA, eluants contained moderately purified agglutinins at a purification factor of 12.8. Potassium thiocyanate, 1 M, also with EDTA, was the most efficient eluant, with a purification factor of 105.2 (Table 2). DISCUSSION Invertebrate bod¢ fluids, almost without exception, contain agghitinins that react with erythrocytes and/or bacteria (Stein et aL, 1981, 1986). Coelomic fluid of the earthworm, Lumbricus terrestris normally

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Logarithms of molecular weights of proteins comprise the abscissa. Positions of both marker proteins and main agglutinin peaks are indicated. contains low levels of agglutinins, but these levels can be increased up to 7-fold by injecting worms with erythrocytes or bacteria. RRBC and several E. cell species are the most effective found so far at increasing agglutinin levels (Stein et al., 1982, 1986). Previous investigations of Lumbricus agglutinins have shown that several agglutinins, with differing physio. chemical properties, may be present in induced fluid (Stein et al., 1982, 1989) but this has never been confirmed by separation procedures. Coelomic fluid of the polychaete annelid, Nereis virens, contains multiple agglutinins, with at least 10 already isolated (Russell and Lai, 1986; Lai and Russell, 1987). Separation of Lumbricus coelomic fluid agglutinins on the basis of molecular size, using Sephadex G-200 gel filtration, revealed a single agglutinin peak for naturally occurring fluid. This peak, of 30,000 mol. wt, retained agglutinating activity after heating at 60 and 100°C. Induced fluid, however, was separated into four peaks of widely differing molecular sizes at

4000, 15,000, 65,000 and 400,000, and a 5th partial peak at 30,000, occurring as a "shoulder" of the 15,000 mol. wt peak. Of these, the 30,000 and 400,000 mol. wt peaks were unaffected by heating at 60 and 100°C, while the 4000 mol. wt peak was minimally inactivated at 100°C. The 15,000 and 65,000 mol. wt peaks were both inactivated at 100°C by 73.8 and 62.5%, respectively. While the chemical composition of these peaks has not been determined, those inactivated by heating are most probably protein. The heat stable agglutinins, however, are probably not proteins, but may be lipids or lipid micelles, since lipid agglutinins have been isolated from both naturally occurring and induced Lumbricus coelomic fluid (Stein et al., 1989). Lipid extracts of coelomic fluid contain agglutinins, and glycolipid and phospholipid fractions possess the highest activity. The 30,000 mol. wt agglutinins from naturally occurring and induced fluid are both unaffected by heating and are probably lipid. The 400,000 mol. wt

Table 1. Thermalsensitivityof naturallyoccurringand inducedagglutininsseparated by gel filtration Molecular Per cent reductionin titer Type of Peak size (k Da) followingheatingat* coleomicfluid number 100°C 60°C 100°C Naturally occurring 1 30 0 0 Induced l 400 0 0 2 65 25 62.5 3a 30 0 0 3b 15 68.4 73.8 4 4 25 31.3 *Heating for 30rain. Reductionin titers calculatedas: [(titer untreated fraction)- (titer heated fraction)+ (titeruntreatedfraction)]× 100.Fractionstested fromeluatesof three chromatographs, 40-50 worms each chromatograph.

Lumbricus agglutinins

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Table 2. Affinitychromatographyof inducedLumbricus coelomicfluid Slmeificactivity* of original Procedu~ Eluant coelomic fluid Eluate RRBC stroma 1M NaCI 312 267 pH 3 glycinebuffer 312 288 BSM-Sepharose 1M NaCI 70 6507 BSM-Affigel-I5 1M NaCI 71 914 KSCN 71 7467 *Specificactivity= titer/mg prot. tPurificationfactor= (specificactivityeluate)+ (specificacitivitycoelomicfluid). agglutinin was also unaffected by heat, and may represent an aggregate of smaller lipid units. The 4000 mol. wt agglutinin was only minimally affected by heat and is also most likely a lipid. Since three of the peaks appear to be primarily lipid in composition, their molecular size as determined by Sephadex G-200 may be only an approximation, since this type of size determination is most accurate for proteins. Three different affinity chromatography procedures were used to purify agglutinins from induced coelomic fluid. Rabbit erythrocyte stroma effectively removed agglutinins from coelomic fluid, but eluates contained considerable protein, apparently leached from the stroma, so that no increase in activity was achieved. Bovine submaxillary mucin was used as a conjugate with Sepharose 4B and Affigel-15, since BSM had previously been found to be a highly effective inhibitor of Lumbricus agglutinins (Stein et al., 1982). Both of these latter substrates produced moderate to good purification. BSM-Sepharose, with a high salt (1 M NaC1) eluant, achieved a 93-fold increase in activity. The same eluant with BSMAffigel-15 was less effective, giving a purification factor of 12.8. However the same substrate using a chaotropic eluant, KSCN, was most effective and produced a 105-fold purification factor. Agglutinins, multivalent substances found in body fluids such as serum, can bind to cell surfaces and particles causing their aggregation. Agglutinins are ubiquitous in the animal and plant kingdom, occurring on the surfaces of viruses and bacteria and in primitive and advanced plants and animals. With respect to distribution, they may be membranebound or free, having been shed into body fluids. The present study demonstrated a significant increase in agglutinin containing peaks in worms after immunization when compared to non-immunized ones. Since agglutinins are involved in receptor-mediated recognition, it is safe to predict that our results are

Purification factort 0 0 93.0 12.8 105.2

important when we consider the role of agglutinins in immunodefense. REFERENCES

Hartree E. F. (1972) Determination of protein: a modification of the Lowry method that gives a linear photometric response. Analyt. Biochem. 48, 422-427. Kabat A. E. and Mayer M. M. (1961) Experimental Immunochemistry, 2nd edn. Charles C. Thomas, Springfield. Lai P. S. and Russell C. S. (1987) Lipid agglutinins in Nereis coelomic fluid. Fedn Proc. 46, 1998. Pharmacia Handbook (1979) Gel Filtration: Theory and Practice. Pharmacia Fine Chemicals, Uppsala, Sweden. Pharmacia Handbook (1979) Affinity Chromatography: Principles and Methods. Pharmacia Fine Chemicals, Uppsala, Sweden. Russell C. S. and Lai P. S. (1986) Hemagglutinins in Nereis coelomic fluid. Ann. N.Y. Acad. Sci. '463, 131-134. Stein E, A., Wojdani A. and Cooper E, L. (1982) Agglutinins in the earthworm, Lumbricus terrestris: naturally occurring and induced. Devl comp. lmmunol. 6, 407-422. Stein E. A. and Cooper E. L. (1983) Carbohydrate and glycoprotein inhibitors of naturally occurring and induced aggiutinins in the earthworm Lumbricus terrestris. Comp. Biochem. Physiol. 7611, 197-206. Stein E. A. and Cooper E. L. (1988) In vitro agglutinin production by earthworm leukocytes. Devl comp. ImmunoL 12, 531-548. Stein E. A., Morovati A., Rahimian P. and Cooper E. L. (1989) Lipid agglutinins from coelomic fluid of the earthworm Lumbricus terrestris. Comp. Biochem. Physiol. 94B, 703-707. Stein E. A., Younai S. and Cooper E. L. (1986) Bacterial agglutinins from the earthworm, Lumbricus terrestris. Comp. Biochem. Physiol. 84B, 409-415. Wojdani A., Stein E. A. and Cooper E. L. (1982) Agglutinins and proteins in the earthworm, Lumbricus terrestris, before and after injection of erythrocytes, carbohydrates and other materials. Devl comp. lmmunol. 6, 613--624.