Isolation and characterization of a hemagglutinin with affinity for lipopolysaccharides from plasma of the crayfish Pacifastacus leniusculus

Developmental and ComparativeImmunology,Vol. 17, pp. 407-418, 1993 0145-305X/93$6.00 + .00 Printed in the USA. All rights reserved. Copyright © 1993 PergamonPress Ltd.

ISOLATION AND CHARACTERIZATION OF A HEMAGGLUTININ WITH AFFINITY FOR LIPOPOLYSACCHARIDES FROM PLASMA OF THE CRAYFISH Pacifastacus leniusculus Petr Kop&~ek, Libor Grubhoffer, and Kenneth S6derh&ll Department of Physiological Botany, University of Uppsala, Villav~lgen 6, 752 36, Uppsala, Sweden

(Submitted September 1992;Accepted July 1993) DAbstract--A hemagglutinin with a high specific activity against trypsinized rabbit erythrocytes was identified in plasma of the freshwater crayfish Pacifastacus leniusculus. The activity of this crayfish hemagglutinin could be inhibited by sialoglycoproteins such as porcine stomach mucin, bovine submaxillary mucin, fetuin, and ovalbumin. However, the involvement of sialic acid in its binding specificity could not be unambiguously proven. Furthermore, the hemagglutinating activity in the crayf'tsh plasma could be specifically inhibited by lipopolysaccharide from E. coHK-235, which might indicate a recognition role for this hemagglutinin. This hemagglutinin, which accounts for less then 0.01% of the total plasma protein, was purified to near homogeneity using affinity chromatography on a Fetuin-Sepharose 4B column. The molecular mass of the unreduced protein as revealed by sodium dodecyl sulphate electrophoresis in polyacrylamide gel was found to be 420,000 Da. Upon reduction with dithiothreitol the hemagglutinin dissociated to several subunits with masses ranging from 65,000 to 80,000 Da. Affinoblotting with peroxidase labelled lectins indicated that the hemagglutinin was likely to be a glycoprotein. DKeywords--Crayfish plasma; Hemagglutinin; Sialic acid; Lipopolysaccharide.

Nomenclature BSM DTT HA kDa LPS

bovine submaxillary mucin dithiothreitol hemagglutinationactivity kilodalton lipopolysaccharide

Address correspondence to Kenneth SOderh~ill.

PAGE PLH PSM RBC R-T SDS UP

polyacrylamide gel electrophoresis Pacifastacus leniusculus hemagglutinin porcine stomach rnucin red blood cells trypsinized rabbit erythrocytes sodium dodecyl sulphate ultracentrifuged crayfish plasma

Introduction H e m a g g l u t i n i n s (lectins) are ubiquitously detected among the Arthropoda both as humoral or membrane associated components (1). The physiological functions of lectins in invertebrates still remain obscure (2,3), but these proteins are considered to be involved in defense reactions of invertebrates against pathogen invasion (1-3); in a few cases lectins have been shown to function as opsonins (4,5). Previously, numerous arthropod lectins have been d e s c r i b e d and several have been purified and characterized [for reviews see (6,7)]. Lectins purified from the Merostomata and Crustacea are best represented by limulin from the horseshoe crab Limulus polyphemus (8-10); carcinoscorpin from the Indian horses h o e c r a b Carcinoscorpius rotunda cauda (11); and agglutinins from the Japanese horseshoe crab Tachypleus tridentatus (12); the marine crab Cancer antennarius (13); and Lagl and Lag2 from the lobster Homarus americanus (14,15). Most of these proteins o c c u r in their native form as high molecular weight aggregates of noncovalently bound subunits and, in general, are reported to be specific for sialic acid or its derivatives (16).

407

408

P. Kop~_(~ek, L. Grubhoffer, and K. SOderha_ll

Little is known about lectins in crayfish. The hemolymph of the crayfish Procambarus clarkii was reported to contain an agglutinin with a high molecular mass (>150 kDa) capable of agglutinating marine bacteria as well as vertebrate erythrocytes (17). In this paper we report the identification, partial purification, and characterization of a hemagglutinin termed P L H from plasma of the crayfish Pacifastacus

2 - a c e t a m i d o - 2 - d e o x y - 3 - O-13-D-galactosyl-D-galacto-pyranose (13-D-GaI[1->3]D-GaI-NAc), 2-keto-3-deoxyoctonate (KDO), and other mono- and disaccharides (Table 1), fetuin (type III), bovine submaxillary mucin (BSM, type I), porcine stomach mucin (PSM, type III), hyaluronic acid, laminarin as well as all lipopolysaccharides (phenol extracted preparations) were p u r c h a s e d from Sigma (St. Louis, MO). Ovalbumin, ovomucoid, and peroxidase were from Serva (Heidelberg, Germany). Asialo-BSM and asialo-fetuin were prepared by acidic hydrolysis with 0.05 M H2SO4 for 1 h at 80°C followed by dialysis against phosphate buffered saline. N-Acetylneuraminic acid content was determined using the thiobarbituric acid method according to Aminoff (18).

leniusculus.

Materials and Methods

Animals Freshwater crayfish Pacifastacus leniusculus were collected from Lake HalmsjOn, Uppland, Sweden and kept in aquaria in aerated tap water at 10°C. Only intermoult animals were used.

Chemical Reagents N-Acetylneuraminic acid, N-acetyllactosamine, N-acetylneuraminyl-lactose,

Erythrocyte Preparation and Treatment E r y t h r o c y t e s were kept in sterile 3.8% (w/v) Na3-citrate and prior to use were washed 3 x in 0.15 M NaCl. Trypsinisation of rabbit RBC was per-

Table 1. Inhibition of Hemagglutination Activity of Crayfish Plasma by Various Carbohydrates.

Inhibitor

Concentration Required for 50% Inhibition* (mM)

D-g IUcose D-glucosamine o-glucuronic acid Trehalose L-fucose Cellobiose N-acetylneuraminic acid N-acetyllactosamine N-acetylneuraminyl-lactose I~-D-GaI[1 ->3]-o-GaI-NAc 2-keto-3-deoxyoctonate

500 500 500 500 500 333 >200 >50 >50 >50 >20

Carbohydrates found to be noninhibitory at 500 mM concentration: D-galactose, D-mannose, D-mannosamine, D-lactose, D-galacturonic acid, N-acetyl-~-D-mannosamine, N-acetyI-Dglucosamine, N-acetyl-galactosamine, methyl-~x-D-glucoside, methyl-e-D-mannoside. * Inhibition tests were performed with UP adjusted to 1 HA unit and with R-T erythrocytes. Consistent results were achieved with R-T, Nb, and R-T, Nv erythrocytes (see Table 2).

Hemagglutinin from crayfish plasma

formed with 0.01% (w/v) trypsin (type III, Sigma) under slow rotation for 1 h at 20°C. Pronase treatment of mouse RBC was with 0.01% (w/v) pronase (KochLight Ltd., England) for 2 h at 20°C. For neuraminidase treatment the erythrocytes were incubated with a 1% (v/v) solution of bacterial neuraminidase (Nb) from Vibrio cholerae or 1% (v/v) viral neuraminidase (Nv) from mumps virus (SEVAC, Prague, Czech Republic) for 2 h at 37°C. Under these conditions the neuraminidases cleaved 4.5 ixmol and 12.9 ixmol of N-acetylneuraminic acid from fetuin used as substrate per 1 mg of Nb and Nv, respectively. Glutaraldehyde fixation of RBC was achieved with 2.5% (v/v) glutaraldehyde in phosphate buffered saline. All treatment procedures were followed by 3 × washing in 0.15 M NaC1.

Hemagglutination and Hemagglutination Inhibition Assays The determination of hemagglutination activity (HA) was performed in 96well U-shaped microtitration plates by serial two-fold dilution of 50-~L samples in 50-p~L hemagglutination buffer (HT, consisting of 0.05 M Tris-HCl, 0.15 M NaCI, and 0.1 M CaClz, pH 8.0) after which 50 ixL of 1% (v/v) suspension of native or treated erythrocytes in 0.15 M NaCI was added. The titer of HA was evaluated after 2 h incubation at 20°C and expressed as the reciprocal of the last sample dilution causing visible agglutination. The amount of hemagglutinin in the last test well with positive hemagglutination was defined as 1 HA unit. The hemagglutination inhibition test was performed to determine the inhibitor concentration that caused a 50% decrease of HA (HIT 50%). Fifty microliters of inhibitor (sugar, glycoconjugate) were serially diluted in the HT buffer to which 50 ixL of ultracentrifuged plasma

409

(see below) diluted to contain 1 HA unit and 50 ixL of 1% (v/v) suspension of erythrocytes were added. The HIT 50% concentrations were evaluated after 1 h at 20°C from the lowest inhibitor concentration that completely inhibited the activity of 1 HA unit.

Plasma Preparation Hemolymph from 15 crayfish (0.5-1.0 mL/animal) was collected with 1.2-mm needles in ice-cold buffer (0.01 M Nacacodylate, 0.25 M sucrose, and 0.1 M CaCI2, pH 7.0). The hemocytes were centrifuged at 800 × g for 10 rain (4°C), and the supernatant (plasma) stored at -25°C until used. Thawed plasma was first assayed for its HA titer and batches with titers of/> 128 were pooled and further processed by dialysis overnight against distilled water at 4°C. The precipitate formed during dialysis was removed by centrifugation at 2,500 × g for 15 min, 10°C. Then the supernatant was ultracentrifuged at 200,000 x g for 2.5 h at 4°C using a Beckman 70.1 Ti rotor to remove the majority of the hemocyanin. The resulting clarified supernatant, referred to as ultracentrifuged plasma (UP), was used as the starting material for hemagglutinin purification. For longterm storage the UP was concentrated approximately 10-fold by ultrafiltration on an Amicon PM I0 membrane, freezedried, and kept at 4°C until used.

Purification of Crayfish Hemagglutinin A Fetuin-Sepharose 4B affinity column was prepared as follows: 90 mg of fetuin was coupled to 3 g of CNBractivated Sepharose 4B (Pharmacia, Uppsala, Sweden) according to manufacturer's instruction. The coupling efficiency was determined to be 98%. The sorbent was equilibrated with Ca-TN3

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P. Kop~(~ek, L. Grubhoffer, and K. S6derh&ll

buffer consisting of 0.033 M Tris/HCl, 0.5 M NaCI, and 0.01 M CaCI2, pH 8.0, and a 1 x l0 cm column was poured. Sufficient solid ammonium sulfate was added to the 200 mL of pooled ultracentrifuged plasma (see above) to achieve 50% saturation and mixed overnight at 4°C. The precipitate was collected by centrifugation (2,500 × g, 15 rain) and dissolved in 2 mL of distilled water. The composition of the solution was adjusted to be equal to the Ca-TN3 buffer, pH 8.0, and the resulting volume was divided into three aliquots. They were then chromatographed in three consecutive runs on the Fetuin affinity column preequilibrated with the Ca-TN3 buffer under a flow rate of 20 mL/h. After sample application the column was extensively washed with Ca-TN3 buffer and bound material was sequentially eluted with 0.01 M and 0.025 M EDTA in 0.033 M Tris/HCl, 0.5 M NaCl, pH 8.0 buffer (TN3). Fractions of 0.7 mL were collected and assayed for HA as described above. Fractions containing HA were pooled, dialyzed against 0.15 M ammonium carbonate buffer, pH 8.0, and freeze-dried.

Coomassie Brilliant Blue R-250 or overstained with silver nitrate according to GOrget al. (20). Lectin affinoblotting was performed as d e s c r i b e d (21) with p e r o x i d a s e labelled lectins from the jack bean (Con A), garden pea (PSA), peanut (PNA), wheat germ (WGA), and garden snail (HPA).

SDS-PAGE and Lectin Affinoblotting SDS-PAGE was performed according to Laemmli (19) in gradient 2-12.5% gels without stacking gel. Reduction of samples was achieved by boiling for 3 min with 0.5% (w/v) dithiothreitol (DTT) in the presence of 1% (w/v) SDS. After cooling to room temperature, iodoacetamide was added to a final concentration of 0.1 M. For the determination of molecular masses Sigma high molecular weight standards consisting of myosin (205 kDa), [3-galactosidase (116 kDa), phosphorylase (97 kDa), bovine serum albumin (66 kDa), egg ovalbumin (45 kDa), and carbonic anhydrase (29 kDa) were used. The gels were stained with

Protein Determination Protein concentrations were determined with the Bradford method (22) using bovine serum albumin as a standard. Low protein concentrations of purified PLH fractions were estimated after 50fold concentration using dialysis and freeze-drying.

Results

Hemagglutination Activity and Binding Specificity of PLH The ability of crayfish plasma to hemagglutinate red blood cells (RBC) of different vertebrates is shown in Table 2. The HA titers were highest using rabbit erythrocytes, which could be remarkably increased by treatment with trypsin. Further treatment of nontrypsinized or trypsinised rabbit erythrocytes (R-T) with both bacterial or viral neuraminidases did not significantly influence HA titers. Therefore, R-T erythrocytes were used in HA assays throughout the work as a standard. The HA titers did not appear to be strictly dependent on the presence of calcium in the HT buffer. However, in the absence of calcium the HA of PLH was strongly inhibited by EDTA (0.025 mM) and EGTA (0.1 mM). Moreover, the readability and reproducibility of HA results were more consistent if calcium was added to the HT buffer. The hemagglutination activity of crayfish plasma was heat labile. The titer of

Hemagglutinin from crayfish plasma

411

Table 2. Hemagglutlnation Activity of Crayfish Plasma Against Different Erythrocyfes. E ryth rocyte/Treatment

Hemagglutination Activity

Human A1 A2 B 0 Horse Bovine Pig Chick Mouse Rabbit Mouse/pronase Rabbit/trypsin (R-T) Rabbit/bacterial neuraminidase (R-Nb) Rabbit/viral neuraminidase (R-Nv) Rabbit/trypsin, b. neuraminidase (R-T, Nb) Rabbit/trypsin, v. neuraminidase (R-T, Nv) Rabbit/glutaraldehyde (R-GA)

4 4 4 3 2 1 2 2 6 64 64 512 64 64 1024 1024 128

Ultracentrifugad crayfish plasma(UP), protein concentration 1.0 mg/mL,was used in the HA assay.

plasma heated at 40°C for 15 min was 32-fold decreased in comparison to samples that were kept for the same time at 20 or 30°C0 Incubation at 50°C for 15 min completely destroyed the HA of crayfish plasma. The results of inhibition tests of crayfish plasma with different carbohydrates are shown in Table 1. Only high concentrations (of about 500 mM) of D-glucose, D - g l u c o s a m i n e , D - g l u c u r o n i c acid, trehalose, L-fucose, and ceUobiose were found to be capable of inhibiting the HA of P L H . Other sugars, including Nacetylneuraminic acid, 2-keto-3-deoxyoctonate (KDO) as well as saccharides present in the sugar moiety of fetuin (23), were not inhibitory at the concentrations tested. The hemagglutination activity of PLH could be more significantly inhibited with the sialoglycoproteins fetuin, mucin, and ovalbumin (Table 3) among which porcine stomach mucin was the strongest inhibitor. Desialization decreased the inhibitory capacity of BSM 2 - 3 - f o l d whereas fetuin and a s i a l o fetuin gave the same results. Among the glycoconjugates tested, inhibition of

PLH with hyaluronic acid, but not colominic (polysialic) acid or laminarin (131,3-glucan) could be observed. The HA of P L H was found to be strongly and specifically inhibited with the lipopolysaccharide (LPS) from E. coli K-235. Other types of LPSs tested had little or no inhibitory effects (Table 4).

Purification and Characterization of P L n The P L H could be highly enriched from the crayfish plasma using affinity chromatography on Fetuin-Sepharose 4B in a buffer of high ionic strength (0.5 M NaC1). The purification scheme is summarized in Table 5. During the initial step involving ultracentrifugation of plasma, 50% of the total HA was lost. This procedure was employed to remove most of the hemocyanin that represents about 90% of total protein in the crude plasma. A typical elution profile of P L H on a Fetuin-Sepharose 4B column is shown in Figure 1. After loading the sample and washing with Ca-TN3 buffer,

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P. Kopa~ek, L. Grubhoffer, and K. S6derh&ll

Table 3. Inhibition of Hemagglutlnation Activity of Crayfish Plasma by Various Glycoproteins and Glycoconjugates,

Inhibitor

C o n c e n t r a t i o n Required for 50% Inhibition* (mg/mL)

Porcine stomach mucin (PSM) Bovine s u b m a x i l l a r y mucin (BSM) A s i a l o - BSM Fetuin A s i a l o - Fetuin Ovalbumin Ovomucoid P e r o x i d a s e (424 U/mg) H y a l u r o n i c acid Laminarin

0.010 0.156 0.416 0.312 0.312 0.416 5.0 5.0 0.312 >5.0

* Inhibition tests were performed with UP adjusted to 1 HA unit and with R-T erythrocytes. Consistent results were achieved with R-T, Nb; R-T, Nv; and R-GA erythrocytes (see Table 2). Hyaluronic acid inhibited at 0.078 mg/mL with R-T, Nv erythrocytes.

the proteins retained on the column were eluted with EDTA. In the first elution step with 0.01 M EDTA, a peak containing about 5% of total HA applied on the column was obtained (peak I). In spite of a relatively high absorbance at 280 nm, the protein concentration in peak I was below the detection limit of the Bradford method (<~5 ~g/mL). The major part of PLH was eluted with 0.025 M EDTA (peak II) with a maximum absorbance at 280 nm of 0.01. No additional HA was eluted with 0.05 M EDTA. As shown in Table 5, the amount of purified PLH was about 7 p.g from 1043 mg of plasma proteins and the yield of total HA was as low as 8%. From these values we could conclude that the original concentration

of PLH in plasma was less then 0.01% of the total plasma protein content. The SDS-PAGE analysis of individual steps of PLH purification is shown in Figure 2. Nonreduced PLH (Fig. 2, lane 4) appeared as one major band with a molecular mass around 420 kDa as estimated from extrapolation of the calibration line based on molecular markers used. Upon reduction of PLH with dithiothreitol (Fig. 2, lane 6) several weak bands in the range of 65-80 kDa were detected. The specific detection for glycoproteins using affinoblotting with PSA-peroxidase conjugate (Fig. 3) revealed that the protein with a molecular mass of about 75 kDa in the reduced PLH fraction was a glycoprotein of the

Table 4. Inhibition of Hemagglutination Activity of Crayfish Plasma by Lipopolyeaccharides (LPS).

Inhibitor LPS-E. coli K-235 LPS-E. coli, serotype 0111 :B4 LPS-E. coli, serotype 0127:B8

LPS-Salmonella typhosa LPS-Salmonella abortus LPS-Serratia marcescens

C o n c e n t r a t i o n Required f o r 50% Inhibition* (mg/mL) 0.009 >5.0 >5.0 0.625 2.5 0.5

* inhibition tests were performed with UP adjusted to 1 HA unit and with R-T erythrocytes. Consistent results were achieved with R-T, Nb; R-T, Nv; and R-GA erythrocytes (see Table 2).

Hemagglutinin from crayfish plasma

413

Table 5. Purification Scheme for Pacifastacus lenlusculus Hemagglutinin. Total Volume (mL)

Purification Step 1. 2. 3. 4.

Crude plasma Ultracentr. plasma (NH4)2SO4 precipit. 3 × Fetuin-Sepharose 4B~:

210 200 11.5 33.8

Total Protein (mg)

Total HA* (Ha Units)

Specific HA (HA Units/mg)

Purification (-fold)

13440 6400 5880 1072

12.9 42.5 191 150000t

1 3.3 14.8 12800t

1043 150 30.8 0.007t

* Hemagglutination activity was determined with R-T erythrocytes (see Table 2). t Three consecutive passages on affinity columns were done, corresponding fractions were pooled and the results were summarized (see text). :~The values are approximations estimated from a protein concentration of 10 i~g/mL determined in 50-fold concentrated, pooled fractions of peak II (see text).

same type as nonreduced PLH suggesting that this protein may be a subunit of the PLH. This method permitted us to distinguish proteins and subunits belonging to the P L H from contaminating hemocyanin subunits, that is, hemocyanin is not stained as a glycoprotein with PSA-peroxidase (Fig. 3, lane 1).

Characterization of the glycosidic moiety of nonreduced P L H by affinoblotting with five peroxidase-labelled lectins is shown in Figure 4. PLH reacted mainly with the lectins Con A and PSA, marginally with WGA and not at all with PNA and HPA. This interaction pattern resembles that expected of high

A b s o r b a n c e (280 nm)

H e m a g g l u t i n a t i o n activity

0.5

100

0.4

80

0.3

60

25 mM EDTA

0.2

rtJ

0.1

I I

0 0

IL

I

i

20

40

40

,

,

I

I

60

80

20

0

100

Volume (mL)

Figure 1. Affinity chromatography of crayfish plasma on Fetuin-Sepharose 4B. Four milliliters of ammonium sulphate fraction (0-50%) precipitated from 70 mL of ultracentrifuged crayfish plasma and dissolved in Ca-TN3 buffer was applied on the column (1 × 10 cm) preequilibrated and washed with the same buffer. Stepwise elution of PLH was achieved with 0.01 M and 0.025 M EDTA in TN3 buffer, respectively. Three similar runs were performed, fractions of 0.7 mL were assayed for HA activity, appropriate fractions were pooled, dialyzed against 0.15 M ammonium carbonate and freeze-dried. The curve represents the profile of absorbance at 280 nm; bars indicate the value of HA detected in individual fractions.

414

P. Kop&(~ek, L. Grubhoffer, and K. SSderh&ll

M.M.

1

2

3

4

5

6

kDa

205 116 _ 97664529Figure 2. Purification of PLH from crayfish plasma analysed by SDS-PAGE in 2-12.5% gradient gels. M.M., molecular weight markers (see text); lane 1, crude crayfish plasma (5 txg of protein); lane 2, ammonium sulphate fraction (0-50%) of UP applied on the affinity column (8 ~g); lane 3, peak I eluted with 0.01 M EDTA, 50× concentrated (cca 0.5 ixg), unreduced; lane 4, peak II eluted with 0.025 M EDTA (PLH), 50x concentrated (cca 0.1-0.2 t~g); lane 5, the same as lane 3, reduced with DTT; lane 6, the same as lane 4 (PLH), reduced with DTT. M.M. and lanes 1 and 2 stained with Coomassie blue, lanes 3 - 6 , overstained with silver nitrate. For details see Materials and Methods.

mannose or complex N-glycans such as commonly seen in eucaryotic cell glycoproteins (24).

Discussion The agglutination pattern of PLH with various untreated vertebrate erythrocytes demonstrated a high specificity for rabbit erythrocytes that could be further enhanced by proteolytic treatment of the RBC with trypsin. This procedure removes surface proteins and consequently probably renders receptor structures specific for PLH binding more accessible. Sialic acid does not seem to be involved in the specific receptors for P L H on the surface of RBC. Rabbit erythrocytes are rich in terminal galactose and thus it might be reasonable to suggest that the main role in the PLH binding to RBC involves glycolipids and/

or gangliosides with high content of galactose. To confirm this notion it would be necessary to isolate and characterize the PLH specific receptor from the rabbit RBC. The inhibition studies performed using mono- or oligosaccharides did not show any significant binding specifity of PLH even if the oligosaccharides present in inhibitory fetuin (N-acetyllactosamine, N-acetylneuraminyl-lactose, and 13-D-Gal[1->3]-D-GaI-NAc) were tested (23). In general, the PLH could be inhibited by sialoglycoproteins but the inhibition potency of these proteins does not reflect the content of sialic acid in the glycosidic moiety of these proteins. For instance, PSM poor in sialic acid was a much more potent inhibitor than highly sialized BSM. Asialo-BSM was found to be 2-3-fold weaker as an inhibitor than BSM; however, there were no differences in inhibitory ability between

Hemagglutinin from crayfish plasma

M.M.

415

1

2

3

4

5

6

kDa

205 -

O

116 97664529Figure 3. Purification of PLH from crayfish plasma analysed by affinoblotting using peroxidaselabelled PSA lectin. The fractions were separated by SDS-PAGE (see Fig. 2) and electroblotted on the nitrocellulose paper. After saturation with 1.5% BSA in phosphate buffer saline with 0.05% Tween 20 overnight, the membrane was incubated with PSA-peroxidase conjugate diluted 1:400 for 1 h and developed with diaminobenzidine as substrate. M.M., molecular weight markers, stained with Amidoblack 10 B; Lane 1, crude plasma (5 ixg); lane 2, ammonium sulfate fraction (0-50%) of UP (8 Ixg); lane 3, peak I, unreduced; lane 4, peak II (PLH), unreduced; lane 5, peak I, reduced; lane 6, peak II (PLH), reduced.

asialo-fetuin and fetuin. Fetuin, BSM, ovalbumin, and ovomucoid comprised different distinct types of N- and/or O-glycosylation as well as o~(2,3) and/or 0t(2,6) linkage of the terminal sialic acid (23,25,26); however, no apparent specificity in this respect was observed. Taken together, the involvement of sialic acid in the binding specificity of PLH is much less certain than for other crustacean or merostomid agglutinins (16). The results achieved here with sugar and glycoconjugate inhibitors imply that a glycan of complex structure could be a natural P L H ligand and its sialization does not seem to be essential for binding to the PLH. The finding of a strong and specific inhibition of P L H with LPS from E. coli K-235 could be of particular interest. The involvement of LPS in the induction of the prophenoloxidase activating system in crayfish hemocyte lysate has been

described (27) as well as the capability of hemolymph from crayfish Procambarus clarkii to agglutinate some marine bacteria (17). In Limulidae a factor responsible for LPS-mediated activation of the hemolymph coagulation cascade was isolated and characterized (28) and its binding specificity for the lipid-A region of LPS demonstrated (29). The common sugar constituent of the LPS core region, that is, the KDO, which structurally resembles the sialic acid (30), was shown to be recognized by sialic acid-binding limulin (31) and carcinoscorpin (32). Recently, a novel LPS-binding hemagglutinin that agglutinated both Gram-negative and Gram-positive bacteria was isolated from hemocytes of the ascidian, Halocynthia roretzi (33). During the preparation of this manuscript the isolation of another crustacean LPS-binding agglutinin from plasma of brown shrimp (Penaeus californiensis Holmes) was re-

416

P. K o p ~ e k , L. Grubhoffer, and K. S6derh&ll

ConA

PSA

PNA

HPA

WGA

kDa

205

-

116

-

97664529Figure 4. Carbohydrate analysis of unreduced PLH (peak il) using affinoblotting with peroxidase-labelled lectins. The concentrated and unreduced PLH (peak II) was separated by SDSPAGE (the same as lane 4 in Figs. 2 and 3), electroblotted on the nitrocellulose paper and incubated with different peroxidase-labelled lectins from jack bean, Con A (1:400); garden pea, PSA (1:400); peanut, PNA (1:150); garden snail, HPA (1:150); wheat germ, WGA (1:200). For details see Fig. 3 and Materials and Methods section.

ported (34). A possible relation of LPSbinding proteins and agglutinins in invertebrates is shown by the LPSbinding protein or lectin purified from the h e m o l y m p h o f the c o c k r o a c h Periplaneta americana that recognized a specific carbohydrate structure of E. coli LPS (35). The purification of the protein responsible for the hemagglutination activity of the plasma of the freshwater crayfish Pacifastacus leniusculus was found to be complicated for several reasons. First, no mono- or oligosaccharides were

found to be potent inhibitors of PLH. Second, the HA in semipurified fractions of PLH was very sensitive to common auxiliary steps such as concentration, desalting, or buffer exchange, probably due to the adherence of this protein to the surface of various materials. Third, the concentration of PLH in crayfish plasma was very low (0.01% of total plasma protein). The molecular mass of PLH determined to be approximately 420 kDa is similar to that of lectins found in marine invertebrates, such as limulin from Limulus polyphemus (7), carcinoscorpin (10), or lectins Lagl and Lag2 from the lobster Homarus americanus (14) and the LPS-binding protein from the insect Periplaneta americana (35). In contrast to these lectins, which are multimeric molecules composed of noncovalently bound subunits, the subunits of PLH appear to be disulfide bonded. Reduced PLH seems to dissociate into several subunits in the range of 65-80 kDa. Among these a subunit of 75 kDa was detected as a glycoprotein of the same type as present in unreduced PLH. The possible interpretation of this result is that the native PLH is a complex molecule composed of different subunits from which only 75 kDa subunits seem to be glycosylated. However, this remains to be more conclusively shown.

Acknowledgement--This work

has been supported by the Swedish Natural Science Research Council. L.G.'s stay in Uppsala was supported by the Swedish Agricultural Research Science Council and P.K. by a postdoctoral fellowship from the University of Uppsala.

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