Isolation and characterization of agglutinins from the hemolymph of an acorn barnacle, Megabalanus volcano

Isolation and characterization of agglutinins from the hemolymph of an acorn barnacle, Megabalanus volcano

DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. Ii, pp. 297-307, 1987. 0145-305X87 $3.00 + .00 Printed in the USA. Copyright (c) 1987 Pergamon Journals...

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DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. Ii, pp. 297-307, 1987. 0145-305X87 $3.00 + .00 Printed in the USA. Copyright (c) 1987 Pergamon Journals. All rights reserved.

I S O L A T I O N AND CHARACTERIZATION OF A G G L U T I N I N S H E M O L Y M P H OF AN A C O R N B A R N A C L E , M E G A B A L A N U S

FROM THE VOLCANO

H. K a m i y a , K. M u r a m o t o , R. G o t o School of Fisheries Sciences, Kitasato University, Sanriku-cho, Kesen-gun, Iwate 022-01, Japan

ABSTRACT

TwO a g g l u t i n i n s . MVA-1 a n d MVA-2, w e r e i s o l a t e d from the hemolymph of the acorn barnacle. Megabalanus volcano. They agglutinated human erythrocyte5 irrespective o f t h e ABO b l o o d g r o u p a n d a l s o r a b b i t and sheep blood cells. Lactose and fetuin strongly inhibited the hemagolutinating activity. D-qalactose, D-arabinose and N-acetylneuramlnic acid were also moderate inhibitors. In sodium dodecyl sulfatepolyacrylamide gel electrophoresis, both Mvg-I and MVA-2 g a v e a s i n g l e band corresponding to 38,000 daltons. It split into one major band with a molecular weight of 23.000 in the presence of 2-mercaptoethanol. The two aqqlutinins showed the same apparent molecular weight of 116,000 by gel filtration. In isoelectric focusing MVA-I s h o w e d o n e b a n d a t pH 4 . 8 , w h e r e a s MVA-2 g a v e a m a i n b a n d a t pH 4 . 4 w i t h f e w f a i n t ones in the r a n g e b e t w e e n pH 4 . 0 a n d 4 . 8 . The aqglutinins were 91ycoproteins containing D-mannose and L-fucose as carbohydrate components. No p r e c i p i t a t i o n reaction was observed in Ouchterlony immuno-diffusion tests using rabbit antisera against the agglutinins from the phylogenetically related Megabalanus rosa.

INTRODUCTION Hema991utinins have been demonstrated variety of marine invertebrates (1,2). tion of these humoral agqlutinins has understood. They may play a role in materials like vertebrate antibodies in lular and humoral defense systems. They Ca++-transport, sugar-transport or could

in the hemolymph of a The physiological funcnot yet been fully recognition of foreign cooperation with celcould also function in maintain +h~ ~ + . . . . .

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thetic symbionts in shellfishes (3-8). In spite of the interesting physiological nature of marine invertebrate agglutinins, only a limited number of molecules have been purified and characterized up to date. In the c o u r s e of our s c r e e n i n g for a g g l u t i n i n s in m a r i n e i n v e r t e b r a t e s , we found hemagglutinating a c t i v i t y in the h e m o l y m p h of a c o r n b a r n a c l e s (9). We h a v e isolated m u l t i p l e a g g l u t i n i n s s h o w i n g an o p s o n i n p r o p e r t y from the coelomic f l u i d of the a c o r n b a r n a c l e M e g a b a l a n u s rosa These a g g l u t i n i n s are u n i q u e in their subunit structures containing b o t h inter- and i n t r a c h a i n d i s u l f i d e b r i d g e s (10-12). Recently. we h a v e d e t e r m i n e d the c o m p l e t e a m i n o - a c i d s e q u e n c e (138 amino acid r e s i d u e s ) of o n e of these m u l t i p l e a g g l u t i n i n s , B R A - 3 (13). It is of interest to know w h e t h e r or not other acorn b a r n a c l e of the same genus possesses humoral agglutinins of similar molecular structures. T h e p u r p o s e of the p r e s e n t s t u d y was to isolate and characterize agglutinins from the h e m o l y m p h of M e g a b a l a n u s volcano.

MATERIALS AND METHODS A n i m a l s and c o l l e c t i o n of h e m o l y m p h . Megabalanus volcano was c o l l e c t e d at O k i - n o - s h i m a Island. Kochi, J a p a n in July, 1985. T h e h e m o l y m p h was o b t a i n e d at the c o l l e c t i o n s i t e by mechanical damage to the calcarious base of animals and a l l o w i n g the h e m o l y m p h to flow into a plastic bag. The hemolymph was centrifuged at 10,000 r p m for 30 min, and the s u p e r n a t a n t was kept in a f r o z e n state.

Purification of agglutinins. For the purification of the agglutinins, I00 ml of h e m o l y m p h was a p p l i e d to a c o l u m n (4.4 x 32 cm) of S e p h a r o s e 4B {Pharmacia} w h i c h had b e e n treated with HCI and s u b s e q u e n t l y e q u i l i b r a t e d w i t h 50 mM T r i s - H C l b u f f e r ( p H 8.0} c o n t a i n i n g 10 mM CaCI 2 and 0.85 % NaCI. The column was washed thoroughly with the same buffer to remove unbound proteins. The agglutinins were then eluted with 0.2 M Dg a l a c t o s e in the buffer. E a c h f r a c t i o n was a n a l y z e d for a b s o r p tion at 280 nm and for h e m a g g l u t i n a t i n g a c t i v i t y against rabbit erythrocytes. The agglutinins eluted were ultrafiltered, d i a l y z e d a g a i n s t 50 mM a m m o n i u m bicarbonate and freeze-dried. H i g h s p e e d gel f i l t r a t i o n was c a r r i e d out on a c o l u m n (2.16 x 60 cm) of T S K G - 3 0 0 0 S W (Toyo Soda) w i t h 0.25 M p h o s p h a t e buffer, pH 7.0, at a flow r a t e of 2 ml/min. The a c t i v e f r a c t i o n was further separated by anion exchange chromatography on Mono-Q (Pharmacia). Samples d i s s o l v e d in 50 mM p h o s p h a t e buffer" (pH 6.8) w e r e a p p l i e d to a M o n o - Q HR5/5 c o l u m n (0.5 x 5 cm) equil i b r a t e d w i t h the s a m e buffer. A g g l u t i n i n s w e r e e l u t e d from the c o l u m n w i t h a g r a d i e n t of 0 - 0 . 2 M NaCI (10 ml), 0.2 M NaCI(10 ml), and then 0.2-0.5 M NaC1 (10 ml) at a flow r a t e of 0.5 ml/min. T h e e l u a t e was m o n i t o r e d by a b s o r p t i o n at 280 nm and t e s t e d for h e m a g g l u t i n a t i n g a c t i v i t y a g a i n s t r a b b i t e r y t h r o c y t e s . A n a l y t i c a l methods. P r o t e i n c o n c e n t r a t i o n was e s t i m a t e d by the m e t h o d of L o w r y et al. (14) u s i n g b o v i n e serum albumin as a standard. C a r b o h y d r a t e c o n t e n t was d e t e r m i n e d as d e s c r i b e d by D u b o i s (15) u s i n g g l u c o s e as a s t a n d a r d . Amino acid analysis

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was c a r r i e d out according to our method using 1 p9 of each of the purified agglutinins (16). Tryptophan was d e t e r m i n e d by acid hydrolysis in the presence of phenol (17). Carbohydrate composition was d e t e r m i n e d by high-performance liquid chromatography (Hypersil ODS, 3 pm p a r t i c l e , 0.46 x 50 cm) of their chromophoric hydrazones (18). Briefly, purified agglutinins (10-20 p9) were hydrolyzed w i t h 2M t r i f l u o r o a c e t i c acid for 4 hours at 105~ and the released sugars were derivatized with dabsyl hydrazide. Analytical electrophoresis was performed on 7~ polyacrylamide gel (PAGE) i n T r i s - H C 1 g l y c i n e buffer, pH 8 . 3 . The molecular weights of agglutinins and their subunits were determined by fast protein liquid chromatography (FPLC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) using standard proteins (Oriental Yeast Co. and Pharmacia). FPLC o n a S u p e r o s e 12 H R 1 6 / 3 0 c o l u m n ( 1 . 6 x 30 cm, Pharmacia) was performed with 0.2 M ammonium bicarbonate at a flow rate of 0.5 ml/min. SDS-PAGE was d o n e i n 1 2 . 5 % gel with and without 2-mercaptoethanol. Electrofocusing o n 7X g e l was carried out at 200 V for 6 hours using Bio-Lyte 3/10 as carrier ampholite at a concentration o f 2X. Gels were stained with Coomassie brilliant blue R-250. Hemagglutination test. Two f o l d s e r i a l dilutions of hemolymph or purified agglutinins were made in micro-titer plates. Estimation of agglutinating activity against various erythrocytes was c a r r i e d o u t u s i n g a 2% s u s p e n s i o n in 0.85~ saline containing 10 mM C a C I 2 . The results were expressed as the maximum titer value for positive agglutination. Hemagglutination inhibition tests. Inhibition tests by simple sugars W e r e d o n e u s i n g t h e h e m o l y m p h 35 w e l l a s t h e p u r i f i e d agglutinins diluted to a titer of 1:2 °. The solutions were allo~ed to react with various concentrations of carbohydrate for one hour, and 2X r a b b i t erythrocyte suspension washed with saline was t h e n a d d e d t o t h e m i x t u r e and the test read after another one hour. Test for heat stability. Heat stability tests were performed by incubating the hemolymph at 40, 60 a n d 80~ for 15 minutes. After cooling to room temperature the hemagglutination titer was determined as described above. Test for Ca++-dependency of the hemaqqIutinatinq activity. One milliliter of hemolymph was d i a l y z e d against ! liter o f 25 mM EDTA i n 0 . 2 M T r i s - H C 1 b u f f e r t p H 8.0) for 24 hours, and then against 0.85X saline f o r 48 h o u r s . An a l i q u o t was t e s t e d for hemagglutinatin9 activity in the presence o f lO mM C a C 1 2 , MgCI 2 , o r MnCI 2 , a n d i n c a l c i u m - f r e e saline. Immuno-diffusion tests. Ouchterlony immuno-diffusion tests were carried out using antisera against aggIutinins isoIated from M. rosa.

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RESULTS

Hemaqqlutinating activity of hemolymph. Pooled hemolymph ~gglutinated rabbit (titer 2 -/) a n d s h e e p e r y t h r o c y t e s (titer 2 ), and also reacted with human blood cells irrespective of the ABO blood group (titer 21Oll). The agglutinins were heat-labile. The initial hemagglutinating activity of 1:210 for human AB erythrocytes was lowered to 1:22 by heating at 60~ f o r 15 minutes. I t was c o m p l e t e l y destroyed at 80~ . The hemagglutinating activity o f M. volcano agglutinins was d e p e n d e n t on the presence o f Ca + + . EDTA-treated hemolymph did not show any hemagglutinating activity in the absense o f Ca + + . The addition o f 10 mM o f C a C I 2 t o t h e r e a c t i o ~ mixture restored the original hemagglutinating activity. Mg 4+ a n d Mn ++ c o u l d n o t s u b s t i t u t e f o r Ca ++ . The specificity of agglutination was i n v e s t i g a t e d by absorption of hemolymph with erythrocytes. Absorption with h u m a n AB e r y t h r o c y t e s greatly reduced hemagglutinating activity to rabbit erythrocytes and vice versa. The activity of the hemolymph and the purified agglutinins against rabbit blood cells was s t r o n g l y inhibited by lactose and fetuin (Table 1). D-galactose, L-arabinose, and Nacetylneuraminic acid were also moderate inhibitors. Other simple sugars, such as D-glucose, D-xylose, and N-acetyl-Dgalactosamine also showed weak inhibitory activity against the hemagglutination of the hemolymph.

TABLE 1 Hemagglutination-Inhibition Inhibitor

D-galactose D-glucose L-arabinose D-xylose lactose N-acetylneuraminic acid N-acetyl-Dgalactosamine fetuin Meffabalanus were diluted to

Initial Cone.

Assay

Maximum I n h i b i t i n g Hemolymph

Sugar MVA-1

Dilution MVA-2

200mM 200mM 200mM 200mM 200mM 200mM

1:8 1:2 1:8 1:2 1:64 1:8

1:8 1:2 1:64 1:4

1:8 1:2 1:64 1:4

200mM

1:2

1:

1:

0.1~

1:4

1:4

volcano titer of

hemolymph and the 8 for these test.

purified

1

I

1:4 agglutinins

Purification of aqqlutinins. Agglutinins were purified by affinity chromatography on acid-treated Sepharose 4B. After applying 100 ml o f t h e h e m o l y m p h ( 1 . 1 g p r o t e i n ) to the column and washing with Tris-HC1 buffer, the agglutinins retained were eluted with 0.2 M D-galactose in the buffer. The hemagglutinat ~ ing activity for rabbit erythrocytes was c o i n c i d e d well with the protein peak as shown in Figure 1, and 14.3 mg o f the agglutinins was r e c o v e r e d in the galactose solution. About 80~ of the initial hemagglutinating activity for rabbit blood cells ~as

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A230 Titer "200

2.0-

1.0-

-100 !

b,Q

! t t

i i i

l

50

100

190

Fraction Number FIG. 1 Affinity chromatoqraphy of ~ vplcano a g g l u t i n i n s on acid-treated Sepharose 4B. 100 ml o f t h e h e m o l y m p h w a s a p plied t o a c o l u m n ( 3 . 2 x 38 c m ) . The column was washed with 0.2 M D-galactose in the buffer (indicated by arrows). Fractions of 8 ml were analyzed for UV a b s o r b a n c e (*--~) and for bemag91utinatin9 activity against rabbit erythrocytes (~-~) The fractions 192-197 were pooled.

FIG. 2 High speed gel filtration of ~ v o l c a n o aqglutinins. 7 m9 o f aqqlutinins were applied t o a c o l u m n o f TSK G-3OOOSW ( 2 . 1 6 x 60 c m ) . Elution was carried out with 0.2 M phosphate buffer (pH 7 . 0 1 a t a f l o w rate of 2 ml/min. A main active fraction (horizontal bar) was pooled.

U ¢-

<

!

5O

rain 75

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MVA-I

ol 0.3 1

MVA-2

I~I

M NaCI -0.5

0.2

0.1 0.1 10

30

50

70

rain

90

FIG.3 Fast protein liquid chromatography of Megabalanus volcano agqlutinins on Mono-O. 1 . 3 mg o f a g g l u t i n i n s was a p p l i e d to a column of Mono-O HR 5 / 5 e q u i l i b r a t e d w i t h 50 mM o f p h o s p h a t e buffer (pH 6 . 8 } . Agqlutinins were eluted from the column with a gradient o f 0 - 0 . 2 M, 0 . 2 M, a n d 0 . 2 - 0 . 5 M NaC1 i n t h e b u f f e r at a flow rate of 0.5 ml/min.

recovered in this fraction. Susequent separation on a TSK G3000SW column gave one major protein p e a k ( 9 . 5 mg) a n d t w o faster-eluting minor peaks (Figure 2}. As s h o w n i n Figure 3, FPLC of the major peak on a Mono-O column gave two active fractions, ~VA-1 a n d MVA-2. MVA-1 a n d MVA-2 w e r e e l u t e d with 0.2 M NaC1 a n d 0 . 3 7 M NaC1 i n p h o s p h a t e buffer, respectively. In a typical run, 3 0 0 ml o f t h e h e m o l y m p h g a v e 4 . 9 mg o f MVA-1 a n d 6 . 5 m9 o f MVA-2. Overall yields of these agglutinins in the h e m o l y B p h o f M. volcano w e r e 0 . 1 4 % f o r MVA-1 a n d 0 . 1 9 % f o r MVA2, respectively. Chemical Properties. To test the purity o f MVA-I a n d MVA-2. polyacrylamide 9el electrophoresis was p e r f o r m e d . I n SDS-PAGE, both MVA-1 a n d MVA-2 g a v e a s i n g l e band corresponding to 38,000 daltons in the absence of 2-mercaptoethanol. It split into one major band of 23,000 daltons under reduced conditions {Figure 4). The apparent molecular weight calculated b a s e d o n FPLC o n a Superose 12 c o l u m n w a s 1 1 6 , 0 0 0 f o r b o t h i n t a c t agqlutinins. In

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analytical isoelectric focusing MVA-I g a v e a someuhat diffused b a n d a t pH 4 . 8 w h i l e MVA-2 s h o w e d a m a j o r b a n d a t pH 4 . 4 a n d f e w faint b a n d s i n t h e pH r a n g e o f 4 . 0 t o 4 . 8 . The similar amino acid compositions of the two agglutinins were observed as shown in Table 2.

a

d

e

f

koi 94 67 43 W 30

,-, D

20j

gll

--

FIG. 4 SDS-Polyacrylamide gel electrophoresis of Meqabalanus volcano agglutinins. a.d, standard proteins: b,e, MVA-1, c , f , MVA-2; a - c , reduced with 2-mercaptoethanol. Standard proteins used: phosphorylase b (94 kD), albumin (64 kD), ovalbumin (43 kD), carbonic anhydrase (30 kD), trypsin inhibitor (20.1 kD), ~-lactoalbumin (14.4 kD).

Ilt

14./, MVA-1 c o n t a i n e d 3.5~ of carbohydrate by weight, and sugar components analyzed were D-mannose and L-fucose in an equal ratio. MVA-2 ( 3 . 1 ~ c a r b o h y d r a t e by weight) also showed the same results. In immuno-diffusion tests, M. v o l c a n o agglutinins were found not to cross-react with rabbit antisera against agglutinins isolated f r o m M. r o s a .

Amino Acid Agglutinins Amino Asp Glu Set Thr Gly Ala Pro Val Arg

Acid

Composition (Mol%).

MVA-1 8.3 9.5 7.3 5.4 4.0 8.5 5.1 6.8 4.6

MVA-2 9.0 10.0 7.3 5.4 6.1 7.8 5.3 6.4 4.0

TABLE 2 of Megabalanus Amino Met Ile Leu Trp Phe Cys Lys His Tyr

Acid

(half)

volcano MVA-I

MVA-2

3.2 5.6 5.8 5.9 3.4 3.5 4.5 3.6 5.0

1.1 5.0 5.9 4.7 3.0 3.5 5.0 4.4 6.3

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DISCUSSION The aqqlutinins o f M. v o l c a n o were purified effectively by affinity chromatography using acid-treated Sepharose 4B a 5 a b sorbent and 0.2 M D-galactose for elution, as in the purification of M. rosa aqqlutinins. Separation on a Mono-Q column resulted in two major components, MVA-1 a n d MVA-2. These aq9Iutinin5 are similar to each other in their moIecular structures and binding sites, but have different isoelectric points. "Isolectins" which are composed of a variety of different isoeIectric points of lectins have been already found in the horseshoe crab, Limulus poIyphemus (19) and in the sponge, haptos papilata(20). The apparent molecular ueiqht o f M. volcano aqglutinins were estimated to be ll6,000 b y FPLC o n S u p e r o s e 12. On t h e b a s i s of SDS-PAGE described here, we p r o p o s e that both MVA-I a n d MVA-2 a r e c o n s t i t u t e d of three of the identicaI dimers having the same subunits (23,000 daltons) cross-linked by disulfide bond(s). These molecular structures o f M. v o I c a n o aq91utinins are quite unique among the marine invertebrate aqglutinins which are rarely known to contain interchain disulfide bonds (1). On t h e o t h e r hand, the structures o f M. v o l c a n o aq9Iutinins are similar to those of the 9alactose-bindin9 aqqlutinin isolated from another species of the same genus, M. rosa with a similar molecular weight. BRA-2 ( 1 4 0 , 0 0 0 daltons), a major component o f M. rosa a991utinins consists of subunits (22,000 daltons) cross-linked by disulfide bonds (12). In spite of the simiIarity of the molecular structures, there may b e c o n siderable differences in the amino-acid sequences, since no common a n t i g e n i c i t y was detected by Ouchterlony immuno-diffusion tests. In inhibition tests with simple sugars, lactose, Dgalactose and N-acetylneuraminic acid showed inhibitory activity against a991utinins from the two species. These sugars have been aiso reported to inhibit a variety of marine invertebrate a991utinins from the butter cIam (21), oyster (22), lobster (23), crab (24)and others. D-mannose, however, which was found to inhibit the hema991utinatin9 activity o f M. rosa a99Iutinins did not affect hemagglutination by M. volcano aqglutinins. Hemagglutinating activity o f M. volcano agglutinins was dependent on the presence o f Ca ++ , and no hemagglutination was observed in the calcium-free saline. Mg ++ a n d Mn +÷ c o u l d n o t s u b stitute f o r Ca ++ . Thus, in the early stage of this study we failed to detect hemaggIutinating activity of the hemolymph against rabbit erythrocytes w h e n we adopted 2X suspension in hlsever's solution which contains sodium citrate and citric acid, chelating agents f o r Ca ++ a n d Mg + + . In contrast, M. rosa agglutinins, in a strict sense, are not dependent on the presence o f Ca ++ f o r their hemagglutinating activity. They could not bind to rabbit erythrocyte ghosts in the absence of Ca ++ , b u t b e c a m e b o u n d t o s o m e e x t e n t after the addition o f NaCI to the reaction mixture (12). It is of interest t h a t M. volcano contains "isolectins" as major agglutinins and only trace amounts of agglutinins with different molecular weights in its hemolymph, ~hile M. r o s a p o s sesses multiple agglutinins having molecular weights ranging from 64,000 to 330,000 daltons (12). Among B a l a n o m o r p h a only acorn barnacles belonging to the genus Megabalanus so far seem

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to to have D-galactose-binding agglutinins of unique molecular structures in their hemolymph. Balanus balanoides also has an agglutinin with an opsonic property. The molecular structure and binding property of this agglutinin are, however, quite differnt from those of Megabalnus agglutinins (11). In the amino-acid sequence determination of BRA-3, a M. rosa agglutinin, we h a v e f o u n d t h a t it exhibited significant sequence homology with the amino-terminal half of Sarcophaga pereqrina lectin. In particular, the location of four half-cystine residues of both lectins are apparently well conserved. We h a v e also recognized the Cys-Pro-Pro-Cys sequence involved in the interchain disulfide bridges in the subunits (12). It is worth noting that this hinge segment is common to the inter-heavy chain disulfide bridges of human immunoglobulin IgG1. It is our future aim to determine whether or not these sequences are maintained in other Meqabalanus agglutinins.

ACKNOWLEDGEMENTS The a u t h o r s express t h e i r thanks t o Dr. S. Ikegami, F a c u l t y of Applied B i o l o g i c a l Science, Hiroshina U n i v e r s i t y , f o r g i v i n g us a chanse t o j o i n t h e 8th Research Cruise of Toyoshio-Maru, a r e s e a r c h v e s s e l of Hiroshima U n i v e s i t y in 1985. We a r e a l s o indebted t o Dr. R. K a d o , School of F i s h e r i e s S c i e n c e s , Kitasato U n i v e r s i t y , f o r i d e n t i f i c a t i o n of t h e specimens.

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Received: March, 1987 Accepted: March, 1987

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