From the
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
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),
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.
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
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.
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
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) .
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.
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
= 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.
(
)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.
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
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).
-terminal Sequence
Analyses
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 NH
HCO
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).
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.
-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.
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.
-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.
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
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.