©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Type of limulus Lectin-L6
PURIFICATION, PRIMARY STRUCTURE, AND ANTIBACTERIAL ACTIVITY (*)

Tetsu Saito (1), Shun-ichiro Kawabata (1) (2), Michimasa Hirata (3), Sadaaki Iwanaga (1) (2)(§)

From the (1)Department of Molecular Biology, Graduate School of Medical Science, the (2)Department of Biology, Faculty of Science, Kyushu University 33, Fukuoka 812-81, and the (3)Department of Bacteriology, School of Medicine, Iwate Medical University, Morioka, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Lipopolysaccharide (LPS)-binding protein(s) was first screened in the detergent extract of horseshoe crab (limulus) hemocytes, using LPS-immobilized agarose. A protein, designated L6 (M = 27,000), was found to bind to LPS-agarose and was eluted with EDTA or o-phenanthroline. The L6 protein, however, did not inhibit the LPS-mediated activation of a limulus serine protease zymogen factor C. L6 had an affinity to the matrix of Sepharose CL-6B itself, and it could be eluted with high concentrations of monosaccharides (0.5-1.0 M), such as glucose, mannose, and galactose, suggesting a lectin-like nature. The entire amino acid sequence of L6 was determined by sequencing peptides derived from CNBr and enzymatic cleavages. L6 contained 7 half-cystines, and 1 cysteine residue at position 201 had a free SH-group. In addition, positions of the remaining three intrachain disulfide bonds were assigned by amino acid and sequence analyses of three cystinyl peptides produced by lysyl endopeptidase digestion. These results indicated that the entire sequence of L6 consisted of 221 residues with no N-linked sugar and was composed of six tandem repeats, each consisting of 33-38 amino acid residues. Inductively coupled plasma spectrometry of L6 indicated the presence of 0.75 mol zinc/mol of protein. No significant sequence homology was observed between L6 and other proteins, including various animal lectins and LPS-binding proteins. However, L6 showed agglutinating activity on LPS-coated sheep erythrocytes and Gram-negative and Gram-positive bacteria, it inhibited the growth of Gram-negative bacteria, and thus it presumably recognizes carbohydrate components in the cell wall of bacteria.


INTRODUCTION

Invertebrates have characteristic host defense systems which differ from those of mammalian immune systems(1) . In the horseshoe crab (limulus), this defense system is carried by the hemolymph, which contains a type of cell, called amebocyte or hemocyte(2) . The hemocytes are extremely sensitive to bacterial endotoxins, lipopolysaccharides (LPS),()which are a major outer membrane component of Gram-negative bacteria. When hemocytes contact Gram-negative bacteria or LPS, they begin to degranulate, and the resulting granular components initiate hemolymph coagulation(2, 3, 4) . This response is thought to be important for host defense in engulfing invading microbes, in addition to preventing the leakage of hemolymph. Thus, the sensitivity of the hemocytes to LPS suggests that there are probably specific, high affinity interactions between LPS and circulating hemocytes. Three LPS-binding proteins have so far been detected in hemocytes lysates of American Limulus polyphemus; an LPS-binding protein of 82 kDa, which is characterized as a negative regulator of Limulus coagulation cascade(5) , a membrane-associated LPS-binding protein of 80 kDa(6) , and an LPS-binding protein with protease-inhibitory activity(7) . However, little is known of which portions of LPS consisting of an external polysaccharide, containing O-specific chain and core region and lipid A region containing D-glucosamine disaccharide backbone, are required for binding of these proteins to LPS. Furthermore, no structural data on these LPS-binding proteins, except for the report of Minetti et al.(7) , are available.

To elucidate the structure and biological function of LPS-binding proteins in limulus hemocytes, our intention was to isolate the purified material and to characterize it biochemically. The present studies were initiated to examine whether hemocyte-derived membrane proteins that are capable of the specific binding of LPS can be identified. We first screened LPS-binding protein(s) from detergent extracts of the hemocyte debris, using an LPS-immobilized agarose, and found evidence for a novel 27-kDa protein, designated L6, which interacts probably with an external polysaccharide portion of LPS or a cell wall component of Gram-negative bacteria. We describe here the purification, covalent structure, and antibacterial activity of this L6 protein.


EXPERIMENTAL PROCEDURES

Materials

LPS purified from Escherichia coli O111:B4, Salmonella minnesota (smooth) and Salmonella minnesota R595 (Re mutant) from List Biological Laboratories, Inc., Campbell, CA, epoxy-activated Sepharose CL-6B and molecular weight standards from Pharmacia LKB Biotechnology, Uppsala, lysyl endopeptidase from Wako Pure Chemical Industries, Ltd., Tokyo, endoproteinase Asp-N from Boehringer Mannheim, and ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) and (4-aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) from Dojindo Laboratories, Kumamoto, were used. The zymogen factor C was purified from the hemocyte lysate, as described elsewhere(8) . All other chemicals were of the highest quality commercially available.

Preparation of LPS-immobilized Agarose

LPS (5 mg) was sonicated in 20 ml of 0.1 M sodium carbonate, pH 11.0, and mixed with epoxy-activated Sepharose CL-6B (10 g). The coupling and blocking reactions were done according to the manufacturer's instructions (Pharmacia).

Preparation of Lubrol Extract from Horseshoe Crab Hemocytes

The washed precipitate, debris of hemocyte homogenate of Japanese horseshoe crab (Tachypleus tridentatus), was prepared as previously described(9, 10) . The debris (20 g) was homogenized in a Waring blender for 3 min in 500 ml of 20 mM Tris-HCl buffer, pH 7.5, containing 10 mM CaCl, and Lubrol was added to give a final concentration of 0.5%. The mixture was then stirred for 3 h at 4 °C and dialyzed overnight against 20 mM Tris-HCl buffer, pH 7.5, containing 0.5 M NaCl and 10 mM CaCl. After centrifugation at 12,000 g for 30 min, the resultant supernatant was used as the starting Lubrol extract.

Purification of L6

The Lubrol extract (450 ml) from the hemocyte debris (10) was applied to an LPS-agarose column (gel volume, 10 ml), equilibrated with 20 mM Tris-HCl buffer, pH 7.5, containing 0.5 M NaCl and 10 mM CaCl. After extensively washing the column with equilibration buffer, elution was performed with 20 mM Tris-HCl buffer, pH 7.5, containing 0.1 M NaCl and 25 mM EDTA at a flow rate of 10 ml/h. The conditions used for separation of the Lubrol extract on a Sepharose CL-6B column (gel volume, 10 ml) were the same as those described above.

Amino Acid and NH-terminal Sequence Analyses

For amino acid analysis, samples were hydrolyzed in 6 M HCl in evacuated and sealed tubes at 110 °C for 24, 48, and 72 h. The hydrolyzates were analyzed using a Hitachi L-8500 amino acid analyzer with chemicals and program supplied by the manufacturer. Tryptophan was determined by hydrolysis in 3 M mercaptoethanesulfonic acid(11) . The amino acid analyses of peptides obtained by chemical and enzymatic cleavages were analyzed using a Pico-Tag system (Waters), according to the method of Heinrikson and Meredith(12) , after hydrolysis with 6 M HCl containing 1% phenol at 110 °C for 20 h. Amino acid sequence analysis was done using gas-phase model 477A and 473A sequencers (Applied Biosystems).

Cyanogen Bromide and Proteolytic Digestions

The protein was reduced, S-alkylated with iodoacetamide (13) and treated with a 100-fold molar excess over methionine residues of CNBr in 70% (v/v) formic acid under nitrogen, and incubated at 22 °C for 24 h in the dark. After lyophilization, the reaction products were dissolved in 200 µl of 6 M guanidine-HCl and separated by reversed-phase HPLC using a µBondasphere S-5 C8 300A (2.1 150 mm, Nihon Waters Ltd., Osaka). Of the three fragments, CN2 and CN3 (not CN1) were further purified on a Phenyl-5PW RP column (4.6 75 mm, TOSOH, Tokyo) (data not shown). The resulting CNBr fragments were digested with endoproteinase Asp-N (E/S = 1/50, w/w) in 50 mM Tris-HCl, pH 7.5, containing 2 M urea at 37 °C for 18 h. The S-alkylated L6 protein was also digested with lysyl endopeptidase (E/S = 1/100, w/w) in 0.1 M NHHCO containing 2 M urea at 37 °C for 24 h. The generated peptides were separated by reversed-phase HPLC using Chemcosorb 5-ODS-H column (2.1 150 mm, Chemco Scientific Co., Ltd., Osaka), and µBondasphere S-5 C8 300A was also used for rechromatography of the peptides. Peptides were eluted from the columns with a linear gradient of 0-80% acetonitrile in 0.06% trifluoroacetic acid at a flow rate of 0.2 ml/min. Absorbance was monitored at 210 nm.

Determination of COOH-terminal Residue

The L6 protein (1 nmol) was dried overnight in vacuo over phosphorus pentoxide, and vapor-phase hydrazinolysis was done, as previously described(14) . The amino acid released from the COOH terminus was identified by amino acid analysis using the Pico-Tag system.

Determination of SH-Cys

The sample (200 µg) was mixed with 200 molar excess of ABD-F over the protein in 0.1 M sodium borate buffer, pH 7.0, containing 6 M guanidine-HCl and 5 mM EDTA, and incubated at 60 °C for 20 min(15, 16) . After dialysis against 50 mM Tris-HCl, pH 8.0, containing 2 M urea and 5 mM EDTA, the modified protein was digested with lysyl endopeptidase (E/S = 1/50, w/w) at 37 °C for 24 h. The resulting peptides were separated by reversed-phase HPLC on a YMC S-5 120A ODS column (4.6 130 mm, Yamamura Chemical Laboratories Co., Ltd., Kyoto). Absorbance was monitored at both 210 and 380 nm, using a diode array detector (Beckman).

Determination of Disulfide Bonds

The protein (250 µg) was dissolved in 0.2 M Tris-HCl, pH 6.8, containing 2 M urea and digested with lysyl endopeptidase (E/S = 1/25, w/w) at 37 °C for 36 h. The peptides generated were separated by reversed-phase HPLC on a YMC C4 column (4.6 150 mm, Yamamura Chemical Laboratories Co., Ltd., Kyoto). Each peptide peak was used for quantification of disulfide bonds with SBD-F, as previously reported (17) and for amino acid and sequence analyses.

Computer-assisted Analysis of Sequence Data

The amino acid sequence was compared with all entries in the data base of Swiss-Plot (release 26.0 August, 1994) with Gene Works system (IntelliGenetics, Inc., Mountain View, CA).

SDS-PAGE

SDS-PAGE was performed in 15% slab gels, according to Laemmli(18) . The gels were stained with Coomassie Brilliant Blue R-250. The reference proteins for molecular weight estimation were phosphorylase b (M = 94,000), bovine serum albumin(67, 0) , ovalbumin(43, 0) , carbonic anhydrase(30, 0) , soybean trypsin inhibitor(20, 0) , and -lactoalbumin(14, 400) .

Effect of L6 on the LPS-mediated Activation of Zymogen Factor C

The sample was preincubated with LPS (15 ng) in 100 µl of 50 mM Tris-HCl buffer, pH 8.0, containing 0.1 M NaCl, 10 mM CaCl, and 0.05% human serum albumin for 5 min at 37 °C. Factor C (0.3 µg) was added, and the mixture was further incubated for 15 min. A fluorogenic substrate, t-butoxycarbonyl-Val-Pro-Arg-4-methylcoumaryl-7-amide(8) , was then added to give a final concentration of 0.2 mM and the initial rate of hydrolysis was measured fluorometrically, as described elsewhere(10) .

LPS Binding Activity

One ml of 1.0% sheep erythrocytes was sensitized with 0.2 ml of LPS from S. minnesota (smooth) or S. minnesota R595 (Re mutant) solution (100 µg/ml) and incubated at 37 °C for 30 min, followed by washing with 20 mM sodium phosphate-buffered saline (pH 7.0). Fifty µl of 1.0% suspension of the sensitized erythrocytes were mixed with 50 µl of a 2-fold serial dilution of the purified L6 in a U-bottomed microtiter plate and incubated at 37 °C for 1 h. The LPS binding activity was expressed as the minimum agglutinating concentration(19) .

Inhibition of LPS Binding Activity by Free LPS

Fifty µl of L6 solution were preincubated with 50 µl of serial 2-fold dilution of LPS from S. minnesota (smooth) or S. minnesota R595 (Re mutant) for 30 min at 37 °C, and each reaction mixture was added to LPS-sensitized sheep erythrocytes described above in a U-bottomed microtiter plate and incubated at 37 °C for 1 h. The inhibition of LPS binding activity was expressed as minimum inhibitory concentration(19) .

Agglutinating Activity of L6 against Various Bacteria

The following strains were used for determination of the agglutination activity; E. coli K12, E. coli B, Staphylococcus saprophyticus KD, Staphylococcus aureus 209P, Micrococcus luteus ATCC 4698, and Enterococcus hirae. Twenty-five µl of suspension of the each bacteria (absorbance at 600 nm was 10) were mixed with 25 µl of a 2-fold serial dilution of the purified L6 in a U-bottomed microtiter plate and incubated at room temperature for overnight. The agglutinating activity was expressed as minimum agglutinating concentration.

Antibacterial Activity

All strains were grown in Tryptosoy broth (Eiken Co., Tokyo). Salmonella typhimurium LT2 (smooth), S. typhimurium TV160 (Rb mutant), S. minnesota R595 (Re), E. coli O9:K39 (K), and Klebsiella pneumoniae were plated on nutrient agar (Eiken Co.). Bacterial cultures were collected at the logarithmic phase of growth, washed twice with 10 mM phosphate buffer, pH 7.0, and adjusted to a final concentration of 5 10 to 1 10 cells/ml. To 450 µl of bacterial suspension, 50 µl each of samples were added, and the mixture was incubated at 37 °C for 1 h and 100 µl of the reaction mixture was then put onto the agar plate. After 24 h of incubation at 37 °C, the number of colony-forming units was determined. As a control experiment, phosphate-buffered saline was added to the bacterial suspension and the mixture was incubated for 1 h, plated on agar, and cultured. For some experiments, the percentage of the control colony-forming units was determined(20) .

Antiserum and Immunoblotting

An antiserum against L6 was raised in rabbits (male, Japanese White, 2.5 kg) as described by Harlow and Lane(21) . The purified L6 (200 µg) was emulsified in Freund's complete adjuvant and given intradermally. After 4 weeks, a booster with the same amount of L6 in Freund's complete adjuvant was given. Blood samples were taken 2 weeks after the third injection, and the serum was stored at -80 °C. Immunoblotting was performed as described previously(22) .

Metal Analysis

L6 was dialyzed against water and then lyophilized. The dried protein was dissolved in water (0.084 mg/ml). Standard solutions containing metal ions (Ca, Fe, Zn, Mg, Cu, and Mn) were prepared in 0.3% HCl at 0.2, 0.5, 1.0, and 10 ppm. Quantification of metals of the sample was performed by an inductively coupled plasma spectrometer, ICPS-1000 III (Shimadzu Co. Ltd., Kyoto).


RESULTS

Purification of L6

The Lubrol extract prepared from 60 g (wet weight) of the hemocyte debris (10) was applied to an LPS-agarose column in the presence of 10 mM Ca. Protein(s) bound to the column was then eluted with 25 mM EDTA according to the method used for purification of the 80-kDa LPS-binding protein of the American horseshoe crab, L. polyphemus(6) . A typical elution profile is shown in Fig. 1. The pooled fractions indicated by a solid bar gave a single protein band (M = 27,000) on SDS-PAGE, under reducing conditions (Fig. 2). Furthermore, the tailing fraction of chromatography contained the same protein as eluted in the peak fraction. This protein was also eluted with 1 mMo-phenanthroline (data not shown), suggesting that divalent transition metal ions are required for the binding of L6 to LPS-agarose. Furthermore, when the Lubrol extract was applied to a Sepharose CL-6B column free from LPS-ligand, the 27-kDa protein was adsorbed on the column and eluted with not only 25 mM EDTA but also 0.5-1.0 M each of glucose, mannose, or galactose, suggesting its lectin-like nature. Galactose was the most effective sugar to elute it, and glucose was the next (data not shown). Through all the procedures described above, the 27-kDa protein was purified reproducibly by one step of the affinity chromatography with the yield of 2-3 mg from 60 g of the hemocyte debris.


Figure 1: Isolation of limulus lectin-L6 on an LPS-agarose column. The Lubrol extract prepared from 60 g (wet weight) of the T. tridentatus hemocyte debris was applied to the column and eluted with 20 mM Tris-HCl, pH 7.5, containing 0.1 M NaCl and 25 mM EDTA at a flow rate of 1.0 ml/min, after washing extensively with the same buffer containing 0.5 M NaCl and 10 mM CaCl without EDTA. Fractions indicated by a solid bar were collected.




Figure 2: SDS-PAGE of purified limulus lectin-L6. The molecular masses (kDa) of marker proteins are given on the right.



Amino Acid Sequence Determination of L6

The purified 27-kDa protein (100 pmol) was subjected to amino acid sequence analysis. The partial sequence, NH-VQWHQIPGKLMHITATPHFLWGVNSNQQIY- revealed that it is virtually identical to that of a protein component contained in large granules of the hemocytes, named L6 (the first 17 amino acid residues have been established), previously reported(23) . The identity of the 27-kDa protein to L6 was further supported by its amino acid composition (see ). Therefore, the name L6 was used. For the COOH-terminal residue of L6, glycine was determined with the 62% yield by the vapor-phase hydrazinolysis method, as described elsewhere(14) . The extinction coefficient of L6 at 280 nm for 0.1% solution in Tris-HCl buffer (pH 7.5) was calculated from the data on amino acid analysis. The value of 24.0 was used for subsequent determination of L6 concentration.

Based on the amino acid composition, L6 contained two Met residues (), therefore three CNBr fragments were expected. The CNBr cleavage of S-alkylated L6 was performed and the digest was separated, as described under ``Experimental Procedures.'' Three homogeneous peptides CN1, CN2, and CN3 were obtained, and the amino acid compositions of L6 could be accounted for by the sum of the total residues in the three peptides (data not shown). The NH-terminal sequences of the peptides are shown in Fig. 3. The peptide CN1, which was derived from the NH-terminal part of L6, contained 11 residues, and sequence analysis led to the identification of up to 10 residues, except for the COOH-terminal end. The sequence analysis of CN2 established the first 35 residues, except for cycles 25, 30, 31, and 34. The peptide CN3 did not contain homoserine, therefore it constituted the COOH-terminal part of L6. Determination of the sequence of CN3 revealed up to 30 residues (Fig. 3).


Figure 3: The entire amino acid sequence of limulus lectin-L6 and its fragments. Amino acid residues are given in single-letter code. Arrows represent amino acid residues determined by Edman degradation. K, lysyl endopeptidase-digested peptides; CN, cyanogen bromide-cleaved peptides; D, endoproteinase Asp-N digested peptides.



From subdigestions of the L6-derived CN2 and CN3 with endoproteinase Asp-N, six and three peptides were obtained, respectively, and their sequences are shown in Fig. 3. By sequencing CN2-D1 and CN2-D2, the unidentified residues at cycles 25, 30, 31, and 34 in CN2 were identified as Cys, Trp, Thr, and Ser. The sequence of CN2-D4 established the 16 residues, except for cycle 3, and the unidentified residue was determined as Ser by sequencing CN2-D5. The unidentified residues in CN2-D5 and CN3-D1 were determined by sequencing peptides derived from lysyl endopeptidase digest, as described below.

To obtain overlaps of the two CNBr fragments (CN2 and CN3) and the peptides derived from their subdigestions, the S-alkylated L6 was digested with lysyl endopeptidase. Nine peptides were isolated and their sequences are also shown in Fig. 3. K3 overlapped CN2-D2, CN2-D3, and CN2-D4; K5 overlapped CN2-D5 and CN2-D6; K7 overlapped CN2-D6, located at the COOH-terminal region of CN2 and CN3; K8 overlapped CN3 and CN3-D1; and finally CN3-D1, CN3-D2, and CN3-D3 were linked by sequencing K9 and K10.

The results described above made way for alignment of all the peptides and complete amino acid sequence of L6 shown in Fig. 3. There is no potential N-linked sugar binding site in the sequence, and no amino sugar was detected in the amino acid analysis of L6.

Determination of the Position of SH-Cys

The L6 protein contained seven half-cystine residues (), indicating the presence of at least one cysteine residue free from the disulfide bond. To identify the position of such SH-Cys, the intact L6 was modified with iodoacetamide, under non-reducing conditions and in the presence of 5 mM EDTA and 6 M guanidine-HCl. Amino acid analysis under this condition revealed that only one cysteine residue was S-alkylated (data not shown). When L6 was treated with ABD-F, a fluorogenic reagent for thiols, an ABD-containing peptide (Fig. 4, peak A) was obtained after digestion with lysyl endopeptidase. This peptide was separated on HPLC by monitoring the absorbance at 380 nm. The amino acid and sequence analyses of peak A showed the presence of a SH-Cys residue at position 201 as a reactive thiol to ABD-F (data not shown).


Figure 4: Detection and isolation of an ABD-labeled peptide after lysyl endopeptidase digestion. The ABD-labeled L6 protein was digested with lysyl endopeptidase, and the resulting peptides were separated by reversed-phase HPLC on a YMC S-5 120A ODS column (4.6 130 mm). Elution was performed with a linear gradient of acetonitrile containing 0.06% trifluoroacetic acid at a flow rate of 0.5 ml/min. A Peak A indicated by an arrow was collected.



Assignment of Disulfide Bonds in L6

The intact L6 was cleaved at pH 6.8 with lysyl endopeptidase, and the digest was separated into 18 major peaks by HPLC (Fig. 5). Disulfide-containing peptides were detected by fluorometric assay, as described under ``Experimental Procedures.'' The three isolated cystinyl peptides with high fluorescence intensity were subjected to amino acid analysis (). Each peptide contained about 2 cysteic acids/peptide, after performic acid oxidation, and peaks 6, 14, and 16 were found to correspond to amino acid residues 109-125, 162-199, and 19-49, respectively. Furthermore, the three peptides were sequenced with the detection of a single phenylthiohydantoin-derivative in each cycle and with no phenylthiohydantoin-derivative at Cys positions (data not shown). Based on these results, the following disulfide linkages in L6 were assigned, Cys-Cys, Cys-Cys, and Cys-Cys.


Figure 5: Separation of disulfide containing peptides by reversed-phase HPLC. The lysyl endopeptidase digest of intact L6 protein was applied to a YMC C4 column (4.6 150 mm) and eluted with a linear gradient of acetonitrile containing 0.06% trifluoroacetic acid at a flow rate of 0.5 ml/min. The disulfide-containing peptides were detected using the SBD-F method as described elsewhere (17). The peptides (peaks 6, 14, and 16) reactive to SBD-F were collected and subjected to amino acid sequence analyses.



LPS Binding Activity of L6

Anti-LPS factor and tachyplesin isolated from horseshoe crab hemocytes strongly inhibit the LPS-mediated autocatalytic activation of factor C, a serine protease zymogen in a horseshoe crab coagulation cascade(4) . To assess the LPS binding activity of L6, LPS was preincubated with an excess amount of L6 and tested for effects on the activation of zymogen factor C. The L6 protein had no effect on activation of zymogen factor C mediated with LPS (Fig. 6), thereby suggesting that the lipid A portion of LPS does not interact with L6 protein. It seemed likely that L6 would bind to LPS-agarose through the matrix, not a ligand LPS. There is also the possibility that the L6 protein interacts with a core portion of LPS, such as 2-keto-3-deoxy-D-manno-octanate. To examine this possibility, the LPS binding activity of L6 was determined by measuring its potential to agglutinate erythrocytes coated with LPS purified from S. minnesota (smooth) and S. minnesota R595 (Re mutant). L6 significantly agglutinated sensitized sheep erythrocytes. The minimum agglutinating concentration was 25 µg/ml (I), thereby suggesting an interaction between L6 and the core polysaccharide portion of LPS. This agglutinating activity was inhibited by free LPS, and the minimum inhibitory concentration was 62.5 and 125 µg/ml for LPS derived from S. minnesota (smooth) and S. minnesota R595 (Re mutant), respectively (). Furthermore, L6 was found to agglutinate both Gram-negative and Gram-positive bacteria, and the activity was more efficient for Gram-negative bacteria than for Gram-positive ones (). These data indicate that L6 recognizes the bacterial cell wall components such as LPS.


Figure 6: Inhibitory effect of L6 on the LPS-mediated activation of limulus factor C zymogen. LPS (0.2 µg/ml) was preincubated with various concentrations of L6 (), tachyplesin (), or anti-LPS factor (), in a total volume of 100 µl of 50 mM Tris-HCl buffer, pH 8.0, containing human serum albumin (0.5 mg/ml). Then, 100 µl of factor C zymogen (0.3 µg/ml) was added and the factor C activation was assayed as described elsewhere (9). The relative activation of zymogen factor C was expressed, taking the amidase activity of factor C generated in the absence of the inhibitors as 100%. The data of tachyplesin and anti-LPS factor were taken from Miyata et al. (9).



Antibacterial Activity of L6

shows concentrations of L6 which inhibit growth of E. coli, S. minnesota, K. pneumoniae, and S. aureus strains. L6 had significant antibacterial activity against Gram-negative bacteria, including E. coli O9:K39 (K), S. minnesota R595 (Re mutant), and K. pneumoniae, but not the Gram-positive Staphylococcus strain.

Subcellular Localization of L6

Separation of granular components of the hemocytes by a combination of sucrose density gradient centrifugation and reversed-phase HPLC indicated the localization of L6 in the large granules(23) . To confirm the localization, antiserum raised against purified L6 was used for immunoblotting. Large and small granules prepared from hemocytes were first treated with 1% SDS at 100 °C for 2 min and subjected to SDS-PAGE, under reducing conditions. The anti-L6 antiserum recognized the 27-kDa protein in the extract of large granules, whereas immunoreactive materials were not found in small granules (Fig. 7). These observations confirm localization of L6 in large granules of the hemocytes. Plasma prepared from the hemolymph was also analyzed, but immunoreactive materials were not found (data not shown).


Figure 7: Immunoblotting of L and S granules with antibody against L6. Small granules and large granules containing 5 µg protein were subjected to SDS-PAGE. Immunoblotting was carried out as described under ``Experimental Procedures.'' Lane L, large granules; lane S, small granules.



Metal Analysis of L6

Inductively coupled plasma spectrometry of L6 indicated the presence of zinc at 0.75 mol/mol protein and calcium at 0.05 mol/mol protein but other metals including Fe, Mg, Cu, and Mn were not detected. This result indicates that L6 contains approximately 1 mol of zinc per mol of protein.


DISCUSSION

In the present study, a novel type of limulus lectin-L6 was purified by affinity chromatography of LPS-agarose, and the entire covalent structure was determined. Although L6 was first purified using LPS-agarose, it was later found to bind to agarose itself, and L6 was eluted with high concentrations of monosaccharides of 0.5-1 M, such as glucose, mannose, and galactose. However, it showed no binding activity for p-nitrophenyl derivatives of monosaccharides including Gal, Man, or GlcNAc at low concentration (10 µM) with equilibration dialysis (24) (data not shown). On the other hand, purified L6 showed an LPS binding potential which agglutinates sheep erythrocytes coated with LPS (I), and the activity was inhibited by the addition of free LPS (). However, it apparently had no hemagglutinin activity for sheep and rabbit red blood cells, and human A, B, and O types of red blood cells. These results would suggest that L6 may recognize an oligosaccharide portion of LPS. Inductively coupled plasma spectrometry suggested the presence of 1 mol of zinc per protein mole of protein. EDTA or o-phenanthroline is an effective eluent and, therefore, the zinc ion must have an important role for the sugar binding property of L6. L6 has antibacterial activity toward Gram-negative but not Gram-positive bacteria (). L6 has also more effective agglutinating activity toward Gram-negative than Gram-positive ones (). Lectins purified mainly from limulus hemolymph plasma(25) , include sialic acid-lectin, called limulin (26, 27) or C-reactive protein(28, 29) , polyphemin(30) , carcinoscorpin (31), and others(32, 33, 34) . However, the chemical structures of these lectins (except for C-reactive protein) have not been determined. Of these lectins, Limulus C-reactive protein present in hemolymph plasma reacts directly with bacterial coat oligosaccharides in a Ca-dependent manner to initiate a humoral defense system, including agglutination of the microbes(29) . This property seems similar to that of L6, but as the amino acid sequences between C-reactive protein and L6, in addition to their molecular weights and localizations differ, L6 is a novel type of lectin located exclusively in large granules of hemocytes. The existence of high concentrations of L6 in the large secretory granules (23) suggests that L6 serves synergistically as a defense molecule for invading microbes, together with the anti-LPS factor(35, 36) , tachyplesins(9, 10) , and coagulation factors(4) , all of which are secreted into the extracellular fluid upon stimulation with LPS.

L6 is a single-chain protein consisting of 221 amino acids with no N-linked sugar chain and contains three intrachain disulfide bonds and one SH-Cys. The calculated molecular weight of 24,383 is lower than that estimated by SDS-PAGE on a 15% gel. Based on a hydropathy plot(37) , L6 is primarily a hydrophilic protein (Fig. 8). It is noteworthy that L6 is rich in Trp (9 residues) and positively charged amino acids (27 residues: 11 Lys, 10 Arg, and 6 His), as compared to negatively charged amino acids (16 residues: 14 Asp and 2 Glu). The isoelectric point was calculated to be 9.69(38) . Moreover, an outstanding structural feature of L6 is that it consists of six tandem repeats, each one containing 33-38 amino acids with 32-61% internal sequence identities (Fig. 9). Two short consensus sequences, -GXWXQIXGXLK- and -GVNSNDXIY- are highly conserved in each repeat. It is also of interest that the three disulfide small loops consisting of five amino acid residues are present in every two repeats. A search of Swiss plots showed no significant sequence similarity between L6 and other proteins, including various animal and plant lectins and LPS-binding proteins, such as Limulus endotoxin-binding protein-protease inhibitor (7) and mammalian plasma LPS-binding proteins(39) .


Figure 8: Hydropathy profile of limulus lectin-L6. The hydropathic index is the mean value of 9 successive residues.




Figure 9: Internal sequence similarity and disulfide bond locations of limulus lectin-L6. Amino acid residues are given in single letter code. Consensus amino acids in six tandem repeats are indicated in bold and large letters, at least four of the six amino acids are identical. A free SH group at Cys-201 is indicated by an asterisk.



The carbohydrate binding potential and the agglutinating activity toward bacteria of L6 indicate involvement in invertebrate defense systems. Further investigations are in progress to define the binding specificity of L6 to oligosaccharides and polysaccharides, studies which will shed light on the significance of L6 in host defense mechanisms.

  
Table: Amino acid composition of limulus L6


  
Table: 0p4in ND, not determined.(119)

  
Table: LPS-sensitized erythrocyte agglutination of L6


  
Table: Inhibition of L6-mediated LPS-sensitized erythrocyte agglutination by free LPS


  
Table: Agglutination activity of limulus L6 against various bacteria


  
Table: Antimicrobial activity of limulus-L6 against various bacteria

S. aureus was insensitive to limulus-L6 (1.64 µM dose). The standard deviations in the table were calculated from the data of three experiments.



FOOTNOTES

*
This work was supported in part by Grants-in Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Dept. of Biology, Faculty of Science, Kyushu University 33, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-81, Japan. Tel.: 092-641-1101 (ext. 4428); Fax: 81-92-632-2742.

The abbreviations used are: LPS, lipopolysaccharide; ABD-F, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; SBD-F, ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We express our thanks to professor N. Yamasaki (Kyushu University) for measurements of the hemagglutinin activity, to professor S. Hase and Dr. T. Mega (Osaka University) for the kind gift of p-nitrophenyl derivatives of monosaccharides, to Dr. S. Umeda (Kyushu University) for the kind gift of bacteria, to H. Hashimoto and C. Yano for excellent technical assistance in amino acid analysis and peptide sequencing, to Dr. T. Sato (Tanabe Seiyaku Co., Ltd.) for metal analysis, to S. Matsumura for expert secretarial assistance, and to M. Ohara for pertinent comments.


REFERENCES
  1. Brehélin, M. (ed)(1986) Immunity in Invertebrates, Springer-Verlag, Berlin
  2. Levin, J., and Bang, F. B.(1964) Bull. Johns Hopkins Hosp.115, 265-274
  3. Ornberg, R. L., and Reese, T. S.(1979) Prog. Clin. Biol. Res.29, 125-130 [Medline] [Order article via Infotrieve]
  4. Iwanaga, S., Miyata, T., Tokunaga, F., and Muta, T.(1992) Thromb. Res.68, 1-32 [Medline] [Order article via Infotrieve]
  5. Roth, R. I., and Tobias, P. S.(1993) Infect. Immun.61, 1033-1039 [Abstract]
  6. Liang, S.-M., Sakmar, T. P., and Liu, T.-Y.(1980) J. Biol. Chem.255, 5586-5590 [Abstract/Free Full Text]
  7. Minetti, C. A. S. A., Lin, Y., Cislo, T., and Liu, T.-Y.(1991) J. Biol. Chem.266, 20773-20780 [Abstract/Free Full Text]
  8. Nakamura, T., Morita, T., and Iwanaga, S.(1986) Eur. J. Biochem.154, 511-521 [Abstract]
  9. Miyata, T., Tokunaga, F., Yoneya, T., Yoshikawa, K., Iwanaga, S., Niwa, M., Takao, T., and Shimonishi, Y.(1989) J. Biochem. (Tokyo) 106, 663-668 [Abstract]
  10. Nakamura, T., Furunaka, H., Miyata, T., Tokunaga. F. Muta, T., Iwanaga, S., Niwa, M., Takao, T., and Shimonishi, Y.(1988) J. Biol. Chem.263, 16709-16713 [Abstract/Free Full Text]
  11. Penke, B., Ferenczi, R., and Kovacs, K.(1974) Anal. Biochem.60, 45-50 [Medline] [Order article via Infotrieve]
  12. Heinrikson, R. L., and Meredith, S. C.(1984) Anal. Biochem.136, 65-74 [Medline] [Order article via Infotrieve]
  13. Stone, K. L., LoPresti, M. B., Crawford, J. M., DeAngelis, R., and Williams, K. R.(1989) in A Practical Guide to Protein and Peptide Purification for Microsequencing (Matsudaira, P. T., ed) pp. 31-47, Academic Press, San Diego, CA
  14. Yamamoto, A., Toda. H., and Sakiyama, F.(1989) J. Biochem. (Tokyo) 106, 552-554 [Abstract]
  15. Toyo'oka, T., and Imai, K.(1984) Anal. Chem.56, 2461-2464
  16. Toyo'oka, T., and Imai, K.(1985) Anal. Chem.57, 1931-1937 [Medline] [Order article via Infotrieve]
  17. Sueyoshi, T., Miyata, T., Iwanaga, S., Toyo'oka, T., and Imai, K. (1985) J. Biochem. (Tokyo) 97, 1811-1813 [Abstract]
  18. Laemmli, U. K.(1970) Nature227, 680-685 [Medline] [Order article via Infotrieve]
  19. Hirata, M., Yoshida, M., Inada, K., and Kirikae, T.(1990) Adv. Exp. Med. Biol.256, 287-299 [Medline] [Order article via Infotrieve]
  20. Larrick, J. W., Hirata, M., Shimomura, Y., Yoshida, M., Zheng, H., Zhong, J., and Wright, S. C.(1993) Antimicrob. Agents Chemother.37, 2534- 2539 [Abstract]
  21. Harlow, E., and Lane, D.(1989) Antibodies: A Laboratory Manual, pp. 471-510, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  22. Miura, Y., Kawabata, S., and Iwanaga, S.(1994) J. Biol. Chem.269, 542-547 [Abstract/Free Full Text]
  23. Shigenaga, T., Takayenoki, Y., Kawasaki, S., Seki, N., Muta, T., Toh, Y., Ito, A., and Iwanaga, S.(1993) J. Biochem. (Tokyo) 114, 307-316 [Abstract]
  24. Mega, T., and Hase, S.(1991) J. Biochem. (Tokyo) 109, 600-603 [Abstract]
  25. Armstrong, P. B.(1991) in Immunology of Insects and Other Arthropods (Gupta, A. P., ed) pp. 4-17, CRC Press, London
  26. Marchalonis, J. J., and Edelman, G. M.(1968) J. Mol. Biol.32, 467-469 [Medline] [Order article via Infotrieve]
  27. Roche, A.-C., and Monsigny, M.(1974) Biochim. Biophys. Acta371, 242-254 [Medline] [Order article via Infotrieve]
  28. Robey, F., and Liu, T.-Y.(1981) J. Biol. Chem.256, 969-975 [Free Full Text]
  29. Nguyen, N. Y., Suzuki, A., Boykins, R. A., and Liu, T.-Y.(1986) J. Biol. Chem.261, 10456-10465 [Abstract/Free Full Text]
  30. Brandin, E. R., and Pistole, T. G.(1983) Biochem. Biophys. Res. Commun.113, 611-617 [Medline] [Order article via Infotrieve]
  31. Srimal, S., Dorai, D. T., Somasundaran, M., Bachhwat, B. K., and Miyata, T.(1985) Biochem. J.230, 321-327 [Medline] [Order article via Infotrieve]
  32. Shimizu, S., Ito, M., and Niwa, M.(1977) Biochim. Biophys. Acta500, 71-79 [Medline] [Order article via Infotrieve]
  33. Fujii, N., Minetti, C. A. S. A., Nakhasi, H. L., Chen, S.-W., Barbehenn, E., Nunes, P. H., and Nguyen, N. Y.(1992) J. Biol. Chem.267, 22452-22459 [Abstract/Free Full Text]
  34. Oppenheim, J. D., Nachbar, M. S., Salton, M. R. J., and Aull, F.(1974) Biochem. Biophys. Res. Commun.58, 1127-1134 [Medline] [Order article via Infotrieve]
  35. Morita, T., Ohtsubo, S., Nakamura, T., Tanaka, S., Iwanaga, S., Ohashi, K., and Niwa, M.(1985) J. Biochem. (Tokyo) 97, 1611-1620 [Abstract]
  36. Aketagawa, J., Miyata, T., Ohtsubo, S., Nakamura, T., Morita, T., Hayashida, H., Miyata, T., Iwanaga, S., Takao, T., and Shimonishi, Y. (1986) J. Biol. Chem.261, 7357-7365 [Abstract/Free Full Text]
  37. Kyte, J., and Doolittle, R. F.(1982) J. Mol. Biol.157, 105-132 [Medline] [Order article via Infotrieve]
  38. Skoog, B., and Wichman, A.(1986) Trends Anal. Chem.5, 82-83 [CrossRef]
  39. Schumann, R. R., Leong, S. R., Flaggs, G. W., Gray, P. W., Wright, S. D., Mathison, J. C., Tobias, P. S., and Ulevitch, R. J.(1990) Science249, 1429-1431 [Medline] [Order article via Infotrieve]

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