Silkworm, Bombyx mori - Journal of Biological Chemistry

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Aug 25, 2018 - Purification of a &1,3-Glucan Recognition Protein in the. Prophenoloxidase Activating System from Hemolymph of the. Silkworm, Bombyx mori*.

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263,No.24, Issue of August 25, pp. 12056-12062,1988 Printed in V.S.A.

Purification of a &1,3-Glucan Recognition Protein in the Prophenoloxidase Activating System from Hemolymph of the Silkworm, Bombyx mori* (Received for publication, November 16, 1987)

Masanori Ochiaiand MasaakiAshidaS From the Biochemical Laboratory, The Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan

The plasma fraction (referred toas plasma-CPB) of glucan recognition protein bound to /3-1,3-glucan did silkworm hemolymph, from which a protein with af- not hydrolyze appreciably any of the 26 commercially finity toB- 1,3-glucan was specifically removed accordavailable peptidyl-7-amino-4-methylcoumarins, subing to Yoshida et al. (Yoshida, H., Ochiai, M., and strates for variousproteases. Ashida, M. (1986) Biochem. Biophys. Res. Commun. 141, 1177-1184), was used to develop a method for quantitating the 0- 1,3-glucan recognition protein of The prophenoloxidase activating system, a cascade, in inthe prophenoloxidase activating system. In principle, a sample was judged to contain &1,3-glucan recogni- sect hemolymph is triggered by elicitors such as @-1,3-glucan tion protein whenthat sample could restore the ability or peptidoglycan (1).At least two zymogens of serine enzyme 8- 1,3- and prophenoloxidase are activated when the system is trigof the system in plasma-CPBto be triggered by glucan. Purificationproceduresforthe recognition gered (1).It has been suggested that the system functions to protein from silkworm hemolymph consisted of frac- recognize foreignness, generate opsonin and hemokinetic factionation with ammonium sulfate, chromatography on tors, and activate fatbody to synthesize immune proteins (2DEAE-Toyopearl, Affi-Gel-heparin, and Mono Q and 5). Thus, thesystem appears to be an essential component of Superose 12 on the fast protein liquid chromatography insect defense mechanisms. system of Pharmacia LKB Biotechnology Inc. About In various biological systems other than insect, @-glucans 2.03 mg of j3-1,3-glucan recognition protein was ob- containing @-1,3-glycosidiclinkages display a diverse array of tained from 300 ml of hemolymph. activities including potentiation of the immune system of The purified &1,3-glucan recognition protein was mammals, activation of the blood coagulation system of the homogeneous as judgedby sodium dodecyl sulfate- horseshoe crab, activation of the alternative complement polyacrylamide gel electrophoresis and isoelectric fo- pathway, and induction of phytoalexin synthesis in plants(6cusing-polyacrylamide gel electrophoresis. B- 1,3-Glucan recognition protein had a molecular mass of 62 10). It is plausible that molecules specifically recognizing 8kDa composed of a single polypeptideand an isoelectric 1,3-glucans at theprimary site of action are presentas receppoint of pH 4.3. It bound to curdlan beads (composed tors at the cell surface or as “free floating” recognition proof B- 1,3-glucan with average particle size of 80 rm) in teins outside of cells. However, their properties are poorly the absence of divalent cation, whereas its binding to understood. C3b, which is involved in activation of the alter1 6)-glycosidic linkages native complement pathway, hasa broad specificity and glucans [email protected](1+ 4)- orCY( was not detected under the experimental conditions. should not be considered a recognition protein for @-1,3Elution of the @-1,3-glucan recognitionprotein bound glucan (11).Thus, no specificreceptors or recognition proteins for @-1,3-glucanhave been isolated and, in many cases, even to curdlan beads could beachieved under strongly denaturing conditions (after incubation of the beads their existence is speculative. In the light of these considerawith sodium dodecyl sulfate and 8-mercaptoethanol in tions, it is desirable to develop a method for isolating ,8-1,3boiling water for 6 min), but elution a t room tempera- glucan receptor or recognition protein from biological systems. ture waspoor. Knowledge of the properties of the molecule and the nature Since &1,3-glucan recognition protein is the only of its interaction with @-1,3-glucanwould advance our underprotein in silkworm plasma with strong affinity to B- standing about the mechanisms of action of this important 1,3-glucan and endows the prophenoloxidase activat- molecule in many living organisms. ing system inplasma-CPB with the ability tobe trigWe have previously suggested that the @-1,3-glucanand gered by &1,3-glucan, it was concluded that binding peptidoglycan recognition proteins’ occur as separate entities of the purified @-1,3-glucan recognition protein with and that the interaction of the recognition protein with the /3-1,3-glucan causes the triggering of the prophenol- respective elicitors triggers the prophenoloxidase activating oxidase activating system in silkworm plasma. How- system in silkworm plasma (12). We also described a method is generated as the ever, the nature of the activity that result of binding is not yet known.The purifiedB-1,3- to specifically remove the @-1,3-glucanrecognition protein from the prophenoloxidase activating system of silkworm * Results of this investigation were presented in part at the Inter- plasma using beads composed of @-1,3-glucan.Plasma, denational Society for Developmental and Comparative Immunology prived of the recognition protein (referred to asplasma-CPB),


Conference, West Berlin, Federal Republic of Germany, September 28-30,1986 (26). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $ To whom correspondence should be addressed.

In our previous papers (12, 26) we referred to recognition protein as &1,8-glucan receptor and peptidoglycan receptor, respectively. However, we adopted the present terms to designate the same molecules because the term ”receptor” usually implies the localization of the molecules at thesurface or within the cytosol of cells.


&1,3-Glucan Recognition Protein in Insect Hemolymph


indicated with a horizontal bar in the figure were pooled and dialyzed against 1000 ml of K-P buffer overnight, followed by centrifugation to remove flocculent materials. The supernatant was applied to an Affi-Gel-heparin column (1.4 X 22 cm), previously equilibrated with K-P buffer. After washing the column with 150 ml of the buffer, bound materials were eluted at a flow rate of 30 ml/h using a 600-ml linear salt gradient, 0 to 0.25 M KCI, established in K-P buffer. Five-ml fractions were collected. The active fractions as indicated with a horizontal bar in Fig. 3 were pooled and dialyzed against 1,000 mlof BTP buffer (20 mM bis-Tris2 propane buffer (pH 6.5)), followed by centrifugation a t 30,000 X g for MATERIALS ANDMETHODS 10 min. Animals-Silkworm (Bombyx mori) larvae were reared on an artiThe following fast protein liquid chromatography was performed ficial diet as described (1). at room temperature. The supernatant obtained in a preceding step Preparation of Silkworm Plasma (Plasma-CPB) for Assaying @-1,3- was applied to a Mono Q column (HR 5/51, equilibrated to BTP Glucan RecognitionProtein-The plasma fraction of hemolymph was buffer, and adsorbed proteins were eluted with a linear salt gradient prepared as described previously (12, 13). The plasma fraction was established in the same buffer (Fig. 4). The flow rate was maintained passed through a column of curdlan-type polysaccharide beads com- at 1 ml/min and 0.5-ml fractions were collected. Fractions (fractions posed of @-1,3-glucan,and theeffluent was concentrated according to 15-16) were pooled. the method of Yoshida et al. (12). The concentrated effluent was A two hundred-pl portion of the pooled fractions was applied to a named plasma-CPB and used for assaying the @-1,3-glucanrecogni- Superose 12 (HR 10/30) column equilibrated with K-P buffer contion protein. The prophenoloxidase activating system in plasma-CPB taining 150 mM NaCl and eluted at a flow rate of 250 pl/min with is triggered with peptidoglycan but not with @-1,3-glucan(12). the same buffer; 0.8-ml fractions were collected. This chromatography Assay of @-1,3-GlucanRecognition Protein Actiuity-The sample was carried out repeatedly to purify all the sample obtained in the solution to be assayed for j3-1,3-glucan recognition protein was serially preceding purification step. Fractions with fl-1,3-glucan recognition diluted, and 10 pl of each diluted solution was added to a mixture of protein activity were combined and used as a purified j3-1,3-glucan 78 p1 of plasma-CPB, 10 p1 of zymosan solution (100 pg of zymosan/ recognition protein preparation. ml of double-distilled water, prepared after Yoshida and Ashida ( l ) ) , Sedimentation Equilibrium Ultracentrifugation-The molecular and 2 pl of 250 mM CaC12, followed by incubation at 25 "C for 120 weight of native &1,3-glucan recognition protein in K-P buffer conmin. After the incubation, phenoloxidase activity of the reaction taining 150 mM NaCl (0.5 mg of protein/ml) was determined by the mixtures was assayed spectrophotometrically (1). To eliminate the method of Yphantis (14) using a Hitachi analytical ultracentrifuge possibility that the observed activation of prophenoloxidase was (Model 282) equipped with Hitachi ultracentrifuge processor (Model independent of p-1,3-glucan action, phenoloxidase activity of the DA-7). reaction mixture devoid of zymosan was always checked after incuSDS-PolyacrylumideGel Electrophoresis (SDS-PAGE) and Isoelecbation. tric Focusing-PolyacrylamideGel Electrophoresis (IEF-PAGE)-SDSThe most dilute solution resulting in more than 18 units of phen- PAGE was carried out in a 1-mm-thickslab gel according to Laemmli oloxidase activity in the reaction mixture was determined, and the (15). Samples were incubated in the presence of 82 mM Tris-HC1 reciprocal of the dilution factor was used to express the amountof 8- buffer (pH 8.8), containing 1%SDS, 1% 8-mercaptoethanol, 30% 1,3-glucan recognition protein activity/ml of sample solution. One glycerol, and 0.01% bromphenol blue (SDS-PAGE solubilizing buffer) unit is defined as the amount of fl-1,3-glucan recognition protein in boiling water for 5 rnin. contained in 1 ml of P-1,3-glucan recognition protein solution for IEF-PAGE was performed according to the method of Wrigley which the reciprocal of the dilution factor is 1. (16). Gels were stained for proteins with Coomassie Brilliant Blue Rj3-1,3-Glucanrecognition protein activity determined as above pro- 250. vides a relative rather than quantitative estimation, since different Procedures for Examining Ability of Plasma Proteinsor Purified 0activity values were obtained for a given @-1,3-glucanrecognition 1,3-GlucanRecognition Protein to Bind to Insoluble Glucans-Hemoprotein solution when different lotsof plasma-CPB preparationswere lymph of four silkworm larvae (5th instar 5-day) was collected into 4 used. Thus, a single preparation of plasma-CPB was used throughout the purification of @-1,3-glucanrecognition protein described in the ml of T-M buffer (10 mM Tris maleate buffer (pH 6.5), containing 150 mM NaC1) containing 0.1 mM p-nitrophenyl p'-guanidinobenzopresent study. ate. Hemocytes were removedby centrifugation at 800 X g for 30 min Purification of @-1,3-Glucan RecognitionProtein-Fifth instar larvae of silkworm (B. nori) at the 5thor 6th day were bled by cutting at 2 "C. The remaining supernatant was used as plasma. Twenty mg ( d r y weight) of beads of curdlan-type polysaccharide abdominal legs with scissors. Hemolymph was immediately mixed with saturated ammonium sulfate (pH 6.5) under vigorous stirring. (@-1,3-glucan)and a mixture of Sephadex G-100 (20 mg)and cellulose Three hundred ml of hemolymph from about 650 larvae was collected (20 mg) suspended in double-distilled water were packed into columns into 650 ml of saturated ammonium sulfate and stored at 4 "C until (inner diameter 8 mm) and equilibrated to T-M buffer. Plasma (2 ml) was applied to each column. After washing the columns with 10use. All subsequent procedures were performed at 0-4 "C and centrifu- ml portions of T-M buffer or consecutively with 10-ml portions of gation carried outat 12,300 X g for 30 min, unless otherwise specified. the buffer and 8 M urea, the washing solution was drained from The hemolymph preparation was centrifuged and theprecipitated glucans as thoroughly as possible. Protein(s) adsorbed on beads of pmaterials dissolved into 20% saturated ammonium sulfate containing 1,3-glucan and a mixture of Sephadex G-100 and cellulose was exK-P buffer (10 mM potassium phosphate buffer (pH 6.5)), 1 mM tracted with 25 and 50 pl, respectively, of SDS-PAGE solubilizing buffer in boiling water for 5 min. In thecase of purified @-1,3-gJucan phenylmethylsulfonyl fluoride, 0.1 mM p-nitrophenyl p'-guanidinobenzoate, 5 mM phenylthiourea, and 5 mM EDTA to make the total recognition protein, 10 pgof protein in 50 pl of T-M buffer was volume 300 ml. After centrifugation of the solution, the supernatant applied to glucan columns and extracted as above. Two pg of @-1,3was brought to 35% saturation by addition of saturated ammonium glucan recognition protein, 10 pl of the extracts from beads of @-1,3sulfate solution (pH 6.5) and left overnight. The resulting precipitate glucan, or 20 pl of extracts from the mixtures of the glucans was was collected by centrifugation, dissolved in 50 mlof K-P buffer subjected to SDS-PAGE to examine if any of the extractscontained containing 1mM phenylmethylsulfonyl fluoride and 5 mM phenylthi- protein with the same electrophoretic mobility as thatof @-1,3-glucan ourea, and dialyzed against 2 liters of the same buffer for 48 h with recognition protein. Assay of AmidaseActivity of @-1,3-GLucanRecognition Protein six changes of the buffer. The dialyzed solution was used as ammoIncubated with Zymosan-Amidase activity of @-1,3-glucanrecogninium sulfate fraction aftercentrifugation. The ammonium sulfate fractionwas applied to a DEAE-Toyopearl tion protein or the molecule bound to zymosan was assayed using column (2.5 X 55 cm)pre-equilibrated with K-P buffer. After washing the column with 400 ml of the same buffer, adsorbed proteins were The abbreviations used are: bis-Tris, 2-[bis(2-hydroxyeluted by applying a linear salt gradient (0-0.3 M KC1 in K-P buffer) ethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; SDS, sodium doin a total volume of 1300 ml. The flow rate was maintained a t 80 ml/ decyl sulfate; IEF, isoelectric focusing; PAGE, polyacrylamide gel h and 9-ml fractions were collected. An elution profile is shown in electrophoresis; MCA, methylcoumarin; Boc, t-butoxycarbonyl-; SUC, Fig. 2. Fractions with @-1,3-glucanrecognition protein activity as succinyl.

may then be used to assay for the P-1,3-glucan recognition protein. The present communication describes an assay method for the @-1,3-glucanrecognition protein of the prophenoloxidase activating system in silkworm plasma and a procedure for obtaining ahomogeneous and functionally active @-1,3-glucan recognition protein preparation, together with a preliminary characterization of the molecule,


/3-1,3-Glucan Recognition Protein

fluorogenic substrates, peptidyl-7-amino-4-methylcoumarins(referred to aspeptidyl-MCAs). Preincubation mixtures, comprising 5 volumes of @-1,3-glucanrecognition protein solution (0.3 mgof protein/ml of K-P buffer containing 150 mM NaCl) and 1 volume of zymosan solution (0.1 mg/ml of double-distilled water) or 5 volumes of the P-1,3-glucanrecognition protein solution and 1 volume of double-distilled water, were incubated at 25 “C. After 10 min incubation, 10-pl aliquots of the preincubation mixtures were assayed for amidase activity. The reaction mixture for the assay consisted of 480 pl of T-M buffer containing 5 mM CaC12,10 p1 of 5 mM fluorogenic substrate, and 10 pl of the above preincubation mixture. After incubation of the mixtures at 30 “C for 120 min, 500 p1 of 50% (v/v) acetic acid was added to terminate enzyme reaction. The amount of liberated 7-amino-4-methylcoumarin was determined after Kojima et al. (17) from fluorescence intensity read a t 460 nm with excitation a t 380 nm, using a Hitachi 204-A fluorescence spectrophotometer. For controls the same preincubation mixtures except for the P-1,3-glucanrecognition protein was prepared and their amidase activity was assayed as above. PeptidylMCAs used were as follows: Arg-MCA, benzoyl-Arg-MCA,Boc-GluLys-Lys-MCA,Boc-Glu(0-benzyl)-Gly-Arg-MCA,Boc-Gln-Arg-ArgMCA, Boc-Ile-Glu-Gly-Arg-MCA, Boc-Leu-Gly-Arg-MCA,Boc-LeuSer-Thr-Arg-MCA, Boc-Leu-Thr-Arg-MCA,Boc-Phe-Ser-Arg-MAC, Boc-Val-Leu-Lys-MCA, Boc-Val-Pro-Arg-MCA, glutalyl-Gly-ArgMCA, Gly-Pro-MCA, Leu-MCA, Lys-Ala-MCA, Pro-Phe-Arg-MCA, L-pyroglutamicacid-MCA, Suc-Ala-Pro-Ala-MCA, Suc-Ala-Ala-ProPhe-MCA, Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-MCA, Suc-GlyPro-Leu-Gly-Pro-MCA, Suc-Gly-Pro-MCA, Suc-Leu-Leu-Val-TyrMCA, Z-Arg-Arg-MCA,and Z-Phe-Arg-MCA. These substrates were dissolved in either double-distilled water, dimethyl sulfoxide, or dimethyl formamide according to manufacturer’s instruction. Analyses of Amino Acid Composition-Purified @-1,3-glucanrecognition protein was dialyzed against double-distilled water and lyophilized. The lyophilized powder (about 0.5 mg)was hydrolyzed with constant boiling point HCl in sealed ampules at 110 “C for 24 h. Amino acids in the hydrolysate were analyzed on a Hitachi835 amino acid analyzer. Tryptophan content was determined separately after hydrolysis of sample with 4 N methanesulfonic acid at 115 “C for 24 h (18). Determination of Protein-Protein was determined by the method of Lowry et al. (19) using bovine serum albumin as thestandard. Chemicals-Chemicals were obtained from the following sources: beads of curdlan-type polysaccharide 13140 (0-1,3-glucan produced by Alcaligenesfaecalis var. myxogenes I F 0 13140 (20))was a gift from Dr. Y. Nakao of Applied Microbiology Labs., Takeda Chemical Ind. Ltd.(Osaka); zymosan and phenylmethanefsulfonyl fluoride were from Sigma; molecular weight standards and Affi-Gel-heparin were from Bio-Rad; Ampholine for pH range 3-6 was from Pharmacia LKB Biotechnology Inc.; DEAE-Toyopearl and cellulose powder were from Advantec Toyo Co. Ltd. (Kanda, Tokyo); standard proteins for isoelectric point calibration, Sephadex G-100, and pre-packed columns (Mono Q column, HR 5/5 and Superose 12 column, HR 10/30) were from Pharmacia LKB Biotechnology Inc.; p-nitrophenyl p ‘ guanidinobenzoate was from Vega Chemicals (Tuscon, AZ); peptidyl7-amino-4-methylcoumarins and 7-amino-4-methylcoumarinwere from Peptide Institute Inc. (Minoshi, Osaka). Other chemicals used were the highest grade commercially available.

i n Hemolymph Insect

y g protein / ml FIG. 1. Relation between the amount of @-1,3-glucanrecognition protein and its activity in an assay for &1,3-glucan recognition protein activity. Purified P-1,3-glucan recognition protein (Superose 12 fraction) was used and its activity assayed as describedunder “Materialsand Methods.” Abscissa and ordinnte show the protein concentrationof @-1,3-glucanrecognition protein solution subjected to activity assay and the @-l,l-glucanrecognition protein activity of the solution, respectively.


“ 2



Fraction Number


FIG. 2. DEAE-Toyopearl column chromatographyof @-1,3glucan recognition protein. Conditions are described under “Maactivity of terials and Methods.” 0-0, absorbance at 280 nm; 0-0, 8-1,3-glucan recognition protein. Broken line shows a gradient of KC1 concentration. The horizontal bar indicates fractions that were pooled and subjected to next step purification.

Assay of P-l,d-Glucan Recognition Protein-The procedure for assay of @-1,3-glucanrecognition protein in the prophenoloxidase activating system was developed as described under “Materials and Methods” and used in the course of the purification of the recognition protein from larval hemolymph of the silkworm, B. mori. As shown in Fig. 1,a linear relation was observed between the amount of P-1,3-glucan recognition protein andthe activity, but thelinear curve is displaced from the origin. Another feature of the assay method is that thelower limit of the detectable concentration of ,8-1,3-glucan recognition protein depends on the availability of plasma-CPB. For example, when separate preparationsof plasma-CPB were used, the concentration was found to be 2.5 and 1pg of recognition protein/ml of reaction mixture in Figs. 1 and 7, respectively.

Therefore, in using the method to quantitate P-1,3-glucan recognition protein, it is important to recognize the limit. In a series of purifications of P-1,3-glucan recognition protein described below, a single preparation of plasma-CPB was employed for the assay of recognition protein activity. Purification of ,8-1,3-GlucanRecognitionProtein-,8-1,3Glucan recognition protein was purified from 300 ml of larval silkworm hemolymph. The purification procedures consisted of ammonium sulfate fractionation and column chromatography on DEAE-Toyopearl, Affi-Gel-heparin, Mono Q, and Superose 12. Elution profiles of proteins and P-1,3-glucan recognition protein are presented in Figs. 2-5. Major and minor peaks of P-1,3-glucan recognition protein activity were detected by chromatography on Affi-Gel-heparin and Mono

Hemolymph Insect P-l,3-Glucan Recognition Prbotein in


chromatography /3-1,3-glucanrecognition protein activity was eluted at theposition corresponding to a major protein peak (Fig. 5). The major active fraction was used as purified 8-1,3glucan recognition protein. Typical data on the purification process of 8-1,3-glucan recognition protein are summarized in Table I. About 2.03 mg of P-1,3-glucan recognition protein was obtained from 300 ml of hemolymph. Homogeneity of Purified @-1,3-GlucanRecognition Protein and Preliminary Characterization of the Protein-Purified p1,3-glucan recognition protein migrated as a single band to the position corresponding to that of 62-kDa polypeptide in SDS-PAGE under reduced conditions (Fig. 6a). InIEF-PAGE the recognition protein preparation gave a single band, the position of which corresponded to PI 4.3 (Fig. 66). The amino acid composition of P-1,3-glucan recognition protein is preFraction Number sented in Table 11, from which partial specific volume was FIG. 3. Affi-Gel-heparin column chromatography of &1,3- calculated to be 0.736 ml/g (21). glucan recognition protein.Conditions are described under “MaMolecules of native /3-1,3-glucan recognition protein were terials and Methods.” Symbols are the same as in Fig. 2. sedimented to equilibrium at 10,000 rpm. A plot of ln(Azso) uersus (radius)’ gave a straight line, the slope of which together with a partial specific volume, 0.736ml/g,gave a molecular weight of 61,000. Thus, itis obvious that thenative molecule is composed of a single polypeptide of about 62 kDa. E 1.5. Purified /3-1,3-glucan recognition protein could restore the W activity of the prophenoloxidase activating systemin plasmaN CPB with respect to p-1,3-glucan as shown in Fig. 7. Under the experimental conditions, the concentration of p-1,3-glucan recognition protein in plasma-CPB influences the lag period of prophenoloxidase activation, whereas maximum velocity of prophenoloxidase activation was constant regardless of the concentration. Almost no difference of the lag period was observed in a range of concentrations higher than 5 pg of P-1,S-glucan recognition protein/ml of plasma-CPB. The proO t phenoloxidase activating system in plasma-CPB could not be 10 triggered at concentrations of /3-1,3-glucan recognition protein F r a c t i o n Number lower than 1pg/ml of plasma-CPB. FIG. 4. Mono Q columnchromatographyof @-1,3-glucan The purified (3-1,3-glucan recognition protein binds to precognition protein. Conditions are described under“Materials and Methods.” Solid line, broken line, and vertical bars show absorb- 1,3-glucan. The bound recognition protein did not dissociate ance at 280 nm, NaCl concentration, and P-1,3-glucan recognition appreciably at neutral pH even in a high salt concentration protein activity, respectively. (3.0 M NaCl, data not shown) or8 M urea (Fig. 8, lane 3))but dissociated in thepresence of 1% SDS, 1% 6-mercaptoethanol at 100 “C (Fig. 8). The dissociated @-1,3-glucanrecognition protein did not differ in terms of its polypeptide molecular mass from that of the native molecule. E The strong and specific affinity of P-1,3-glucan recognition C 0 protein to p-1,3-glucan enabled the detection of the @-1,3Eo glucan recognition protein without assaying its activity. As cu shown in lane 6 of Fig. 8, polypeptide with relative molecular m mass of 62 kDa was practically the only protein with the same al 0 degree of affinity as thatof purified /3-1,3-glucan recognition C m protein to fi-1,3-glucan among those present in the silkworm n L 0 plasma under the experimental conditions. p-1,3-Glucan recn ognition protein did not bind to Sephadex G-100 or cellulose, a which are composed of a-(1+ 6) (with minor part of ~ ( 1 2), a41 + 3), anda-(1-+4)) or (341+ 4)-glycosidic linkages, respectively, suggesting that the recognition protein has a Elution Volume (mll specific affinity to ,8-(143)-glycosidic linkages (Fig. 8). FIG. 5. Superose 12 column chromatography of j3-1,3-gluAmidase activity of ,f3-1,3-glucan recognition protein bound Sample was injected a t time 0 as indicated can recognition protein. with an arrow. For other details see “Materialsand Methods.” Sym- to zymosan was examined using 26 commercially available bols are the same as in Fig. 4. peptidyl-MCAs aslistedunder“Materials and Methods.” None of the substrateswas hydrolyzed appreciably by bound Q. However, in the present purification, the recognition pro- B-1,3-glucan recognition protein under the experimental contein in the minor peaks was discarded and only the recognition ditions, suggesting that @-1,3-glucanrecognition protein is protein in the major peak was purified. In Superose 12 column not an inactive form of protease. 1

d .



in Hemolymph Insect

/3-1,3-Glucan Recognition Protein


TABLE I Summary of purification of P-I,3-glucan recognition protein from larval hemolymph of the silkworm,B. mori Total volume



Specific activity







Recovery“ % I

300 Hemolymphb85.0 Ammonium sulfate 155 60.0 1 100 4.55 4.85 91.0 DEAE-Toyopearl 6.18 Affi-Gel-heparin2.71 71.0 106 2.70 1.00 44.7 73.0 185 68.5 Mono Q 29.8 Superose 12 0.38 5.34 ‘Purification fold and recovery was calculated based on the specific activity and total activity, respectively, of DEAE-Toyopearl fraction. * The volume of hemolymph collected from about 650 silkworm larvae. e Because hemolymph and ammonium sulfate precipitate contained unidentified factor(s) which causes activation of prophenoloxidase in plasma-CPB without P-1,3-glucan and inhibitors for protease and phenoloxidase, respectively, &1,3-glucan recognition protein activity in these fractionscould not be quantitated.

- 3.50

TABLEI1 Amino acid composition of P-1,3-glucan recognition protein Amino acid

92,5K66.2 -

Recovered amino acid” mo1/1000 mol

Asx 110 Thr 44 Ser 61 111 Glx G~Y 90 65 Ala %Cys 5 57 Val Met 10 Ile 59 Leu 74 42 TYr Phe 53 LYS 67 14 His 19 Trp ‘4% 41 Pro 78 “Values except for tryptophan are means of the results of three determinations in which separate samples were analyzed. Content of FIG. 6. SDS-PAGE and IEF-PAGE of purified @-1,3-glucan tryptophan was determined two times after hydrolyses with methrecognition protein. About 1.5 pg of protein was subjected to SDS- anesulfonic acid of separate samples as described under “Materials PAGE or IEF-PAGE. Other experimental detailsare described under and Methods,” and mean of the values is presented here. “Materials and Methods.” a, SDS-PAGE; b, IEF-PAGE. In SDSPAGE, gel was calibrated with the following protein molecular mass protein was successfully employed to quantitate the recognimarkers and their weights in kilodaltons (K) are indicated at theleft tion protein as shown in Fig. 1. The method is sufficiently of a: phosphorylase b (92,500);bovine serum albumin (66,200); ovalbumin (45,000);carbonic anhydrase (31,000); soybean trypsin inhib- sensitive to allow detection of 0.1-0.25 pg of the recognition itor (21,500);lysozyme (14,400). In IEF-PAGE, the gel was calibrated protein,although it is influenced by unknownfactors as with the following isoelectric pointmarkers andtheir isoelectric shown by the fact that thesensitivity of the method depends points are indicated at the right of b: amyloglucosidase (PI 3.50); on thequality of plasma-CPB (Figs. 1 and 7). glucose oxidase (PI 4.15); soybean trypsininhibitor (PI 4.55); pProphenoloxidase in insect hemolymph can be activated lactoglobulin A (PI 5.20); bovine carbonic anhydrase B (PI 5.85).



- 4,15



- 4,55



- 5,20 - 5,85

14,4 -


In a previous report we proposed that theprophenoloxidase activating system in insect hemolymph has two entry sites where putative /3-1,3-glucan recognition protein and peptidoglycan recognition protein are located (12). We also speculated that interaction of these molecules with P-1,3-glucan or peptidoglycan, respectively, initiates activation of the cascade. In the present investigation we have established a method for assaying P-1,3-glucan recognition protein activity and confirmed the existence of a putatative @-1,3-glucanrecognition protein. A proteinaceous molecule has been purified with specific affinity to @-1,3-glucanand the ability to make the prophenoloxidase activating system reactive to /3-1,3-glucan. A new method for the assay of P-1,3-glucan recognition

not only by the action of P-1,3-glucan or peptidoglycan, but also by a-chymotrypsin (22) orotherunidentifiedsubstance(s); thisproperty becomes evident when hemolymph is collected by the conventional methodof cutting larvalabdominal legs (23). Silkworm hemolymph for purification of P-1,3-glucan recognition protein was collected by the conventional method, which may explain why hemolymph activated prophenoloxidase in plasma-CPB without P-1,3-glucan (Table I), thereby making the assay method unapplicable to hemolymph. It is likely that the substance(s) responsible for the P-1,3-glucanindependent prophenoloxidase activation was separated from P-1,3-glucan recognition protein during ammonium sulfate fractionation and chromatography on Mono Q column. We tried to elute native P-1,3-glucan recognition protein from curdlan-type polysaccharide beads to achieve a one-step

Protein in Hemolymph Insect

P-1,3-Glucan Recognition





60 Time


FIG. 7. Activation of prophenoloxidase activating system by j3-1,3-glucan in plasma-CPB supplemented with purified j3-1,3-glucan recognition protein. Each reaction mixture consisted of 25 pl of @-1,3-glucanrecognition protein solution, 200 pl of plasma-CPB containing 5 mM CaC12,and 25 pl of zymosan solution (100 pg/ml). Reaction mixtures were incubated at 25 “C, and at intervals an aliquot was assayed for phenoloxidase activity to monitor the activation of prophenoloxidase activating system. Concentrations of P-1,3-glucan recognition protein in reaction mixtures (pg of protein/ml of reaction mixture): 0-0,10 pg; 0-0, 5 pg; A-A, 1 pg; 0-0, 0.1 pg.








92.5 K 66.2


45 31 21,5 14.4


FIG. 8. Examination of binding of j3-1,3-glucan recognition protein to insoluble glucans. Procedures for binding of plasma proteins and purified P-1,3-glucan recognition protein onto beads of curdlan-type polysaccharide (P-1,3-glucan) and a mixture of Sephadex G-100 and cellulose and for elution of the adsorbed proteins from the glucans are described under “Materials and Methods.” Samples subjected to SDS-PAGE: 1,purified P-1,3-glucan recognition protein; 2, protein elutedfrom beads of curdlan-type polysaccharide previously treated with purified P-1,3-glucan recognition protein and washed with T-M buffer; 3, same as in lane 2 except that beads of curdlantype polysaccharide were washed sequentially with T-M buffer and 8 M urea; 4 , same as in lane 3 except that a mixture of Sephadex G-100 and cellulose was used instead of beads of curdlan-type polysaccharide; 5, plasma (2 pl); 6, protein eluted from beads of curdlan-type polysaccharide previously treated with plasma (2 ml) and washed as in lane 3; 7, same as in lane 5 except that a mixture of Sephadex G100 and cellulose was employed.

purification of the molecule. But solutions, which could elute @-1,3-glucan recognition protein from the beads, always caused the inactivation of the molecule. Therefore, for the purification of P-1,3-glucan recognition protein, larval silkworm hemolymph was fractionated by ammonium sulfateand chromatography on DEAE-Toyopearl, Affi-Gel-heparin, and Mono Q and Superose 12 in the fast protein liquid chroma-


tography system of Pharmacia LKB Biotechnology Inc. The final product of the purification was a homogeneous preparation of protein (Fig. 6). The purified protein is the only molecule with affinity to P-1,3-glucan among the proteins present in plasma under the experimental conditions (Fig. 8) and makes the prophenoloxidase activating system reactive to @-1,3-glucan(Fig. 7). Therefore, the P-1,3-glucan recognition protein appearsto be the molecule predictedin our previous report (12). In addition to the major peak of /3-1,3glucan recognition protein activity, minor activitypeaks were eluted from Affi-Gel-heparin or Mono Q columns (Figs. 3 and 4). Itis not clear whether the P-1,3-glucan recognition protein in the minor peaksis artifact generated during purification or if more than one kind of /?-1,3-glucan recognition protein occurs in vivo. Preliminarycharacterization of /3-1,3-glucan recognition protein indicated that the molecule is a single polypeptide, because molecular weight determined under nondenaturedor denatured (in the presence of 1%SDS) conditions coincides well (61,000 and 62,000, respectively). No amino sugars were detected in theamino acid analysis of the recognition protein, suggesting that themolecule is not a glycoprotein. @-1,3-Glucan recognition protein showed specific and strong affinity to /3-1,3-glucan (Fig. 8). In the light of our previous observation (24) that only glycans with ,8-1,3-glycosidic linkages could trigger the prophenoloxidase activating system in silkworm plasma, the demonstrated specificity of binding of the purified /3-1,3-glucan recognition protein indicates that binding of the recognition protein to P-1,3-glucan is a necessary condition for the molecule to display its potential activity for triggering the prophenoloxidase activating system. When the prophenoloxidase activating system in plasmaCPB supplemented withP-1,3-glucan recognition protein was triggered with P-1,3-glucan, the evolution of phenoloxidase activity was influenced in sucha way that thehigher concentration of the recognition protein caused a shorter period of lag time. However, above the concentration of 5 pg of recognition protein/mlof plasma-CPB, the lag period did not vary appreciably. If the concentration of /3-1,3-glucan recognition protein in plasma-CPB was decreased to 0.1 pg of proteinlml, the prophenoloxidase activating system was no longer triggered by /3-1,3-glucan under the experimental conditions(Fig. 7). These phenomena appear to relate to the mode of action of /3-1,3-glucan recognition protein in triggering the system. In the horseshoe crab, the well known blood coagulation system is triggered by /3-1,3-glucan or lipopolysaccharide. A protein (factor C) has been shown to be activated through interaction with lipopolysaccharide and the activated factor C to have amidase activity (25). The possibility of a similar mechanism operating in the activation of the prophenoloxidase activating system by P-1,3-glucan was investigated. /31,3-Glucan recognition proteinbound to zymosan did not show appreciable amidase activity to any of the peptidylMCAs examined. A feature of the prophenoloxidase activating system that contrastswith the alternativecomplement pathway, a cascade present in mammalian blood, is that every component of the system is not inactivated by 1 (1)or 10 mM3 diisopropyl fluorophosphate before being triggered by elicitor. This indicates that all the serine enzymes in the prophenoloxidase activating system (an esterase hydrolyzing benzoylL-arginine ethyl ester, prophenoloxidase activating enzyme, and maybe yet unknown serine enzymes) are activated from zymogens as a consequence of binding of P-1,3-glucan recognition protein to /3-1,3-glucan.It is probable that /3-1,3-glucan M. Ochiai and M. Ashida, unpublished observations.


,8-1,3-GlucanRecognition Protein inInsect Hemolymph

recognition protein bound to /3-1,3-glucan offers an environment in which one of the above zymogens is activated. Obviously, much remains to be studied on the physicochemistry of /3-1,3-glucan recognition protein and on the mode of action of the molecule in the activation of the prophenoloxidase activating system by P-1,3-glucan. As asubstantial amount of homogeneous /3-1,3-glucan recognition protein is now available, detailed studies of these topics are in progress in our laboratory and will be reported elsewhere. The results of such studies will help advance our understanding of the effects exerted by /3-1,3-glucan in various biological systems other than insects. The /3-1,3-glucan recognition protein in the insect prophenoloxidase activating system is the first molecule purified with specific affinity to /3-1,3-glucan and proposed to be located at theprimary site of its action. Acknowledgements-We wish to extend sincere gratitude to Dr. Y. Nakao of Applied Microbiology Labs., Takeda Chemical Ind. Ltd., Osaka, for providing us with curdlan-type polysaccharide beads and Prof. R. G . H. Downer of Waterloo University for reading the manuscript. REFERENCES 1. Yoshida, H., and Ashida, M. (1986) Insect Biochem. 16,539-545 2. Soderhall, K. (1982) Deu. Comp. Immunol. 6,601-611 3. Ratcliffe, N. A., Leonard, C. M., and Rowley, A. F.(1984) Science 226,557-559 4. Huxham, I. M., and Lackie, A. M. (1988) Cell Tissue Res. 2 5 1 , 677-684 5. Ashida, M., Iwama, R., Iwahana, H., and Yoshida, H. (1982) Proceedings of the 3rd International Colloquium on Invertebrate Pathology, Vol. 3, pp. 81-86, University of Sussex, Brighton, U. K.

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