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Hemagglutinin activity in Nereis coelomic fluid
HEMAGGLUTININ ACTIVITY IN NEREZS COELOMIC FLUID C. S. RUSSELL, J. RODRIGUEZ
and P.-S. LAI
Department of Chemistry, City University of New York, The City College, New York. NY 10031, USA (Receiced 23 August 1982) Abstract-l. Nereis coelomic fluid agglutinates rat, mouse, chicken, guinea pig and rhesus monkey erythrocytes (RBC). 2. Lipid fractions of the particulate matter from coelomic fluid are hemagglutinins exhibiting different activity inhibition profiles with complex polysaccharides. 3. The high mol. wt hemagglutinin from coelomic fluid supernatant is not a protein and is inhibited by bovine submaxillary mucin (BSM), thyroglobulin, transferrin and their asialo derivatives. 4. Coelomic fluid supernatant has a population of low mol. wt protein hemagglutinins inhibited by BSM. fetuin. antiserum to coelomic fluid and some mannan preparations. 5. Hemagglutination by lipids characterized by RBC specificity and specificity for inhibition by carbohydrate is noteworthy and may be significant in studies of cellular interactions and immunity in invertebrates.
(1981) reported that oleic acid, dioleylphosphatidic acid, a polar lipid fraction from calf thymocytes and certain bovine brain lipids have some lectin-like activity, i.e. agglutination specificity for certain RBC and specific inhibition of activity by complex carbohydrates. However, unlike most lectin activity due to proteins, lipid-mediated agglutination is heat-insensitive and unaffected by a wide variety of monosaccharides. In an investigation of lectin activity in Nereis Grens coelomic fluid we observed that it retarded the growth of Ehrlich ascites tumors in mice (Tarnowski, Garte and Russell, unpublished results; Tabrah et al., 1970) and that rat RBC were agglutinated both by the supernatant and by the particulate fraction. The latter activity and part of the former was not diminished by heating, suggesting that much of the activity is due to non-protein material (Russell et ul., 1982). Reported
INTRODUCTION
Among the lectins from the invertebrates which have been characterized are those from the horseshoe crab (Limulus polyphenzus) (Marchalonis & Edelman, 1968; Finstad et al., 1972: Roche & Monsigny, 1974), the American oyster (Cra.ssostrea cirginica) (Acton et al., 1962; Li & Flemming, 1967; McDade & Tripp, 1967; Acton et al., 1973), the elongated clam (Tridacna maxima) (Bald0 & Uhlenbruck, 1975a,b; Eichmann & Uhlenbruck, 1976), the spiny lobster (Pan&us argus) (Acton ef al., 1973: Acton & Weinheimer. 1974), the marine coral (Leptogorgia uirgulata) (Lesniak & Liu, 1982), the American lobster (Homarus americanus) (Cornick & Stewart, 1973; Hall & Rowlands, 1974a,b), the sea hare (Aplysiu culjfornica) (Pauley et trl., 1971), the starfish (Asterius ,jbrhesi) (Roche & Monsigny, 1974), Amphitrite ornatu (Carte & Russell, 1976), the garden snail (H&Y pomatia) (Prokop et a/., 1965; Hammarstrom & Kabat, 1969; Oshiyama et a/., 1973) and the snails, He/i.\- hortensia (Prokop et al., 1964). Otula lactea (Boyd & Brown, 1965; Bhatia & Boyd, 1967) and Euphadra periomphalu (Ishiyama & Takatsu, 1970). Invertebrates lack that immune system of vertebrates, characterized by the production of antibodies. Therefore, the protective mechanisms of invertebrates consist of lysis and agglutination by components in the coelomic fluid and phagocytosis by coelomocytes. Although the recognition of self and non-self by coelomocytes is well-documented, the mechanism of action of this discriminatory property is poorly understood (Cohen & Uhlenbruck, 1974; Baskin, 1974). It has been proposed that agglutinins participate in the defense mechanism of invertebrates by aiding phagocytosis (Hall & Rowlands, 1974a,b). Stone (1946) observed that mixtures of pure lipids with specific stoichiometries showed agglutinatin properties towards fowl RBC similar to that of lipid extracts of a wide variety of tissues. Tsivion & Sharon
here are observations nol extracts of the
made when chloroform-methaparticulate residue from Nereis
coelomic fluid were tested for lectin activity and the properties of fractions of this extract obtained by silicic acid chromatography. In addition, evidence is presented that coelomic fluid supernatant contains at least two agglutinin activities, one heat and antiserum-insensitive and presumably non-protein and one heat- and antiserum-sensitive and reactive with some yeast mannan preparations and presumably protein. MATERIALS
AND
METHODS
Cl7emicals
Enzymes. glycoproteins, mucopolysaccharides, and complex polysaccharides were from Sigma (St Louis, MO). Mannan was Sigma M-7504, Lot No. 77C-0238 and Lot No. 90F-3864. Fetuin (Spiro method) was from GIBCO (Grand Island. NB). Monosaccharides were from EM Chemicals (Darmstadt, West Germany) and Sigma. Sephadex gels. protein mol. wt standards, and Blue Dextran 2000 57
58
C. S. RUSSELL et ul.
were from Pharmacia (Piscataway, NJ). BioGel P-100 was from Bio-Rad (Richmond, CA). All other chemicals were reagent grade or better and were obtained from standard sources. Dialysis tubing, SpectraPor 1, 2 and 3 was from Fisher (Springfield, NJ). Live N. cirrus were purchased from Bait Wholesalers (New Rochelle, NY). Rat RBC were obtained from live Wistar rats by cardiac puncture using 3.8% citrate as an anticoagulant. Sheep, rhesus monkey and guinea pig RBCs were purchased from M. A. Bioproducts (Rockville, MA). Erythrocytes from chicken and mice were provided by Dr G. Cooper, Department of Biology, City College. Human RBCs groups A, B, AB and 0 (with and without papain treatment) were a gift of the New York Blood Center. Instrumentation All centrifugations, except for RBCs, were done at 4°C in a Beckman J-21 refrigerated centrifuge with rotors JA-10, JA-14 and JA-20. Lipids were solubilized with a Bronson Sonicator Model W-185 and a Vortex-Genie mixer Microtiter
assay fbr hemagglutination
and inhibition studies
Erythrocytes were washed at room temperature (RT) three times in a table-top International Clinical Centrifuge with 0.9”,, NaCI-1 mM phosphate, pH 7.220.01x NaN, (PBS) and suspended in PBS to make a 5% suspension. Ahquots of 50 ~1 of PBS or test inhibitor solutions, consisting of glycoproteins, mucins or monosaccharides dissolved in PBS, were added to each well of a row of the Microtiter plate (M.A. Bioproducts). Aliquots of 25 ~1 or 50 ~1 of the sample to be assayed were diluted serially starting with the second well of a row. The first wells served as controls. A second 50~1 aliquot of PBS or test solution was added to each well, making a total of 100~1. When a test solution was used, it was allowed to preincubate for 30 min at RT. Then a 25 ~1 aliquot of a 5% RBC suspension was added to each well. The plates were sealed (M.A. Bioproducts) and shaken for 3 min on a Tektator plate shaker (Scientific Products) and observed after a half hour and one hour at RT. and after standing overnight in the refrigerator (779 C). The plates were read by tilting. In a positive result, the RBCs formed a button at the bottom of the well and did not flow. The titer is defined as the reciprocal of the highest dilution of sample which showed agglutination. Since twofold dilutions were made, the titers are reported as powers of two. The titers reported here are overnight results for rat RBC unless stated otherwise. Po/yacr~~lamide disc-gel electrophoresis
(PAGE)
The method of Davis (1964) was followed. The stacking gel consisted of 4’2, polyacrylamide polymerized at pH 6.7. The separating gel consisted of 79/, polyacrylamide-at pH 8.9. The electrophoresis buffer was alvcine-Tris, PH 8.3. Preparative disc-gel electrophore% was done in 8 mm internal diameter x 85 mm long tubes with samples of 100-200~~1 (with l-2 drops of glycerol). The gels were run at 5 mA (constant current) for about 60 min, using bromophenol (BPB) as marker. A Buechler 3-1500 constant power supply and jacketed electrophoresis apparatus were used with a Lauda K-21R refrigerated circulator maintained at 1O‘C. After electrophoresis, the gels were cut into 5 mm slices and each soaked in 300 ~1 of PBS. The gels were crushed by freezing and thawing three times. The supernatant was dialyzed against PBS. A 100 pi aliquot of the dialyzate was tested with 25 ~1 of 5% rat suspension. One gel of each run was stained with Coomassie blue. Method.5 qf concentration Materials were concentrated in SpectraPor 1 dialysis tubing (68 kD mol. wt cut-off), embedded in polyethylene glycol (PEG. Sigma 20,OOOmol wt) until the desired volume was reached. The concentrated material was dialyzed
against deionized water (dH,O) and then agamst PBS. Ultrafiltrations were done using Amicon filter units and UM-2 (Amicon) membranes with a mol. wt cut-off of 2 kD. Analytical
methods
Protein concentration was determined by the method of Bradford (1976) using Bio-Rad reagent and bovine y elobulin as standard. Hex&e concentra%on was deter&id by the phenol-sulfuric micro-assay method of Dubois ct (11. (1956) using glucose or Dextran as a standard. Immunod$kion Rabbit antiserum to coelomic fluid was obtained by the method described by Cooper (1977). Immunodiffusion plates were from Miles Laboratories (Elkhart, Ind.). Aliquots of 30-50~1 of test solutions and antiserum were used. Silicic acid chromatography
of lipid ,fiactions
All solvents were glass distilled before use. Sihcic acid (Mallinckrodt, 100 mesh powder) was heated at 12O’C for 2 hr. Following the method of Kates (1972) the samples were applied to the column in chloroform and eluted with chloroform (10 column volumes), acetone (40 column volumes) and methanol (10 column volumes). Before loading the samples, 1Oml aliquots of each solvent were run through the column. The three fractions collected were evaporated to dryness and the inside of the containers were washed with l-2 ml of PBS by vortexing. These washes were titered using 5”,, rat RBC suspensions. No activity was observed. Preparation
qf afinity chromatography
columns
Hog gastric mucin polyleucine (HGMPL). Hog gastric mucin (45 g, Sigma Type II, crude) was purified following the method of Poretz (1973). The nurified material was immobilized on polyleucine by the method of Kaplan & Kabat (1966). Fetuin (Spiro)-Sepharose 48 (FS). Fetuin (Spiro. 500 mg) was immobilized with 15g of CNBr-activated Sepharose 4B (Pharmacia) following the method of Sela et al. (1975). Desialylation qf mucopolysaccharides (MPS). (a) Mucopolysaccharides (10 mg) were treated with 1 ml of 0.1 N HZSOL and incubated at 8O’C, in a water bath, for one hour (Kieda et al., 1978). The solutions were neutralized by adding NaHCO, crystals slowly until no more CO, gas evolved; at this point the pH was approx. 7.5. PBS was added to bring the solutions to 1 mg/ml. (b) Bovine submaxillary mucin (BSM, Sigma Type II. 20 mg) was dissolved in 19 ml of PBS and treated with 1 ml of neuraminidase (Sigma, 2 units/ml). This mixture was incubated for I hr at 37°C. Then it was heated on the steam bath for 10min. In the control the denatured enzyme was added to 20 mg of BSM in 19 ml of PBS. This mixture was heated on the steam bath for IO min. Treatments
with Phospholipase-C
(a) Phospholipase-C (Sigma, Type IX, 125 units) was reconstituted with 1.5 ml of dH20. A control aliquot was heated on the steam bath for 50 min. Aliquots of each test sample (1.0 or 1.5 ml) were treated with 0.3 ml of reconstituted enzyme (25 units), or 0.3 ml of denatured enzyme, or 0.3 ml of buffer. After mixing, the solutions were incubated for 3 hr at 37°C. To stop the reaction, the solutions were heated on the steam bath for 1 hr. The precipitate which appeared was removed by centrifugation. The supernatants were tested for hemagglutinating activity. (b) Phospholipase-C (Sigma, Type X, 25 units) was reconstituted with 250 ~1 of dH*O. The method followed is modified from MacFarlane & Knight (1941). The test solution consisted of: 2.0 ml buffer (0.904 NaCl&1 mM Tris. pH 7.221 mM CaCl,). 0.5 ml of 0.05 N CaCl,, 1.5 ml of substrate, 0.9 ml of 1 mg/ml bovine serum albumin (in
Hemagglutinin
in Nereis coelomic
activity
0.05 M Tris-maleate. pH 7.3) and 0.1 ml of enzyme. In the control solution, no enzyme was added, the amount of buffer was 2.1 ml, and all the other solutions were the same. The solutions were incubated at 37’ C for 30 min and heated in the steam bath for 30 min. The precipitate which appeared was removed as before, and the supernatant was tested for activity. Gel filtration
chromatography
Ail columns (Pharmacia) had a 2.6cm i.d. and were 25-30 cm high. The void volume of the Sephadex gels was determined with 2 ml of Blue Dextran 2000 (1.5 mg/ml in PBS); for the BioGel, 0.5 ml of thyroglobulin (36 mg/ml) was used. The mol. wt standards were aldolase (rabbit muscle) (158 kD). albumin (67 kD), ovalbumin (43 kD). cc-chymotrypsinogen A (bovine pancreas) (25 kD) and ribonuclease (13.7 kD). For the Sephadex gels, the standards were loaded in 2 ml aliquots of 15 mg of each protein per ml; for the BioGel, 0.5 ml aliquots at 36 mg/ml were used. The buffer was PBS unless stated otherwise. and fractions were collected using an LKB Ultrarac fraction collector with elution monitored at 254 nm and recorded by an LKB 8300 Uvicord II monitor and recorder.
Preparations Coelomic fluid. Coelomic fluid (CF) was harvested by electric stimulation of the worms (Roth, 1979). It was centrifuged at 2 kr.p.m. (JA-20) for 15 min. The particulate fraction was stored in sea water. The supernatant was fractionated by adding ammonium sulfate to make the solution 55Y/, saturated. After centrifugation [I5 kr.p.m.
fluid
59
(JA-20) for 1 hr] the precipitate obtained was taken up in PBS and dialyzed against PBS overnight. Eitraction cf lipids. The particulate fraction from I25 ml coelomic fluid was stored in 35 ml of sea water. The Folch (1941) procedure was used to extract the lipid components (Scheme 1). A mixture of CHC13-CH,OH (44ml:88 ml) was added to the particulate fraction in sea water and shaken by hand for 30min at RT. This was centrifuged at 12 kr.p.m. (JA-20) for 30 min. To the supernatant CHCl,Hz0 (84ml:84 ml) was added. This separated into two layers: the organic (CM) extract (118 ml), which had 89 mg of lipid and an aqueous layer (227 ml). A 10 ml aliquot of aqueous layer from which the methanol had been blown off with N, had a residue concentration of 16 mg.iml. titer: 23. A 10 ml aliquot of CM containing 8 mg of material, was dried with NZ gas and taken up in 2 ml of PBS by vortexing for 30 min. This material had a titer of 2’. A 50ml aliquot of the CM extract was evaporated to dryness with N,, the residue (42 mg) taken up in 2 ml chloroform and put on a silicic acid column (1.5 cm i.d. x 5.6 cm). The fractions eluted were: 19 mg of neutral lipids with chloroform, 8 mg of glycolipids with acetonc. and 14mg of phospholipids with methanol, The solvents were removed by rotary evaporation. The residues were suspended in 0.9”” NaCl-I mM Tris. pH 7.2~O.l”,, Triton X-100 by sonicating for 3-12 min and vortexing. When the concentration of each material was 4 mgjml. the toters were 2s for the chloroform eluate. 2’ for the acetone eluate. and 2” for the methanol eluate. HGMPL
chromatoyraph~
The dialyzate (lOOmI) of a 3&55”,, ammonium sulfate precipitate (AS) from coelomic fluid was mixed wrth Zg
Coelomic fluid from 78 Nereis Supernatant IPartic?lates (Stored in sea water)
CM extract 118 ml (89 mg residue)
Aqueous extract 227 ml (1.76 g residue) Titer: 22
CM (50 ml aliquot:42 mg residue)
,
Eluates:
CHC13
lsilijl. acid chromatograph,y
Acetone
(19 m9) Titers (4 mg/ml)*
*Buffer:
(8 mg)
28
2?
CH30H (14 mg) 29
0.9% NaCl - 1 mM Tris, pH 7.2 - 0.1% Triton X-100.
Scheme
1
60
et 01.
C. S. RUSSELL
HGMPL and stirredovernight at 4‘C (Scheme 2). The mixture was centrifuged at 14 kr.p.m. (JA-14) for 45 min. The supernatant had a titer of 2‘). The precipitate was washed 8 times with 100ml of PBS per wash until the absorbance at 280 nm was 0. The washes were pooled, concentrated on PEG to 33 ml and dialyzed against dH,O and then PBS. The activity of this fraction was 2”. The material adsorbed on the HGMPL was removed by washing with 50 ml So”,, ethylene glycol-50”” PBS (v/v) (EC-PBS) 7 times until there was 0 absorbance at 280 nm. The supernatants. from centrifugation at 14 kr.p.m. for 1 hr. were dialyzed against dHZO to remove the EC. The dialyzates were concentrated over PEG and redialyzed against dHzO and then PBS. Since some particles were present, the dialyzates were pooled and centrifuged at 14 kr.p.m. for 2 hr. The precipitate (I) was put aside. The supernatant (248 ml) had a titer of 2”. It was taken to 55”;, saturation with ammonmm sulfate. After stirring overnight (4 C). an oily layer (I) appeared. This material was lyophilized. Preclpltate I was washed 7 times with 10 ml PBS each time, centrifuging each time at 14 kr.p.m. (JA-14) for 30 min. The super-
natants were pooled and concentrated over PEG and dlalyzed against dH1O. The final dialyzate (33 ml) was taken to 55”$ with ammonium sulfate. After stirring overnight (4 C), an oily layer (II) appeared. It was taken up in 5 ml PBS which was added to the lyophilizate of oily layer 1. This solution was dialyzed against PBS overnight and had a titer of 2”.
The coelomic lluid from approx. 300 Nervis was fractionated with ammonium sulfate. The 3&55”,, ammonium sulfate precipitate was taken up in 75 ml of PBS and dialyzed against PBS. Its titer was 2”. A 50 ml aliquot of this sample was added to FS prepared as above from 500 mg fetuin and this was mixed overnight at 26 C on an endover-end rotator (Kraft Rugged Rotator). This mixture was poured mto a 2.6 cm i.d. column to a height of 25 cm. PBS wash was collected until absorbance at 254 came down to baseline (fractions I- 20). The material adsorbed was eluted by washmg with EC-PBS. These fractions were pooled and concentrated (9.9 ml). and 55”,, ammonium sulfate was
Coelomic fluid from 500 Nereis
I
-
+)2s04
Supernatant
Precipitate 1. +lOO ml PBS 2. dialysis 3. +2g HGMPL
I
I
Supernatant
Adsorbent (HGMPL)
Titer: 2'
Wash with 100 ml PBS, 8X
I
I
Supernatant
Adsorbent (HGMPL)
concentration and dialysis
Wash with 50 ml
Dialysate. 33 ml Titer: 2' Supernatant 1. dialysis 2. concentration 3. centfifugation
Precipitate (I)
Adsorbent (HGMPL)
Supernatant, 248 ml
1. Wash 10 ml PBS, 7X
Titer: 212
3. 55% (NH~)*s~~ 2. Concentration Oily layer II
I
551 (NH~)*S~~
Oily layer (I) lyophilized
1 Dialysis I
2'2
Titer:
I
Sephadex G-100, Bio Gel P-100 Scheme
2
Hemagglutinin
activity
in ,Vrwis coelomic
fluid
Coelomic fluid from 300 Nereis
1. + 75 ml PBS 2. dialysis 3. 50 ml aliquot + FS 4. Wash with PBS r----l Eluate
Adsorbent (FS)
([;; ii;; with pBs
I
Supernatant + wash
Adsorbent (FS)
,
Eluate (216 ml)
Adsorbent
Concentrate (10 ml)
Wash with EG-H20 (1:l)
(370 t 420 ml)
, ( :;?B$::T)
Titer: Z7 55% (NH~)~S~~
I Concentrate (37 ml) Titer: 2"
Eluate (330 ml) oily layer
I Concentrate (10 ml)
1. lyophilized 2. acetone pptn 3. 2 ml PBS
Titer: 27
Titer: 26 \
Scheme
added to 55”,, saturation. After stirring for 48 hr (4’C). a brown oily layer was observed. The material was lyophilized to dryness and cold acetone (2 ml) was added. The resulting precipitate was taken up in 2 ml of PBS and dialyzed against PBS, overnight at 4’C. The dialyzate had a titer of 2’. Since fractions 1~20 from the FS chromatography had activity. they were remixed for 4X hr with the FS as before. The mixture was transferred to a column and washed with 370ml PBS until the eluate had 0 absorbance of 280nm. Concentration to l/IO the volume gave a titer of 2l'.The column was then eluted with 330ml EC-PBS which was concentrated and redialyzed as before. The final dialyzate (IO ml) had particles which were removed by centrifugation. The titer of the supernatant was 2’. Detwminution
of’mol.
/ Sephadex G-100 3
The Folch extract (CM) of coelomic fluid particulates when taken to dryness and taken up in saline or saline-Triton X-100 gave the RBC agglutination specificity profile shown in Table I. The extract was active against rat, mouse, chicken. guinea pig and rhesus monkey RBC in decreasing order of titer but not against sheep. Heating the test solution on the steam bath for one or two hours did not diminish
Table I. Specificity of coelomic fluid supernatant extracts for erythrocytes (RBC) from different
wt.\ of the hemagglutinins
Aliquots of the following samples were put on Sephadex G-100: coelomic fluid supernatant (A), 55”” ammonium sulfate precipitate of A (B). material desorbed from FS (C), material not absorbed on FS (D) and material desorbed from HGMPL (Et. All samples showed agglutinin activity at void volume. B and D had an active low mol. wt fraction at -20 kD while C and E showed activity at z 11 and s 13 kD respectively. E was rechromatographed on BioGel P-100 and gave an active peak at 13 kD.
Erythrocytes” Rat Mouse Chicken Guinea pig Rhesus Sheep Humanh Rabbit Hamster
Coelomic fluid supernatant
+++++ +++ ++ + N.D. _ _ _ _
and hptd species
CM extract and fractions from silicic acid chromatography
+++++ +++ ++ + + _ N.D. N.D. N.D.
RESULTS AND DISCUSSION
Schemes 1, 2 and 3 show typical extraction fractionation procedures for Nereis coelomic particulates and supernatant respectively.
and fluid
’ 5Y<,suspensions in PBS. ‘Human RBC included groups A, B, AB and 0 (with and without papain treatment). + relative activity of agglutination; - : no agglutination; N.D.: not determined.
:
Table 2. Heat sensitivity and interaction with mannan active fractions from Nereis coelomic fluid
Fraction
Control
Portrcrrlurc~ 1,CM extract 2. Aqueous extract 3. Methanol fraction from silicic acid Slf/~f~~l~(ifl~f?f 1. tintreated 5, High MW peak from G-100 6. Desorhed from HGMPL 7. (61 from G-100 (low MW)
Titer Heated4
Mannan effect
2’2
2” 25
No No No
2’ 712 L
2’
2” 2’* 2’ ZK
for
2’ 21’
No
0 0
Yes Yes
NO
,’Samples were heated for l-2 hr on the steam bath. Table 3. The effects of phosphohpase tluid
C on Nerh
coelomic
Titer Fractron
Control
I. CM extract” 2. Methanol fraction from silicic acid” 3. Coelomic Ruid supernatanth ii Phospholipase h Phospholipase
Test
78
74
” ;8
2‘r 2”
C. type IX. 25 units (method a). C. type X. 10 units (method h).
Table 4. The effects of glycoproteins on hemagglutinin titer of ,Vcwi,\ coelomic fluid supernatant and CM extract
Glycoprotein Control (PBS) BSM Asialo BSM,’ HGM Asialo HGM,’ Fetuin Astalo fctuin,’ Thyroglobulin (TG) Asialo T-G” Transferrin (TF) Asialo TF”
Coelomic fluid supernatant 2h 0 0 2” t9 f8 ;,., 22 0 24 72
CM extract 2’ 0 2h N.D. 2’ 25 23 2’ 2& 25 2”
The CM extract had a concentration of 3.8 mgjml. The concentration of all the glycoproteins was 1 mg/ml in PBS. N.D.: not determined. “Desialylation by method (a).
(Table 2). Table 3 shows that phospholipas~ C treatment lowered the titer against rat RBC in the CM and coelomic supernatant and suggests that phospholipid is partly responsible for agglutination. CM gave no precipitin band with rabbit antiserum to homogenized coelomic fluid nor did it interact with mannan. CM was fractionated on a silicic acid column (Kates. 1972) and the chloroform (neutral lipids), acetone (glycolipids) and methanol (phospholipids) fractions all showed heat-insensitive agglutinin activity against rat RBC but with different inhibitor sensitivities (Tables 4 and 5). Chloroform fraction activity was inhibited completely by bovine submaxillary mucin (BSM) and partially by fetuin but not by sialic acid (NANA) or sialy~-(NANA) lactose. Heated BSM activity
(control for neuraminidase treatment) was not as effective an inhibitor, suggesting that conformation and/or the protein moiety of BSM is required for inhibition. Acetone fraction was inhibited by BSM, but not by NANA. Inhibition was not affected by pre-treatment of BSM with neuraminidase or heat, The methanol fraction was inhibited by fetuin, NANA and by heated BSM. Untreated BSM was an inhibitor at higher con~ntrations of the methanol fraction but not at lower ones (see footnote, Table 5). Most of the aggiutinin activity in coelomic Ruid supernatant (RBC specificity in Table 1) eluted at void volume from Sephadex G-100 or G-200 chromatography. This fraction was heat- and mannaninsensitive and did not precipitate with rabbit antiserum to coelomic lluid. Hemagglutinin activity with rat RBC was inhibited by BSM, thyroglobulin and transferrin in that order of effectiveness and by their asialo derivatives (Table 4). It appears that this material is not a protein. Because phospholipase C diminishes its activity, it is possible that some of the activity is due to phospholipid. Hemagglutinin activity due to protein (lectins) in coelomic fluid supernatant was very low and was detected and partially characterized in the following experiments. When a 55”;, ammonium sulfate precipitate (AS) from coelomic tluid was chromatographed on Sephadex G-100. activity peaked at void volume and at a fraction corresponding to -20 kD mol. wt. When AS was adsorbed onto the affinity media, hog gastric mucin-polyleucine (Scheme 2) or fetuinSepharose (Scheme 3) the material desorbed with 50’!<, ethylene glycol--PBS (EG-PBS) gave an oily layer when saturated with ammonium sulfate to 55”,,. Lyophilization and/or acetone precipitation was followed by gel chromatography on Sephadex G-100 or BioGei P- 100. The activity peak corresponded to a mol. wt of _ 13kD. Two closely spaced bands at Rr -0.7 (compared to BPB) appeared in PAGE. For the 20 kD and 13 kD proteins, titer and protein concentration peaks coincided. One continuous precipitin line with rabbit Table 5. The hemagglutination
effects of carbohydrate titer of lipid fractions chromatography
compounds on from silictc acid
Trter Additions (I mg:ml) Control (PSS) BSM Fetuin NANA NANA-lactose Control (PBS) BSM BSM-Neur BSM-Neur-control
Fractions: chloroform 2H 0 ?d 27 ZK 7 ‘2 0 ?3 A. 2”
acetone 2’ F 2’ N.D. 76 ,I2 23 2” 2&
methanol 29 27.‘ 0 72 78 ;,I 28h 0 0
The CM extract was fractionated by silicic acid chromatography. The fractions of the eluates were made to -BS. BSM was treated with neur4.2 mg/ml in Tris-Cazi aminidase by method (b). ND: not determined. ,’ Wells 2-S were sometimes negative; wells 6-7 were positive. hWells 2-4 were negative; wells 5-8 were positive.
Hemagglutinin activity in Nrreis coelomic fluid
63
Table 6. Yeast mannan interacts with Nereis low mol. wt hemagglutinins Titer Time 1 hr 2 hr 3 hr 4 hr overnight
Control + + -I+ +
22
23
+ + + + +
+ f + + +
24
25
26
2’
2s
2”
2’0
211
2’2
_
+ + + + +
+
-
-
+ +
+
-
_ -
_ -
-
_ -
+
+
+
+
+
+
+ +
+ + +
Mannan alone and the low mol. wt h~magglutinins alone agglutinate RBC, but at certain dilutions of hemagglutinin they interact and agglutination does not take place. Mannan concentration was 25 mg/ ml. The hemagglutinin was diluted serially and it was incubated with the mannan for 30 min. before the 5”;, rat RBC suspension was added to each well.
antiserum to coelomic fluid was obtained for both materials, Hemagglutinin activity and precipitin lines were abolished if the 13 kD and 20 kD species were heated first. In both cases, antiserum inhibited hemagglutination. Heated antiserum did not. Both lectins interacted with mannan (Table 6); that is, while each alone and mannan alone a~lutinated rat RBC, at some dilutions they interacted with mannan and agglutination was delayed. This effect was eliminated by heating, The mannan interaction was positive for Sigma mannan M7504 Lot No. 77C-0238 but not Lot No. 90F-3864. Some, but not other, strains of Saccharomvces cerecisiae were agglutinated by these lectins. Rat, mouse and chicken RBC were agglutinated in descending order of titer. Activity was inhibited by BSM and fetuin but not by a large number of monosaccharides. The 13 kD protein may be present in very small amounts in coelomic fluid and may be concentrated by preferential adsorption on HGMPL and FS or it may be the deglycosylated protein from the 20 kD lectin. The latter has 48% hexose and the 13 kD has little or none. It is concluded from these results that (I) different lipid fractions from the particulate fraction of Nereis coelomic fluid exhibit lectin-like activity which is specific but heat-insensitive, (2) the supernatant fraction of the coelomic fluid has a heat-insensitive, lectin-like component inhibited by BSM, thyroglobulin and transferrin but not by mannan, and protein lectins, 20 kD and 13 kD, which are heat-sensitive, inhibited by fetuin and BSM and interactive with some mannan preparations. The agglutination specificities of different fractions of ,Vereis coelomic fluid is noteworthy and further characterization of them is in progress. The lectin-like behavior of what appear to be different lipid components or mixtures of lipids corroborate and amplify previous reports. The implications of these results for studies of lipid involvement in mechanisms of cellular interactions is apparent.
ACTON
R. T. & WEINHEIMERP. F. (1974) Contemporar! Topics in Immunology. Vol. IV (Edited by COOPERE. L.) pp. 271-282. Plenum Press, New York. ACTONR. T., WEINHEIMER P. F. & NIEDERMEIER W. (1973) The carbohydrate composition of invertebrate hemaggiutinin subunjts isolated from the lobster. Pant&as argus and the oyster, Crassostrea rirg~}~jca. Crimp. Bio&em. P~~~s~5~. 44B, 185-l 89. BALDOB. A. & UHLENBRUCKG. (1975a) Purification of Tridacnin, a novel anti- -(l-6)-Digalactobiose precipitin from the haemolymph of Tridacna maxima (Roeding). FEBS Lett. 55, 25-29. BALDO B. A. & UHLENBR~JCKG. (1975b) Quantitative precipitin studies on the specificity of an extract from Tridacna maxima (Roeding). Carhohydr. Res. 40, 143151. BASKIN D. G. (1974) The coelomocytes of Nereid polychaetes. In Contemporary Topics in lmmunolog~~, Vol. IV (Edited by COOPER E. L.). Plenum Press. New York. BHATIAH. M., BOYDW. C. & BROWNR. (1967) Serological and immuno~hemicai studies of snail (Ofaia iocfvfl) anti-A: a simple purification method. Tran~~u.~i~~~(Philadeliria) 7, 53-59. BOYD W: C. & BROWNR. (1965) Immunology: a specific agglutinin in the snail, Ott& (H&v) Itrc~uu..&;itrure208, 593-594. BRADFORDH. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anu/J,r. Biochem.
72. 248-254. PESCE A. Experimental Techniques
BREWERSJ. M.,
J. & ASHWORTHR. Q. (1974) in Biochemistr); pp. 225-221. Prentice Hall, New York. COHENE. & UHLENBRUCKG. (1974) Biomedical perspectives of agglutinins of invertebrates and plant origin. Ann. N.Y. Acad. 5%. 234. CoOPER T. G. (1977) The ?bols elf’ ~~~)~~7~~~7~.~t~~ pp. 359-364. John Wiley. New York. CORNICK J. W. & S’I‘EWART J. E. (1973) Partial characteritation of a natural agglutinin in the hemolymph of the lobster, Homarus americanus. J. lncertehr. Pctthol. 2t, 225-262. DAVIS B. (1964) Disc electrophoresis~ll. Method and application to human serum proteins. Ann. N.Y. Acud. Sci. 121, 404427. Ackno~lrdgernent-This work was supported in part by DUBOISM., GILLESK. A., HAMILTONJ. K., REBERSP. A. & NlH Grant GM 07639 and NIH Grant 1 SO6 RR08168. SMITHF. (1956) Coiorimetric method for determination of sugars and related substances. Analvr. Ckem. 28. 350-356. REFERENCES EICHMANNK. & UHLENBRUCX G. (1976) Similar combining ACTON R. T.,BENNETTJ. C., EVANS F. E. & SCHROHENspecificities of invertebrate precipitins and mouse myeLONERR. E. (1969) Physical and chemical characterizloma protein 5539 for ~-~1,6)-galactans. ~fn~~~noc/ien~i,str~ ation of an oyster hemagglutinin. .I. b&l. Chem. 244, 13, l-6. 4128-4135. FINSTADC. L., L~TMAN G. W. & GOCIDR. (1972) The evolu-
64
C. S. RUSSELL. J. RODRIGUFZ and P.-S. LAI
tion of the immune response: XIII. The characterization of purified erythrocyte agglutinins from two invertebrate species. d. Immun. 108, 1074171 I. CARTE S. J. & RUSSELL C. S. (1976) Isolation and characterization of a hemagglutinin from Amphirrirr orrtatu. a polychaetous annelid. Biopl1y.y. Acra 439, 36%379. HALL J. L. & ROWLANDS D. T. (1974a) Heterogeneity of lobster agglutinins. I. Purification and physiochemical characterigation. Biochrmi.str~~ 13, 811 GSZ?. HALL J. L. & ROWLANDS D. T. (1974b) Heterogeneity of lobster agglutinins. II. Specificity of agglutinin-erythrocyte binding. Biochni.srry 13, Q&83?. HAWARSTROM S. & KABA~ E. A. (1969) Purification and characterization of a blood group A reactive hemagglutinin from the snail. He/i.\- pornatlcr and a study of its combining site. /3ioclwmisfr~~ 8. 2696-2705. ISHIYAMA I.. DIETZ E. & UHLENBRU~I< G. (1973) Comparative studies of anti-A agglutinins from various snails of the genus Helix (Hrli.~ pomaria and Hcliz aspc’rsa). Cornp. Biochern. Ph~xiol. 44B, 529%.547. ISHIYAMA1. & TAKATSU A. (1970) Investigations on enzyme absorption on the red cell surface. I. Investigation on blood group A-decomposing enzyme from CIosrridiun~ certium. I/o\- Sang. 19, 14GlSO. KAPLAP~ M. E. & KARAT E. A. (1966) Studies on human antibodies. IV. Purification and properties of anti-A and anti-B obtained by absorption and elution from insoluble blood group substances. J. rrp. Med. 123, 1061~1081. KATES M. (1972) Trc~hr~iqur !), Lipidolog? p. 351. North Holland/American Elsevier, New York. KIEDA C. M.. BOWLES D. J.. RAVID A. & SHARON N. (1978) Lectins in lymphocyte membranes. FEBS L~rt. 94, 391~~396. LESNIAK A. P. & LIU E. H. (1982) Characterization of a hemagglutinin from Leptogorgia r~irgulata (marine coral). Co,np. Biochrm. Phy,siol. 71 B, 302 308. LI M. F. & FLEMMING C. (1967) Hemagglutinins from oyster hemolymph. Can. J. Zool. 45, 1225-1234. MA~FARLANE M. G. & KNIGHT S. D. (1941) The biochemistry of bacterial toxins. I. The lecithinase activity of CI. rvelchii toxins. Biocllenl. J. 35, 884-902. MARCIIALOVIS I. J. & EDELMAN G. M. (196X) Isolation and
characterization of a hemagglutinin from Li~~rtr/[r\ p0/1,phrmus. J. molec. Bid. 32, 453-465. MCDADE J. E. & TRIPP M. R. (1967) Mechanism of agglutination of red blood cells by oyster hemolymph. .1. /~lrc,rr&r. Par/ml. 9, 523-530. PAUI.EY G. B., GRANCSK G. A. & KKASS~~K S. N. (19711 Characterization of a natural agglutinin present in the hemolymph of the California sea hare. .Ap/~,vu c~crliforn~tr. J. Inrwrdw. Par/d. 18, 207 218. Po~trz R. D. (1973) Preparation of purified hog gastric mucin. Preparation of polyleucyl hog gastric mucIn Merh. E~Izw&. 28, 352 353. PROKOP 0.. RACKWITZ A. & SCHLI~SI%C~EK0. (1965) A new human blood group receptor Ahel tested with saline extracts from Heliu horretlsit (garden snail). .I. Forcz,~\~c, $frd. 12, 108-I IO PKOKOP 0.. SCHLL:SI\C;EK 0. & RACKWIIZ A. (1965) Uber das Vorkommen van P I-Blutgruppensubstanz bei elnpen Metazoen. ins. besondere A vc,uri.\ \u~~,?1und [.I~)IIhricus rcwmrrrs. Uber eine thermostabile “antibodk-like substance” (Anti-Ahell bei He/i\- pomaria und deren- Herkunft. Z. Inzrrnlrlfbr\(,II.129. 344-354. 402 412. ROCH P. (1979) Protein analysis of earthworm coelomic fluid: I. Polymorphic system of the natural hemolysin of
properties of limulin: a lectin (agglutinin) from hemolymph of Lirnullr\ pol~pl~rrmr,c. Bioc,him. hioplt~x -lc,rtr 371, 242-254. RUSSELL C. S.. ROI>KIG~EL J. & LAI P.-S. (19X1)Lectln activity in a lipid extract of coelomocytes from &‘~~r~,i\. Fcdrl Proc. Fe&. Am Sots. KYp. Bid. 41, 1033. SFLA B. M.. WAYG J. L. & ED~LMAN G. M. (1975) Isolation of lectins of ditrerent specificities on a single affinity adsorbent. J. Biochetn. 250. 7535-753X. Smhl J. D. (1946) Lipid haemagglutinins. rlrf\r,.. .I. c’\,>. Biol. & Med. 24, 197 205. TABRAH F. L.. KASIIIWA(;I M. &. No~ro\ T. R. (1970) Antitumor activity in mice of tentacles of two tropical sea annelids. Scirncr 170, IX I 183. TSIVION Y. & SHARON N. (1981) Lipid-medlated hemagglutination and its relevance to lectin-mediated agglutlnation. Bioc,hrnt. bionhw Am 642. 336 344.
and P.-S. LAI
Department of Chemistry, City University of New York, The City College, New York. NY 10031, USA (Receiced 23 August 1982) Abstract-l. Nereis coelomic fluid agglutinates rat, mouse, chicken, guinea pig and rhesus monkey erythrocytes (RBC). 2. Lipid fractions of the particulate matter from coelomic fluid are hemagglutinins exhibiting different activity inhibition profiles with complex polysaccharides. 3. The high mol. wt hemagglutinin from coelomic fluid supernatant is not a protein and is inhibited by bovine submaxillary mucin (BSM), thyroglobulin, transferrin and their asialo derivatives. 4. Coelomic fluid supernatant has a population of low mol. wt protein hemagglutinins inhibited by BSM. fetuin. antiserum to coelomic fluid and some mannan preparations. 5. Hemagglutination by lipids characterized by RBC specificity and specificity for inhibition by carbohydrate is noteworthy and may be significant in studies of cellular interactions and immunity in invertebrates.
(1981) reported that oleic acid, dioleylphosphatidic acid, a polar lipid fraction from calf thymocytes and certain bovine brain lipids have some lectin-like activity, i.e. agglutination specificity for certain RBC and specific inhibition of activity by complex carbohydrates. However, unlike most lectin activity due to proteins, lipid-mediated agglutination is heat-insensitive and unaffected by a wide variety of monosaccharides. In an investigation of lectin activity in Nereis Grens coelomic fluid we observed that it retarded the growth of Ehrlich ascites tumors in mice (Tarnowski, Garte and Russell, unpublished results; Tabrah et al., 1970) and that rat RBC were agglutinated both by the supernatant and by the particulate fraction. The latter activity and part of the former was not diminished by heating, suggesting that much of the activity is due to non-protein material (Russell et ul., 1982). Reported
INTRODUCTION
Among the lectins from the invertebrates which have been characterized are those from the horseshoe crab (Limulus polyphenzus) (Marchalonis & Edelman, 1968; Finstad et al., 1972: Roche & Monsigny, 1974), the American oyster (Cra.ssostrea cirginica) (Acton et al., 1962; Li & Flemming, 1967; McDade & Tripp, 1967; Acton et al., 1973), the elongated clam (Tridacna maxima) (Bald0 & Uhlenbruck, 1975a,b; Eichmann & Uhlenbruck, 1976), the spiny lobster (Pan&us argus) (Acton ef al., 1973: Acton & Weinheimer. 1974), the marine coral (Leptogorgia uirgulata) (Lesniak & Liu, 1982), the American lobster (Homarus americanus) (Cornick & Stewart, 1973; Hall & Rowlands, 1974a,b), the sea hare (Aplysiu culjfornica) (Pauley et trl., 1971), the starfish (Asterius ,jbrhesi) (Roche & Monsigny, 1974), Amphitrite ornatu (Carte & Russell, 1976), the garden snail (H&Y pomatia) (Prokop et a/., 1965; Hammarstrom & Kabat, 1969; Oshiyama et a/., 1973) and the snails, He/i.\- hortensia (Prokop et al., 1964). Otula lactea (Boyd & Brown, 1965; Bhatia & Boyd, 1967) and Euphadra periomphalu (Ishiyama & Takatsu, 1970). Invertebrates lack that immune system of vertebrates, characterized by the production of antibodies. Therefore, the protective mechanisms of invertebrates consist of lysis and agglutination by components in the coelomic fluid and phagocytosis by coelomocytes. Although the recognition of self and non-self by coelomocytes is well-documented, the mechanism of action of this discriminatory property is poorly understood (Cohen & Uhlenbruck, 1974; Baskin, 1974). It has been proposed that agglutinins participate in the defense mechanism of invertebrates by aiding phagocytosis (Hall & Rowlands, 1974a,b). Stone (1946) observed that mixtures of pure lipids with specific stoichiometries showed agglutinatin properties towards fowl RBC similar to that of lipid extracts of a wide variety of tissues. Tsivion & Sharon
here are observations nol extracts of the
made when chloroform-methaparticulate residue from Nereis
coelomic fluid were tested for lectin activity and the properties of fractions of this extract obtained by silicic acid chromatography. In addition, evidence is presented that coelomic fluid supernatant contains at least two agglutinin activities, one heat and antiserum-insensitive and presumably non-protein and one heat- and antiserum-sensitive and reactive with some yeast mannan preparations and presumably protein. MATERIALS
AND
METHODS
Cl7emicals
Enzymes. glycoproteins, mucopolysaccharides, and complex polysaccharides were from Sigma (St Louis, MO). Mannan was Sigma M-7504, Lot No. 77C-0238 and Lot No. 90F-3864. Fetuin (Spiro method) was from GIBCO (Grand Island. NB). Monosaccharides were from EM Chemicals (Darmstadt, West Germany) and Sigma. Sephadex gels. protein mol. wt standards, and Blue Dextran 2000 57
58
C. S. RUSSELL et ul.
were from Pharmacia (Piscataway, NJ). BioGel P-100 was from Bio-Rad (Richmond, CA). All other chemicals were reagent grade or better and were obtained from standard sources. Dialysis tubing, SpectraPor 1, 2 and 3 was from Fisher (Springfield, NJ). Live N. cirrus were purchased from Bait Wholesalers (New Rochelle, NY). Rat RBC were obtained from live Wistar rats by cardiac puncture using 3.8% citrate as an anticoagulant. Sheep, rhesus monkey and guinea pig RBCs were purchased from M. A. Bioproducts (Rockville, MA). Erythrocytes from chicken and mice were provided by Dr G. Cooper, Department of Biology, City College. Human RBCs groups A, B, AB and 0 (with and without papain treatment) were a gift of the New York Blood Center. Instrumentation All centrifugations, except for RBCs, were done at 4°C in a Beckman J-21 refrigerated centrifuge with rotors JA-10, JA-14 and JA-20. Lipids were solubilized with a Bronson Sonicator Model W-185 and a Vortex-Genie mixer Microtiter
assay fbr hemagglutination
and inhibition studies
Erythrocytes were washed at room temperature (RT) three times in a table-top International Clinical Centrifuge with 0.9”,, NaCI-1 mM phosphate, pH 7.220.01x NaN, (PBS) and suspended in PBS to make a 5% suspension. Ahquots of 50 ~1 of PBS or test inhibitor solutions, consisting of glycoproteins, mucins or monosaccharides dissolved in PBS, were added to each well of a row of the Microtiter plate (M.A. Bioproducts). Aliquots of 25 ~1 or 50 ~1 of the sample to be assayed were diluted serially starting with the second well of a row. The first wells served as controls. A second 50~1 aliquot of PBS or test solution was added to each well, making a total of 100~1. When a test solution was used, it was allowed to preincubate for 30 min at RT. Then a 25 ~1 aliquot of a 5% RBC suspension was added to each well. The plates were sealed (M.A. Bioproducts) and shaken for 3 min on a Tektator plate shaker (Scientific Products) and observed after a half hour and one hour at RT. and after standing overnight in the refrigerator (779 C). The plates were read by tilting. In a positive result, the RBCs formed a button at the bottom of the well and did not flow. The titer is defined as the reciprocal of the highest dilution of sample which showed agglutination. Since twofold dilutions were made, the titers are reported as powers of two. The titers reported here are overnight results for rat RBC unless stated otherwise. Po/yacr~~lamide disc-gel electrophoresis
(PAGE)
The method of Davis (1964) was followed. The stacking gel consisted of 4’2, polyacrylamide polymerized at pH 6.7. The separating gel consisted of 79/, polyacrylamide-at pH 8.9. The electrophoresis buffer was alvcine-Tris, PH 8.3. Preparative disc-gel electrophore% was done in 8 mm internal diameter x 85 mm long tubes with samples of 100-200~~1 (with l-2 drops of glycerol). The gels were run at 5 mA (constant current) for about 60 min, using bromophenol (BPB) as marker. A Buechler 3-1500 constant power supply and jacketed electrophoresis apparatus were used with a Lauda K-21R refrigerated circulator maintained at 1O‘C. After electrophoresis, the gels were cut into 5 mm slices and each soaked in 300 ~1 of PBS. The gels were crushed by freezing and thawing three times. The supernatant was dialyzed against PBS. A 100 pi aliquot of the dialyzate was tested with 25 ~1 of 5% rat suspension. One gel of each run was stained with Coomassie blue. Method.5 qf concentration Materials were concentrated in SpectraPor 1 dialysis tubing (68 kD mol. wt cut-off), embedded in polyethylene glycol (PEG. Sigma 20,OOOmol wt) until the desired volume was reached. The concentrated material was dialyzed
against deionized water (dH,O) and then agamst PBS. Ultrafiltrations were done using Amicon filter units and UM-2 (Amicon) membranes with a mol. wt cut-off of 2 kD. Analytical
methods
Protein concentration was determined by the method of Bradford (1976) using Bio-Rad reagent and bovine y elobulin as standard. Hex&e concentra%on was deter&id by the phenol-sulfuric micro-assay method of Dubois ct (11. (1956) using glucose or Dextran as a standard. Immunod$kion Rabbit antiserum to coelomic fluid was obtained by the method described by Cooper (1977). Immunodiffusion plates were from Miles Laboratories (Elkhart, Ind.). Aliquots of 30-50~1 of test solutions and antiserum were used. Silicic acid chromatography
of lipid ,fiactions
All solvents were glass distilled before use. Sihcic acid (Mallinckrodt, 100 mesh powder) was heated at 12O’C for 2 hr. Following the method of Kates (1972) the samples were applied to the column in chloroform and eluted with chloroform (10 column volumes), acetone (40 column volumes) and methanol (10 column volumes). Before loading the samples, 1Oml aliquots of each solvent were run through the column. The three fractions collected were evaporated to dryness and the inside of the containers were washed with l-2 ml of PBS by vortexing. These washes were titered using 5”,, rat RBC suspensions. No activity was observed. Preparation
qf afinity chromatography
columns
Hog gastric mucin polyleucine (HGMPL). Hog gastric mucin (45 g, Sigma Type II, crude) was purified following the method of Poretz (1973). The nurified material was immobilized on polyleucine by the method of Kaplan & Kabat (1966). Fetuin (Spiro)-Sepharose 48 (FS). Fetuin (Spiro. 500 mg) was immobilized with 15g of CNBr-activated Sepharose 4B (Pharmacia) following the method of Sela et al. (1975). Desialylation qf mucopolysaccharides (MPS). (a) Mucopolysaccharides (10 mg) were treated with 1 ml of 0.1 N HZSOL and incubated at 8O’C, in a water bath, for one hour (Kieda et al., 1978). The solutions were neutralized by adding NaHCO, crystals slowly until no more CO, gas evolved; at this point the pH was approx. 7.5. PBS was added to bring the solutions to 1 mg/ml. (b) Bovine submaxillary mucin (BSM, Sigma Type II. 20 mg) was dissolved in 19 ml of PBS and treated with 1 ml of neuraminidase (Sigma, 2 units/ml). This mixture was incubated for I hr at 37°C. Then it was heated on the steam bath for 10min. In the control the denatured enzyme was added to 20 mg of BSM in 19 ml of PBS. This mixture was heated on the steam bath for IO min. Treatments
with Phospholipase-C
(a) Phospholipase-C (Sigma, Type IX, 125 units) was reconstituted with 1.5 ml of dH20. A control aliquot was heated on the steam bath for 50 min. Aliquots of each test sample (1.0 or 1.5 ml) were treated with 0.3 ml of reconstituted enzyme (25 units), or 0.3 ml of denatured enzyme, or 0.3 ml of buffer. After mixing, the solutions were incubated for 3 hr at 37°C. To stop the reaction, the solutions were heated on the steam bath for 1 hr. The precipitate which appeared was removed by centrifugation. The supernatants were tested for hemagglutinating activity. (b) Phospholipase-C (Sigma, Type X, 25 units) was reconstituted with 250 ~1 of dH*O. The method followed is modified from MacFarlane & Knight (1941). The test solution consisted of: 2.0 ml buffer (0.904 NaCl&1 mM Tris. pH 7.221 mM CaCl,). 0.5 ml of 0.05 N CaCl,, 1.5 ml of substrate, 0.9 ml of 1 mg/ml bovine serum albumin (in
Hemagglutinin
in Nereis coelomic
activity
0.05 M Tris-maleate. pH 7.3) and 0.1 ml of enzyme. In the control solution, no enzyme was added, the amount of buffer was 2.1 ml, and all the other solutions were the same. The solutions were incubated at 37’ C for 30 min and heated in the steam bath for 30 min. The precipitate which appeared was removed as before, and the supernatant was tested for activity. Gel filtration
chromatography
Ail columns (Pharmacia) had a 2.6cm i.d. and were 25-30 cm high. The void volume of the Sephadex gels was determined with 2 ml of Blue Dextran 2000 (1.5 mg/ml in PBS); for the BioGel, 0.5 ml of thyroglobulin (36 mg/ml) was used. The mol. wt standards were aldolase (rabbit muscle) (158 kD). albumin (67 kD), ovalbumin (43 kD). cc-chymotrypsinogen A (bovine pancreas) (25 kD) and ribonuclease (13.7 kD). For the Sephadex gels, the standards were loaded in 2 ml aliquots of 15 mg of each protein per ml; for the BioGel, 0.5 ml aliquots at 36 mg/ml were used. The buffer was PBS unless stated otherwise. and fractions were collected using an LKB Ultrarac fraction collector with elution monitored at 254 nm and recorded by an LKB 8300 Uvicord II monitor and recorder.
Preparations Coelomic fluid. Coelomic fluid (CF) was harvested by electric stimulation of the worms (Roth, 1979). It was centrifuged at 2 kr.p.m. (JA-20) for 15 min. The particulate fraction was stored in sea water. The supernatant was fractionated by adding ammonium sulfate to make the solution 55Y/, saturated. After centrifugation [I5 kr.p.m.
fluid
59
(JA-20) for 1 hr] the precipitate obtained was taken up in PBS and dialyzed against PBS overnight. Eitraction cf lipids. The particulate fraction from I25 ml coelomic fluid was stored in 35 ml of sea water. The Folch (1941) procedure was used to extract the lipid components (Scheme 1). A mixture of CHC13-CH,OH (44ml:88 ml) was added to the particulate fraction in sea water and shaken by hand for 30min at RT. This was centrifuged at 12 kr.p.m. (JA-20) for 30 min. To the supernatant CHCl,Hz0 (84ml:84 ml) was added. This separated into two layers: the organic (CM) extract (118 ml), which had 89 mg of lipid and an aqueous layer (227 ml). A 10 ml aliquot of aqueous layer from which the methanol had been blown off with N, had a residue concentration of 16 mg.iml. titer: 23. A 10 ml aliquot of CM containing 8 mg of material, was dried with NZ gas and taken up in 2 ml of PBS by vortexing for 30 min. This material had a titer of 2’. A 50ml aliquot of the CM extract was evaporated to dryness with N,, the residue (42 mg) taken up in 2 ml chloroform and put on a silicic acid column (1.5 cm i.d. x 5.6 cm). The fractions eluted were: 19 mg of neutral lipids with chloroform, 8 mg of glycolipids with acetonc. and 14mg of phospholipids with methanol, The solvents were removed by rotary evaporation. The residues were suspended in 0.9”” NaCl-I mM Tris. pH 7.2~O.l”,, Triton X-100 by sonicating for 3-12 min and vortexing. When the concentration of each material was 4 mgjml. the toters were 2s for the chloroform eluate. 2’ for the acetone eluate. and 2” for the methanol eluate. HGMPL
chromatoyraph~
The dialyzate (lOOmI) of a 3&55”,, ammonium sulfate precipitate (AS) from coelomic fluid was mixed wrth Zg
Coelomic fluid from 78 Nereis Supernatant IPartic?lates (Stored in sea water)
CM extract 118 ml (89 mg residue)
Aqueous extract 227 ml (1.76 g residue) Titer: 22
CM (50 ml aliquot:42 mg residue)
,
Eluates:
CHC13
lsilijl. acid chromatograph,y
Acetone
(19 m9) Titers (4 mg/ml)*
*Buffer:
(8 mg)
28
2?
CH30H (14 mg) 29
0.9% NaCl - 1 mM Tris, pH 7.2 - 0.1% Triton X-100.
Scheme
1
60
et 01.
C. S. RUSSELL
HGMPL and stirredovernight at 4‘C (Scheme 2). The mixture was centrifuged at 14 kr.p.m. (JA-14) for 45 min. The supernatant had a titer of 2‘). The precipitate was washed 8 times with 100ml of PBS per wash until the absorbance at 280 nm was 0. The washes were pooled, concentrated on PEG to 33 ml and dialyzed against dH,O and then PBS. The activity of this fraction was 2”. The material adsorbed on the HGMPL was removed by washing with 50 ml So”,, ethylene glycol-50”” PBS (v/v) (EC-PBS) 7 times until there was 0 absorbance at 280 nm. The supernatants. from centrifugation at 14 kr.p.m. for 1 hr. were dialyzed against dHZO to remove the EC. The dialyzates were concentrated over PEG and redialyzed against dHzO and then PBS. Since some particles were present, the dialyzates were pooled and centrifuged at 14 kr.p.m. for 2 hr. The precipitate (I) was put aside. The supernatant (248 ml) had a titer of 2”. It was taken to 55”;, saturation with ammonmm sulfate. After stirring overnight (4 C). an oily layer (I) appeared. This material was lyophilized. Preclpltate I was washed 7 times with 10 ml PBS each time, centrifuging each time at 14 kr.p.m. (JA-14) for 30 min. The super-
natants were pooled and concentrated over PEG and dlalyzed against dH1O. The final dialyzate (33 ml) was taken to 55”$ with ammonium sulfate. After stirring overnight (4 C), an oily layer (II) appeared. It was taken up in 5 ml PBS which was added to the lyophilizate of oily layer 1. This solution was dialyzed against PBS overnight and had a titer of 2”.
The coelomic lluid from approx. 300 Nervis was fractionated with ammonium sulfate. The 3&55”,, ammonium sulfate precipitate was taken up in 75 ml of PBS and dialyzed against PBS. Its titer was 2”. A 50 ml aliquot of this sample was added to FS prepared as above from 500 mg fetuin and this was mixed overnight at 26 C on an endover-end rotator (Kraft Rugged Rotator). This mixture was poured mto a 2.6 cm i.d. column to a height of 25 cm. PBS wash was collected until absorbance at 254 came down to baseline (fractions I- 20). The material adsorbed was eluted by washmg with EC-PBS. These fractions were pooled and concentrated (9.9 ml). and 55”,, ammonium sulfate was
Coelomic fluid from 500 Nereis
I
-
+)2s04
Supernatant
Precipitate 1. +lOO ml PBS 2. dialysis 3. +2g HGMPL
I
I
Supernatant
Adsorbent (HGMPL)
Titer: 2'
Wash with 100 ml PBS, 8X
I
I
Supernatant
Adsorbent (HGMPL)
concentration and dialysis
Wash with 50 ml
Dialysate. 33 ml Titer: 2' Supernatant 1. dialysis 2. concentration 3. centfifugation
Precipitate (I)
Adsorbent (HGMPL)
Supernatant, 248 ml
1. Wash 10 ml PBS, 7X
Titer: 212
3. 55% (NH~)*s~~ 2. Concentration Oily layer II
I
551 (NH~)*S~~
Oily layer (I) lyophilized
1 Dialysis I
2'2
Titer:
I
Sephadex G-100, Bio Gel P-100 Scheme
2
Hemagglutinin
activity
in ,Vrwis coelomic
fluid
Coelomic fluid from 300 Nereis
1. + 75 ml PBS 2. dialysis 3. 50 ml aliquot + FS 4. Wash with PBS r----l Eluate
Adsorbent (FS)
([;; ii;; with pBs
I
Supernatant + wash
Adsorbent (FS)
,
Eluate (216 ml)
Adsorbent
Concentrate (10 ml)
Wash with EG-H20 (1:l)
(370 t 420 ml)
, ( :;?B$::T)
Titer: Z7 55% (NH~)~S~~
I Concentrate (37 ml) Titer: 2"
Eluate (330 ml) oily layer
I Concentrate (10 ml)
1. lyophilized 2. acetone pptn 3. 2 ml PBS
Titer: 27
Titer: 26 \
Scheme
added to 55”,, saturation. After stirring for 48 hr (4’C). a brown oily layer was observed. The material was lyophilized to dryness and cold acetone (2 ml) was added. The resulting precipitate was taken up in 2 ml of PBS and dialyzed against PBS, overnight at 4’C. The dialyzate had a titer of 2’. Since fractions 1~20 from the FS chromatography had activity. they were remixed for 4X hr with the FS as before. The mixture was transferred to a column and washed with 370ml PBS until the eluate had 0 absorbance of 280nm. Concentration to l/IO the volume gave a titer of 2l'.The column was then eluted with 330ml EC-PBS which was concentrated and redialyzed as before. The final dialyzate (IO ml) had particles which were removed by centrifugation. The titer of the supernatant was 2’. Detwminution
of’mol.
/ Sephadex G-100 3
The Folch extract (CM) of coelomic fluid particulates when taken to dryness and taken up in saline or saline-Triton X-100 gave the RBC agglutination specificity profile shown in Table I. The extract was active against rat, mouse, chicken. guinea pig and rhesus monkey RBC in decreasing order of titer but not against sheep. Heating the test solution on the steam bath for one or two hours did not diminish
Table I. Specificity of coelomic fluid supernatant extracts for erythrocytes (RBC) from different
wt.\ of the hemagglutinins
Aliquots of the following samples were put on Sephadex G-100: coelomic fluid supernatant (A), 55”” ammonium sulfate precipitate of A (B). material desorbed from FS (C), material not absorbed on FS (D) and material desorbed from HGMPL (Et. All samples showed agglutinin activity at void volume. B and D had an active low mol. wt fraction at -20 kD while C and E showed activity at z 11 and s 13 kD respectively. E was rechromatographed on BioGel P-100 and gave an active peak at 13 kD.
Erythrocytes” Rat Mouse Chicken Guinea pig Rhesus Sheep Humanh Rabbit Hamster
Coelomic fluid supernatant
+++++ +++ ++ + N.D. _ _ _ _
and hptd species
CM extract and fractions from silicic acid chromatography
+++++ +++ ++ + + _ N.D. N.D. N.D.
RESULTS AND DISCUSSION
Schemes 1, 2 and 3 show typical extraction fractionation procedures for Nereis coelomic particulates and supernatant respectively.
and fluid
’ 5Y<,suspensions in PBS. ‘Human RBC included groups A, B, AB and 0 (with and without papain treatment). + relative activity of agglutination; - : no agglutination; N.D.: not determined.
:
Table 2. Heat sensitivity and interaction with mannan active fractions from Nereis coelomic fluid
Fraction
Control
Portrcrrlurc~ 1,CM extract 2. Aqueous extract 3. Methanol fraction from silicic acid Slf/~f~~l~(ifl~f?f 1. tintreated 5, High MW peak from G-100 6. Desorhed from HGMPL 7. (61 from G-100 (low MW)
Titer Heated4
Mannan effect
2’2
2” 25
No No No
2’ 712 L
2’
2” 2’* 2’ ZK
for
2’ 21’
No
0 0
Yes Yes
NO
,’Samples were heated for l-2 hr on the steam bath. Table 3. The effects of phosphohpase tluid
C on Nerh
coelomic
Titer Fractron
Control
I. CM extract” 2. Methanol fraction from silicic acid” 3. Coelomic Ruid supernatanth ii Phospholipase h Phospholipase
Test
78
74
” ;8
2‘r 2”
C. type IX. 25 units (method a). C. type X. 10 units (method h).
Table 4. The effects of glycoproteins on hemagglutinin titer of ,Vcwi,\ coelomic fluid supernatant and CM extract
Glycoprotein Control (PBS) BSM Asialo BSM,’ HGM Asialo HGM,’ Fetuin Astalo fctuin,’ Thyroglobulin (TG) Asialo T-G” Transferrin (TF) Asialo TF”
Coelomic fluid supernatant 2h 0 0 2” t9 f8 ;,., 22 0 24 72
CM extract 2’ 0 2h N.D. 2’ 25 23 2’ 2& 25 2”
The CM extract had a concentration of 3.8 mgjml. The concentration of all the glycoproteins was 1 mg/ml in PBS. N.D.: not determined. “Desialylation by method (a).
(Table 2). Table 3 shows that phospholipas~ C treatment lowered the titer against rat RBC in the CM and coelomic supernatant and suggests that phospholipid is partly responsible for agglutination. CM gave no precipitin band with rabbit antiserum to homogenized coelomic fluid nor did it interact with mannan. CM was fractionated on a silicic acid column (Kates. 1972) and the chloroform (neutral lipids), acetone (glycolipids) and methanol (phospholipids) fractions all showed heat-insensitive agglutinin activity against rat RBC but with different inhibitor sensitivities (Tables 4 and 5). Chloroform fraction activity was inhibited completely by bovine submaxillary mucin (BSM) and partially by fetuin but not by sialic acid (NANA) or sialy~-(NANA) lactose. Heated BSM activity
(control for neuraminidase treatment) was not as effective an inhibitor, suggesting that conformation and/or the protein moiety of BSM is required for inhibition. Acetone fraction was inhibited by BSM, but not by NANA. Inhibition was not affected by pre-treatment of BSM with neuraminidase or heat, The methanol fraction was inhibited by fetuin, NANA and by heated BSM. Untreated BSM was an inhibitor at higher con~ntrations of the methanol fraction but not at lower ones (see footnote, Table 5). Most of the aggiutinin activity in coelomic Ruid supernatant (RBC specificity in Table 1) eluted at void volume from Sephadex G-100 or G-200 chromatography. This fraction was heat- and mannaninsensitive and did not precipitate with rabbit antiserum to coelomic lluid. Hemagglutinin activity with rat RBC was inhibited by BSM, thyroglobulin and transferrin in that order of effectiveness and by their asialo derivatives (Table 4). It appears that this material is not a protein. Because phospholipase C diminishes its activity, it is possible that some of the activity is due to phospholipid. Hemagglutinin activity due to protein (lectins) in coelomic fluid supernatant was very low and was detected and partially characterized in the following experiments. When a 55”;, ammonium sulfate precipitate (AS) from coelomic tluid was chromatographed on Sephadex G-100. activity peaked at void volume and at a fraction corresponding to -20 kD mol. wt. When AS was adsorbed onto the affinity media, hog gastric mucin-polyleucine (Scheme 2) or fetuinSepharose (Scheme 3) the material desorbed with 50’!<, ethylene glycol--PBS (EG-PBS) gave an oily layer when saturated with ammonium sulfate to 55”,,. Lyophilization and/or acetone precipitation was followed by gel chromatography on Sephadex G-100 or BioGei P- 100. The activity peak corresponded to a mol. wt of _ 13kD. Two closely spaced bands at Rr -0.7 (compared to BPB) appeared in PAGE. For the 20 kD and 13 kD proteins, titer and protein concentration peaks coincided. One continuous precipitin line with rabbit Table 5. The hemagglutination
effects of carbohydrate titer of lipid fractions chromatography
compounds on from silictc acid
Trter Additions (I mg:ml) Control (PSS) BSM Fetuin NANA NANA-lactose Control (PBS) BSM BSM-Neur BSM-Neur-control
Fractions: chloroform 2H 0 ?d 27 ZK 7 ‘2 0 ?3 A. 2”
acetone 2’ F 2’ N.D. 76 ,I2 23 2” 2&
methanol 29 27.‘ 0 72 78 ;,I 28h 0 0
The CM extract was fractionated by silicic acid chromatography. The fractions of the eluates were made to -BS. BSM was treated with neur4.2 mg/ml in Tris-Cazi aminidase by method (b). ND: not determined. ,’ Wells 2-S were sometimes negative; wells 6-7 were positive. hWells 2-4 were negative; wells 5-8 were positive.
Hemagglutinin activity in Nrreis coelomic fluid
63
Table 6. Yeast mannan interacts with Nereis low mol. wt hemagglutinins Titer Time 1 hr 2 hr 3 hr 4 hr overnight
Control + + -I+ +
22
23
+ + + + +
+ f + + +
24
25
26
2’
2s
2”
2’0
211
2’2
_
+ + + + +
+
-
-
+ +
+
-
_ -
_ -
-
_ -
+
+
+
+
+
+
+ +
+ + +
Mannan alone and the low mol. wt h~magglutinins alone agglutinate RBC, but at certain dilutions of hemagglutinin they interact and agglutination does not take place. Mannan concentration was 25 mg/ ml. The hemagglutinin was diluted serially and it was incubated with the mannan for 30 min. before the 5”;, rat RBC suspension was added to each well.
antiserum to coelomic fluid was obtained for both materials, Hemagglutinin activity and precipitin lines were abolished if the 13 kD and 20 kD species were heated first. In both cases, antiserum inhibited hemagglutination. Heated antiserum did not. Both lectins interacted with mannan (Table 6); that is, while each alone and mannan alone a~lutinated rat RBC, at some dilutions they interacted with mannan and agglutination was delayed. This effect was eliminated by heating, The mannan interaction was positive for Sigma mannan M7504 Lot No. 77C-0238 but not Lot No. 90F-3864. Some, but not other, strains of Saccharomvces cerecisiae were agglutinated by these lectins. Rat, mouse and chicken RBC were agglutinated in descending order of titer. Activity was inhibited by BSM and fetuin but not by a large number of monosaccharides. The 13 kD protein may be present in very small amounts in coelomic fluid and may be concentrated by preferential adsorption on HGMPL and FS or it may be the deglycosylated protein from the 20 kD lectin. The latter has 48% hexose and the 13 kD has little or none. It is concluded from these results that (I) different lipid fractions from the particulate fraction of Nereis coelomic fluid exhibit lectin-like activity which is specific but heat-insensitive, (2) the supernatant fraction of the coelomic fluid has a heat-insensitive, lectin-like component inhibited by BSM, thyroglobulin and transferrin but not by mannan, and protein lectins, 20 kD and 13 kD, which are heat-sensitive, inhibited by fetuin and BSM and interactive with some mannan preparations. The agglutination specificities of different fractions of ,Vereis coelomic fluid is noteworthy and further characterization of them is in progress. The lectin-like behavior of what appear to be different lipid components or mixtures of lipids corroborate and amplify previous reports. The implications of these results for studies of lipid involvement in mechanisms of cellular interactions is apparent.
ACTON
R. T. & WEINHEIMERP. F. (1974) Contemporar! Topics in Immunology. Vol. IV (Edited by COOPERE. L.) pp. 271-282. Plenum Press, New York. ACTONR. T., WEINHEIMER P. F. & NIEDERMEIER W. (1973) The carbohydrate composition of invertebrate hemaggiutinin subunjts isolated from the lobster. Pant&as argus and the oyster, Crassostrea rirg~}~jca. Crimp. Bio&em. P~~~s~5~. 44B, 185-l 89. BALDOB. A. & UHLENBRUCKG. (1975a) Purification of Tridacnin, a novel anti- -(l-6)-Digalactobiose precipitin from the haemolymph of Tridacna maxima (Roeding). FEBS Lett. 55, 25-29. BALDO B. A. & UHLENBR~JCKG. (1975b) Quantitative precipitin studies on the specificity of an extract from Tridacna maxima (Roeding). Carhohydr. Res. 40, 143151. BASKIN D. G. (1974) The coelomocytes of Nereid polychaetes. In Contemporary Topics in lmmunolog~~, Vol. IV (Edited by COOPER E. L.). Plenum Press. New York. BHATIAH. M., BOYDW. C. & BROWNR. (1967) Serological and immuno~hemicai studies of snail (Ofaia iocfvfl) anti-A: a simple purification method. Tran~~u.~i~~~(Philadeliria) 7, 53-59. BOYD W: C. & BROWNR. (1965) Immunology: a specific agglutinin in the snail, Ott& (H&v) Itrc~uu..&;itrure208, 593-594. BRADFORDH. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anu/J,r. Biochem.
72. 248-254. PESCE A. Experimental Techniques
BREWERSJ. M.,
J. & ASHWORTHR. Q. (1974) in Biochemistr); pp. 225-221. Prentice Hall, New York. COHENE. & UHLENBRUCKG. (1974) Biomedical perspectives of agglutinins of invertebrates and plant origin. Ann. N.Y. Acad. 5%. 234. CoOPER T. G. (1977) The ?bols elf’ ~~~)~~7~~~7~.~t~~ pp. 359-364. John Wiley. New York. CORNICK J. W. & S’I‘EWART J. E. (1973) Partial characteritation of a natural agglutinin in the hemolymph of the lobster, Homarus americanus. J. lncertehr. Pctthol. 2t, 225-262. DAVIS B. (1964) Disc electrophoresis~ll. Method and application to human serum proteins. Ann. N.Y. Acud. Sci. 121, 404427. Ackno~lrdgernent-This work was supported in part by DUBOISM., GILLESK. A., HAMILTONJ. K., REBERSP. A. & NlH Grant GM 07639 and NIH Grant 1 SO6 RR08168. SMITHF. (1956) Coiorimetric method for determination of sugars and related substances. Analvr. Ckem. 28. 350-356. REFERENCES EICHMANNK. & UHLENBRUCX G. (1976) Similar combining ACTON R. T.,BENNETTJ. C., EVANS F. E. & SCHROHENspecificities of invertebrate precipitins and mouse myeLONERR. E. (1969) Physical and chemical characterizloma protein 5539 for ~-~1,6)-galactans. ~fn~~~noc/ien~i,str~ ation of an oyster hemagglutinin. .I. b&l. Chem. 244, 13, l-6. 4128-4135. FINSTADC. L., L~TMAN G. W. & GOCIDR. (1972) The evolu-
64
C. S. RUSSELL. J. RODRIGUFZ and P.-S. LAI
tion of the immune response: XIII. The characterization of purified erythrocyte agglutinins from two invertebrate species. d. Immun. 108, 1074171 I. CARTE S. J. & RUSSELL C. S. (1976) Isolation and characterization of a hemagglutinin from Amphirrirr orrtatu. a polychaetous annelid. Biopl1y.y. Acra 439, 36%379. HALL J. L. & ROWLANDS D. T. (1974a) Heterogeneity of lobster agglutinins. I. Purification and physiochemical characterigation. Biochrmi.str~~ 13, 811 GSZ?. HALL J. L. & ROWLANDS D. T. (1974b) Heterogeneity of lobster agglutinins. II. Specificity of agglutinin-erythrocyte binding. Biochni.srry 13, Q&83?. HAWARSTROM S. & KABA~ E. A. (1969) Purification and characterization of a blood group A reactive hemagglutinin from the snail. He/i.\- pornatlcr and a study of its combining site. /3ioclwmisfr~~ 8. 2696-2705. ISHIYAMA I.. DIETZ E. & UHLENBRU~I< G. (1973) Comparative studies of anti-A agglutinins from various snails of the genus Helix (Hrli.~ pomaria and Hcliz aspc’rsa). Cornp. Biochern. Ph~xiol. 44B, 529%.547. ISHIYAMA1. & TAKATSU A. (1970) Investigations on enzyme absorption on the red cell surface. I. Investigation on blood group A-decomposing enzyme from CIosrridiun~ certium. I/o\- Sang. 19, 14GlSO. KAPLAP~ M. E. & KARAT E. A. (1966) Studies on human antibodies. IV. Purification and properties of anti-A and anti-B obtained by absorption and elution from insoluble blood group substances. J. rrp. Med. 123, 1061~1081. KATES M. (1972) Trc~hr~iqur !), Lipidolog? p. 351. North Holland/American Elsevier, New York. KIEDA C. M.. BOWLES D. J.. RAVID A. & SHARON N. (1978) Lectins in lymphocyte membranes. FEBS L~rt. 94, 391~~396. LESNIAK A. P. & LIU E. H. (1982) Characterization of a hemagglutinin from Leptogorgia r~irgulata (marine coral). Co,np. Biochrm. Phy,siol. 71 B, 302 308. LI M. F. & FLEMMING C. (1967) Hemagglutinins from oyster hemolymph. Can. J. Zool. 45, 1225-1234. MA~FARLANE M. G. & KNIGHT S. D. (1941) The biochemistry of bacterial toxins. I. The lecithinase activity of CI. rvelchii toxins. Biocllenl. J. 35, 884-902. MARCIIALOVIS I. J. & EDELMAN G. M. (196X) Isolation and
characterization of a hemagglutinin from Li~~rtr/[r\ p0/1,phrmus. J. molec. Bid. 32, 453-465. MCDADE J. E. & TRIPP M. R. (1967) Mechanism of agglutination of red blood cells by oyster hemolymph. .1. /~lrc,rr&r. Par/ml. 9, 523-530. PAUI.EY G. B., GRANCSK G. A. & KKASS~~K S. N. (19711 Characterization of a natural agglutinin present in the hemolymph of the California sea hare. .Ap/~,vu c~crliforn~tr. J. Inrwrdw. Par/d. 18, 207 218. Po~trz R. D. (1973) Preparation of purified hog gastric mucin. Preparation of polyleucyl hog gastric mucIn Merh. E~Izw&. 28, 352 353. PROKOP 0.. RACKWITZ A. & SCHLI~SI%C~EK0. (1965) A new human blood group receptor Ahel tested with saline extracts from Heliu horretlsit (garden snail). .I. Forcz,~\~c, $frd. 12, 108-I IO PKOKOP 0.. SCHLL:SI\C;EK 0. & RACKWIIZ A. (1965) Uber das Vorkommen van P I-Blutgruppensubstanz bei elnpen Metazoen. ins. besondere A vc,uri.\ \u~~,?1und [.I~)IIhricus rcwmrrrs. Uber eine thermostabile “antibodk-like substance” (Anti-Ahell bei He/i\- pomaria und deren- Herkunft. Z. Inzrrnlrlfbr\(,II.129. 344-354. 402 412. ROCH P. (1979) Protein analysis of earthworm coelomic fluid: I. Polymorphic system of the natural hemolysin of
properties of limulin: a lectin (agglutinin) from hemolymph of Lirnullr\ pol~pl~rrmr,c. Bioc,him. hioplt~x -lc,rtr 371, 242-254. RUSSELL C. S.. ROI>KIG~EL J. & LAI P.-S. (19X1)Lectln activity in a lipid extract of coelomocytes from &‘~~r~,i\. Fcdrl Proc. Fe&. Am Sots. KYp. Bid. 41, 1033. SFLA B. M.. WAYG J. L. & ED~LMAN G. M. (1975) Isolation of lectins of ditrerent specificities on a single affinity adsorbent. J. Biochetn. 250. 7535-753X. Smhl J. D. (1946) Lipid haemagglutinins. rlrf\r,.. .I. c’\,>. Biol. & Med. 24, 197 205. TABRAH F. L.. KASIIIWA(;I M. &. No~ro\ T. R. (1970) Antitumor activity in mice of tentacles of two tropical sea annelids. Scirncr 170, IX I 183. TSIVION Y. & SHARON N. (1981) Lipid-medlated hemagglutination and its relevance to lectin-mediated agglutlnation. Bioc,hrnt. bionhw Am 642. 336 344.