Purification of a peptidoglycan recognition protein from hemolymph of the silkworm, Bombyx mori.

A method was developed for obtaining a homogeneous silkworm hemolymph protein (peptidoglycan recognition protein, PGRP) which has affinity for peptidoglycan and the ability to trigger the prophenoloxidase cascade upon its binding to peptidoglycan. The purified PGRP had a molecular mass of about 19 kDa and is composed of a single polypeptide with an isoelectric point of 6.5. It bound to peptidoglycan in the absence of divalent cation, whereas its binding to β1,3-glucan and chitin was not detected. N-Acetyl-D-glucosaminyl-(β1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine did not inhibit purified PGRP to bind insoluble peptidoglycan, but fragmented soluble peptidoglycan did. PGRP seemed to require peptidoglycan as a possible ligand to keep its glycan portion consisting of at least two or more of the repeating unit. PGRP did not have any detectable lysozyme activity, and its amino acid composition and amino-terminal sequence of 20 amino acid residues were shown to be different from those of silkworm lysozyme. PGRP seems to be a hitherto unknown protein. In the absence of PGRP, the prophenoloxidase cascade in the plasma fraction of hemolymph could not be triggered by peptidoglycan, indicating that some type of activity, capable of activating the cascade, is generated upon their binding. However, the exact nature of this activity is not yet known. The purified PGRP bound to peptidoglycan did not hydrolyze significantly any of the 26 commercially available peptidyl-7-amino-4-methylcoumarins, substrates for various proteases.

Insects have effective defense mechanisms against microorganisms such as bacteria and fungi. The major defense mechanisms in the hemocoel are either cellular (phagocytosis, hemocyte aggregation (nodule formation) and formation of multicellular hemocytic capsules (encapsulation)) (1) or humoral effected by immune proteins, lectins, and the prophenoloxidase cascade (2)(3)(4)(5)(6). Recognition of microorganisms as non-self is apparently involved when the insect defense mechanisms are set in motion. As stated by Janeway (7), clonally selected recognition molecules like immunogloblins in higher vertebrates have not evolved in insects, but molecules for nonclonal pattern recognition play a central role in discrimination of self from non-self. Thus, a number of molecules with affinity to particular structures of bacterial or fungal cell walls have been reported from insect hemolymph and they are thought to be potential recognition molecules for foreignness. It is reasonable that recognition molecules for foreignness have to be expressed constitutively and to be present in the plasma or cell surface before invasion by foreign objects. Hemolin (8,9), lectins with various ligand specificities (4, 10 -13) and proteins with affinity to ␤1,3-glucan (14 -16), lipopolysaccharide (17), and peptidoglycan (18) have been suggested as recognition molecules. However, the biological activities of these proteins are far from being fully understood. Proteins with affinity to ␤1,3-glucan and peptidoglycan were originally discovered in the course of studies on prophenoloxidase cascade.
The prophenoloxidase cascade is present in insect and crustacean hemolymph (5,6). Very recently the chitinous cuticle of the insect exoskeleton was also found to contain a prophenoloxidase cascade (19). The hemolymph prophenoloxidase cascade consists of prophenoloxidase, serine protease zymogens, and proteins with specific affinity to bacterial or fungal cell wall components. It is activated by various non-self materials naturally or artificially introduced into the hemocoel. Under the conditions where the cascade is triggered, increased phagocytosis and hemocyte movement have been observed (20,21). After activation, phenol oxidase catalyzes melanin synthesis from phenolic substances. Melanization is thought to facilitate sequestration of pathogens in nodules or multicellular capsules (22), and more recently it has been speculated that intermediate compounds (quinones) in melanin synthesis from mono-or diphenols are highly toxic to living cells including infectious microorganisms (23). Thus, the prophenoloxidase cascade is considered an integral part of insect defense mechanisms.
Previously we predicted the presence of two kinds of hemolymph molecules which have specific affinity for peptidoglycan (a bacterial cell wall component) and ␤1,3-glucan (a fungal cell wall component), respectively (18). Both molecules have been supposed to have ability to trigger prophenoloxidase cascade upon binding to their respective ligands. We proposed to name them ␤1,3-glucan recognition protein (␤GRP) 1 and peptidoglycan recognition protein (PGRP) (14). The postulated ␤GRP has been isolated from silkworm, cockroach, and crayfish (14 -16). cDNA of crayfish ␤1,3-glucan binding protein (which has been shown to have similar function to silkworm ␤GRP) was cloned and the deduced amino acid sequence of the protein was reported (24).
Peptidoglycan has been shown to have various biological functions both in mammals (25) and insects (26). Specific binding proteins or receptors have been reported from mammals (27)(28)(29), but such molecules have not been isolated from invertebrates. Previously, a plasma fraction of silkworm hemolymph, which had been passed through Sepharose 4B coupled to peptidoglycan in the absence of divalent cation, was shown to have all prophenoloxidase cascade components except for the putative PGRP. This plasma fraction (referred to as plasma-PG) was proposed to be used for assaying PGRP in the course of its purification (18).
We now describe a method for assaying PGRP by using plasma-PG and a procedure for obtaining homogeneous PGRP, as well as preliminary characterization of the molecule.

MATERIALS AND METHODS
Animals-Silkworm (Bombyx mori) larvae were reared on an artificial diet as described (30).
Preparation of Peptidoglycan and Soluble Fragmented Peptidoglycan-Peptidoglycan was prepared from Micrococcus luteus cell walls according to the method of Araki et al. (31).
For the preparation of soluble fragmented peptidoglycan, peptidoglycan was digested with egg white lysozyme (chicken) and fractionated by chromatography on a Sephadex G-50 SF column as described (18). Fractions (numbers 57 to 81) obtained in the chromatography were pooled and lyophilized. The lyophilized powder was dissolved in a small volume of distilled water and centrifuged at 89,000 ϫ g for 30 min to remove flocculent materials and the supernatant was again lyophilized. The lyophilized powder thus obtained was dissolved in 10 mM Tris maleate buffer, pH 6.5, containing 0.15 M NaCl (T-M buffer) at a concentration of 2 mg/ml and used as a soluble fragmented peptidoglycan preparation.
Preparation of Silkworm Plasma (Plasma-PG) for Assaying PGRP-The plasma fraction of hemolymph was prepared as described previously (30,32). The plasma fraction was passed through a peptidoglycan-Sepharose 4B column in the presence of EDTA according to Yoshida et al. (18). The effluent was named plasma-PG and used for assaying PGRP. The prophenoloxidase cascade in plasma-PG was triggered with ␤1,3-glucan but not with peptidoglycan (18), which indicates that PGRP had been removed.
Assay for PGRP Activity-The sample to be assayed for PGRP was serially diluted, and 10 l of each dilution was added to a mixture of 5 l of 80 mM CaCl 2 , 50 l of plasma-PG, and 5 l of peptidoglycan (1 mg of peptidoglycan/ml distilled deionized water, prepared after Yoshida et al. (18)), followed by incubation at 25°C for 120 min. At the end of the incubation, phenol oxidase activity of the reaction mixture was assayed spectrophotometrically (30). To eliminate the possibility that the observed activation of prophenoloxidase was independent of peptidoglycan action, phenol oxidase activity of the reaction mixture devoid of peptidoglycan was always checked after incubation.
The greatest dilution of PGRP giving more than 30 units of phenol oxidase activity in the reaction mixture during the incubation period of 120 min was determined, and the reciprocal of the dilution factor used as a tentative measure for quantifying the amount of PGRP. The reciprocal is expressed as the number of units of PGRP activity/ml of sample solution in the present study.
Purification of PGRP-Silkworm larvae on the 5th or 6th day of the fifth instar were bled by cutting abdominal legs with scissors. Hemolymph was immediately mixed with saturated ammonium sulfate, pH 6.5, under vigorous stirring. Two hundred-fifty ml of hemolymph from about 400 larvae was collected into 440 ml of saturated ammonium sulfate and stored at 4°C until use. All subsequent procedures were performed at 0 -4°C and centrifugation was carried out at 12,000 ϫ g for 20 min unless otherwise specified.
The preparation was centrifuged and the precipitate was suspended in 390 ml of 0.2 M potassium phosphate buffer, pH 6.5, containing 1 mM EDTA, 1 mM 1,10-phenanthroline, 1 mM phenylmethanesulfonyl fluoride, 5 mM phenylthiourea, and 1% ethanol. The suspension was stirred for 2 h, followed by centrifugation at 4,800 ϫ g for 20 min. Ammonium sulfate was added to the supernatant (69 g/500 ml of the supernatant) and the mixture was stirred for 2 h. Precipitated material was then collected by centrifugation and dissolved in 100 ml of 0.1 M potassium phosphate buffer, pH 6.5, containing additives as above. The solution was dialyzed for 30 h against the same buffer (1.9 liter) followed by dialysis against two changes of 0.1 M potassium phosphate buffer, pH 6.5.
The dialyzed solution was applied at a flow rate of 20 ml/h to a peptidoglycan-Sepharose 4B column (5 ϫ 2.5-cm inner diameter) according to the method of Yoshida et al. (18) except that lysozymedigested peptidoglycan was used without fractionation by column chromatography on Sephadex G-50. The column was then sequentially eluted at 20 ml/h with the following eluants: 50 ml of 0.1 M potassium phosphate buffer, pH 6.5; a linear gradient of KCl from 0 to 2 M in a total volume of 120 ml of 0.1 M potassium phosphate buffer, pH 6.5; 60 ml of 5 mM MES, pH 5.5, containing 2 M KCl. The final elution was carried out with 150 ml of 5 mM acetate buffer, pH 4.5, containing 2 M KCl at a flow rate 220 ml/h. Thirty-ml fractions were collected in containers containing 1.2 ml of 0.5 M Pipes, pH 7.0. All of the fractions were dialyzed separately against 3 liters of 10 mM potassium phosphate buffer, pH 6.5, for 18 h with a change of buffer.
The following column chromatography was performed at room temperature on a fast protein liquid chromatography system (FPLC; Pharmacia LKB Biotechnology Inc.). The active fractions (numbers 8 -12 in Table I) obtained in the previous step were applied at a flow rate of 1 ml/min to a hydroxyapatite column (100 ϫ 7.8-mm inner diameter; Koken Ltd., Tokyo) for high pressure liquid chromatography, previously equilibrated with 10 mM potassium phosphate buffer, pH 6.5, followed by washing the column with 10 ml of the same buffer. The adsorbed proteins were eluted at a flow rate of 1.0 ml/min with two consecutive linear gradients of potassium phosphate buffer, pH 6.5, from 10 to 144 mM and from 144 to 1 M with concentration incremental rates of 2.48 and 93 mM/min, respectively. Fraction volume was 1.5 ml. Fractions eluted between 190 and 198 min from the beginning of the application of phosphate gradient were pooled ( Fig. 1) and dialyzed overnight against 2 liter of 10 mM triethanolamine-HCl buffer, pH 7.5.
The dialyzed fractions were applied to a Mono Q column (HR 5/5) (Pharmacia LKB Biotechnology Inc.), equilibrated with the same buffer as that used for dialysis. Adsorbed proteins were eluted with a linear salt gradient in the same buffer (Fig. 2). The flow rate was maintained at 1 ml/min and 1.5-ml fractions were collected. The fraction with the highest PGRP activity (Fig. 2) was used as the PGRP preparation for study.
For the amino acid sequence analyses, the purified PGRP was passed through a reversed phase cyanopropyl-derived silica high performance liquid chromatography column (4.6 mm inner diameter ϫ 250 mm, pore size ϭ 300 Å) as follows: 0.5 ml of PGRP solution (about 40 g of protein/ml of 10 mM Tris-HCl buffer, pH 7.5, containing 0.1 M NaCl) was applied to the column which was then eluted with a gradient of CH 3 CN from 30 to 70% in 0.1% CF 3 COOH/H 2 O at a flow rate of 0.4 ml/min. It took 50 min to finish the gradient. The only protein peak appeared at 29.69 min from the beginning of the gradient elution. Protein contained in the peak was pooled and lyophilized.
Purification of Silkworm Lysozyme-Lysozyme activity was assayed by measuring turbidity of Micrococcus lysodeikticus (Seikagaku Kogyo Ltd., Tokyo) suspension according to the manufacturer's instruction manual.
The supernatant at 65% saturation of the ammonium sulfate fractionation of silkworm hemolymph during the first step of PGRP purification was dialyzed against 2.5 liters of 10 mM phosphate buffer, pH 6.5, for 40 h with three changes of the buffer. The dialyzed solution was applied to a CM-cellulose column (180 ϫ 21-mm inner diameter) equilibrated with the same buffer at a flow rate of 100 ml/h. Adsorbed proteins were eluted at a flow rate 10 ml/h with a linear gradient from 0 to 1.0 M KCl in 200 ml of 10 mM potassium phosphate buffer, pH 6.5. Active fractions eluted at about 0.5 M KCl were pooled and dialyzed against 3 liters of 3 mM potassium phosphate buffer, pH 6.5, for 24 h with a change of the buffer, followed by chromatography on hydroxyapatite column (100 ϫ 7.8-mm inner diameter; Koken Ltd., Tokyo) in the fast protein liquid chromatography. A gradient with an incremental rate of phosphate buffer, pH 6.5, concentration, 1 mM/min was applied at a flow rate of 1 ml/min to the column. A major peak eluted at 0.28 M was used as purified silkworm lysozyme.
Determination of the Molecular Weight of Native PGRP-The molecular weight of native PGRP was estimated with two methods.
The PGRP (A 280 nm ϭ 0.5) in 40 mM potassium phosphate buffer, pH 6.5, containing 0.2 M NaCl was subjected to sedimentation equilibrium ultracentrifugation which was conducted by the method of Yphantis (33) using a Hitachi analytical ultracentrifuge (Model 282 equipped with a Hitachi ultracentrifuge processor (Model-7).
The native PGRP (15 g) was chromatographed at a flow rate of 0.5 ml/min on Superose 12 column equilibrated with 10 mM Tris-HCl buffer, pH 7.5, containing 0.15 M NaCl. Egg white ovalbumin (chicken, 45 kDa), myoglobin (horse skeletal muscle, 17 kDa), and cytochrome c (horse heart, 12.4 kDa) were used as proteins for molecular mass standard. Two-hundred l of each standard protein solution containing 200 g of protein was subjected to chromatography on the Superose 12 column under the same conditions as PGRP. A plot of their retention times against logarithms of their molecular masses gave a straight line. The molecular mass corresponding to the retention time of PGRP was read from the line.

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and
Isoelectric Focusing-Polyacrylamide Gel Electrophoresis (IEF-PAGE)-SDS-PAGE was carried out in a 1-mm thick slab gel according to Laemmli (34), with 12% acrylamide in the separation gel.
Tests for Ability of Purified PGRP to Bind Peptidoglycan, Chitin, and ␤1,3-Glucan and the Effect of Some Constituents of Peptidoglycan and Soluble Fragmented Peptidoglycan on the Ability-One mg of peptidoglycan was dispersed in 1 ml of T-M buffer and washed with 1 ml of this buffer by four cycles of sedimentation and suspension by centrifugation at 12,600 ϫ g for 5 min. The sedimented peptidoglycan was suspended in 0.5 ml of T-M buffer. Forty-l of purified PGRP (73 g of protein/ml of 10 mM triethanolamine-HCl buffer, pH 7.5, containing 0.04 M NaCl) was mixed with 120 l of T-M buffer and 160 l of the peptidoglycan suspension, and incubated at 4°C for 30 min. The mixture was then centrifuged at 12,600 ϫ g for 5 min. The sedimented peptidoglycan was washed with 300 l of T-M buffer as above except that sedimented peptidoglycan was transferred to a new vessel before the final washing. The washed peptidoglycan was extracted with 80 l of solubilizing solution (0.0625 M Tris-HCl buffer, pH 6.8, containing 2% SDS, 19% glycerol, and 5% ␤-mercaptoethanol). The supernatant (20 l) of the mixture of PGRP and peptidoglycan suspension and extract (20 l) of the sedimented peptidoglycan were subjected to SDS-PAGE together with a known amount of purified PGRP. Binding of purified PGRP to chitin (colloidal) and ␤1,3-glucan (insoluble beads of ␤1,3glucan) was examined as above except that peptidoglycan was replaced with chitin and ␤1,3-glucan, respectively.
To investigate the effect of N-acetyl-D-glucosaminyl-(␤1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP), N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), N-acetylmuramic acid, N-acetylglucosamine, and soluble fragmented peptidoglycan on ability of purified PGRP to bind to peptidoglycan was individually examined as above except that purified PGRP was preincubated for 30 min at 4°C with the compounds at 2.19 mM and the soluble fragmented peptidoglycan at 1.55 mg/ml for 30 min at 4°C in T-M buffer. The preincubated PGRP was then examined for its ability to bind to peptidoglycan as above, except that all procedures until the end of washing of peptidoglycan were performed in the presence of the constituents at 1.41 mM and soluble fragmented peptidoglycan at 1 mg/ml.
Search for Proteins in 0 -50 Fraction with Affinity to Peptidoglycan-A plasma fraction (0 -50 fraction) was prepared from hemolymph of silkworm larvae as described (36). The 0 -50 fraction did not contain lysozyme activity which is known to be detected in silkworm hemolymph. One hundred-forty l of 0 -50 fraction (7.41 mg of protein/ml) was treated with 160 l of peptidoglycan suspension in the presence of 1 mM of (p-amidinophenyl)methanesulfonyl fluoride. The mixture was then centrifuged and the sedimented peptidoglycan was washed with T-M buffer followed by extraction of proteins with 80 l of solubilizing solution as described in the preceding section. The extract (20 l) was subjected to SDS-PAGE.
Examination of Reactivity of PGRP to Lectins-The reactivity was examined (37) using peroxidase-conjugated lectins. Briefly, purified PGRP (1.5 g of protein) was run on SDS-PAGE as described above and transferred to polyvinylidine difluoride membranes which were subsequently blotted with peroxidase-conjugated lectins (concanavalin A, Lentil seed agglutinin A, Dolichos biflorus agglutinin, Phaeaseolus vulgalis agglutinin E4, Arachis hypogaea agglutinin, Ulex europaeus agglutinin 1, and wheat germ agglutinin). The blots were visualized by incubation with 4-chloronaphtol and H 2 O 2 .
Determination of NH 2 -terminal Amino Acid Sequence of PGRP-PGRP preparation purified on cyanopropyl-derived silica HPLC column was analyzed for its NH 2 -terminal amino acid sequence by automated Edman degradation with a protein sequencer (model 477A, Applied Biosystems) (38). The sequence was verified by analyzing about 300 pmol of the PGRP 4 times.
Preincubation mixtures, comprising 5 volumes of PGRP solution (0.3 mg of protein/ml in 10 mM potassium phosphate buffer, pH 6.5, containing 150 mM NaCl) and 1 volume of peptidoglycan suspension (0.1 mg/ml distilled deionized water) or 5 volumes of the PGRP solution and 1 volume of distilled deionized water, were incubated at 25°C. After 10 min incubation, 10-l samples of the preincubation mixtures were assayed for amidase activity. The reaction mixture for the assay consisted of 480 l of T-M buffer containing 5 mM CaCl 2 , 10 l of 5 mM fluorogenic substrate, and 10 l of the above preincubation mixture. After incubation at 30°C for 120 min, 500 l of 50% (v/v) acetic acid was added to terminate the enzyme reaction. The amount of liberated 7-amino-4-methyl-coumarin was determined after Kojima et al. (39) from fluorescence at 460 nm with excitation at 380 nm, using a Hitachi 204-A fluorescence spectrophotometer. For controls the same preincubation mixtures except for the PGRP were prepared and their amidase activity was assayed as above. The peptidyl-NH-Mecs used were the same as those in our previous report (14). The substrates were dissolved in distilled deionized water, dimethyl sulfoxide, or dimethylformamide according to the manufacturer's instruction.
Analyses of Amino Acid Composition-Purified PGRP was dialyzed against distilled deionized water and lyophilized. The lyophilized powder (about 0.2 mg) was hydrolyzed with 4 N methane sulfonic acid at 115°C for 24 h (40), and amino acids were analyzed on a Hitachi 835 amino acid analyzer.
Determination of Protein-Protein was determined by the method of Lowry et al. (41) with bovine serum albumin as the standard.

RESULTS
Assay of PGRP-The assay procedure for PGRP in plasma-PG was developed as described under "Materials and Methods" and used in the chromatographic purification of the recognition protein from larval hemolymph of the silkworm, B. mori (Table I, Figs. 1 and 2). The assay was not applicable to crude preparations of PGRP such as the ammonium sulfate fraction, which contained substance(s) causing the activation of prophenoloxidase in plasma-PG in the absence of peptidoglycan (Table II). When the PGRP concentration was above 90 ng of protein/ml in plasma-PG, it could be detected (Fig. 4).
Purification of PGRP-PGRP was purified from 250 ml of larval silkworm hemolymph. The purification procedure consisted of ammonium sulfate fractionation, column chromatography on peptidoglycan-Sepharose 4B, hydroxyaptite, and Mono Q. Elution profiles of proteins and PGRP are shown in Table I and Figs. 1 and 2. As PGRP could not be quantitated in hemolymph and the ammonium sulfate fraction, the yield of PGRP in the first steps could not be calculated. Nonetheless, the effectiveness of the affinity chromatography is evident considering that the initial amount of hemolymph protein was reduced to about 1/20,000 in the peptidoglycan-Sepharose 4B active fraction. After three chromatographic steps, 283 g of protein with PGRP activity was obtained (Table II).
Homogeneity of Purified PGRP and Preliminary Character-ization of the Protein-In SDS-PAGE under reducing conditions, purified PGRP migrated as a single band to the position corresponding to that of the 19-kDa polypeptide (Fig. 3, lane a). Lysozyme purified from the same hemolymph showed a smaller molecular mass (16.5 kDa) in the SDS-PAGE (Fig. 3,  lane b).
In IEF-PAGE, the PGRP preparation gave a single band, the position of which corresponded to about pI 6.5 (data not shown). The amino acid composition of PGRP is presented in Table III together with that of silkworm lysozyme for comparison. No amino sugar was detected in the amino acid analysis of PGRP. Peroxidase-conjugated lectins (concanavalin A, Lentil seed agglutinin A, D. biflorus agglutinin, P. vulgalis agglutinin E4, A. hypogaea agglutinin, U. europaeus agglutinin 1, and wheat germ agglutinin) were not reactive to PGRP under the experimental conditions as described under "Materials and Methods." The absence of amino sugar and the non-reactivity of the lectins corroborate the possibility that PGRP is not a glycoprotein, although more thorough studies are necessary to demonstrate unambiguously the absence of sugar moiety in PGRP.
Native PGRP was sedimented to equilibrium at 23,000 rpm. A plot of ln(A 280 ) versus (radius) 2 gave a straight line. If the partial specific volume was assumed to be 0.75 ml/g, the slope of the line corresponded to that of protein with a molecular mass of 14 kDa. Native PGRP eluted as a symmetrical peak from the Superose 12 column with a retention time corresponding to that of a protein of 17 kDa when the column was calibrated with ovalbumin (chicken), myoglobin (horse skeletal muscle), and cytochrome c (horse heart) for molecular mass determination (data not shown). These values are smaller than the molecular mass of PGRP observed in SDS-PAGE. The reason for the inconsistency was unclear, but we concluded that native PGRP is likely to exist as monomer.
The restoration of reactivity of the prophenoloxidase cascade to peptidoglycan in plasma-PG supplemented with varied amounts of purified PGRP is shown in Fig. 4. A decreasing lag period was observed as the concentration of PGRP increased. Once activation of prophenoloxidase is initiated, however, the rate of conversion of prophenoloxidase to phenol oxidase seems to be independent of the amount of PGRP added. The kinetics for the activation of prophenoloxidase must reflect the underlying molecular mechanism for triggering prophenoloxidase cascade by peptidoglycan.
Purified PGRP was shown to bind to peptidoglycan, but not to chitin or ␤1,3-glucan (Fig. 5A). GMDP, MDP, N-acetylmuramic acid, and N-acetylglucosamine, all of which are the constituents of peptidoglycan, did not inhibit appreciably the binding of PGRP to peptidoglycan. However, soluble fragmented peptidoglycan inhibited appreciably for PGRP to bind to insoluble peptidoglycan (Fig. 5B). As our preliminary experiments indicated that the glycan portion of peptidoglycan has the ability to trigger prophenoloxidase cascade, 2 it is probable that PGRP binds to the glycan portion of peptidoglycan.
A fraction (0 -50 fraction), which has been shown to have all the components of prophenoloxidase cascade except prophenoloxidase, was prepared to examine whether proteins other than PGRP with affinity to peptidoglycan are present in silkworm hemolymph. A few polypeptides in the fraction were detected to bound to peptidoglycan (Fig. 6). It remains, however, to be investigated whether each of them can be adsorbed directly onto peptidoglycan by itself.
The sequence of 20 amino acid residues from the NH 2 terminus of PGRP was determined as:  second residue could not be identified.
Amidase activity of PGRP bound to peptidoglycan was examined using 26 commercially available peptidyl-NH-Mecs. None of the substrates were hydrolyzed significantly (data not shown), suggesting that PGRP is not an inactive protease which becomes active after binding to peptidoglycan. DISCUSSION We have previously proposed that the insect hemolymph prophenoloxidase cascade includes a ␤GRP and a PGRP, which have specific affinity to ␤1,3-glucan and peptidoglycan, respectively (18). These molecules were proposed to trigger the cascade upon binding to their respective ligands. Ochiai and Ashida (14) reported a method to obtain a homogeneous preparation of the putative ␤GRP. The postulated PGRP, capable of binding to peptidoglycan has been demonstrated, purified, and characterized in the present study.
The purified PGRP preparation was shown to be homogeneous by SDS-PAGE, IEF-PAGE, reversed-phase HPLC on cyanopropyl-derived silica column, and the determination of a 20-amino acid NH 2 -terminal sequence. The molecules are capable of restoring the reactivity of the prophenoloxidase cascade to peptidoglycan in plasma-PG which is assumed to contain all components of prophenoloxidase cascade except for PGRP (Fig. 4). These results indicate that the purified protein is PGRP. The molecular weight, isoelectric point, and amino acid composition of PGRP are different from those of ␤GRP (14). So, it is now proved unambiguously that the prophenoloxidase cascade in insect hemolymph has two points of initiation where PGRP and ␤GRP interact with their respective ligands.  a Yield was calculated based on the total activity of peptidoglycan-Sepharose 4B fraction. b Because hemolymph and ammonium sulfate precipitate contained unidentified factor(s) which causes activation of prophenoloxidase in plasma-PG in the absence of peptidoglycan, PGRP activity in these fractions could not be quantified.
c Not determined.   Asx  100  142  Thr  33  54  Ser  71  78  Glx  111  70  Gly  115  78  Ala  69  45  Cys/2  21  58  Val  90  35  Met  10  8  Ile  48  32  Leu  82  66  Tyr  37  31  Phe  23  35  Lys  28  107  His  39  41  Trp  16  28  Arg  63  65  Pro  43  25 a PGRP and lysozyme were analyzed for amino acid composition as described under "Materials and Methods." The assay for PGRP activity by using plasma-PG enabled us to detect PGRP at concentrations as low as 90 ng/ml (Fig. 4). However, prophenoloxidase in plasma-PG can be activated without peptidoglycan by unknown factor(s), in hemolymph or the ammonium sulfate fraction. Such nonspecific activation of prophenoloxidase was experienced in the assay of ␤GRP and the reason for it discussed (14). To ensure that we really assayed PGRP, the effect of a given sample on the prophenoloxidase cascade in plasma-PG was examined both with and without peptidoglycan and a given sample was judged to contain PGRP only when it could trigger the prophenoloxidase cascade in plasma-PG with, but not without peptidoglycan. Another inconvenience of the assay method for PGRP is that different preparations of plasma-PG give different values for the amount of PGRP contained in a given sample. Knowing this, we used a single plasma-PG preparation throughout the purification described in the present study.
Insect hemolymph contains lysozyme. We reported that egg white lysozyme reduced the activity of peptidoglycan as an elicitor for triggering the prophenoloxidase cascade (30). The activation kinetics of the prophenoloxidase cascade in plasma are likely to be complicated as they are influenced by concentrations of PGRP, lysozyme, and peptidoglycan. The roles of each of these components in the kinetics remains to be studied.
PGRP and lysozyme both possess affinity to peptidoglycan and one may expect that they share some common properties. However, molecular weight, amino acid composition, isoelectric point, and NH 2 -terminal sequence of PGRP were all different from those of insect lysozyme (43). The purified PGRP preparation did not show any appreciable lysozyme activity. 2 We are currently undertaking by molecular cloning the entire primary structure of PGRP, to determine whether PGRP and lysozyme are related. When the prophenoloxidase cascade in plasma-PG supplemented with PGRP was triggered with peptidoglycan, prophenoloxidase was activated in such a way that higher concentrations of the recognition protein reduced the lag time. However, the maximum velocity of prophenoloxidase activation seemed not to depend on the PGRP concentration (Fig. 4). A similar relationship between concentration of ␤GRP and activation of prophenoloxidase in plasma-CPB (plasma devoid of ␤GRP) was observed (14). Furthermore, as was observed for ␤GRP, PGRP bound to peptidoglycan did not show any significant activity to hydrolyze 26 commercially available substrates for proteases. These observations suggest that the basic mechanisms for the activation of prophenoloxidase cascade by peptidoglycan and ␤1,3-glucan are similar. It would be necessary to isolate the components and to reconstruct the prophenoloxidase cascade in vitro to analyze the mechanism by which it is triggered by peptidoglycan or ␤1,3-glucan.
Proteins with ability to bind to peptidoglycan were looked for by using a fraction (0 -50 fraction) prepared from silkworm hemolymph plasma. The 0 -50 fraction had advantages over non-fractionated plasma in such an investigation. 1) It did not have lysozyme activity which was likely to interfere with the present method for examining the binding of protein to insoluble petidoglycan. 2) The fraction has been shown to contain all the components of prophenoloxidase cascade except prophenoloxidase (36) of which activation in plasma is known to cause the formation of aggregates of proteins (44). As is seen in Fig. 6, some polypeptides including the one with mobility corresponding to that of PGRP seemed to bind to peptidoglycan. This result, however, does not necessarily mean that each of them independently has ability to bind to peptidoglycan by FIG. 5. Binding of PGRP to M. luteus peptidoglycan, chitin, and ␤1,3-glucan (A) and the effect of some constituents of peptidoglycan and soluble fragmented peptidoglycan on the binding (B). A, purified PGRP (2.92 g) was incubated separately with 0.32 mg of insoluble peptidoglycan, chitin, and ␤1,3-glucan in a total volume of 320 l and adsorbed protein on the insoluble materials was separated from unadsorbed protein as described under "Materials and Methods." One-fourth of the adsorbed and one-sixteenth of unadsorbed PGRPs were subjected to SDS-PAGE together with purified PGRP and stained by Coomassie Brilliant Blue. a, 1.2 g of PGRP; b, adsorbed PGRP onto peptidoglycan; c, non-adsorbed fraction from a mixture of PGRP and peptidoglycan; d, adsorbed PGRP onto chitin; e, adsorbed PGRP onto ␤1,3-glucan; m, marker proteins (upper band, carbonic anhydrase; lower band, soybean trypsin inhibitor). No protein band was detectable in lane c after the gel was silver-stained (data not shown). B, purified PGRP (2.92 g) was preincubated separately with 2.19 mM GMDP, MDP, N-acetylglucosamine, and N-acetylmuramic acid with 1.55 mg/ml soluble fragmented petidoglycan before binding of the PGRP to insoluble peptidoglycan was tested as described under "Materials and Methods." One-fourth of the adsorbed PGRP was subjected to SDS-PAGE together with purified PGRP and stained by Coomassie Brilliant Blue. a and k, 1.2 g of PGRP and 0.73 g of PGRP, respectively; f, control (adsorbed PGRP in the absence of the test compounds); g, h-j, and l, adsorbed PGRPs in the presence of GMDP, MDP, and N-acetylglucosamine, N-acetylmuramic acid, and soluble fragmented peptidoglycan, respectively. m, the same marker proteins as in A.
FIG. 6. Search for proteins in plasma with affinity to M. luteus peptidoglycan. A fraction (referred to as 0 -50 fraction), which contains all prophenoloxidase cascade components except prophenoloxidase, was prepared from silkworm plasma (36). Proteins adsorbed onto insoluble peptidoglycan in the fraction were investigated as described under "Materials and Methods." The adsorbed proteins were subjected to SDS-PAGE together with 0 -50 fraction. a, adsorbed proteins from 260 g of protein of 0 -50 fraction; b, 104 g of 0 -50 fraction; m, marker proteins (see Fig. 3).
itself. It should be noted that we have observed that a purified polypeptide from silkworm hemolymph did not bind to peptidoglycan, but it did in the presence of PGRP. 3 Thus, there remains a possibility that the polypeptides other than PGRP shown to have apparent affinity to peptidoglycan in Fig. 6 did not bind directly to peptidoglycan.
The inability of PGRP to bind to ␤1,3-glucan and chitin and the fact that the PGRP which had been incubated with soluble fragmented peptidoglycan barely bound to insoluble peptidoglycan corroborate our contention that the binding of PGRP to peptidoglycan is specific. Furthermore, the present observation is in accordance with our previous survey on the substances with elicitor activity for triggering prophenoloxidase cascade in silkworm plasma (45). No constituents of peptidoglycan (GMDP, MDP, N-acetylglucosamine, and N-acetylmuramic acid), however, inhibited appreciably PGRP to bind to insoluble peptidoglycan. This result seems to indicate that structure with two or more repeating units of the glycan portion of peptidoglycan is recognized by PGRP. It is desirable to study further the structural requirements for PGRP to bind to peptidoglycan for our understanding on the mechanism of activation of prophenoloxidase cascade by peptidoglycan.
Peptidoglycan is known to have various biological activities, such as potentiation of the immune system, production of fever, promotion of slow wave sleep (25), and macrophage activation in mammals (27) and induction of synthesis of bactericidal substance in insect (46 -48). Undoubtedly peptidoglycan interacts with specific ligands or receptors when it elicits such activities in living organisms. Isolation of peptidoglycan receptor on cell surface or peptidoglycan binding protein (or recognition protein) in hemolymph of insect has not been reported until the present study. Specific surface receptors for peptidoglycan in mammalian macrophages and leucocytes have been reported (27). One of them had been claimed to be identified as a 70-kDa, 6.5 pI protein (29). This protein, however, was recently proven not to be necessary for macrophage cell lines, which do not possess it, to be stimulated by peptidoglycan for the production of tumor necrosis factor-␣ (49). Thus, the underlying mode of peptidoglycan action is poorly understood at the molecular level, not only in insect but also in higher vertebrates.
In a few mammalian systems, lipopolysaccharide binding protein in plasma was shown to potentiate cellular response to lipopolysaccharide by facilitating binding of the complex of lipopolysaccharide binding protein and lipopolysaccharide to the plasma membrane protein, CD14, which serves as an lipopolysaccharide receptor (50). A protein (factor C) with affinity for lipopolysaccharide has been purified from horseshoe crab (Tachypleus tridentatus) hemocyte lysate (51). The factor C is a component of a blood coagulation cascade and becomes an active serine protease upon interaction with lipopolysaccharide. This protein, however, has not been shown to have any other function than serine protease zymogen. Hence, an arthropod protein with similar function to the mammalian lipopolysaccharide binding protein has not been found in arthropod yet. The recognition protein (binding protein) for ␤1,3-glucan from insect and crustacean hemolymph was originally found as a factor which triggers the prophenoloxidase cascade (14 -16). Interestingly, crayfish ␤1,3-glucan binding protein has recently been shown to stimulate phagocytic uptake of yeast particles by isolated homologous hemocytes (24). It remains to be studied whether PGRP has opsonic activity for Gram-positive bacteria or other biologic activities in addition to its activity to trigger the prophenoloxidase cascade. Such study, together with more detailed physicochemical characterization of the PGRP, will advance our understanding on its special role in insect defense along with molecules such as lectins, hemolin, lipopolysaccharide binding protein, and ␤GRP, all of which are present in insect hemolymph and possess affinities for microbial cell wall components.