- Email: [email protected]
Sponge aggregation factor and sponge hemagglutinin: Possible relationships between two different molecules
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, V01. 3, pp. 399-416, 1979. 0145-305X/79030399-13502.00/0 Printed in the USA. Copyright (e) 1979 Pergamon Press Ltd. All rights reserved.
SPONGE AGGREGATION FACTOR AND SPONGE HEMAGGLUTININ: POSSIBLE RELATIONSHIPS BETWEEN TWO DIFFERENT MOLECULES
Peter Vaith, Gerd Uhlenbruck, Werner E.G. MUller" and Gisela Holz Medizinische Universit~tsklinik K~in, Abteilung fHr Experimentelle Innere Medizin, Kerpener Str. 15, D-5000 K~in 41 (GFR) and •Physiologisch-Chemisches Institut der Johannes Gutenberg Universit~t, Abteilung fur Angewandte Molekularbiologie, Duisbergweg, D-6500 Mainz (GFR)
ABSTRACT.
A lectin from the marine sponge GEODIA CYDONIUM was isolated and characterized. GEODIA lectin (GL) agglutinates human red blood cells i r r e s p e c t i v e of the ABO blood group and precipitates with a variety of D-galactose containing glycosubstances, i.e. certain snail galactans, bovine erythrocyte glycoprotein and PNEUMOCOCCUS type XIV polysac" charide. The only simple sugars inhibiting the GL-mediated hemagglutination were lactose and ~-acetyl-_qD-galactosamine. GL was purified by affinity chromatography on Sepharose 4B almost to homogeneity as tested by polyacrylamide disc gel electrophoresis. Positive staining of the lectin band with Coomassie brilliant blue and PAS suggest that GL is a glycoprotein. Under dissociating conditions GL exhibits an apparent mol. wt. of 12,000 daltons. The elution behaviour of the lectin on Biogel suggests that GL exists as an oligomeric molecule in its native state. Our data obtained with GL demonstrate that the species-specific aggregation factor (AF) from GEODIA is not identical with this lectin. Two major hypotheses for a biological role of GL are discussed: first, the cooperation of GL with AF in sorting-out processes of GEODIA cells and second, the possibility that GL is involved in defense mechanisms. 399
400
SPONGE LECTIN
Vol. 3, No. 3
INTRODUCTION In the foregoing p a p e r (I) we reported on our i n v e s t i g a tions c o n c e r n i n g the i n t e r a c t i o n between a g g r e g a t i o n factor (AF) and a g g r e g a t i o n r e c e p t o r (AR) in the m a r i n e sponge G E O D I A C Y D O N I U M . Evidence for an i m p o r t a n t role of A R - b o u n d ~ - g l u G u r o n i c acid in the A F - m e d i a t e d c e l l u l a r r e a g g r e g a t i o n was given. A c c o r d i n g l y , the s p e c i e s - s p e c i f i c r e a g g r e g a t i o n of G E O D I A cells can be viewed as the " r e c o g n i t i o n " of a ~ - g l u c uronic acid c o n t a i n i n g cell surface r e c e p t o r (AR) by an i n t e r c e l l u l a r p r o t e i n a c e o u s AF m a c r o m o l e c u l e (I). During our exp e r i m e n t s c o n c e r n e d w i t h the s e r o l o g i c a l a c t i v i t y of the agg r e g a t i o n r e c e p t o r (AR) we tested in a d d i t i o n to d i f f e r e n t lectins the c r u d e a g g r e g a t i o n factor (AF) for h e m a g g l u t i n a t i o n ; a very r e m a r k a b l e a g g l u t i n a t i o n of human red blood c e l l s could be observed. To our surprise, the AR, which has p r e v i o u s l y been shown to form a c o m p l e x with the AF d u r i n g G E O D I A cell reagg r e g a t i o n (2), i n h i b i t e d this h e m a g g l u t i n a t i o n only scarcely. Hence, we supposed that the a g g l u t i n a t i n g a c t i v i t y in the c r u d e AF e x t r a c t was not due to the AF itself. Our s u g g e s t i o n could be s u b s t a n t i a t e d by the i s o l a t i o n of a lectin responsible for this h e m a g g l u t i n a t i n g a c t i v i t y . G E O D I A lectin (GL) was found to a g g l u t i n a t e human e r y t h r o c y t e s i r r e s p e c t i v e of the ABO type. The a g g l u t i n a t i o n titre could be e n h a n c e d by p r i o r n e u r a m i n i d a s e and even m o r e by p r o n a s e t r e a t m e n t of the cells. In addition, e r y t h r o c y t e s from several d i f f e r e n t species w e r e found to be a g g l u t i n a t e d by G L (3). The p u r i f i e d lectin is also m i t o g e n i c for human l y m p h o c y t e s (4). The aim of this p a p e r is first, to report on the i s o l a t i o n and i m m u n o c h e m i c a l c h a r a c terization of this new lectin, and second, to p r e s e n t our h y p o theses on the b i o l o g i c a l function of this lectin in GEODIA. The h i s t o r y of h e t e r o a g g l u t i n i n s in sponges d a t e s back to G A L T S O F F (1929), who d e s c r i b e d a lyric and a g g l u t i n a t i n g a c t i o n of sponge e x t r a c t s upon c e l l s of o t h e r sponge species (5). The property of sponge e x t r a c t s to a g g l u t i n a t e red blood cells too, was first reported by M a c L E N N A N and DODD in 1967 (6), s u p p o s i n g a r e l a t i o n s h i p between h e r e t o - and h e m a g g l u t i n i n s . S u b s e q u e n t l y several lectins from d i f f e r e n t sponges have been i s o l a t e d and purified in the last y e a r s (7). An e x c e l l e n t d i s c u s s i o n on the b i o l o g i c a l role of sponge a g g r e g a t i o n factors on the one hand, and sponge h e t e r o - or hema g g l u t i n i n s on the other, has been given by M a c L E N N A N in 1974, who proposed a S e l f - N o t - S e l f r e c o g n i t i o n function for both molecules (8). An exact c o m p a r i s o n b e t w e e n a g g r e g a t i o n factor and h e m a g g l u t i n i n , h o w e v e r , has been impeded by the fact that the a c t i v i t i e s c o m p a r e d w e r e d e r i v e d from d i f f e r e n t sponge species as yet: e.g. AF from M I C R O C I O N A and h e m a g g l u t i n i n from A X I N E L L A (7,8). Our d i s c o v e r y of a lectin in the sponge G E O D I A C Y D O N I U M enables us now to i n v e s t i g a t e the r e l a t i o n s h i p b e t w e e n a lectin and an a g g r e g a t i o n f a c t o r in the same sponge species. A m a j o r a d v a n t a g e for this study is the fact that n u m e r o u s m o l e c u l a r p r o p e r t i e s involved in the r e a g g r e g a t i o n p r o c e s s of G E O D I A cells have been a n a l y z e d p r e v i o u s l y by W. M O L L E R and c o w o r k e r s (9).
Vol. 3, NOo 3
SPONGE LECTIN
401
MATERIAL AND METHODS Chemicals. Sugars used were of highest purity commercially available. Mono- and oligosaccharides were obtained from Serva Ltd, Heidelberg (GFR)~ E. Merck, Darmstadt (GFR)~ Sigma Chemical Co, St. Louis (USA)~ Calbiochem, San Diego (USA)~ Baker Chemicals, Deventer (Holland)~ and Koch-Light Laboratories Ltd, Colnbrook (UK). Protein marker substances were purchased from Serva. Blue dextran 2,000 from Pharmacia, Uppsala (Sweden) was used. Polysaccharides and glycoproteins. The origin and preparation of snail galactans from HELIX POMATIA, HELIX HORTENSIS and ACHATINA FULICA (Madagaskar) as well as PNEUMOCOCCUS type XIV polysaccharide have been described (10-13). Arabin~galactan (larch) was purchased from Serva. Bovine erythrocyte mucoid was prepared as described by UHLENBRUCK and SCHMID (14). Source of GEODIA substances. These materials were derived from the siliceous sponge GEODIA CYDONIUM (Tetractinellida) collected in the vicinity of Rovinj (Yugoslavia). The crude aggregation factor (AF) was prepared as described (15). Briefly, the sponge was cut into cubes of 2 mm . Fifty grams material were stirred for 2 h with 50 ml of calcium- and magnesiumfree artificial sea water, pH 8.2, containing 20 mM EDTA. The supernatant collected after centrifugation at I0,000 ~ for 30 min. will be termed crude AF~ protein content was 6 mg/ml. GEODIA polysaccharide was prepared by phenol-saline (90% v/v phenol, 1:1) extraction of dried GEODIA tissue. As a source of lectin, crude AF and dried GEODIA tissue were employed. In the latter case, 100 g GEODIA tissue was homogenized in a blender and extracted with I 1 phosphate-buffered saline (PBS) pH 7.2 overnight at 4°C under magnetic stirring. After centrifugation at 4,000 rpm for 30 min and at 27,000 rpm (Beckman ultracentrifuge, SW 27 rotor) for 3 hours at 4°C the clear brown supernatant ~as used as a crude lectin extract. Aliquots were stored at -20 C or alternatively after dialysis in a lyophilized state. Affinity chromatography of the crude GEODIA lectin. Crude AF extract or PBS-extracted GEODIA tissue were chromatographed on Sepharose 4B (Pharmacia). In a typical experiment 10 ml crude AF was layered onto a Sepharose column (47.0 x 2.5 cm) and eluted with Tris-NaCl buffer (0.5 M NaCI, 0.05 M Tris-HCl buffer, pH 8.2) at 4°C. The column was washed until the optical density at 280 nm reached zero. Specific elution of the lectin was carried out by the addition of 0.01 M ~-galactose to the eluting buffer. The specifically eluted fractions were combined, dialyzed against 0.01 M NH4HCO 3 buffer, pH 8.2, and lyophilized. Gel filtration experiments. Instead of dialysis, lectin fractions from the latter procedure were alternatively freed of galactose containing buffer by gel filtration on Biogel P 100 (Biorad). A Biogel column (92.0 x 2.6 cm) was equilibrated with 0.01 M NH4HCO 3 buffer, ph 8.2. Lectin activity appeared
402
SPONGE LECTIN
Vol. 3, No. 3
in one symmetrical peak near the void volume. An identical elution b e h a v i o u r was observed when the lectin dissolved in PBS or in Tris-NaCl b u f f e r was eluted from Biogel P I00~ previously equilibrated with the respective buffer. A Biogel P 300 column (17.0 x 1.5 cm) was employed for gel filtration of the crude AF material. Flow rate: I ml per hour in Tris-NaCl buffer; fraction volume: I ml. C a l i b r a t i o n of the column was performed by measuring elution volumes of blue dextran and of m a r k e r proteins as described by DETERMANN (16). The ratios of the elution volumes of the m a r k e r proteins (V e) to the void volume ( V ) were: Human transferrin (mol. wt. 80,000) = 1.64; b o v i n e ~erum albumin (mol. wt. 67,000) = 1.9; ovalbumin (mol. wt. 45,000) = 2.19; c h y m o t r y p s i n o g e n (mol. wt. 25,000) = 2.54. Analytical methods. Protein was determined after LOWRY et al. (17) with bovine serum albumin as a standard. Neutral sugars were assessed by DUBOIS' method (18) with D-galactose as a reference substance. P o l y a c r y l a m i d e disc gel e l e c t r o p h o r e s i s was performed according to LAEMMLI (19); 7.5, 10, and 12.5 % gels were employed at pH 8.8 of the separating gel. For mol. wt. d e t e r m i n a t i o n s the method of W E B E R and OSBORN (20) was applied. M a r k e r proteins consisted of a m i x t u r e of bovine serum albumin, ovalbumin, c h y m o t r y p s i n o g e n A, and c y t o c h r o m e C (mol. wt. 12,400). In some experiments, ovalbumin was substituted by rabbit aldolase (mol. wt. of subunits 40,000). Bromophenol blue or Pyronin G were used as tracing dyes, respectively. The gels were stained for protein with C o o m a s s i e b r i l l i a n t blue and for c a r b o h y d r a t e with the PAS method as described by DAHR et al. (21). H e m a g g l u t i n a t i o n inhibition tests were done as already reported (I). Generally, n e u r a m i n i d a s e treated O e r y t h r o c y t e s were used as test cells. As a source of agglutinin we employed crude AF, crude lectin after P B S - e x t r a c t i o n of GEODIA tissue~ and affinity purified GL. O u c h t e r l o n y e x p e r i m e n t s were performed in agarose (I %) (Serva) as d e s c r i b e d (I). A g a r gels were prepared in saline or in Tris-NaCl b u f f e r c o n t a i n i n g ~ - g a l a c t o s e (0.05 M) in order to prevent binding of the lectin to agarose. The same GEODIA m a t e r i a l s were used as described for the h e m a g g l u t i n a t i o n experiments. The p a r t i c u l a r source of GL activity used in h e m a g g l u t i n a t i o n inhibition and agar gel experiments will be stated under 'Results'. Formaldehyde treatment of sheep red blood cells (SRB~s). A 2% solution of p a r a f o r m a l d e h y d e in PBS was heated at 60-C for 30 min. After cooling, 10 ml of a 10% suspension of SRBCs (Behringwerke, MarbUrg, GFR) was added to 90 gl p a r a f o r m a l d e hyde solution and incubated for 48 hours at 4°C, followed by washing of the cells in Tris-NaCl buffer. Absorption of GEODIA lectin on formalinized SRBCs. The hemagglutinating fractions separated on Biogel P 300 from crude AF extract (fractions 20-27, Fig. 4) were combined and one aliquot ( 8 0 ~ g protein) was incubated with SRBCs at room temperature until no agglutinin activity was found in the supernatant. As a control, SRBCs were tested with b u f f e r alone. Both supernarants and an unabsorbed lectin sample (8o ~ g protein) were dialyzed against distilled water, m i c r o p o r e filtered and lyo-
Vol. 3, No. 3
SPONGE LECTIN
philized. Subsequently, amide gels.
403
the samples were run on SDS polyacryl-
Test for Ca++-dependency Of the hema~@lutinatin~ activity. 5 ml of crude AF extract was dialyzed against 5 1 0.$ M EDTAsaline solution for two days, and then for three days against saline at 4 C. An aliquot (~.8 ml) was mixed with 0.2 ml 0.1 M CaCl2-saline solution, and another With 0.2 ml calcium-free saline. These solutions were tested for hemagglutinating activity. RESULTS Occurence of lectin activity in GEODIA. Our initial hypothesis that the crude AF extract contains a lectin activity independent of the AF activity was supported by Ouchterlony experiments. As shown in Fig. I, strong precipitin lines were obtained, when crude AF was tested against several glycosubstances, i.e. certain snail galactans, bovine erythrocyte mucoid or PNEUMOCOCCUS type XIV polysaccharide. No reaction was seen, however, with a polysaccharide material extracted from GEODIA. FIG. I Precipitin Reactions with the GEODIA Lectin
Substances are arranged clockwise from 12 o'clock on; in the center is crude aggregation factor (AF). ~= Bovine erythrocyte mucoid, 2= HELIX POMATIA galactan, 3= HELIX HORTENSIS galactan, 4~ GEODIA polysaccharide, 5= PNEUMOCOCCUS type XIV p01ysaccharide, 6= ACHATINA FULICA galactan. .L
To, test this reactivity against distinct glycosUbstances further, we performed hemagg!utination inhibition experiments. Many different simple Sugars and polysacch~arides were tested ~s potential inhibitors of the GEODIA hemagglutinin, (Table I). Clearly there is a strong reactivity of the hemagglotinating
404
SPONGE LECTIN
TABLE
Hemagglutination
Vol. 3, No. 3
I
I n h i b i t i o n of G E O D I A Different Sugars
Hemagglutinin
by
Sugar
IT
Sugar
IT
L_.-f u c o s e
-
~i-acety l-D--ga l a c t o s a m i n e
22
D__-f u c o s e
-
~ - a c e ty l-D--g l u c o s a m i n e
-
D--ga l a c t o s e
-
D-ga lacturonic
-
D-glucose
-
D-g lucuronic
D--mannose
-
/~-acety i- D--neu r a m i n i c
L-arabinose
-
lactose
22
L-rhamnose
-
melibiose
-
D - x y lose
-
treha lose
-
1-/~-me thy I- t&-D-ga I ac to se
-
sucrose
1-/l-me thy l-B- D-ga lac rose
-
raffinose
1-11-me thy i-~- ~ g luco se
-
cel!obiose
1-Q-methy l-S-D-g lucose
-
ma I tose
D-galactosamine
-
ACHATINA
HCI
acid
FULICA
D-glucosamine
HCI
-
HELIX
D-mannosamine
HCI
-
arabinogalactan
GEODIA
-
AR
acid
POMATIA
acid
galactan
galactan (larch)
-
29 29 -
21
V a l u e s are e x p r e s s e d as t h e i r r e c i p r o c a l s ; they r e p r e s e n t the m i n i m u m a m o u n t of inhibitor required to b r i n g a b o u t i n h i b i t i o n of f o u r a g g l u t i n a t i o n d o s e s of a g g l u t i n i n . C r u d e AF w a s u s e d as a s o u r c e of a g g l u t i n i n . The same i n h i b i t i o n p a t t e r n - e x c e p t the u n r e a c t i v i t y w i t h G E O D I A - A R - w a s o b t a i n e d w i t h p u r i f i e d G L as an a g g l u t i n i n . M o n o - and o l i g o s a c c h a r i d e s u s e d w e r e 0.05 m o l a r in T r i s - N a C l b u f f e r ; p o l y s a c c h a r i d e s w e r e t e s t e d as I% s o l u t i o n s in the same b u f f e r . IT = I n h i b i t i o n titre.
material against certain galactosyl containing polymers, while D - g a l a c t o s e i t s e l f w a s i n a c t i v e at the c o n c e n t r a t i o n s tested. The h e m a g g l u t i n a t i o n i n h i b i t i o n e x e r t e d by ~ - a c e t y l - D - g a l a c t o s a m i n e and D - l a c t o s e shows that the g a l a c t o s y l c o n f i g u r a t i o n of the O H - g r o u p at C 4 is f a v o u r e d c o m p a r e d w i t h the g l u c o s y l c o n f i g u r a t i o n s i n c e n e i t h e r ~-acetyl-D__-glucosamine nor c e l l o b i o s e w e r e found i n h i b i t o r y . In a d d i t i o n to a t e r m i n a l g a l a c t o s e residue, the l i n k a g e type to the s u b t e r m i n a l s u g a r s t r o n g l y i n f l u e n c e s the r e a c t i v i t y of G E O D I A a g g l u t i n i n , for m e l i b i o s e and r a f f i n o s e w i t h an ~ - I - 6 linked D - g a l a c t o s e at the n o n r e d u c i n g end did not i n h i b i t . A l l of the t e s t e d o l i g o s a c c h a r i d e s w i t h D--glucose at the n o n r e d u c i n g end w e r e n o n i n h i b i t o r y .
Vol. 3, No. 3
SPONGE LECTIN
405
Isolation of GEODIA lectin. The crude AF material was chrom a t o g r a p h e d on Sep-harose 4B. This procedure has previously been applied as a purification step in the isolation of the AF from GEODIA. The AF containing m a c r o m o l e c u l e is eluted near the void volume, followed by a second, broad peak without aggregation promoting activity. The eluted fractions were tested for agglutinating activity. (Fig. 2). FIG.
2
Isolation of G E O D I A Agglutinin by Affinity C h r o m a t o g r a p h y on Sepharose 4B
oo 21~o nm ~5,
)
/
I
/\.
,
_
-
...-.,_.,.
..... __ ........
Crude AF was applied to a column of Sepharose 4B, as described under 'Methods'. The arrow marks the addition of 0.01 M ~ - g a lactose to the eluting buffer. Each fraction was examined for protein (X) by measuring the absorbance at 280 nm and for hema g g l u t i n a t i n g activity (O) against n e u r a m i n i d a s e treated O red blood cells. Flow rate: 15 ml per hour, fraction volume: 5 ml.
The relatively low h e m a g g l u t i n a t i n g activity found within and between the two protein peaks, respectively, was further investigated by h e m a g g l u t i n a t i o n inhibition experiments. Using the combined fractions of the first, AF containing peak no inhibition was obtained with all saccharides listed in Table C. To our surprise the AR, however, was now inhibitory up to a dilution of 1 : 16, as we already documented (1). The specificity
406
SPONGE LECTIN
Vol. 3, No. 3
of the interaction of the enriched AF fractions with erythrocytes is not clear. We believe this to be a nonspecific aggregation of cells, possibly via calcium-bridges, rather than a true agglutination. From reaggregation e x p e r i m e n t s a calciumdependency of GEODIA AF is well established. The a g g l u t i n a t i n g activity detected in the ascending part of the second Sepharose peak gave the same inhibition pattern as the crude AF extract, but was too low to account for the total activity of the starting material. The a n t i - g a l a c t o s y l specificity of GEODIA lectin, however, lent support to the idea that part of it bound to the Sepharose beads by interaction with ~ - g a l a c t o s e groupings of the gel. C o n s e q u e n t l y we added 0.01 M ~ - g a l a c t o s e to the elution buffer, after the two protein peaks were eluted from the column. A small protein peak appeared with a powerful a g g l u t i n a t i o n activity; the agglutinating fractions were combined and used for the following experiments. A t t e m p t s to elute the lectin by 0.0C M D-glucose were negative. A purification factor of a p p r o x i m a t e l y 45 can be calculated from the increase in specific titre (titre/ protein content). The overall yield of purified lectin was about 9 % relative to protein. The lectin activity detected in the ascending part of the second Sepharose peak d e m o n s t r a t e s that the lectin binding capacity of the gel was probably exceeded; at the same time, the elution b e h a v i o u r of this activity gives an idea of the lectin's size. This will be referred to, below. C h a r a c t e r i z a t i o n of GEODIA lectin. Affinity purified GL reacted with the same g l y c o s u b s t a n c e s as the crude AF extract (Fig.1 and Table I) or the PBS-extracted GEODIA tissue. The aggregation receptor (AR), however, was u n r e a c t i v e with purified GL in h e m a g g l u t i n a t i o n inhibition experiments. To test the affinity purified GL for homogeneity, polyacrylamide disc gel e l e c t r o p h o r e s i s was applled.(Fig. 3). One major C o o m a s s i e positive band was obtained, when affinity purified GL was run on gels both in the presence and a b s e n c e of SDS (Fig. 3, No. 3,4 and 8). The sharp m a j o r band seen in SDS gels migrated with the m a r k e r dye (Fig. 3, No. 3 and 4), w h i l s t a broad band located in the m i d d l e of the gel was found in the absence of SDS (Fig. 3, No. 8). The d i f f u s e band on the top of gel No. I, containing the crude AF extract, can be resolved by comparison with gels No. 3 and 4 (purified GL) and gel No. 2, where a fraction of the first Sepharose peak (enriched AF) was separated. That broad band in the crude AF material is obviously comprised of a fast moving band c o r r e s p o n d i n g to GL and a band next to the m a r k e r dye, which possibly represents the AF molecule (mol. wt. between 16,000 and 23,000) (22). The absence of a band c o r r e s p o n d i n g to GL in the enriched AF fractions is also ascertained in gels w i t h o u t SDS (Fig. 3, No. 7). This finding is strong evidence for a n o n - i d e n t i t y of GL and AF; furthermore it can be seen clearly that GL is not associated with the AF containing m a c r o m o l e c u l e obtained from the first Sepharose peak. When an SDS gel was overloaded with purified GL, two additional minor, slow moving bands were discerned b e s i d e s the ma-
Vol. 3, No. 3
SPONGE LECTI~
407
FIG. 3 Polyacrylamide Disc Gel Electrophoresis of Several GEODIA Lectin Fractions
1
Gels are numbered from the left to the right. Gels 1-5 were run in the presence and gels 6-8 in the absence of SDS. I= crude AF extract ( 5 0 ~ g ) ; 2= first Sepharose 4B peak ( 5 0 ~ g ) ; 3= affinity purified GL ( 5 0 ~ g ) ; 4= affinity purified GL ( 1 0 ~ g ) ; 5= marker proteins ( 5 ~ g , each); 6= same as I) without SDS; 7= same as 2) without SDS; 8= same as 3) without SDS. Polyacrylamide concentration of the separating gels was 10 %. Gels were stained for protein with Coomassie brilliant blue. Values in brackets are the respective amounts of protein added per gel.
jor band on top of the gell the nature of these two bands was not investigated further. Evidence for an identity of the major, fast moving band with the lectin activity comes from absorption studies on formalinized sheep red blood cells (SRBCs). This procedure has been reported for the purification of an anti-galactosyl lectin from slime molds (23). We absorbed the agglu£inating fractions eluted from Biogel P 300 (see Fig. 4, fractions 20-27) on neuraminidase treated formalinized SRBCs until no agglutinating activity could be found in the supernatant. This absorbed material showed only a trace of the major band in
408
SPONGE LECTIN
VOI, 3, NO.
SDS gel electrophoresis; the relative intensities of the slower moving bands compared with the major band, however, were significantly increased. This result can be taken as evidence that the slow moving bands were not associated with GEODIA lectin activity. The m o l e c u l a r weight of GL in the presence of SDS and dithioerythritol, as determined after W E B E R and OSBORN, appears to be 12,000 ~ 1,000 daltons. Staining of the gels by the PAS method suggests that GL is a glycoprotein. A neutral sugar content of about 5 % was found in affinity purified GL using DUBOIS' method. No lectin activity associated with such a low mol. wt. substance was detectable in gel filtration experiments. As already shown in Fig. 2, the agglutinin activity not bound to Sepharose was eluted in the ascending part of the second protein peak. Further spreading of this elution pattern was obtained, when the crude AF material was subjected to gel filtration on Biogel P 3001 three protein peaks appeared now, the intermediate peak contained all the a g g l u t i n a t i n g activity, (Fig. 4). FIG.
4
Gel Filtration of the Crude AF Extract on Biogel P 300
Oil 2110 hi'. i i-ll
O,.10-
!
. ll
.27
:
!
32-1
hill,
0.22. 0.18,
O.1/..
it ... J
0.10.
GO6.
~
OJDi, |
S"
m
i
•
•
li
X
iI
z
%\ a
a
~
,"
li
•
li
i
~
C r u d e AF (0.5 ml) was applied to the column as described u n d e r 'Methods'. Each fraction was examined for protein (x) by measuring the a b s o r b a n c e at 280 nm. All the a g g l u t i n a t i n g activity was found within the fractions of the i n t e r m e d i a t e peak, fractions 20-27. The arrow marks the void volume, as d e t e r m i n e d with dextran blue. For calibration data of the column, see under 'Methods'.
Vol. 3, No. 3
SPONGE LECTIN
402
The elution volumes of h e m a g g l u t i n a t i n g fractions 20-27 roughly correspond to a mol. wt. range between 30,000 and 70,000 daltons with a peak at about 45,000. Based upon these values one could assume a tetrameric m o l e c u l e composed of subunits with a mol. wt. of 12,000 daltons, but aggregates of different size are equally possible. Attempts to estimate the m o l e c u l a r size of affinity purified GL by gel filtration on Biogel yielded inconclusive results. The elution b e h a v i o u r of GL changed in a wide range from experiment to experiment, presumably due to different s e l f - a g g r e g a t i o n states of GL subunits. A few additional e x p e r i m e n t s were performed to confirm the d i f f e r e n c e s between AF and GL. The affinity purified ~L was tested for heat sensitivity; heating for 3 min. at 60 C reduced the agglutinin titre by I: 8~ while at 80°C a titre reduction of I : 64 was found. All activity was lost, when GL was boiled for 3 min. Though a substantial loss of activity occured upon heating, the lectin activity appeared to be relatively resistant compared with the AF, the activity of ~hich was completely destroyed after heating for I min. at 60 C (15). A n o t h e r d i f f e r e n c e between AF and GL was observed in the strong c a l c i u m - d e p e n d e n c y of the AF activity. The agglutinating power of GL was~ however, not reduced at all after extensive dialysis of the crude AF extract against 0.1 M EDTA. DISCUSSION One aim of our study was to c h a r a c t e r i z e the lectin activity in GEODIA. This h e m a g g l u t i n a t i n g and p r e c i p i t a t i n g activity was traced in the crude AF extract as well as in GEODIA tissue after saline extraction. Serological specificity of GEODIA lectin (GL). GL reacts with certain snail galactans from HELIX POMATIA (HP), HELIX HORTENSIS (HH) and A C H A T I N A FULICA (AFU). These galactose containing p o l y s a c c h a r i d e s have previously been shown to interact with a series of lectins, termed a n t i - g a l a c t a n s (24). HP-galactan was reported to p r e c i p i t a t e with a lectin from the clam TRIDACNA MAXIMA, and A F U - g a l a c t a n with an agglutinin extract from the sponge A X I N E L L A POLYPOIDES (10), but not vice versa. The lectin from GEODIA, however, apparently recognized structures common to both galactans. A third type of galactan tested, arabinogalactan from larch, was u n r e a c t i v e with GL. This non-reactivity may be explained by the scarcity of galactose side chains in this galactan (24). A X I N E L L A was also reported not to precipitate with a r a b i n o g a l a c t a n (10). Evidence that GL interacts with certain terminal, non-reducing linked B-_.D-galactosyl groups comes from our experiments with lactose and PNEUMOCOCCUS type XIV polysaccharide. As neit h e r e - nor B - m e t h y l - g a l a c t o s e were found inhibitory, the combining site of GL seems to recognize also the glycosidic linkage t y p e to the subterminal sugar, i.e. the 1-4 linkage to glucose, as in lactose and perhaps in type XIV too (25). This limited reactivity with certain terminal galactose residues is further supported by the negative precipitin reaction between anti-
410
SPONGE LECTIN
Vol. 3, No. 3
freeze glycoprotein from an antarctic fish (3). The latter glycoprotein is characterized by the presence of multiple disaccharide units, the structure of which has been identified as B-_qD-galactosyl-1-3-/~-acetyl-_q-galactosamine (26). Melibiose and raffinose, both contain terminal ~ - I - 6 linked galactose residues, were also non-inhibitory. Interestingly, ~-acetyl-D-galactosamine inhibited as well as lactose; hence it seems that the presence of an acetylated amino group at C 2 of galactose enables the lectin to accomodate this free sugar. None of the tested oligosaccharides having a terminal D_.-glucose residue was found to inhibit the GL-mediated hemagglutination. The strong precipitin reaction of GL with bovine erythrocyte mucoid reflects the agglutinability of red blood cells by GL. The agglutination of human erythrocytes was independent of the ABO group and could be enhanced by prior neuraminidase and even more by pronase treatment of the cells (3). A comparison between the agglutinability of erythrocytes from different animal species by GL will be given elsewhere (3). The erythrocyte receptor for GL is not known; the hemagglutination inhibition data suggest, however, that GL receptors will most probably carry terminal, non-reducing B-linked ~-galactose residues or ~-acetyl-_DD-galactosamine termini. The latter monosaccharide can practically be excluded as a determinant for GL in bovine mucoid, since bovine erythrocyte glycoproteins do not react with different agglutinins directed against ~-acetyl-_D-galactosamine (27,28). On the other hand, bovine erythrocyte glycoproteins contain significantly more ~-galactose and ~-acetyl-_qD-glucosamine residues than erythrocyte glycoproteins from other species and react strongly (after neuraminidase treatment) with anti-type XIV antiserum (27,28). Hence, we suppose that GL acts upon terminal, non-reducing bound lactosamine- or upon B-galactosyl-~-6-galactose groups; the occurence of the latter has been shown previously in bovine red cell glycoproteins (24). The specificity of GL resembles in some way that of agglutinin I and II from the marine sponge AXINELLA POLYPOIDES (29). First, the isolation by affinity chromatography on Sepharose is likewise applicable for the lectins from both sponges, and second, hapten inhibition studies indicate some similarity of combining sites (7,10,29). Both AXINELLA agglutinins are best inhibited by terminal, non-reducing ~-galactose residues linked by B-I-6 glycosidic bonds. The preference for certain 8-1inked galactose residues has also been demonstrated for GL. As no oligosaccharides with B-I-6 linked terminal galactose residues were available, such a specificity could not be shown conclusively. The reactivity of GL as well as of AXINELLA with snail galactans containing this disaccharide unit, however, support a similar reactivity of both sponge agglutinins. A clearcut difference between AXINELLA agglutinins and GL can be seen in the inhibiting effect of ~-galactose and ~-acetyl-_qD-galactosamine, respectively: the latter monosaccharide was reported to be much less active than ~-galactose for AXINELLA (7,10,29), a reverse relationship being true for GEODIA lectin.
Vol. 3, No. 3
411
SPONGE LECTIN
Biochemical characteristics of GEODIA lectin. The affinity purified lectin proved to be almost homogeneous when examined by polyacrylamide disc gel electrophoresis. In the presence of SDS and dithioerythritol, GL exhibited an apparent mol. wt. of 12,000 Z 1,000 daltons. The elution behaviour of hemagglutinating fractions on Biogel P 300 suggests that the unreduced lectin exists in an oligomeric state. Staining of the polyacrylamide gels for protein and carbohydrate, respectively, point to a glycoprotein nature of GL. In contrast to the AF, which occurs as a subunit of a large proteid particle (22), GL is not assoc%ated with this aggregation promoting macromolecule (see Fig. 3 and 4). GL is relatively resistant to heat, presumably because of its carbohydrate content. The lectin activity is apparently independent of calcium ions. Possible biolo@ical role of GEODIA lectin. Besides the characterization of this new lectin, a second aim of our study was to compare GL with AF. The AF has previously been isolated and characterized structurally and functionally (I,15,22,30, 31). In Table 2 several major characteristics of both molecules are summarized. Based upon these data, there is no doubt that GL and AF represent two distinct entities.
TABLE 2 Comparison between AF and GL Na tu re
AF protein
Mol. wt. Specificity subunit
(?)
GL glycoprotein
16,00023,000 (?)12,000
Ca ++dependency ÷
GIuA
Gal I
Heatresistance
S
÷/-
)4
Gal I 8 ) 6
(?)
Ga INAc GIuA= ~-glucuronic acid~ Gal= ~-galactose~ galactosamine. For references see text.
GalNAc= ~-acetyl-DD-
Whilst the biological function of GEODIA AF is being increasingly understood (9), that of GL remains open to question. Thus, we want to discuss several hypotheses referring to the possible significance of this lectin in GEODIA. Two major hypotheses will be considered: first, an involvement of GL in sorting-out processes of GEODIA cells, and second, the possibility that GL plays a defensive role against heterologous cells.
412
SPONGE LECTIN
Vol. 3, No. 3
Is GL involved in cellular sorting-out processes ? Considering cellular sorting-out processes, one must distinguish between events leading to a histiotypic rearrangement of dissociated cells within one sponge organism, and a speciesspecific sorting-out between different sponges. This problem has already been discussed in detail by MOSCONA (32). In the following section, sorting-out events occuring between reaggregating GEODIA cells will be considered. The cellular reaggregation of GEODIA cells is mediated by an intercellular, high mol. wt. particle (15), which carries in addition to the AF several functionally different subunits~ among them, a sialyltransferase (33), a glucuronyltransferase (34) and a galactosyltransferase (34) was identified. The high mol. wt. particle hence represents a multiglycosyltransferase system associated with aggregation promoting activity (35). Recently, two extracellularly located glycosidases have been isolated from GEODIA, a B-glucuronidase and a 8-galactosidase, (35). A working hypothesis has thus been postulated to explain the coordinated aggregation and sorting-out processes in GEODIA at the molecular level (35): a) activation of the aggregation receptor (AR) by its enzymic glucuronylation~ b) adhesive recognition of two cells, mediated by the aggregation factor (AF) and the glucuronylated AR~ c) inactivation of the AR by its enzymic deglucuronylation with the membrane bound B-glucuronidase, and d) cell separation due to the loss of the recognition site (glucuronic acid) of the AR for the AF. It is quite possible that GL is involved in these controlled cellular interactions. Such a role would require the existence of a cell surface receptor for GL at least on a subpopulation of GEODIA Cells. A reasonable candidate for this receptor can be seen in the anti-aggregation receptor (AAR), recently isolated from certain non-aggregating GEODIA cells, (34). The AAR was found to carry terminal galactosyl residues, which may represent a binding site for GL. On the other hand, the aggregation receptor (AR), which contains considerable amounts of ~-galactose (I) cannot be excluded as a receptor for GL, provided that after enzymic deglucuronylation subterminal galactose residues become available. An interaction of GL with these cell surface receptors could be an essential event in the controlled sorting-out processes of reaggregating GEODIA cells. Is GL part of a surveillance system against heteroloqous cells? The ability of sponge lectins to agglutinate and to lyse heterologous sponge cells, originally observed in vitro by GALTSOFF (5), may reflect the significance of these heteroagglutinins in vivo. Based upon extensive immunological investigations with a variety of sponge cells, MacLENNAN proposed (e.g. AXINELLA) that sponge heteroagglutinins are " an expression of a true selective Not-Self recognition, rather than of a general reactivity with galactose containing polymers" (8). Several observations made by MacLENNAN with the sponge AXINELLA were confirmed by our work with GEODIA: e.g. non-reactivity between GL and a GEODIA polysaccharide preparation (Fig. I) and no agglu-
Vol. 3, No. 3
SPONGE LECTIN
413
tination of GEODIA cells by GL, contrasted by a strong agglutination brought about by an agglutinin extract from AXINELLA (unpublished). The critical experiment will thus be to show an involvement of GL as part of a surveillance system against pathogens or heterologous sponge cells. Cellular reactions against different heterografts have been investigated in another sponge organism (36). It may be quite reasonable to assume that GL integrated in the membrane of certain GEODIA cells (archaeocytes ?) does mediate such cellular rejection processes. R e c e n t l y , two very elegant hypotheses have been proposed, which draw a phylogenetic line from invertebrate lectins to the products of the major histocompatibility complex (MHC) in mammals (37,38). Taking these arguments into account, two types of recognition factors, one for Self and another for Non-Self determinants, should have evolved from a common ancestral gene by gene duplication and mutation. Enzymes (e.g. glycosyltransferases) or lectins (devoid of enzymatic activity) would represent such recognition factors, the recognized determinants being carbohydrate in nature. Certain similarities between these hypotheses and our experimental findings appear striking: The large circular proteid particle, which carries different glycosyltransferases and the aggregation factor (AF) (35) would be equivalent to the multiglycosyl enzymatic and recognition system (MGER), as termed by ROTHENBERG (38) and to an essentially similar model proposed by PARISH (39). The Self-determinant, enzymatically modified and recognized by this multienzyme-AF-complex would be represented by the aggregation receptor (AR) with a glucuronic acid terminus (I). A second recognition factor identified in GEODIA is GL, described in this paper: GL would represent an anti-Non-Self receptor in this model. If such a reactivity will turn out to be the essential function of GL, a comparison between AF and GL regarding their amino acid sequence should be highly interesting; for, accordi n g t o this hypothesis, they should have evolved from a common precursor molecule (8,37,38). REFERENCES I.
VAITH, P., MULLER, W.E.G., and UHLENBRUCK, G. On the role of ~-glucuronic acid in the aggregation of cells from the marine sponge GEODIA CYDONIUM. Dev. Comp. Immunol. (in press).
.
MULLER, W.E.G., MULLER, I., ZAHN, R.K.~ and KURELEC, B. Species-specific aggregation factor in sponges. VI. Aggregation receptor from the cell surface. J. Cell Sci. 21, 227, 1976.
.
CHATTERJEE, B.P., VAITH, P., CHATTERJEE, S., KARDUCK, D., and UHLENBRUCK, G. Comparative studies of new marker lectins for alkali-labile and alkali-stable carbohydrate chains in glycoproteins. Int. J. Biochem. (in press).
414
4.
SPONGE LECTI~
Vol. 3, No. 3
VAITH, P., MULLER, W.E.G., van MIL, A., and UHLENBRUCK, G. A novel mitogenic lectin from the marine sponge GEODIA CYDONIUM. (in preparation).
.
GALTSOFF, P.S. Heteroagglutination of dissociated sponge cells. Biol. Bull. Mar. Biol. Lab. Woods Hole 57, 250, 1929.
.
MACLENNAN, A.P. and DODD, R.Y. Promoting activity of extracellular materials on sponge cell reaggregation. J. Embryol. Exp. Morph. 18, 473, 1967.
.
GOLD, E.R. and BALDING, P. (eds.) Receptor-Specific Proteins. American Elsevier Publishing Company, Inc., New York 1975, p. 237.
.
MACLENNAN, A.P. The chemical bases of taxon-specific cellular reaggregation and "self,' - "not-self" recognition in sponges. Arch. Biol. (Bruxelles) 85, 53, 1974.
.
MULLER, W.E.G., MULLER, I., and ZAHN, R.K. Aggregation in sponges, Research in Molecular Biology, Vol. 8. Akademie der Wissenschaften und der Literatur, Mainz 1978, p. I.
10.
BALDO, B.A., UHLENBRUCK, G., and STEINHAUSEN, G. Antigalactan agglutinins from the marine sponge AXINELLA POLYPOIDES (Schmidt). Biol. Zbl. 96, 723, 1977.
11.
UHLENBRUCK, G., STEINHAUSEN, G., and PALATNIK, M. Similarity of glycoproteo-galactans in the albumin glands from ACHATINA and BORUS snails. Comp. Biochem. Physiol. 57 B, 335, 1977.
12.
UHLENBRUCK, G., STEINHAUSEN, G., and KAREEM, H.A. Different glycosubstances and galactans in the albumin gland and eggs of ACHATINA FULICA. Z. Immun.-Forsch. 152, 220, 1976.
13.
BALDO, B.A. and UHLENBRUCK, G. Quantitative precipitin studies on the specificity of an extract from TRIDACNA MAXIMA (R~ding). Carbohydr. Res. 40, 143, 1975.
14.
UHLENBRUCK, G. and SCHMID, D.O. Ein Mucoid mit Blutgruppeneigenschaften aus Rindererythrozytenstroma. Z. Immun.Forsch. 123, 466, 1962.
15.
MOLLER, W.E.G. and ZAHN, R.K. Purification and characterization of a species-specific aggregation factor in sponges. Exptl. Cell Res. 80, 95, 1973.
16.
DETERMANN, H. Gelchromatographie. York: Springer-Verlag, 1967.
17.
LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L., and RANDALL, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265, 1951.
Berlin-Heidelberg-New
Vol. 3, No. 3
SPONGE LECT!N
4/5
18.
ASHWELL, G. New colorimetric methods of sugar analysis. In: Methods in Enzymology VIII. Neufeld, E.F. and Ginsburg, V. (eds.). Academic Press New York and London 1966, p. 85.
19.
LAEMMLI, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680, 1970.
20.
WEBER, K. and OSBORN, M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406, 1969.
21.
DAHR, W., UHLENBRUCK, G.~ GUNSON, H.H., and van der Hart, M. Molecular basis of Tn-polyagglutinability. Vox Sang. 29, 36, 1975.
22.
MOLLER, W.E.G., MOLLER, I., and ZAHN, R.K. Two different aggregation principles in reaggregation process of dissociated sponge cells (GEODIA CYDONIUM). Experientia 30, 899, 1974.
23.
SIMPSON, D.L., ROSEN, St.D., and BARONDES, S.H. Discoidin, a developmentally regulated carbohydrate-binding protein from DICTYOSTELIUM DISCOIDEUM. Purification and characterization. Biochemistry 13, 3487, 1974.
24.
UHLENBRUCK, G., STEINHAUSEN, G., and BALDO, B.A. Galactane und Anti-Galactane. Verlag Josef Stippak, Aachen, Postfach 1262, 1975.
25.
LARM, O. and LINDBERG, B. The pneumococcal polysaccharides: A re-examination.°In: Advances in Carbohydrate Chemistry and Biochemistry (Tipson, R.St. and Horton, D., eds.). Academic Press New York-San Francisco-London 1976, p. 295.
26.
GLOCKNER, W.M., NEWMAN, R.A., and UHLENBRUCK, G. Carbohydrate structure and serological behaviour of "antifreeze" glycoproteins from an antarctic fish. Biochem. Biophys. Res. Comm. 66, 701, 1975.
27.
GLOCKNER, W.M., NEWMAN~ R.A., DAHR, W., and UHLENBRUCK, G. Alkali-labile oligosaccharides from glycoproteins of different erythrocyte and milk fat globule membranes. Biochim. Biophys. Acta 443, 402, 1976.
28.
NEWMAN, R.A. and UHLENBRUCK, G. Investigation into the occurence and structure of lectin receptors on human and bovine erythrocyte, milk-fat globule and lymphocyte plasma membrane glycoproteins. Eur. J. Biochem. 76, 149, 1977.
29.
BRETTING, H. and KABAT, E.A. Purification and characterization of the agglutinins from the sponge AXINELLA POLYPOIDES and a study of their combining sites. Biochemistry 15, 3228, 1976.
416
SPONGE LECTIN
YOI. _3~ NO. 3
30.
MOLLER, W.E.G., MOLLER, I., and ZAHN, R.K. Species-specific aggregation factor in sponges. V. Influence on programmed syntheses. Biochim. Biophys. Acta 418, 217, 1976.
31.
MOLLER, W.E.G., BEYER, R., PONDELJAK, V., MOLLER, I., and ZAHN, R.K. Species-specific aggregation factor in sponges. XIII. Entire and core structure of the large circular proteid particle from GEODIA CYDONIUM. Tissue and Cell 10, 191, 1978.
32.
MOSCONA, A.A. Cell aggregation: Properties of specific cell-ligands and their role in the formation of multicellular systems. Dev. Biol. 18, 250, 1968.
33.
MOLLER, W.E.G., ARENDES, J., KURELEC, B., ZAHN, R.K., and MOLLER~ I. Species-specific aggregation factor in sponges. IX. Sialyltransferase associated with aggregation factor. J. Biol. Chem. 252, 3836, 1977.
34.
MULLER, VAITH, XVIII. gation 1978.
35.
MULLER, W.E.G., ZAHN, R.K., KURELEC, B., MOLLER, I., UHLENBRUCK, G., and VAITH, P. Aggregation of sponge cells. A novel mechanism of controlled intercellular adhesion, basing on the interrelation between glycosyltransferases and glycosidases. J. Biol. Chem. 254, 1280, 1979.
36.
CHENG, T.C., YEE, H.W.F., RIFKIN, E., and KRAMER, M.D. Studies on the internal defense mechanism of sponges. III. Cellular reactions in TERPIOS ZETEKI to implanted heterologous biological materials. J. Invert. Pathol. 12, 29, 1968.
37.
KOLB, H. On the phylogenetic origin of the immune system, a hypothesis. Dev. Comp. Immunol. I, 193, 1977.
38.
ROTHENBERG, B.E. The self recognition concept: An active function for the molecules of the major histocompatibility complex based on the complementary interaction of protein and carbohydrate. Dev. Comp. Immunol. 2, 23, 1978.
39.
PARISH, C.R. Simple model for self-non-self-discrimination in invertebrates. Nature 267, 711, 1977.
W.E.G., ZAHN, R.K., KURELEC, B., UHLENBRUCK, G., P., and MULLER, I. Aggregation of sponge cells. Glycosyltransferases associated with the aggrefactor. Hoppe-Seyler's Z. Physiol. Chem. 359, 529,
SPONGE AGGREGATION FACTOR AND SPONGE HEMAGGLUTININ: POSSIBLE RELATIONSHIPS BETWEEN TWO DIFFERENT MOLECULES
Peter Vaith, Gerd Uhlenbruck, Werner E.G. MUller" and Gisela Holz Medizinische Universit~tsklinik K~in, Abteilung fHr Experimentelle Innere Medizin, Kerpener Str. 15, D-5000 K~in 41 (GFR) and •Physiologisch-Chemisches Institut der Johannes Gutenberg Universit~t, Abteilung fur Angewandte Molekularbiologie, Duisbergweg, D-6500 Mainz (GFR)
ABSTRACT.
A lectin from the marine sponge GEODIA CYDONIUM was isolated and characterized. GEODIA lectin (GL) agglutinates human red blood cells i r r e s p e c t i v e of the ABO blood group and precipitates with a variety of D-galactose containing glycosubstances, i.e. certain snail galactans, bovine erythrocyte glycoprotein and PNEUMOCOCCUS type XIV polysac" charide. The only simple sugars inhibiting the GL-mediated hemagglutination were lactose and ~-acetyl-_qD-galactosamine. GL was purified by affinity chromatography on Sepharose 4B almost to homogeneity as tested by polyacrylamide disc gel electrophoresis. Positive staining of the lectin band with Coomassie brilliant blue and PAS suggest that GL is a glycoprotein. Under dissociating conditions GL exhibits an apparent mol. wt. of 12,000 daltons. The elution behaviour of the lectin on Biogel suggests that GL exists as an oligomeric molecule in its native state. Our data obtained with GL demonstrate that the species-specific aggregation factor (AF) from GEODIA is not identical with this lectin. Two major hypotheses for a biological role of GL are discussed: first, the cooperation of GL with AF in sorting-out processes of GEODIA cells and second, the possibility that GL is involved in defense mechanisms. 399
400
SPONGE LECTIN
Vol. 3, No. 3
INTRODUCTION In the foregoing p a p e r (I) we reported on our i n v e s t i g a tions c o n c e r n i n g the i n t e r a c t i o n between a g g r e g a t i o n factor (AF) and a g g r e g a t i o n r e c e p t o r (AR) in the m a r i n e sponge G E O D I A C Y D O N I U M . Evidence for an i m p o r t a n t role of A R - b o u n d ~ - g l u G u r o n i c acid in the A F - m e d i a t e d c e l l u l a r r e a g g r e g a t i o n was given. A c c o r d i n g l y , the s p e c i e s - s p e c i f i c r e a g g r e g a t i o n of G E O D I A cells can be viewed as the " r e c o g n i t i o n " of a ~ - g l u c uronic acid c o n t a i n i n g cell surface r e c e p t o r (AR) by an i n t e r c e l l u l a r p r o t e i n a c e o u s AF m a c r o m o l e c u l e (I). During our exp e r i m e n t s c o n c e r n e d w i t h the s e r o l o g i c a l a c t i v i t y of the agg r e g a t i o n r e c e p t o r (AR) we tested in a d d i t i o n to d i f f e r e n t lectins the c r u d e a g g r e g a t i o n factor (AF) for h e m a g g l u t i n a t i o n ; a very r e m a r k a b l e a g g l u t i n a t i o n of human red blood c e l l s could be observed. To our surprise, the AR, which has p r e v i o u s l y been shown to form a c o m p l e x with the AF d u r i n g G E O D I A cell reagg r e g a t i o n (2), i n h i b i t e d this h e m a g g l u t i n a t i o n only scarcely. Hence, we supposed that the a g g l u t i n a t i n g a c t i v i t y in the c r u d e AF e x t r a c t was not due to the AF itself. Our s u g g e s t i o n could be s u b s t a n t i a t e d by the i s o l a t i o n of a lectin responsible for this h e m a g g l u t i n a t i n g a c t i v i t y . G E O D I A lectin (GL) was found to a g g l u t i n a t e human e r y t h r o c y t e s i r r e s p e c t i v e of the ABO type. The a g g l u t i n a t i o n titre could be e n h a n c e d by p r i o r n e u r a m i n i d a s e and even m o r e by p r o n a s e t r e a t m e n t of the cells. In addition, e r y t h r o c y t e s from several d i f f e r e n t species w e r e found to be a g g l u t i n a t e d by G L (3). The p u r i f i e d lectin is also m i t o g e n i c for human l y m p h o c y t e s (4). The aim of this p a p e r is first, to report on the i s o l a t i o n and i m m u n o c h e m i c a l c h a r a c terization of this new lectin, and second, to p r e s e n t our h y p o theses on the b i o l o g i c a l function of this lectin in GEODIA. The h i s t o r y of h e t e r o a g g l u t i n i n s in sponges d a t e s back to G A L T S O F F (1929), who d e s c r i b e d a lyric and a g g l u t i n a t i n g a c t i o n of sponge e x t r a c t s upon c e l l s of o t h e r sponge species (5). The property of sponge e x t r a c t s to a g g l u t i n a t e red blood cells too, was first reported by M a c L E N N A N and DODD in 1967 (6), s u p p o s i n g a r e l a t i o n s h i p between h e r e t o - and h e m a g g l u t i n i n s . S u b s e q u e n t l y several lectins from d i f f e r e n t sponges have been i s o l a t e d and purified in the last y e a r s (7). An e x c e l l e n t d i s c u s s i o n on the b i o l o g i c a l role of sponge a g g r e g a t i o n factors on the one hand, and sponge h e t e r o - or hema g g l u t i n i n s on the other, has been given by M a c L E N N A N in 1974, who proposed a S e l f - N o t - S e l f r e c o g n i t i o n function for both molecules (8). An exact c o m p a r i s o n b e t w e e n a g g r e g a t i o n factor and h e m a g g l u t i n i n , h o w e v e r , has been impeded by the fact that the a c t i v i t i e s c o m p a r e d w e r e d e r i v e d from d i f f e r e n t sponge species as yet: e.g. AF from M I C R O C I O N A and h e m a g g l u t i n i n from A X I N E L L A (7,8). Our d i s c o v e r y of a lectin in the sponge G E O D I A C Y D O N I U M enables us now to i n v e s t i g a t e the r e l a t i o n s h i p b e t w e e n a lectin and an a g g r e g a t i o n f a c t o r in the same sponge species. A m a j o r a d v a n t a g e for this study is the fact that n u m e r o u s m o l e c u l a r p r o p e r t i e s involved in the r e a g g r e g a t i o n p r o c e s s of G E O D I A cells have been a n a l y z e d p r e v i o u s l y by W. M O L L E R and c o w o r k e r s (9).
Vol. 3, NOo 3
SPONGE LECTIN
401
MATERIAL AND METHODS Chemicals. Sugars used were of highest purity commercially available. Mono- and oligosaccharides were obtained from Serva Ltd, Heidelberg (GFR)~ E. Merck, Darmstadt (GFR)~ Sigma Chemical Co, St. Louis (USA)~ Calbiochem, San Diego (USA)~ Baker Chemicals, Deventer (Holland)~ and Koch-Light Laboratories Ltd, Colnbrook (UK). Protein marker substances were purchased from Serva. Blue dextran 2,000 from Pharmacia, Uppsala (Sweden) was used. Polysaccharides and glycoproteins. The origin and preparation of snail galactans from HELIX POMATIA, HELIX HORTENSIS and ACHATINA FULICA (Madagaskar) as well as PNEUMOCOCCUS type XIV polysaccharide have been described (10-13). Arabin~galactan (larch) was purchased from Serva. Bovine erythrocyte mucoid was prepared as described by UHLENBRUCK and SCHMID (14). Source of GEODIA substances. These materials were derived from the siliceous sponge GEODIA CYDONIUM (Tetractinellida) collected in the vicinity of Rovinj (Yugoslavia). The crude aggregation factor (AF) was prepared as described (15). Briefly, the sponge was cut into cubes of 2 mm . Fifty grams material were stirred for 2 h with 50 ml of calcium- and magnesiumfree artificial sea water, pH 8.2, containing 20 mM EDTA. The supernatant collected after centrifugation at I0,000 ~ for 30 min. will be termed crude AF~ protein content was 6 mg/ml. GEODIA polysaccharide was prepared by phenol-saline (90% v/v phenol, 1:1) extraction of dried GEODIA tissue. As a source of lectin, crude AF and dried GEODIA tissue were employed. In the latter case, 100 g GEODIA tissue was homogenized in a blender and extracted with I 1 phosphate-buffered saline (PBS) pH 7.2 overnight at 4°C under magnetic stirring. After centrifugation at 4,000 rpm for 30 min and at 27,000 rpm (Beckman ultracentrifuge, SW 27 rotor) for 3 hours at 4°C the clear brown supernatant ~as used as a crude lectin extract. Aliquots were stored at -20 C or alternatively after dialysis in a lyophilized state. Affinity chromatography of the crude GEODIA lectin. Crude AF extract or PBS-extracted GEODIA tissue were chromatographed on Sepharose 4B (Pharmacia). In a typical experiment 10 ml crude AF was layered onto a Sepharose column (47.0 x 2.5 cm) and eluted with Tris-NaCl buffer (0.5 M NaCI, 0.05 M Tris-HCl buffer, pH 8.2) at 4°C. The column was washed until the optical density at 280 nm reached zero. Specific elution of the lectin was carried out by the addition of 0.01 M ~-galactose to the eluting buffer. The specifically eluted fractions were combined, dialyzed against 0.01 M NH4HCO 3 buffer, pH 8.2, and lyophilized. Gel filtration experiments. Instead of dialysis, lectin fractions from the latter procedure were alternatively freed of galactose containing buffer by gel filtration on Biogel P 100 (Biorad). A Biogel column (92.0 x 2.6 cm) was equilibrated with 0.01 M NH4HCO 3 buffer, ph 8.2. Lectin activity appeared
402
SPONGE LECTIN
Vol. 3, No. 3
in one symmetrical peak near the void volume. An identical elution b e h a v i o u r was observed when the lectin dissolved in PBS or in Tris-NaCl b u f f e r was eluted from Biogel P I00~ previously equilibrated with the respective buffer. A Biogel P 300 column (17.0 x 1.5 cm) was employed for gel filtration of the crude AF material. Flow rate: I ml per hour in Tris-NaCl buffer; fraction volume: I ml. C a l i b r a t i o n of the column was performed by measuring elution volumes of blue dextran and of m a r k e r proteins as described by DETERMANN (16). The ratios of the elution volumes of the m a r k e r proteins (V e) to the void volume ( V ) were: Human transferrin (mol. wt. 80,000) = 1.64; b o v i n e ~erum albumin (mol. wt. 67,000) = 1.9; ovalbumin (mol. wt. 45,000) = 2.19; c h y m o t r y p s i n o g e n (mol. wt. 25,000) = 2.54. Analytical methods. Protein was determined after LOWRY et al. (17) with bovine serum albumin as a standard. Neutral sugars were assessed by DUBOIS' method (18) with D-galactose as a reference substance. P o l y a c r y l a m i d e disc gel e l e c t r o p h o r e s i s was performed according to LAEMMLI (19); 7.5, 10, and 12.5 % gels were employed at pH 8.8 of the separating gel. For mol. wt. d e t e r m i n a t i o n s the method of W E B E R and OSBORN (20) was applied. M a r k e r proteins consisted of a m i x t u r e of bovine serum albumin, ovalbumin, c h y m o t r y p s i n o g e n A, and c y t o c h r o m e C (mol. wt. 12,400). In some experiments, ovalbumin was substituted by rabbit aldolase (mol. wt. of subunits 40,000). Bromophenol blue or Pyronin G were used as tracing dyes, respectively. The gels were stained for protein with C o o m a s s i e b r i l l i a n t blue and for c a r b o h y d r a t e with the PAS method as described by DAHR et al. (21). H e m a g g l u t i n a t i o n inhibition tests were done as already reported (I). Generally, n e u r a m i n i d a s e treated O e r y t h r o c y t e s were used as test cells. As a source of agglutinin we employed crude AF, crude lectin after P B S - e x t r a c t i o n of GEODIA tissue~ and affinity purified GL. O u c h t e r l o n y e x p e r i m e n t s were performed in agarose (I %) (Serva) as d e s c r i b e d (I). A g a r gels were prepared in saline or in Tris-NaCl b u f f e r c o n t a i n i n g ~ - g a l a c t o s e (0.05 M) in order to prevent binding of the lectin to agarose. The same GEODIA m a t e r i a l s were used as described for the h e m a g g l u t i n a t i o n experiments. The p a r t i c u l a r source of GL activity used in h e m a g g l u t i n a t i o n inhibition and agar gel experiments will be stated under 'Results'. Formaldehyde treatment of sheep red blood cells (SRB~s). A 2% solution of p a r a f o r m a l d e h y d e in PBS was heated at 60-C for 30 min. After cooling, 10 ml of a 10% suspension of SRBCs (Behringwerke, MarbUrg, GFR) was added to 90 gl p a r a f o r m a l d e hyde solution and incubated for 48 hours at 4°C, followed by washing of the cells in Tris-NaCl buffer. Absorption of GEODIA lectin on formalinized SRBCs. The hemagglutinating fractions separated on Biogel P 300 from crude AF extract (fractions 20-27, Fig. 4) were combined and one aliquot ( 8 0 ~ g protein) was incubated with SRBCs at room temperature until no agglutinin activity was found in the supernatant. As a control, SRBCs were tested with b u f f e r alone. Both supernarants and an unabsorbed lectin sample (8o ~ g protein) were dialyzed against distilled water, m i c r o p o r e filtered and lyo-
Vol. 3, No. 3
SPONGE LECTIN
philized. Subsequently, amide gels.
403
the samples were run on SDS polyacryl-
Test for Ca++-dependency Of the hema~@lutinatin~ activity. 5 ml of crude AF extract was dialyzed against 5 1 0.$ M EDTAsaline solution for two days, and then for three days against saline at 4 C. An aliquot (~.8 ml) was mixed with 0.2 ml 0.1 M CaCl2-saline solution, and another With 0.2 ml calcium-free saline. These solutions were tested for hemagglutinating activity. RESULTS Occurence of lectin activity in GEODIA. Our initial hypothesis that the crude AF extract contains a lectin activity independent of the AF activity was supported by Ouchterlony experiments. As shown in Fig. I, strong precipitin lines were obtained, when crude AF was tested against several glycosubstances, i.e. certain snail galactans, bovine erythrocyte mucoid or PNEUMOCOCCUS type XIV polysaccharide. No reaction was seen, however, with a polysaccharide material extracted from GEODIA. FIG. I Precipitin Reactions with the GEODIA Lectin
Substances are arranged clockwise from 12 o'clock on; in the center is crude aggregation factor (AF). ~= Bovine erythrocyte mucoid, 2= HELIX POMATIA galactan, 3= HELIX HORTENSIS galactan, 4~ GEODIA polysaccharide, 5= PNEUMOCOCCUS type XIV p01ysaccharide, 6= ACHATINA FULICA galactan. .L
To, test this reactivity against distinct glycosUbstances further, we performed hemagg!utination inhibition experiments. Many different simple Sugars and polysacch~arides were tested ~s potential inhibitors of the GEODIA hemagglutinin, (Table I). Clearly there is a strong reactivity of the hemagglotinating
404
SPONGE LECTIN
TABLE
Hemagglutination
Vol. 3, No. 3
I
I n h i b i t i o n of G E O D I A Different Sugars
Hemagglutinin
by
Sugar
IT
Sugar
IT
L_.-f u c o s e
-
~i-acety l-D--ga l a c t o s a m i n e
22
D__-f u c o s e
-
~ - a c e ty l-D--g l u c o s a m i n e
-
D--ga l a c t o s e
-
D-ga lacturonic
-
D-glucose
-
D-g lucuronic
D--mannose
-
/~-acety i- D--neu r a m i n i c
L-arabinose
-
lactose
22
L-rhamnose
-
melibiose
-
D - x y lose
-
treha lose
-
1-/~-me thy I- t&-D-ga I ac to se
-
sucrose
1-/l-me thy l-B- D-ga lac rose
-
raffinose
1-11-me thy i-~- ~ g luco se
-
cel!obiose
1-Q-methy l-S-D-g lucose
-
ma I tose
D-galactosamine
-
ACHATINA
HCI
acid
FULICA
D-glucosamine
HCI
-
HELIX
D-mannosamine
HCI
-
arabinogalactan
GEODIA
-
AR
acid
POMATIA
acid
galactan
galactan (larch)
-
29 29 -
21
V a l u e s are e x p r e s s e d as t h e i r r e c i p r o c a l s ; they r e p r e s e n t the m i n i m u m a m o u n t of inhibitor required to b r i n g a b o u t i n h i b i t i o n of f o u r a g g l u t i n a t i o n d o s e s of a g g l u t i n i n . C r u d e AF w a s u s e d as a s o u r c e of a g g l u t i n i n . The same i n h i b i t i o n p a t t e r n - e x c e p t the u n r e a c t i v i t y w i t h G E O D I A - A R - w a s o b t a i n e d w i t h p u r i f i e d G L as an a g g l u t i n i n . M o n o - and o l i g o s a c c h a r i d e s u s e d w e r e 0.05 m o l a r in T r i s - N a C l b u f f e r ; p o l y s a c c h a r i d e s w e r e t e s t e d as I% s o l u t i o n s in the same b u f f e r . IT = I n h i b i t i o n titre.
material against certain galactosyl containing polymers, while D - g a l a c t o s e i t s e l f w a s i n a c t i v e at the c o n c e n t r a t i o n s tested. The h e m a g g l u t i n a t i o n i n h i b i t i o n e x e r t e d by ~ - a c e t y l - D - g a l a c t o s a m i n e and D - l a c t o s e shows that the g a l a c t o s y l c o n f i g u r a t i o n of the O H - g r o u p at C 4 is f a v o u r e d c o m p a r e d w i t h the g l u c o s y l c o n f i g u r a t i o n s i n c e n e i t h e r ~-acetyl-D__-glucosamine nor c e l l o b i o s e w e r e found i n h i b i t o r y . In a d d i t i o n to a t e r m i n a l g a l a c t o s e residue, the l i n k a g e type to the s u b t e r m i n a l s u g a r s t r o n g l y i n f l u e n c e s the r e a c t i v i t y of G E O D I A a g g l u t i n i n , for m e l i b i o s e and r a f f i n o s e w i t h an ~ - I - 6 linked D - g a l a c t o s e at the n o n r e d u c i n g end did not i n h i b i t . A l l of the t e s t e d o l i g o s a c c h a r i d e s w i t h D--glucose at the n o n r e d u c i n g end w e r e n o n i n h i b i t o r y .
Vol. 3, No. 3
SPONGE LECTIN
405
Isolation of GEODIA lectin. The crude AF material was chrom a t o g r a p h e d on Sep-harose 4B. This procedure has previously been applied as a purification step in the isolation of the AF from GEODIA. The AF containing m a c r o m o l e c u l e is eluted near the void volume, followed by a second, broad peak without aggregation promoting activity. The eluted fractions were tested for agglutinating activity. (Fig. 2). FIG.
2
Isolation of G E O D I A Agglutinin by Affinity C h r o m a t o g r a p h y on Sepharose 4B
oo 21~o nm ~5,
)
/
I
/\.
,
_
-
...-.,_.,.
..... __ ........
Crude AF was applied to a column of Sepharose 4B, as described under 'Methods'. The arrow marks the addition of 0.01 M ~ - g a lactose to the eluting buffer. Each fraction was examined for protein (X) by measuring the absorbance at 280 nm and for hema g g l u t i n a t i n g activity (O) against n e u r a m i n i d a s e treated O red blood cells. Flow rate: 15 ml per hour, fraction volume: 5 ml.
The relatively low h e m a g g l u t i n a t i n g activity found within and between the two protein peaks, respectively, was further investigated by h e m a g g l u t i n a t i o n inhibition experiments. Using the combined fractions of the first, AF containing peak no inhibition was obtained with all saccharides listed in Table C. To our surprise the AR, however, was now inhibitory up to a dilution of 1 : 16, as we already documented (1). The specificity
406
SPONGE LECTIN
Vol. 3, No. 3
of the interaction of the enriched AF fractions with erythrocytes is not clear. We believe this to be a nonspecific aggregation of cells, possibly via calcium-bridges, rather than a true agglutination. From reaggregation e x p e r i m e n t s a calciumdependency of GEODIA AF is well established. The a g g l u t i n a t i n g activity detected in the ascending part of the second Sepharose peak gave the same inhibition pattern as the crude AF extract, but was too low to account for the total activity of the starting material. The a n t i - g a l a c t o s y l specificity of GEODIA lectin, however, lent support to the idea that part of it bound to the Sepharose beads by interaction with ~ - g a l a c t o s e groupings of the gel. C o n s e q u e n t l y we added 0.01 M ~ - g a l a c t o s e to the elution buffer, after the two protein peaks were eluted from the column. A small protein peak appeared with a powerful a g g l u t i n a t i o n activity; the agglutinating fractions were combined and used for the following experiments. A t t e m p t s to elute the lectin by 0.0C M D-glucose were negative. A purification factor of a p p r o x i m a t e l y 45 can be calculated from the increase in specific titre (titre/ protein content). The overall yield of purified lectin was about 9 % relative to protein. The lectin activity detected in the ascending part of the second Sepharose peak d e m o n s t r a t e s that the lectin binding capacity of the gel was probably exceeded; at the same time, the elution b e h a v i o u r of this activity gives an idea of the lectin's size. This will be referred to, below. C h a r a c t e r i z a t i o n of GEODIA lectin. Affinity purified GL reacted with the same g l y c o s u b s t a n c e s as the crude AF extract (Fig.1 and Table I) or the PBS-extracted GEODIA tissue. The aggregation receptor (AR), however, was u n r e a c t i v e with purified GL in h e m a g g l u t i n a t i o n inhibition experiments. To test the affinity purified GL for homogeneity, polyacrylamide disc gel e l e c t r o p h o r e s i s was applled.(Fig. 3). One major C o o m a s s i e positive band was obtained, when affinity purified GL was run on gels both in the presence and a b s e n c e of SDS (Fig. 3, No. 3,4 and 8). The sharp m a j o r band seen in SDS gels migrated with the m a r k e r dye (Fig. 3, No. 3 and 4), w h i l s t a broad band located in the m i d d l e of the gel was found in the absence of SDS (Fig. 3, No. 8). The d i f f u s e band on the top of gel No. I, containing the crude AF extract, can be resolved by comparison with gels No. 3 and 4 (purified GL) and gel No. 2, where a fraction of the first Sepharose peak (enriched AF) was separated. That broad band in the crude AF material is obviously comprised of a fast moving band c o r r e s p o n d i n g to GL and a band next to the m a r k e r dye, which possibly represents the AF molecule (mol. wt. between 16,000 and 23,000) (22). The absence of a band c o r r e s p o n d i n g to GL in the enriched AF fractions is also ascertained in gels w i t h o u t SDS (Fig. 3, No. 7). This finding is strong evidence for a n o n - i d e n t i t y of GL and AF; furthermore it can be seen clearly that GL is not associated with the AF containing m a c r o m o l e c u l e obtained from the first Sepharose peak. When an SDS gel was overloaded with purified GL, two additional minor, slow moving bands were discerned b e s i d e s the ma-
Vol. 3, No. 3
SPONGE LECTI~
407
FIG. 3 Polyacrylamide Disc Gel Electrophoresis of Several GEODIA Lectin Fractions
1
Gels are numbered from the left to the right. Gels 1-5 were run in the presence and gels 6-8 in the absence of SDS. I= crude AF extract ( 5 0 ~ g ) ; 2= first Sepharose 4B peak ( 5 0 ~ g ) ; 3= affinity purified GL ( 5 0 ~ g ) ; 4= affinity purified GL ( 1 0 ~ g ) ; 5= marker proteins ( 5 ~ g , each); 6= same as I) without SDS; 7= same as 2) without SDS; 8= same as 3) without SDS. Polyacrylamide concentration of the separating gels was 10 %. Gels were stained for protein with Coomassie brilliant blue. Values in brackets are the respective amounts of protein added per gel.
jor band on top of the gell the nature of these two bands was not investigated further. Evidence for an identity of the major, fast moving band with the lectin activity comes from absorption studies on formalinized sheep red blood cells (SRBCs). This procedure has been reported for the purification of an anti-galactosyl lectin from slime molds (23). We absorbed the agglu£inating fractions eluted from Biogel P 300 (see Fig. 4, fractions 20-27) on neuraminidase treated formalinized SRBCs until no agglutinating activity could be found in the supernatant. This absorbed material showed only a trace of the major band in
408
SPONGE LECTIN
VOI, 3, NO.
SDS gel electrophoresis; the relative intensities of the slower moving bands compared with the major band, however, were significantly increased. This result can be taken as evidence that the slow moving bands were not associated with GEODIA lectin activity. The m o l e c u l a r weight of GL in the presence of SDS and dithioerythritol, as determined after W E B E R and OSBORN, appears to be 12,000 ~ 1,000 daltons. Staining of the gels by the PAS method suggests that GL is a glycoprotein. A neutral sugar content of about 5 % was found in affinity purified GL using DUBOIS' method. No lectin activity associated with such a low mol. wt. substance was detectable in gel filtration experiments. As already shown in Fig. 2, the agglutinin activity not bound to Sepharose was eluted in the ascending part of the second protein peak. Further spreading of this elution pattern was obtained, when the crude AF material was subjected to gel filtration on Biogel P 3001 three protein peaks appeared now, the intermediate peak contained all the a g g l u t i n a t i n g activity, (Fig. 4). FIG.
4
Gel Filtration of the Crude AF Extract on Biogel P 300
Oil 2110 hi'. i i-ll
O,.10-
!
. ll
.27
:
!
32-1
hill,
0.22. 0.18,
O.1/..
it ... J
0.10.
GO6.
~
OJDi, |
S"
m
i
•
•
li
X
iI
z
%\ a
a
~
,"
li
•
li
i
~
C r u d e AF (0.5 ml) was applied to the column as described u n d e r 'Methods'. Each fraction was examined for protein (x) by measuring the a b s o r b a n c e at 280 nm. All the a g g l u t i n a t i n g activity was found within the fractions of the i n t e r m e d i a t e peak, fractions 20-27. The arrow marks the void volume, as d e t e r m i n e d with dextran blue. For calibration data of the column, see under 'Methods'.
Vol. 3, No. 3
SPONGE LECTIN
402
The elution volumes of h e m a g g l u t i n a t i n g fractions 20-27 roughly correspond to a mol. wt. range between 30,000 and 70,000 daltons with a peak at about 45,000. Based upon these values one could assume a tetrameric m o l e c u l e composed of subunits with a mol. wt. of 12,000 daltons, but aggregates of different size are equally possible. Attempts to estimate the m o l e c u l a r size of affinity purified GL by gel filtration on Biogel yielded inconclusive results. The elution b e h a v i o u r of GL changed in a wide range from experiment to experiment, presumably due to different s e l f - a g g r e g a t i o n states of GL subunits. A few additional e x p e r i m e n t s were performed to confirm the d i f f e r e n c e s between AF and GL. The affinity purified ~L was tested for heat sensitivity; heating for 3 min. at 60 C reduced the agglutinin titre by I: 8~ while at 80°C a titre reduction of I : 64 was found. All activity was lost, when GL was boiled for 3 min. Though a substantial loss of activity occured upon heating, the lectin activity appeared to be relatively resistant compared with the AF, the activity of ~hich was completely destroyed after heating for I min. at 60 C (15). A n o t h e r d i f f e r e n c e between AF and GL was observed in the strong c a l c i u m - d e p e n d e n c y of the AF activity. The agglutinating power of GL was~ however, not reduced at all after extensive dialysis of the crude AF extract against 0.1 M EDTA. DISCUSSION One aim of our study was to c h a r a c t e r i z e the lectin activity in GEODIA. This h e m a g g l u t i n a t i n g and p r e c i p i t a t i n g activity was traced in the crude AF extract as well as in GEODIA tissue after saline extraction. Serological specificity of GEODIA lectin (GL). GL reacts with certain snail galactans from HELIX POMATIA (HP), HELIX HORTENSIS (HH) and A C H A T I N A FULICA (AFU). These galactose containing p o l y s a c c h a r i d e s have previously been shown to interact with a series of lectins, termed a n t i - g a l a c t a n s (24). HP-galactan was reported to p r e c i p i t a t e with a lectin from the clam TRIDACNA MAXIMA, and A F U - g a l a c t a n with an agglutinin extract from the sponge A X I N E L L A POLYPOIDES (10), but not vice versa. The lectin from GEODIA, however, apparently recognized structures common to both galactans. A third type of galactan tested, arabinogalactan from larch, was u n r e a c t i v e with GL. This non-reactivity may be explained by the scarcity of galactose side chains in this galactan (24). A X I N E L L A was also reported not to precipitate with a r a b i n o g a l a c t a n (10). Evidence that GL interacts with certain terminal, non-reducing linked B-_.D-galactosyl groups comes from our experiments with lactose and PNEUMOCOCCUS type XIV polysaccharide. As neit h e r e - nor B - m e t h y l - g a l a c t o s e were found inhibitory, the combining site of GL seems to recognize also the glycosidic linkage t y p e to the subterminal sugar, i.e. the 1-4 linkage to glucose, as in lactose and perhaps in type XIV too (25). This limited reactivity with certain terminal galactose residues is further supported by the negative precipitin reaction between anti-
410
SPONGE LECTIN
Vol. 3, No. 3
freeze glycoprotein from an antarctic fish (3). The latter glycoprotein is characterized by the presence of multiple disaccharide units, the structure of which has been identified as B-_qD-galactosyl-1-3-/~-acetyl-_q-galactosamine (26). Melibiose and raffinose, both contain terminal ~ - I - 6 linked galactose residues, were also non-inhibitory. Interestingly, ~-acetyl-D-galactosamine inhibited as well as lactose; hence it seems that the presence of an acetylated amino group at C 2 of galactose enables the lectin to accomodate this free sugar. None of the tested oligosaccharides having a terminal D_.-glucose residue was found to inhibit the GL-mediated hemagglutination. The strong precipitin reaction of GL with bovine erythrocyte mucoid reflects the agglutinability of red blood cells by GL. The agglutination of human erythrocytes was independent of the ABO group and could be enhanced by prior neuraminidase and even more by pronase treatment of the cells (3). A comparison between the agglutinability of erythrocytes from different animal species by GL will be given elsewhere (3). The erythrocyte receptor for GL is not known; the hemagglutination inhibition data suggest, however, that GL receptors will most probably carry terminal, non-reducing B-linked ~-galactose residues or ~-acetyl-_DD-galactosamine termini. The latter monosaccharide can practically be excluded as a determinant for GL in bovine mucoid, since bovine erythrocyte glycoproteins do not react with different agglutinins directed against ~-acetyl-_D-galactosamine (27,28). On the other hand, bovine erythrocyte glycoproteins contain significantly more ~-galactose and ~-acetyl-_qD-glucosamine residues than erythrocyte glycoproteins from other species and react strongly (after neuraminidase treatment) with anti-type XIV antiserum (27,28). Hence, we suppose that GL acts upon terminal, non-reducing bound lactosamine- or upon B-galactosyl-~-6-galactose groups; the occurence of the latter has been shown previously in bovine red cell glycoproteins (24). The specificity of GL resembles in some way that of agglutinin I and II from the marine sponge AXINELLA POLYPOIDES (29). First, the isolation by affinity chromatography on Sepharose is likewise applicable for the lectins from both sponges, and second, hapten inhibition studies indicate some similarity of combining sites (7,10,29). Both AXINELLA agglutinins are best inhibited by terminal, non-reducing ~-galactose residues linked by B-I-6 glycosidic bonds. The preference for certain 8-1inked galactose residues has also been demonstrated for GL. As no oligosaccharides with B-I-6 linked terminal galactose residues were available, such a specificity could not be shown conclusively. The reactivity of GL as well as of AXINELLA with snail galactans containing this disaccharide unit, however, support a similar reactivity of both sponge agglutinins. A clearcut difference between AXINELLA agglutinins and GL can be seen in the inhibiting effect of ~-galactose and ~-acetyl-_qD-galactosamine, respectively: the latter monosaccharide was reported to be much less active than ~-galactose for AXINELLA (7,10,29), a reverse relationship being true for GEODIA lectin.
Vol. 3, No. 3
411
SPONGE LECTIN
Biochemical characteristics of GEODIA lectin. The affinity purified lectin proved to be almost homogeneous when examined by polyacrylamide disc gel electrophoresis. In the presence of SDS and dithioerythritol, GL exhibited an apparent mol. wt. of 12,000 Z 1,000 daltons. The elution behaviour of hemagglutinating fractions on Biogel P 300 suggests that the unreduced lectin exists in an oligomeric state. Staining of the polyacrylamide gels for protein and carbohydrate, respectively, point to a glycoprotein nature of GL. In contrast to the AF, which occurs as a subunit of a large proteid particle (22), GL is not assoc%ated with this aggregation promoting macromolecule (see Fig. 3 and 4). GL is relatively resistant to heat, presumably because of its carbohydrate content. The lectin activity is apparently independent of calcium ions. Possible biolo@ical role of GEODIA lectin. Besides the characterization of this new lectin, a second aim of our study was to compare GL with AF. The AF has previously been isolated and characterized structurally and functionally (I,15,22,30, 31). In Table 2 several major characteristics of both molecules are summarized. Based upon these data, there is no doubt that GL and AF represent two distinct entities.
TABLE 2 Comparison between AF and GL Na tu re
AF protein
Mol. wt. Specificity subunit
(?)
GL glycoprotein
16,00023,000 (?)12,000
Ca ++dependency ÷
GIuA
Gal I
Heatresistance
S
÷/-
)4
Gal I 8 ) 6
(?)
Ga INAc GIuA= ~-glucuronic acid~ Gal= ~-galactose~ galactosamine. For references see text.
GalNAc= ~-acetyl-DD-
Whilst the biological function of GEODIA AF is being increasingly understood (9), that of GL remains open to question. Thus, we want to discuss several hypotheses referring to the possible significance of this lectin in GEODIA. Two major hypotheses will be considered: first, an involvement of GL in sorting-out processes of GEODIA cells, and second, the possibility that GL plays a defensive role against heterologous cells.
412
SPONGE LECTIN
Vol. 3, No. 3
Is GL involved in cellular sorting-out processes ? Considering cellular sorting-out processes, one must distinguish between events leading to a histiotypic rearrangement of dissociated cells within one sponge organism, and a speciesspecific sorting-out between different sponges. This problem has already been discussed in detail by MOSCONA (32). In the following section, sorting-out events occuring between reaggregating GEODIA cells will be considered. The cellular reaggregation of GEODIA cells is mediated by an intercellular, high mol. wt. particle (15), which carries in addition to the AF several functionally different subunits~ among them, a sialyltransferase (33), a glucuronyltransferase (34) and a galactosyltransferase (34) was identified. The high mol. wt. particle hence represents a multiglycosyltransferase system associated with aggregation promoting activity (35). Recently, two extracellularly located glycosidases have been isolated from GEODIA, a B-glucuronidase and a 8-galactosidase, (35). A working hypothesis has thus been postulated to explain the coordinated aggregation and sorting-out processes in GEODIA at the molecular level (35): a) activation of the aggregation receptor (AR) by its enzymic glucuronylation~ b) adhesive recognition of two cells, mediated by the aggregation factor (AF) and the glucuronylated AR~ c) inactivation of the AR by its enzymic deglucuronylation with the membrane bound B-glucuronidase, and d) cell separation due to the loss of the recognition site (glucuronic acid) of the AR for the AF. It is quite possible that GL is involved in these controlled cellular interactions. Such a role would require the existence of a cell surface receptor for GL at least on a subpopulation of GEODIA Cells. A reasonable candidate for this receptor can be seen in the anti-aggregation receptor (AAR), recently isolated from certain non-aggregating GEODIA cells, (34). The AAR was found to carry terminal galactosyl residues, which may represent a binding site for GL. On the other hand, the aggregation receptor (AR), which contains considerable amounts of ~-galactose (I) cannot be excluded as a receptor for GL, provided that after enzymic deglucuronylation subterminal galactose residues become available. An interaction of GL with these cell surface receptors could be an essential event in the controlled sorting-out processes of reaggregating GEODIA cells. Is GL part of a surveillance system against heteroloqous cells? The ability of sponge lectins to agglutinate and to lyse heterologous sponge cells, originally observed in vitro by GALTSOFF (5), may reflect the significance of these heteroagglutinins in vivo. Based upon extensive immunological investigations with a variety of sponge cells, MacLENNAN proposed (e.g. AXINELLA) that sponge heteroagglutinins are " an expression of a true selective Not-Self recognition, rather than of a general reactivity with galactose containing polymers" (8). Several observations made by MacLENNAN with the sponge AXINELLA were confirmed by our work with GEODIA: e.g. non-reactivity between GL and a GEODIA polysaccharide preparation (Fig. I) and no agglu-
Vol. 3, No. 3
SPONGE LECTIN
413
tination of GEODIA cells by GL, contrasted by a strong agglutination brought about by an agglutinin extract from AXINELLA (unpublished). The critical experiment will thus be to show an involvement of GL as part of a surveillance system against pathogens or heterologous sponge cells. Cellular reactions against different heterografts have been investigated in another sponge organism (36). It may be quite reasonable to assume that GL integrated in the membrane of certain GEODIA cells (archaeocytes ?) does mediate such cellular rejection processes. R e c e n t l y , two very elegant hypotheses have been proposed, which draw a phylogenetic line from invertebrate lectins to the products of the major histocompatibility complex (MHC) in mammals (37,38). Taking these arguments into account, two types of recognition factors, one for Self and another for Non-Self determinants, should have evolved from a common ancestral gene by gene duplication and mutation. Enzymes (e.g. glycosyltransferases) or lectins (devoid of enzymatic activity) would represent such recognition factors, the recognized determinants being carbohydrate in nature. Certain similarities between these hypotheses and our experimental findings appear striking: The large circular proteid particle, which carries different glycosyltransferases and the aggregation factor (AF) (35) would be equivalent to the multiglycosyl enzymatic and recognition system (MGER), as termed by ROTHENBERG (38) and to an essentially similar model proposed by PARISH (39). The Self-determinant, enzymatically modified and recognized by this multienzyme-AF-complex would be represented by the aggregation receptor (AR) with a glucuronic acid terminus (I). A second recognition factor identified in GEODIA is GL, described in this paper: GL would represent an anti-Non-Self receptor in this model. If such a reactivity will turn out to be the essential function of GL, a comparison between AF and GL regarding their amino acid sequence should be highly interesting; for, accordi n g t o this hypothesis, they should have evolved from a common precursor molecule (8,37,38). REFERENCES I.
VAITH, P., MULLER, W.E.G., and UHLENBRUCK, G. On the role of ~-glucuronic acid in the aggregation of cells from the marine sponge GEODIA CYDONIUM. Dev. Comp. Immunol. (in press).
.
MULLER, W.E.G., MULLER, I., ZAHN, R.K.~ and KURELEC, B. Species-specific aggregation factor in sponges. VI. Aggregation receptor from the cell surface. J. Cell Sci. 21, 227, 1976.
.
CHATTERJEE, B.P., VAITH, P., CHATTERJEE, S., KARDUCK, D., and UHLENBRUCK, G. Comparative studies of new marker lectins for alkali-labile and alkali-stable carbohydrate chains in glycoproteins. Int. J. Biochem. (in press).
414
4.
SPONGE LECTI~
Vol. 3, No. 3
VAITH, P., MULLER, W.E.G., van MIL, A., and UHLENBRUCK, G. A novel mitogenic lectin from the marine sponge GEODIA CYDONIUM. (in preparation).
.
GALTSOFF, P.S. Heteroagglutination of dissociated sponge cells. Biol. Bull. Mar. Biol. Lab. Woods Hole 57, 250, 1929.
.
MACLENNAN, A.P. and DODD, R.Y. Promoting activity of extracellular materials on sponge cell reaggregation. J. Embryol. Exp. Morph. 18, 473, 1967.
.
GOLD, E.R. and BALDING, P. (eds.) Receptor-Specific Proteins. American Elsevier Publishing Company, Inc., New York 1975, p. 237.
.
MACLENNAN, A.P. The chemical bases of taxon-specific cellular reaggregation and "self,' - "not-self" recognition in sponges. Arch. Biol. (Bruxelles) 85, 53, 1974.
.
MULLER, W.E.G., MULLER, I., and ZAHN, R.K. Aggregation in sponges, Research in Molecular Biology, Vol. 8. Akademie der Wissenschaften und der Literatur, Mainz 1978, p. I.
10.
BALDO, B.A., UHLENBRUCK, G., and STEINHAUSEN, G. Antigalactan agglutinins from the marine sponge AXINELLA POLYPOIDES (Schmidt). Biol. Zbl. 96, 723, 1977.
11.
UHLENBRUCK, G., STEINHAUSEN, G., and PALATNIK, M. Similarity of glycoproteo-galactans in the albumin glands from ACHATINA and BORUS snails. Comp. Biochem. Physiol. 57 B, 335, 1977.
12.
UHLENBRUCK, G., STEINHAUSEN, G., and KAREEM, H.A. Different glycosubstances and galactans in the albumin gland and eggs of ACHATINA FULICA. Z. Immun.-Forsch. 152, 220, 1976.
13.
BALDO, B.A. and UHLENBRUCK, G. Quantitative precipitin studies on the specificity of an extract from TRIDACNA MAXIMA (R~ding). Carbohydr. Res. 40, 143, 1975.
14.
UHLENBRUCK, G. and SCHMID, D.O. Ein Mucoid mit Blutgruppeneigenschaften aus Rindererythrozytenstroma. Z. Immun.Forsch. 123, 466, 1962.
15.
MOLLER, W.E.G. and ZAHN, R.K. Purification and characterization of a species-specific aggregation factor in sponges. Exptl. Cell Res. 80, 95, 1973.
16.
DETERMANN, H. Gelchromatographie. York: Springer-Verlag, 1967.
17.
LOWRY, O.H., ROSEBROUGH, N.J., FARR, A.L., and RANDALL, R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265, 1951.
Berlin-Heidelberg-New
Vol. 3, No. 3
SPONGE LECT!N
4/5
18.
ASHWELL, G. New colorimetric methods of sugar analysis. In: Methods in Enzymology VIII. Neufeld, E.F. and Ginsburg, V. (eds.). Academic Press New York and London 1966, p. 85.
19.
LAEMMLI, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680, 1970.
20.
WEBER, K. and OSBORN, M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406, 1969.
21.
DAHR, W., UHLENBRUCK, G.~ GUNSON, H.H., and van der Hart, M. Molecular basis of Tn-polyagglutinability. Vox Sang. 29, 36, 1975.
22.
MOLLER, W.E.G., MOLLER, I., and ZAHN, R.K. Two different aggregation principles in reaggregation process of dissociated sponge cells (GEODIA CYDONIUM). Experientia 30, 899, 1974.
23.
SIMPSON, D.L., ROSEN, St.D., and BARONDES, S.H. Discoidin, a developmentally regulated carbohydrate-binding protein from DICTYOSTELIUM DISCOIDEUM. Purification and characterization. Biochemistry 13, 3487, 1974.
24.
UHLENBRUCK, G., STEINHAUSEN, G., and BALDO, B.A. Galactane und Anti-Galactane. Verlag Josef Stippak, Aachen, Postfach 1262, 1975.
25.
LARM, O. and LINDBERG, B. The pneumococcal polysaccharides: A re-examination.°In: Advances in Carbohydrate Chemistry and Biochemistry (Tipson, R.St. and Horton, D., eds.). Academic Press New York-San Francisco-London 1976, p. 295.
26.
GLOCKNER, W.M., NEWMAN, R.A., and UHLENBRUCK, G. Carbohydrate structure and serological behaviour of "antifreeze" glycoproteins from an antarctic fish. Biochem. Biophys. Res. Comm. 66, 701, 1975.
27.
GLOCKNER, W.M., NEWMAN~ R.A., DAHR, W., and UHLENBRUCK, G. Alkali-labile oligosaccharides from glycoproteins of different erythrocyte and milk fat globule membranes. Biochim. Biophys. Acta 443, 402, 1976.
28.
NEWMAN, R.A. and UHLENBRUCK, G. Investigation into the occurence and structure of lectin receptors on human and bovine erythrocyte, milk-fat globule and lymphocyte plasma membrane glycoproteins. Eur. J. Biochem. 76, 149, 1977.
29.
BRETTING, H. and KABAT, E.A. Purification and characterization of the agglutinins from the sponge AXINELLA POLYPOIDES and a study of their combining sites. Biochemistry 15, 3228, 1976.
416
SPONGE LECTIN
YOI. _3~ NO. 3
30.
MOLLER, W.E.G., MOLLER, I., and ZAHN, R.K. Species-specific aggregation factor in sponges. V. Influence on programmed syntheses. Biochim. Biophys. Acta 418, 217, 1976.
31.
MOLLER, W.E.G., BEYER, R., PONDELJAK, V., MOLLER, I., and ZAHN, R.K. Species-specific aggregation factor in sponges. XIII. Entire and core structure of the large circular proteid particle from GEODIA CYDONIUM. Tissue and Cell 10, 191, 1978.
32.
MOSCONA, A.A. Cell aggregation: Properties of specific cell-ligands and their role in the formation of multicellular systems. Dev. Biol. 18, 250, 1968.
33.
MOLLER, W.E.G., ARENDES, J., KURELEC, B., ZAHN, R.K., and MOLLER~ I. Species-specific aggregation factor in sponges. IX. Sialyltransferase associated with aggregation factor. J. Biol. Chem. 252, 3836, 1977.
34.
MULLER, VAITH, XVIII. gation 1978.
35.
MULLER, W.E.G., ZAHN, R.K., KURELEC, B., MOLLER, I., UHLENBRUCK, G., and VAITH, P. Aggregation of sponge cells. A novel mechanism of controlled intercellular adhesion, basing on the interrelation between glycosyltransferases and glycosidases. J. Biol. Chem. 254, 1280, 1979.
36.
CHENG, T.C., YEE, H.W.F., RIFKIN, E., and KRAMER, M.D. Studies on the internal defense mechanism of sponges. III. Cellular reactions in TERPIOS ZETEKI to implanted heterologous biological materials. J. Invert. Pathol. 12, 29, 1968.
37.
KOLB, H. On the phylogenetic origin of the immune system, a hypothesis. Dev. Comp. Immunol. I, 193, 1977.
38.
ROTHENBERG, B.E. The self recognition concept: An active function for the molecules of the major histocompatibility complex based on the complementary interaction of protein and carbohydrate. Dev. Comp. Immunol. 2, 23, 1978.
39.
PARISH, C.R. Simple model for self-non-self-discrimination in invertebrates. Nature 267, 711, 1977.
W.E.G., ZAHN, R.K., KURELEC, B., UHLENBRUCK, G., P., and MULLER, I. Aggregation of sponge cells. Glycosyltransferases associated with the aggrefactor. Hoppe-Seyler's Z. Physiol. Chem. 359, 529,