1,3-β-D glucan binding protein (BGBP) from the white shrimp, Penaeus vannamei , is also a heparin binding protein

Fish & Shellfish Immunology (2002) 13, 171–181doi:10.1006/fsim.2001.0392Available online at http://www.idealibrary.com on

1,3-�-D glucan binding protein (BGBP) from the whiteshrimp, Penaeus vannamei, is also a heparin binding

protein

FLOR JIMEuNEZ-VEGA2, ROGERIO R. SOTELO-MUNDO

1, FELIPE ASCENCIO2

AND

FRANCISCO VARGAS-ALBORES1*

1Marine Biotechnology, CIAD, P.O. Box 1735, Hermosillo Son, 83000, Mexico,and 2Marine Pathology, CIBNOR, P.O. Box 128, La Paz, BCS, 23000, Mexico

(Received 25 June 2001, accepted after revision 5 November 2001, publishedelectronically 19 July 2002)

Shrimp BGBP was purified as a 100 kDa glycoprotein by a$nity chroma-tography using immobilised heparin. BGBP bound simple carbohydrates,glycosaminoglycans like heparin sulphate and glycoproteins, but it wasunable to agglutinate erythrocytes. Using an ELISA-based microplate assay,it was shown that simple carbohydrates such as D-glucose and D-mannose arecompetitive inhibitors of heparin sulphate binding to BGBP. Based on theseproperties BGBP is considered as a new type of heparin binding protein.

� 2002 Elsevier Science Ltd. All rights reserved.

Key words: shrimp, prawn, beta-glucan, protein, heparin, heparan-sulphate,glycosaminoglycan, heparinoid, polyanionic, proteoglycans.

*Corresponding author: E-mail: [email protected]

I. Introduction

Crustacean �-glucan binding proteins (BGBPs) are important components ofthe immune system. Although a functionally similar BGBP has also beendescribed in insects, it is significantly di#erent in molecular weight, subunitscomposition and amino acid sequence to the known crustacean BGBPs [1–3].Crustacean BGBPs are monomeric 100 kDa glycoproteins that are present inthe haemolymph of freshwater crayfishes, Pacifastacus leniusculus, Astacusastacus and Procambarus clarkii [4, 5], the marine crab, Carcinus maenas [6];and the marine penaeid shrimps, Penaeus californiensis, P. vannamei and P.stylirostris [7, 8]. All reported crustacean BGBPs have a conserved N-terminusamino acid sequence, carbohydrate content as well as biological role andmechanism.

BGBP binds �-glucan forming a complex that recognises a receptor in thehaemocyte surface, causing degranulation and the subsequent activation ofthe prophenoloxidase (proPO) activating system [9–11]. ProPO activatingsystem is a powerful and complex defence system that is activated by

1711050–4648/02/$-see front matter � 2002 Elsevier Science Ltd. All rights reserved.

172 F. JIMÉNEZ-VEGA ET AL.

microbial compounds eliciting several immune mechanisms including melani-sation, phagocytosis, encapsulation and nodule formation. Since the proPO-activating system involves serine proteinases and is triggered by bacteriallipopolysaccharide (LPS), peptidoglycans and �-glucan, an analogy with thealternative pathway of the vertebrate complement system (C�) has beenproposed [12, 13]. Crustaceans do not have immunoglobulins, but recognitionproteins such as BGBP perform analogue functions through similar mecha-nisms. After reaction with the ligand they activate other immune responsesincluding C� activation (vertebrates) or proPO system activation (inverte-brates), as well as degranulation and phagocytosis.

In shrimp, the LPS-binding agglutinin (LPS-BP) and BGBP have beenproposed as recognition proteins. While LPS-BP increases phagocytosis(opsonisation), BGBP activates degranulation and the proPO system, but onlyafter reaction with the cognate ligand [9]. Noteworthily, shrimp BGBP is thesame protein described as a serum high density lipoprotein (HDL) [14]. Thisdual function, lipid transport and immune recognition, for BGBP/HDL wasfirst reported in the freshwater crayfish [25] and later confirmed in marineshrimp [15]. However, LPS-BP and BGBP apparently have a broad recognitionspectrum for identification and destruction of most of the invading pathogensin shrimp. In this paper BGBP is shown to be a heparin binding protein (HBP)and evidence is presented that binding of BGBP to heparin sulphate (HS) isspecific and can be competed by simple saccharides. Advantage was taken ofthis interaction to purify BGBP using immobilised heparin with better yieldsthan the previously used laminarin a$nity matrix. This is the first reportwhere a marine invertebrate �-glucan binding protein is shown to be also aHBP.

II. Materials and Methods

EXPERIMENTAL ANIMALS

Ten to fifteen grams weight healthy juvenile Pacific white shrimp (P.vannamei) were obtained from an experimental tide pond (Centre for Biologi-cal Research, La Paz, BCS, Mexico). Shrimps were acclimatised in a 1500 lculture fibreglass tank with aeration and filtered (0·2 �m) sea water contain-ing 10 mg l�1 of EDTA . Na2. Filtered sea water was maintained at pH 7·8–8·2,27� C, and 35‰ salinity. Animals were acclimatised for 15 days before theexperiments and fed ad libitum with a commercial pellet.

BGBP PURIFICATION

Haemolymph was extracted from the pleopod base of the first abdominalsegment into two volumes of pre-cooled (10� C) shrimp anticoagulant solution(450 mM NaCl, 10 mM KCl, 10 mM EDTA . Na2, 10 mM HEPES, pH 7·3,850 mOsm kg�1) [16] using a 1 ml syringe and 27 gauge needle. The haemo-lymph was then centrifuged at 800�g for 5 min, and the plasma supernatantseparated.

The plasma was dialysed overnight against double-distilled water and thencentrifuged at 3000�g for 20 min, as previously described for the purification

INTERACTION OF SHRIMP BGBP WITH HEPARIN 173

of penaeid BGBP [7, 8, 17]. The reddish pellet containing BGBP was resus-pended in 50 mM Tris-HCl, pH 7·5. Approximately 1·5 mg of protein wasapplied to a 3 ml A$-Gel-heparin column, previously equilibrated with 50 mM

Tris-HCl, 0·2 M NaCl pH 7·5. The column was washed with equilibration bu#eruntil the protein content was zero (as indicated by Bradford assay). The boundproteins were sequentially eluted (flow rate of 0·1 ml min�1) with 0·5 and 1·0 M

NaCl dissolved in 50 mM Tris-HCl, pH 7·5. BGBP-rich fractions (eluted at 0·5 M

NaCl) were re-chromatographed in the same column, under the same condi-tions, but using a step-wise elution gradient of 0·3 M NaCl to 0·5 M NaCl.During the chromatography, 1 ml fractions were collected and its proteinconcentration was estimated by Bradford reaction [18], using a modificationfor microplates where 25 �l of sample and 250 �l of Bradford reagent areincubated for 5 min before measuring absorbance at 595 nm. The concen-tration was determined using a bovine serum albumin (BSA) standard curve.Purified BGBP was concentrated by ultrafiltration (Microcon 30 kDa cut o#,Millipore), at a final concentration of 1 mg ml�1. BGBP aliquots were storedat �20� C for further analysis.

HAEMAGGLUTINATION ASSAY

Human or animal blood was obtained by venous puncture, collected andstored in sterile Alsever’s solution. Before use, the red blood cells (RBC) werewashed twice by centrifugation (800�g, 10� C, 10 min) with TBS-Ca (50 mM

Tris-HCl, 100 mM NaCl, 10 mM CaCl2, pH 7·5). RBC were resuspended to 2%(v/v) in TBS-Ca. Haemagglutination was performed on U plates (Falcon) usingtwo-fold serial dilutions of BGBP in TBS-Ca (50 �l), then 50 �l of 2% RBC wereadded. The plates were incubated at room temperature (26�2� C) for 1 h andthe haemagglutinating titre was recorded as the reciprocal of the last dilution,showing evidence of agglutination.

SDS-PAGE AND WESTERN BLOT

SDS-PAGE of BGBP (7 �g) was performed at 100 V for 2 h, using a 7·5%polyacrylamide gel under non-reducing conditions [19], in a Mini PROTEANII system (Bio-Rad). Myosin (205 kDa), �-galactosidase (116 kDa), phosphory-lase b (97·4 kDa), fructose 6 phosphate kinase ovoalbumin (45 kDa) andcarbonic anhydrase (29 kDa) were used as standards. After electrophoresis,the gel was stained with silver nitrate [20] or soaked in transfer bu#er (25 mM

Tris, 192 mM glycine, pH 8·8+20% methanol) for 10 min and electrotransferred(1·5 h at 200 mA) to Immobilon P (Millipore) membrane [21].

After transfer, the membrane was incubated in blocking bu#er (PBScontaining 3% BSA and 0·05% Tween 20) for 2 h. Then the membrane wasincubated with anti-shrimp BGBP polyclonal rabbit antibody (1:500), for 1 h[15]. The membrane was washed 3�5 min with PBS, and incubated with goatanti-rabbit IgG conjugated to horseradish peroxidase (HRP) (1:500) (SigmaChem. Co.) for 1 h, washed 4�5 min with PBS before addition of the substrate(3,3�-diaminobenzidine, 3 mg ml�1). Interaction with heparan sulphate was


demonstrated following the same procedure but incubating 1 h withperoxidase-labelled heparan sulphate (see below) instead antibodies.

CONJUGATION OF HEPARAN SULPHATE WITH PEROXIDASE (HS-HRP)

Heparan sulphate-horseradish peroxidase (HS-HRP), was prepared follow-ing the method reported by Harlow & Lane [22]. Four mg of HRP weresuspended in 0·2 ml of 0·1 M NaIO4, incubated 20 min at 22� C and dialysedagainst 1 mM sodium acetate (pH 4·4) at 4� C for 24 h. This solution was mixedwith 1 ml of heparan sulphate (1 mg ml�1, in 10 mM Na2CO3 bu#er, pH 9·4) andincubated at 22� C for 4 h. The reaction was stopped by addition of 0·1 ml ofNaBH4 (4 mg ml�1).

BINDING OF HS-HRP TO IMMOBILISED BGBP

Purified BGBP was immobilised in 96-well assay plates incubating 1 �g perwell of BGBP in sodium carbonate bu#er (15 mM Na2CO3, 35 mM NaHCO3,pH 9·6) overnight at 4� C. The plate was blocked with 300 �l of 2% glycine andwashed three times with PBS. Then 22 �g of HS-HRP (in 100 �l of PBS) wereadded to each well and incubated 1 h at 37� C. The plate was washed threetimes with PBS and 100 �l of OPD-substrate (1 mg ml�1 OPD in 50 mM sodiumcitrate bu#er, pH 5·0) was added to each well and incubated 30 min at 37� C.The reaction was stopped with 50 �l of 1 M H2SO4, and absorbance wasmeasured at 490 nm. A well without BGBP was used as blank.

N-acetyl glucosamine, glucose, sucrose, galactose, ramnose, fucose, fructoseand mannose (100 mM) as well as hyaluronic acid, heparin, �-glucan, lami-narin, fetuin and mucin (1 mg ml�1) were tested as inhibitors. Inhibitionassays were performed by incubating immobilised BGBP in the plate, at 37� Cfor 1 h, with 100 �l of inhibitor dissolved in PBS. After incubation, 100 �l(22 �g) of HS-HRP were added and the plate incubated for an additional houras described above. In other experiments, glucose, heparin, laminarin, dextransulphate and lactoferrin were tested at 1, 10 and 100 �g (in 100 �l of PBS).

III. Results

BGBP PURIFICATION

After extensive dialysis against distilled water, BGBP was recovered as areddish precipitate which was dissolved in 50 mM Tris-HCl and 1·5 mg of totalprotein were loaded to an immobilised-heparin column. The column waswashed with 50 mM Tris-HCl, pH 7·5 containing 0·2 M NaCl before the elutionof bound proteins with 0·5 or 1 M NaCl. BGBP was eluted with 0·5 M NaCl (Fig.1), but an SDS-PAGE analysis of the fractions showed significant contaminantproteins, mainly haemocyanin. BGBP fractions were pooled and loaded to thesame column, but using a step gradient (0·3, 0·4, 0·45 and 0·5 M NaCl inTris-HCl, pH 7·5). BGBP was eluted with 0·3 M NaCl and appeared in SDS-PAGE as a homogeneous 100 kDa protein (Fig. 2). Purified BGBP was kept inrefrigeration as a 1 mg ml�1 solution in Tris-HCl 50 mM, 0·3 NaCl, pH 7·5.

NaC

l (M

)


60

0.8

Fraction no.

(b)

Abs

orba

nce

(59

5 n

m)

0

0.1

0.2

0.3

0.4

0.6

0.7

3010 40200.0

1.0

0.4

0.6

0.8

0.2

0.5

50Fraction no.

(a)

Abs

orba

nce

(59

5 n

m)

0

0.20

0.40

0.60

0.80

1.00

1.20

3010 4020

NaC

l (M

)

0.00

1.00

0.25

0.50

0.75

Fig. 1. Heparin a$nity chromatography from white shrimp plasma. The precipitateobtained by dialysis of plasma against distilled water was resuspended in Tris bu#erand 1·5 mg of protein was (a) loaded to the column and BGBP was eluted with 0·5 M

NaCl. (b) Fractions containing BGBP were pooled and re-loaded to the columnwhere BGBP was eluted with 0·3 M NaCl.

Fig. 2. SDS-PAGE of purified white shrimp BGBP. Molecular weight markers (lane A)are indicated on the left. BGBP precipitate (lane B), Heparin-chromatographyfractions eluted with 0·5 M NaCl (lane C), Pure BGBP obtained with 0·3 M NaCl (laneD), Immunoblot with anti brown shrimp BGBP antiserum (lane E) and Interactionwith heparan sulphate labelled with peroxidase (lane F).

Purified BGBP was separated by SDS-PAGE and electrotransferred toImmobilon membrane (Millipore). Immobilised BGBP was recognised bypolyclonal antibodies anti-P. californiensis BGBP [7, 8]. To test interaction


with a proteoglycan, immobilised BGBP was also incubated with HS-HRP,and the assay followed by detection of the peroxidase attached to thecarbohydrate.

Table 1. Solid phase competition assay for BGBP HS-HRPinteraction. Immobilised BGBP (1 �g per well) was incubatedwith 100 �l of 0·1 M simple sugars or 1 mg ml�1 complex sugarsand glycoproteins. The mixture was incubated with 22 �g ofHS-HRP and the excess eliminated by washing. Bound HS-HRP

was estimated using OPD as substrate, at 490 nm

Inhibitor Percentage of inhibition

N-acetyl glucosamine 83D-galactose 87D-glucose 92D-mannose 93Ramnose 86L-fucose 87D-fructose 21Sucrose 42�-glucan 76Laminarin 62Hyaluronic acid 42Heparin 77Fetuin 60Mucin 48

INTERACTION WITH SUGARS AND GLYCOPROTEINS

Purified BGBP was unable to agglutinate either human or rabbit RBC, evenwhen longer incubation times and di#erent temperatures (10, 25, 35� C) as wellas presence of divalent cations were tested. Thus, for evaluating the interac-tion with saccharides, a solid phase assay was designed, immobilising purifiedBGBP in 96 well microplates and using HS-HRP to quantify the interaction.Although other concentrations (up to 10 �g) of immobilised BGBP were tested,1 �g of protein per well was enough for this assay. After BGBP immobilisation,22 �g of HS-HRP per well was added and the plate was incubated. Then, theplate was washed and the interaction was measured spectrophotometricallyusing OPD as substrate.

The interaction of BGBP with saccharides and glycoproteins was demon-strated inhibiting the reaction of BGBP with HS-HRP. As shown in Table 1,sugars and glycoproteins inhibited up to 93% the BGBP-HS interaction.Hexoses and pentoses presented the highest inhibitory e#ect (93%), exceptfor D-fructose that inhibited only 21%. Hyaluronic acid inhibits 42%,heparin 77%, and glycoproteins as mucin and fetuin inhibited 48% and 60%,respectively.

Dose-dependent inhibition was tested for glucose, heparin, laminarin anddextran sulphate. A direct relationship between ligand concentration and


displacement of HS-HRP was found (Fig. 3), indicating that BGBP recognisessaccharide residues in addition to �-glucans and heparin. Glucose showed thehighest inhibitory e#ect, but complex carbohydrates including sulphatedpolysaccharides as dextran sulphate and glycoproteins like lactoferrine#ectively also blocked the interaction between BGBP and HS.

0100

Concentration (µg)

Per

cen

tage

of

inh

ibit

ion

0

20

40

60

80

100

1 10

Fig. 3. Inhibition of interaction between BGBP and heparan sulphate by haptenicsugars (glucose, —�—; heparin, – –�– –; laminarin, ——; dextran sulphate,– –– –, lactoferrin, · ·�· ·).

IV. Discussion

Many heparin binding proteins have been identified in diverse animalgroups, including proteins of circulatory fluid and those involved in lipidmetabolism and polymerases [23]. They have been extensively characterisedby the distributions of an heparin-binding sequence, although such interac-tion may not have a physiological significance.

BGBP was first characterised as a defence protein, capable of binding�-glucans and enhancing the proPO activating system [7, 8]. Later it wasshown that BGBP is the same protein previously described as an HDL [14],having an important role in lipid transport [24]. The dual role of thiscrustacean protein was first demonstrated in the freshwater crayfish, P.leniusculus [25], and confirmed later in shrimp [15]. The protein has been


purified by two di#erent ways: by a$nity chromatography using immobilisedlaminarin (as BGBP) or by ultracentrifugation (as HDL). Looking for apreparative method for isolation and purification of BGBP, immobilisedheparin was tested, considering that white shrimp BGBP contains almost 13%of basic amino acids [8]. These positively charged amino acids may interactwith the polyanionic groups of the heparin through electrostatic forces [26].As it is shown in this work, BGBP interacts with heparin, allowing its use inpurification, and constituting the first report of a heparin binding proteinfrom shrimp serum. The isoelectric point of BGBP is 5·8 [14] therefore it istempting to propose that at least one positively charged patch is present in thesolvent-accessible surface of the protein that interacts with polyanionicmolecules such as heparin. Supporting the role of electrostatic interactionsbetween BGBP and heparin comes from the fact that the protein is eluted withNaCl as in ion-exchange chromatography.

Proteoglycans play important biological roles like the activation of pancre-atic lipases by heparin [27]. In shrimp, a low molecular weight heparin hasbeen isolated [28], and the author suggests that heparin may be involved indefence mechanism against bacteria and other foreign materials. On the otherhand, HS has been also implicated in cell-cell contact as well as the binding oflipoproteins to cell surfaces [28, 29].

Previous to this report, only the interaction of shrimp BGBP with �-glucanshas been described [7], however, in this work evidence is presented that BGBPinteracts with other carbohydrates which may be physiologically important.Heparin binding was first demonstrated by a$nity chromatography and by theHS-HRP interaction with immobilised BGBP. Competition assays were donein a solid-phase assay to determine whether specificity was involved. Eventhough the interaction of BGBP with monosaccharides and complex sugarswas observed, attempts to determine binding a$nities were unsuccessful,since laminarin, �-glucans and proteoglycans are heterogeneous mixtures anddo not have a defined molecular weight, therefore mass units are reported.

Fungal �-glucans were long known to activate the defence system in thecrayfish [29], and it was later discovered that such mechanism is triggered bya carbohydrate-protein complex [4, 9] However, the fact that mono anddisaccharides compete for the proteoglycan binding site suggests that BGBPmay have other physiological roles besides activation of proPO and lipidtransport, or that its activity may be modulated by physiological changes inthe haemolymph concentration of sugars.

It is also important to mention that HS, heparin, and heparin-like sub-stances have been identified in shrimps, and in a number of di#erent inverte-brate species [30]. Thus, it is more likely that HS proteoglycans, as importantconstituents of the extracellular matrix components, may be important mol-ecules involved in cell–cell communication. Certainly, they have beendescribed as modulators of the immune reactions or other body defencemechanisms [30].

It is possible that P. vannamei BGBP performs other roles aside immuneactivation and lipid transport and the significance of its heparin-bindingproperties remains to be investigated. However, considering that mammalianHBPs can be subdivided into five major groups: i.e. chemokines, cytokines,


extracellular matrix proteins, heparin-dependent growth factors, growthfactor-binding proteins, and a number of enzymes such as superoxide dis-mutase [31], BGBP in marine invertebrates may have additional functions inaddition to targeting Gram-negative bacteria, fungi, and yeast cell wallcomponents. Other HPB as integrins, growth factors and cytokine, areinhibited only by complex sugars, so P. vannamei BGBP may represent adi#erent type or family of HBP.

On the other hand, lectins are carbohydrate-binding proteins that aggluti-nate erythrocytes, and shrimp BGBP was unable to agglutinate either humanor rabbit RBC, although this does not overrule the possibility that BGBPbinds but does not agglutinate erythrocytes, therefore being a monovalentlectin. Similar result has been reported for crayfish BGBP [4]. It is noteworthyto mention important similarities between BGBP and the monovalent deriva-tive of the Maackia amurensis leukoagglutinin (MAL) [32]. This lectin doesnot haemagglutinate, but binds specifically to fetuin and the interaction iscompetitively inhibited by simple sugars (Neu5Ac� 2–3 lactose and lactose)[32]. For BGBP, it was found that glucose is the monosaccharide that inhibitsthe interaction with HS-HRP, which is consistent with the structure oflaminarin as a polymer of glucose units linked by a �,1–3 glucosidic bond.

It is enticing to hypothesise that the interaction of BGBP with saccharidesis physiologically relevant in shrimp, although it requires further work.Nonetheless, identifying the presence of heparin and other complex sugarseither in plasma or as a surface carbohydrate in membrane proteins may helpto elucidate the role of the shrimp BGBP on defence and lipid transport.

We are grateful to Guillermo Portillo and Angel Campa (CIBNOR) for supplying theanimals for experiments and Enrique Villalpando (CIAD) for his technical assistance.Funding was provided by CONACYT (Mexico) grant J-31643B and CIAD-CIBNORinstitutional grants. A graduate scholarship to FJV was awarded by CONACYT(Mexico).

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31 Wadström, T. & Ljungh, Å. (1999). Glycosaminoglycan-binding microbial proteins intissue adhesion and invasion: key events in microbial pathogenicity. Journal ofMedical Microbiology 48, 223–233.

32 Kaku, H., Mori, Y., Goldstein, I. J. & Shibuya, N. (1993). Monomeric, monovalentderivative of Maackia amurensis leukoagglutinin. Preparation and application tothe study of cell surface glycoconjugates by flow cytometry. Journal of BiologicalChemistry 268, 13237–13241.

33 Söderhäll, K. (1981). Fungal cell wall �-1,3-glucans induce clotting and phenoloxi-dase attachment to foreign surface of crayfish hemocyte lysate. Developmental andComparative Immunology 5, 565–573.

1,3-β-D glucan binding protein (BGBP) from the white shrimp, Penaeus vannamei , is also a heparin binding protein

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