From the Institute of Biological Chemistry, Academia Sinica, Nankang Borough, Taipei 115, Taiwan, Republic of China
Received for publication, September 13, 2000, and in revised form, December 7, 2000
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ABSTRACT |
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A Sepharose CL-4B-binding protein,
Tachypleus plasma lectin 1 (TPL-1), and a
lipopolysaccharide (LPS)-binding protein, Tachypleus plasma
lectin-2 (TPL-2), have been isolated from the plasma of Tachypleus tridentatus and biochemically characterized.
Each protein is coded by a homologous family of multigenes. TPL-1 binds
to Sepharose CL-4B and was eluted with buffer containing 0.4 M GlcNAc. The deduced amino acid sequence of TPL-1
consisted of 232 amino acids with an N-glycosylation site,
Asn-Gly-Ser at residues 74-76. It shares a 65% sequence identity and
similar internal repeats of about 20 amino acid motifs with
tachylectin-1. Tachylectin-1 was identified as a
lipopolysaccharide-agarose binding nonglycosylated protein from the
amebocytes of T. tridentatus. TPL-2 was eluted from the
LPS-Sepharose CL-4B affinity column in buffer containing 0.4 M GlcNAc and 2 M KCl. The deduced amino acid
sequence of TPL-2 consisted of 128 amino acids with an
N-glycosylation site, Asn-Cys-Thr, at positions 3-5. It
shares an 80% sequence identity with tachylectin-3, isolated from the
amebocytes of T. tridentatus. TPL-2 purified by
LPS-affinity column from the plasma predominantly exists as a dimer of
a glycoprotein with an apparent molecular mass of 36 kDa. Tachylectin-3
is an intracellular nonglycosylated protein that also exists as a dimer
in solution with an apparent molecular mass of 29 kDa. It recognizes
Gram-negative bacteria through the 0-antigen of LPS. Western blot
analyses showed that, in the plasma, TPL-1 and TPL-2 exist
predominantly as oligomers with molecular masses above 60 kDa. They
both bind to Gram-positive and Gram-negative bacteria, and this binding
is inhibited by GlcNAc. Possible binding site of TPL-1 and TPL-2 to the
bacteria could be at the NAc moiety of GlcNAc-MurNAc of the
peptidoglycan. The physiological function of TPL-1 and TPL-2 is
most likely related to their ability to form a cluster of interlocking
molecules to immobilize and entrap invading organisms.
The innate and the adaptive immunities are the two general systems
that mediate resistance to infectious agents. Although a certain form
of adaptive immunity is present in all vertebrates, the invertebrates
have developed only the innate immune system that has been thought of
as an evolutionary rudiment, whose only function is to limit infection
until adaptive immune response is induced. Recent studies have shown
that the innate immune system has the capacity to induce costimulatory
signals necessary for the activation and differentiation of lymphocytes
(1-3). This finding has renewed interest on the studies of
invertebrate and vertebrate innate immunology.
The innate immune system uses germline-encoded receptors for
recognition of common antigens on the surface of microbial pathogens. This feature distinguishes the innate immune system found in
invertebrates from the adaptive immune system of the vertebrates that
possess a repertoire of specific antigen receptors and antibodies. The conserved constituents or patterns, displayed by microorganisms, are
recognized by pattern recognition molecules or receptors (4). These
patterns, called pathogen-associated molecular patterns, seem to be
shared among groups of pathogens. The lipopolysaccharides (LPS)1 of Gram-negative
bacteria, lipoteichoic acid of Gram-positive bacteria, glycolipids of
mycobacterium, and mannans of yeast are some examples. The innate
defense system is designed to recognize those pathogen-associated
molecular patterns.
The horseshoe crab, an arthropod, has evolved only a nonclonal,
or innate, defense system. The hemolymph and the hemocytes carry this
defense system. Whereas the hemocytes, also named amebocytes, contain
large and small granules that are filled with defense molecules, such
as coagulation factors (5-7), protease inhibitors (8), and
antimicrobial peptides (6), the hemoplymph contains three major
proteins: hemocyanin, C-reactive proteins (CRPs), and
Several lectins with a broad range of specificity have been identified
in the amebocytes of horseshoe crab (12, 13). These lectins have been
proposed to function in concert to defend horseshoe crabs from invading
pathogens. However, since these lectins are present mostly in the
granules of the hemocytes, they are unlikely to be involved in the
immediate-early response of host-pathogen interaction.
The plasma of horseshoe crab also contains lectin-like innate defense
molecules (14, 15). In the previous study from this laboratory, we
described the isolation and characterization of proteins that bind to
Sepharose CL-4B, lipopolysaccharide of Escherichia coli, and
protein A of Staphylococcus aureus from the plasma of Tachypleus tridentatus (15). In the present study, we report biochemical characterization and cDNA cloning of two of the
proteins, the Sepharose CL-4B-binding protein (TPL-1) and the
lipopolysaccharide-binding protein (TPL-2), which we believe are
involved in the innate immunity of horseshoe crabs.
Reagents--
E. coli O55:B5 LPS was purchased
from Sigma. Sepharose CL-4B, CNBr-activated Sepharose CL-4B, molecular
weight standards, and staphylococcal protein A-Sepharose CL-4B were
from Amersham Pharmacia Biotech (Uppsala, Sweden). Trypsin and complete
protease inhibitor tablets were from Roche Molecular Biochemicals.
Streptavidin-agarose and EZ-Link NHC-LC-Biotin were from Pierce. All
other chemicals were of the highest quality commercially available.
Horseshoe Crab and Hemolymph--
T. tridentatus were
captured on the beaches of Quimoi Island, Taiwan. Horseshoe
crabs were bled by cardiac puncture, and hemolymph was collected in a
conical tube containing equal volume of chilled sterile 3% NaCl
supplemented with 2 mM propranolol and protease inhibitor
tablets (1 tablet/50 ml) to maintain the isotonic condition and to
prevent the lysis of amebocyte (16). The amebocytes were separated from
plasma by centrifugation at 140 × g for 15 min at
4 °C. The supernatant was transferred to a new conical tube under
sterile condition, filtered through a 0.2-µm pyrogen-free filter, and
loaded immediately into column.
Preparation of LPS-Sepharose CL-4B Affinity Resin--
LPS
affinity resin was prepared by coupling LPS from E. coli
O55:B5 with CNBr-activated Sepharose CL-4B according to the instruction of manufacturer with the ligand concentration of 2 × 10 Purification of TPL-1 and TPL-2 from Hemolymph--
Five hundred
milliliters of filtered, protease inhibitor-supplemented hemolymph was
passed sequentially through three 10 mm × 10-cm tandemly linked
affinity columns, packed with Sepharose CL-4B, staphylococcal protein
A-Sepharose CL-4B, and LPS-Sepharose CL-4B, respectively. The columns
were pre-equilibrated with initial buffer (10 mM Tris·Cl,
pH 7.4, 150 mM NaCl, 10 mM CaCl2)
and at the end of sample loading, washed with at least 10 column
volumes of the initial buffer containing 1 M KCl until a
steady base line was obtained. The columns were detached from each
other. To recover TPL-1 from the Sepharose CL-4B, which served as the
affinity matrix, the column was eluted with the initial buffer
containing 0.4 M GlcNAc. To recover the
lipopolysaccharide-binding protein (TPL-2), the LPS column was eluted
with the initial buffer containing 0.4 M GlcNac and 2 M KCl. Solid ammonium sulfate was added to the effluent
fractions containing the adsorbed proteins to 50% saturation. The
precipitate was collected by centrifugation at 10,000 × g for 10 min and dissolved in initial buffer. The entire
purification procedure was performed at 4 °C.
Reverse Phase HPLC Analysis--
High performance liquid
chromatography was performed on an HP1100 (Hewlett-Packard) HPLC system
with a C4 column (214TP54, Vydac) using a flow rate of 0.25 ml/min for protein and with a C18 column (218TP52, Vydac)
using a flow rate of 0.15 ml/min for protease-digested peptides. The
compositions of Buffer A and Buffer B were
acetonitrile:water:trifluoroacetic acid at 10:90:0.1 and at 90:10:0.1,
respectively. Proteins and peptides were eluted from columns with
linear gradient of 0-100% Buffer B. Absorbency for proteins and
peptides were monitored at 280 and 214 nm, respectively.
Proteolytic Digestion--
Protein purified by HPLC was
lyophilized and dissolved in 0.4 M
NH4HCO3 containing 8 M urea. After
reduction with dithiothreitol and S-alkylation with
iodoacetamide, three volumes of distilled H2O were added.
The protein was then digested with trypsin (E/S = 1/25, w/w) at
37 °C for 24 h. The peptides generated were separated by
reversed-phase HPLC as described above using C18 column
(218TP52, Vydac).
Sugar Analysis--
Periodic acid-Schiff stain was performed to
assay for glycoprotein. At the end of SDS-PAGE, gel was fixed with
trichloroacetic acid, oxidized with periodic acid, followed by staining
with Schiff's reagent and destaining with acetic acid as described
(17). Monosaccharide contents were analyzed by gas chromatograph-mass
spectroscopy using the Hewlett-Packard model 6890 gas chromatograph,
connected to a Hewlett-Packard 5973 mass selective detector. Samples
for analysis were subjected to methanolysis,
re-N-acetylation, and trimethylsilylation and dissolved in
hexane prior to splitless injection into a HP-5MS fused silica
capillary column (30 m × 0.32 mm, inner diameter,
Hewlett-Packard). The column head pressure was maintained at around
8.2 p.s.i. to give a constant flow rate of 1 ml/min using helium
as carrier gas. Oven temperature was held at 60 °C for 1 min,
increased to 90 °C in 1 min, and then to 290 °C in 25 min. The
trimethylsilyl derivatives were analyzed by gas chromatograph-mass
spectroscopy on the Hewlett-Packard system using a temperature gradient
of 60-140 °C at 25 °C/min, and then increased to 300 °C at
10 °C/min.
Protein Sequencing and Sequence Analysis--
Sequencing of
samples recovered from the reverse-phase HPLC and from
SDS-PAGE/electroblottings were performed on an ABI 492 Procise
automatic protein sequencer (PerkinElmer Life Sciences). The initial
yield ranged from 10 to 20 pmol. The sequences were then analyzed by
the GCG package (Genetics Computer Group Inc.).
Preparation of Anti-TPL-1, Anti-TPL-2 Polyclonal
Antibodies--
To raise antiserum against TPL-1 and TPL-2, proteins
recovered from the eluate of Sepharose CL-4B and LPS-affinity column, respectively, were further purified by HPLC to obtain a 30-kDa TLP-1-species and a 36-kDa TLP-2-species, as judged by SDS-PAGE (15).
Each of the purified protein was mixed with Freund's complete adjuvant
and injected into a female New Zealand White rabbit by the
intrasplenitic route (18). Blood samples were collected after 4 weeks
and subsequently every 7 days for the following 6-8 weeks (50 ml each
time). Sera obtained were stored at Western Blot Analysis--
Proteins were electrophoresed on 12%
SDS-PAGE and transferred electrophoretically to nitrocellulose using an
electroblot apparatus (Hoefer TE70 semidry transfer unit,
Amersham Pharmacia Biotech) with constant current of 0.8 mA/cm2. The membranes were blocked with 5% (w/v) skim milk
in phosphate-buffered saline supplemented with 0.1% Tween 20 and
probed with specific antibody. Blots were incubated with horseradish
peroxidase-conjugated anti-rabbit immunoglobulin (IgG) in the second
step and developed by the enhance chemiluminescence method (ECL system,
Amersham Pharmacia Biotech).
Immunoprecipitation of TPL-1-TPL-2 Heteromer from the Plasma with
Biotinylated Anti-TPL-1 Antibodies Coupled to
Streptavidin-agarose--
Anti-TPL-1 antibodies were biotinylated with
EZ-Link NHS-Biotin (Pierce) as described by the manufacturer. The
biotinylated antibodies were incubated with strepavidin-agarose
(Pierce), pretreated with 1% BSA for 1 h at 4 °C to block
nonspecific binding. The gel (25 µl) was washed with the initial
buffer, and incubated with horseshoe crab plasma (2 µg/50 µl)
overnight at 4 °C. After washing with the initial buffer, the pellet
was re-suspended in the nonreducing Laemmli SDS-PAGE buffer, boiled for
5 min and the supernatant (5 µl) was subjected to Western blot
analysis using anti-TPL-2 antiserum.
Analysis of TPL-1 and TPL-2 Binding to Bacterial Cells by
Enzyme-linked Immunosorbent Assay--
To the enzyme-linked
immunosorbent assay plates (Greiner F-form), suspension of bacteria in
a mixture of chloroform and ethanol 1:9 (v/v), were added (5 × 107 cells/well), and the solvent was evaporated under a
stream of warm air (19). The concentration of bacteria in the culture was determined by measuring the scattered light of the culture at
optical density of 600 nm with a spectrophotometer. The number of
cells/ml was estimated assuming 0.1 opticl density unit is roughly
equivalent to 108 cells/ml. The microplates with the
adsorbed bacteria were washed with wash buffer (0.05% Tween 20 in
phosphate-buffered saline), and the unbound sites were blocked with 1%
BSA dissolved in the wash buffer. A serially diluted TPL-1 or TPL-2 in
diluent buffer (1% BSA in wash buffer) was added to each well and
incubated for 2 h at room temperature. After washing, rabbit
anti-TPL-1 or anti-TPL-2 antiserum were added to each well and
incubated for 2 h at room temperature.
The antiserum against TPL-1 and TPL-2 used were preadsorbed with
immobilized bacteria (Streptococcus pneumoniae R36A, E. coli Bos-12, or Vibrio parahaemoliticus) to minimize
cross-reactivity with these bacteria. After washing, horseradish
peroxidase-linked anti-rabbit immunoglobulin antibody was added to each
well and the plates were incubated for 2 h at room temperature.
After washing with the wash buffer, 0.l ml of 0.1 mg/ml
3,3',5,5-tetramethylbenzidine (Sigma) in substrate buffer was added to
each well and incubated at room temperature for exactly 10 min. The
reaction was terminated by the addition of 0.l ml of 2 M
H2SO4 and the absorbency at 450 NM was read.
Since 0.4 M GlcNAc and 0.4 M GlcNAc plus 2 M KCl inhibit the binding of TPL-1 and TPl-2, respectively,
to bacteria, these samples served as controls for the binding assay.
Mass Spectrometry--
Mass spectrometric analysis of
HPLC-purified TPL-1 and TPL-2 was performed on a model DE-RP MALDI-TOF
(PE Biosystems, Framingham, MA). All samples were dissolved in
3,5-dimethoxy-4-hydroxycinnamic acid (sinipinic acid) at 10 mg/ml and
analyzed in the positive ion mode.
cDNA Synthesis--
Tissues were obtained from an adult male
of T. tridentatus. Immediately after dissection, the
hepatopancreas, muscle, and hemocytes were excised and placed in liquid
nitrogen. Total RNAs were prepared from hepatopancreas, using the
RNAzol B kit (Biotex), and poly(A)+ RNAs were purified
using QuickPrepR Micro mRNA purification kit with
oligo(dT)-cellulose chromatography (Amersham Pharmacia Biotech). The
first strand cDNA synthesis was primed with a hybrid oligo(dT) linker-primer and random primers and was transcribed using moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.).
The synthesized cDNA was used as a template in subsequent PCR.
PCR--
The primers were synthesized as follows: TPL-1,
sense primer 5'-GA(A/G)TGGAC(A/C/G/T)CA(C/T)AT(A/C/T)AA(C/T)AA-3' from
EWTHING (residues 1-7) of N-terminal sequence and antisense primer
5'-TT(A/G)TC(A/C/G/T)GA(C/T)TG(C/T)TT(A/C/G/T)AG(A/G)TA(A/C/ G/T)CC-3'
from GYKQXDN (residues 195-201) of peptide tryp-2; TPL-2, sense primer
5'GA(A/G)GG(A/C/G/T)AA(A/G)(C/T)T(A/C/G/T)ATGAA(A/G)CA(C/T)CC-3' from
EGKLMKHP (residues 13-20) of N- terminal sequence and oligo(dT) as
antisense primer. The PCR of cDNA template was performed in a
Biometra personal cycler with the following program: cycle 1, 96 °C
for 2 min; cycles 2-41, 96 °C for 30 s, 50 °C for 30 s, 72 °C for 1 min; cycle 42, 72 °C for 10 min. Amplified DNA
fragments were analyzed on a 1% agarose gel, ligated into pGEM-T easy
vector (Promega), and transformed into E. coli strain JM109
by the calcium chloride method.
DNA Sequence Analysis--
DNA sequence reaction was performed
using the PRISM Ready Reaction DyeDeoxy Terminator sequencing kit (PE
Applied Biosystems). Samples were subjected to electrophoresis on an
ABI 310 DNA sequencer, read automatically, and recorded using ABI Prism
model version 2.1.1 software (PE Applied Biosystems).
5'-Rapid Amplification of cDNA Ends (RACE) and
3'-RACE--
The sequences of 5' end cDNA of TPL-1 and TPL-2 were
determined by using the MARATHON cDNA cloning system
(CLONTECH) with gene-specific primers (GSPs). For
TPL-1, GSP1 was 5'GTATCC-AACTCCCATTACCATCCACTGG-3' and GSP2 was
5'-GGTACACGGTCGCGTGCACCTGAAGATG-3', corresponding to nucleotides
1063-1090 and 935-962, respectively; for TPL-2, GSP1 was
5'-AAATCACATTTACACAAGAGTTTCTCC-3' and GSP2 was
5'-AAATATTTGATTGAATACATTCCCAGT-3', corresponding to nucleotides
446-474 and 418-444, respectively. The primers for 3' RACE of TPL-1
were synthesized as follows: GSP1, 5'-ATATTTCGTCGACCCGTTGACGG-3'; GSP2,
5'-ATCTTTCGCTGCAAGAAACCTTGC-3', corresponding to nucleotides 1276-1298
and 1390-1413, respectively. All the procedures were performed
according to the manufacturer's recommendations. The amplified
fragments representing overlapping 5' and 3' cDNA for TPL-1 and 5'
cDNA for TPL-2 were gel-purified and cloned as described previously
(20). These cDNAs were subsequently ligated together to create
full-length cDNAs.
Multiple Gene Analysis by PCR--
Genomic DNA was purified from
hepatopancreas as described (20). The primers used for PCR were based
on consensus amino acid sequences (residues 13-20 and 101-108)
between TPL-2 and tachylectin-3 (21): sense primer,
5'-GA(A/G)GG(A/C/G/T)AA(A/G)(C/T)T(A/C/G/T)ATGAA(A/G)CA(C/T)CC-3'; and
antisense primer, 5'-TTGATTTTGTTCCAGTC(A/C/G/T)AAACA-3'. The PCR
reaction was carried out under the following conditions: cycle 1, 96 °C for 2 min; cycles 2-4, 96 °C for 30 s, 55 °C for
30 s, 72 °C for 1 min; cycles 5-7, 96 °C for 30 s,
50 °C for 30 s, 72 °C for 1 min; cycles 8-41, 96 °C
for 30 s, 45 °C for 30 s, 72 °C for 1 min; cycle 42, 72 °C for 15 min. The PCR product was gel-purified, cloned, and
sequenced as described previously (20).
Purification of TPL-1 and TPL-2--
By passing plasma of
horseshoe crabs through tandemly linked affinity columns, two new
lectins were purified and characterized: TPL-1, which binds to
Sepharose CL-4B; and TPL-2, which binds to the LPS of E. coli. The use of Sepharose CL-4B as a "pre-column," prior to
the passage of plasma through the LPS-Sepaharose CL-4B, allowed the
separation of TPL-1 from TPL-2. TPL-1 and TPL-2 could not be eluted
from their respective affinity column with 2 M KCl, EDTA,
galactose, or lactose. In the previous study, both proteins were eluted
from the respective affinity column with buffer (10 mM
Tris·Cl, pH 7.4, 150 mM NaCl) containing 4 M
urea or 2 M guanidium chloride (15). Proteins eluted with 4 M urea or 2 M guanidium chloride gradually
formed irreversible precipitate upon removal of the chaotropic agents.
In the present study, 0.4 M GlcNAc was found effective in
eluting TPL-1 from the Sepharose CL-4B column, while elution of TPL-2
from the LPS-affinity column required 2 M KCl in addition
to 0.4 M GlcNAc. Proteins eluted with GlcNAc remain in
solution after removal of GlcNAc and can be readsorbed to the affinity
column. One passage of the plasma through the affinity columns depleted
all of the proteins that bind to Sepharose CL-4B and LPS-columns.
Subsequent to the elution of the columns with GlcNAc, insignificant
amount of other proteins were eluted with 4 M urea or 2 M guanidium chloride.
The mechanism of the binding of TPL-1 to the Sepharose CL-4B and its
elution by GlcNAc is not known. Sepharose is a polymerized form of
agarose consisting of repeating unit of
Tachylectin-2 was isolated from the amebocyte of horseshoe crabs by
dextransulfate chromatography and shown to exhibit high affinity for
both GlcNAc and GalNAc (23). Tachylectin-P was puritfied from the
perivitelline fluid of horseshoe crab by using an affinity
column consisting of bovine submaxillary gland mucin attached to
Sepharose 4B, and eluted from the column with GlcNAc (24). From the
plasma of horseshoe crabs, tachylectin 5A and 5B were purified, using
an N-acetylated resin and elution of the proteins by GlcNAc
(14). While exhibiting a common specificity of binding to the
N-acetoamido moiety of hexoses, tachylectin-2, tachylectin-P, and tachylectin-5A/B do not share any sequence homology
with each other. Whether these proteins would bind to unmodified
Sepharose CL-4B like TPL-1, and be eluted from it by GlcNAc, is not known.
TPL-2 can be eluted from the LPS-affinity column with buffer containing
0.1% LPS and 2 M KCl. However, the LPS-eluted TPL-2 could
not be completely separated from LPS. GlcNAc is a component of the LPS
used in the preparation of LPS-affinity column. Thus, GlcNAc.was used
to elute LPS-binding protein.
TPL-1 and TPL-2 isolated accounted for about 0.1% and 0.02-0.04%,
respectively, of the total hemolymph proteins. Although the amount of
TPL-1 remained fairly constant, the amount of TPL-2 decreased rapidly
after the animals have been kept in captivity.
Biochemical Properties of TPL-1--
On SDS-PAGE under nonreducing
condition (Fig. 1A,
lane 1), TPL-1 showed two major bands at around
30 kDa, a band at around 52 kDa, two bands at around 66 kDa, and
additional bands at over 100 kDa. Under reducing condition (Fig.
1B, lane 1), protein bands at 30, 52, and 66 kDa were detected. In Western blot analysis, antibodies raised
against the 30-kDa TPL-1, reacted with these protein bands in
nonreducing (Fig. 1C, lane 3) and
reducing (Fig. 1C, lane 7) SDS-PAGE.
Protein bands with even higher molecular masses (52 kDa and above) were
detected in both the pre- and post-column plasma samples, under
nonreducing SDS-PAGE (Fig. 1C, lane 1,
pre-column; lane 2, post-column) and reducing
(Fig. 1C, lane 5, pre-column; lane 6, post-column). TPL-2 did not react with
anti-TPL-1 serum (Fig. 1C, lanes 4 and
8), validating the specificity of the antibodies.
Previously (15), we described a protein band with a molecular mass of
40 kDa as the major protein eluted by 4 M urea from Sepharose CL-4B. With 0.4 M GlcNAc solution as eluent, the
40-kDa protein was not detected (Fig. 1, A (lane
1) and B (lane 1)). The
30-kDa protein (TPL-1) was shown to have identical amino-terminal sequence with the previously published sequence of GBP (15).
The gene sequence data predicted 232 amino acid residues for TPL-1
(Fig. 2). The deduced amino acid sequence
of TPL-1 shares a 65% identity to tachylectin-1 (TL-1) (25),
identified in the large granules of amebocytes of the horseshoe crab,
and 66% identity to tachylectin-P (TL-P) (24), an embryonic lectin in
perivitelline fluid of the horseshoe crab (Fig.
3). A notable difference is the presence
of a potential N-glycosylation site
Asn74-Gly75-Ser76 in TPL-1 and its
absence in the other two intracellular proteins, TL-1 and TL-P.
Although TL-1 and TL-P share 98% sequence homologies with each other,
they manifest different biological and biochemical characteristics, in
hemagglutinating activity, antibacterial activity, and affinity to
other endogenous proteins (24). TPL-1 also shows a 30-65% identity to
tectonin I and tectonin II of myxomycete (Fig. 3) whose function is as
yet not known (26). A sequence homology search showed no significant
similarity between TPL-1 and any other proteins besides TL-1, TL-P,
tectonin I, and tectonin II, including the galectins.
Assuming 8 of the 9 Cys in TPL-1 are involved in disulfide bond
formation (based on sequence homologies and conservation of Cys
positions with tachylectin-1 (25), the calculated molecular mass of
TPL-1 is 25,801.9 Da. This value agrees well with the 25,857.5-Da TPL-1
found by mass spectrometry (Fig.
4A). The calculated pI of
TPL-1 is 8.04, making it a slightly basic protein.
Mass spectrometry analysis showed one other major TPL-1-species with a
molecular mass of 26,699.7 Da, and minor species of 16,595.7, 17,578.6, 49,208.8, 50,45.7, 51,889.4, 52,604.2, and 77,903.2 Da. The mass
difference of 842.2 between the 25,857.5-Da TPL-1 and the 26,699.7-Da
TPL-1 can be attributed to the presence of two HexNac and three hexoses
on the 26,699.7-Da TPL-1. The minor 16,595.7-Da and the 17,578.6-Da
TPL-1 represent proteolytic cleavage products of TPL-1 as reported
previously (15).
The combined SDS-PAGE, Western blot analysis, and the mass spectrometry
data suggest monomer/dimer relationship between the following species
of TPl-1: 24,548.0 Da/49,208.8 Da; 25,857.5 Da/51,889.4 Da; and
26,699.7 Da/52,604.2 Da, respectively. The 77,903.2-Da species
corresponds to a trimer of the 25,857.5-Da TPL-1.
Biochemical Properties of TPL-2--
Upon SDS-PAGE, the purified
TPL-2 showed major protein bands with a mass of about 36 kDa and a
minor band of about 72 kDa, under both nonreducing (Fig. 1A,
lane 2) and reducing conditions (Fig. 1B,
lane 2). In Western blot analysis, these protein
bands reacted with antiserum raised against the HPLC-purified 36-kDa TPL-2 (Fig. 1D, lane 4, NR;
lane 8, R). The plasma samples, before and after passage through the affinity columns, showed protein bands of
72 kDa and higher molecular masses, reacting with anti-TPL-2 serum in
the nonreducing SDS-PAGE (Fig. 1D, lane
1, pre-column; lane 2, post-column),
and mainly of a 66-kDa protein band in the reducing SDS-PAGE (Fig.
1D, lane 5, pre-column;
lane 6, post-column). TPL-1 did not react with
anti-TPL-2 serum (Fig. 1D, lanes 3 and 7), affirming the specificity of anti-TPL-2 antibodies.
The deduced amino acid sequence of TPL-2 (Fig.
5) showed a 68% identity with
conservation of the 6 Cys positions to tachylectin-3 (Fig.
6). Although a potential
N-glycosylation site,
Asn3-Cys4-Thr5 is present in TPL-2,
this site is absent in tachylectin-3. Tachylectin-3 is a
nonglycosylated intracellular protein isolated from the large granule
of the amebocyte.
In our previous report (15), the amino-terminal residues 1, 2, and 3 were left as blank, while residue 4 was shown as Tyr and residue 6 as
Lys. Amino-terminal residue analysis of the GlcNAc eluted TPL-2 showed
(residue number in superscript, recovery of PTH-amino acid (picomoles)
in parentheses, and absence of PTH-amino acid denoted as X):
E1 (152)-D2
(90)-X3-X4-T5
(70)-X6-V7 (75)-T8
(61)-D9 (82)-R10 (61)-S11
(36)-L12 (66)-E13 (43)-G14
(56)-K15 (75)-L16 (46)-M17
(40)-K18 (78)-H19 (25)-P20 (40).
Gene sequence analysis (Fig. 5) predicts residue 3 as Asn and both
residues 4 and 6 as Cys. The sequence analysis shown above corrects and
confirms the earlier sequence analysis of TPL-2 (15). The absence of
PTH-amino acid at Asn3 supports the contention that the
N-glycosylation site of TPL-2 at this position.
The gene sequence data predicted 128 amino acid residues for TPl-2
(Fig. 5). Assuming 6 of the 7 Cys in TPL-2 are engaged in disulfide
bond formation based on sequence homologies and conservation of Cys
positions with tachylectin-3 (21), the calculated molecular mass of
TPL-2 will be 14,295.4 Da. Mass spectrometric analysis (Fig.
4B) showed a major TLP-2-species with a mass of 35,879.4 Da,
and two minor ones with masses of 17,954.4 and 72,088.0 Da. If the
difference of 3,659 Da between the 17.954.4-Da species determined by
mass spectrometry and the calculated mass of 14,295.4 Da could be
attributed to N-glycosylation, a number of possible complex
type glycostructures, consisting of about 20 hexoses, can be
accommodated. The presence of sugars in TPL-2 was confirmed by periodic
acid-Schiff staining (data not shown) and by carbohydrate analysis of
the HPLC-purified TPL-2 eluted by GlcNAc. The molar ratio of hexoses
determined in this study is very similar to the one reported earlier
(15). Although, without knowing the structure of the carbohydrate, it
will be difficult to calculate the exact contribution of carbohydrate
to the molecular mass of a glycoprotein, the molar ratio of hexoses
shows: Man, 3.1; Gal, 1.9; GlcNac, 2.4; and GalNAc, 0.4. Setting GalNAc
as 1.0, it then follows: Man, 7.8; Gal, 4.8; and GlcNAc, 6.0. This
gives rise to a total of 20 hexoses with a calculated molecular mass of
3534, which is close to the difference of 3659 between the 17.954.4-Da
species determined by mass spectrometry and the calculated mass of
14,295.4 Da calculated from the deduced amino acid sequence of the
TPL-2.
The results of SDS-PAGE, Western blot analysis, and mass spectrometric
analysis of TPL-2 strongly suggest that the 17,954.4-Da species
represents the monomer, the 35,879.4-Da species the dimer, and the
72,088.0-Da species the tetramer of TPL-2. TPL-2 contains 7 Cys, with a
free Cys that could form intermolecular disulfide bond. TPL-2 purified
by affinity column exists mainly as a dimer even under denaturing and
reducing condition (Fig. 1B, lane 2). In the plasma, TPL-2 and its isoform exist mainly as oligomers of even
higher molecular masses (Fig. 1D, lanes
1, 2, 5, and 6).
Using primers based on consensus nucleotide sequence between TPL-2 and
tachylectin-3, PCR was performed to examine the possible existence of
multiple genes for TLP-2-like molecules. Of the 23 clones identified, 5 new genes were found to code for proteins with similar but not
identical amino acid sequence to TLP-2 and tachylectin-3 (Fig. 6). The
results indicate that a homologous family of multiple genes code for
TPL-2 and its isoforms.
TPL-2 binds to LPS from E. coli. This binding was the basis
for the purification procedure employing LPS-affinity chromatography. The binding of TPL-2 to LPS is apparently independent of
Ca2+ ion, since sodium citrate or EDTA was not able to
elute TPL-2 from the LPS-affinity matrix. In this respect, TPL-2
differs from the 12-kDa Limulus LPS-binding protein (8)
and tachylectin-1 (25).
Among the LPS-binding proteins, the site of interaction with LPS has
not been identified, although significant sequence homologies were
observed among a number of these proteins (26). TPL-2 does not share
any homology with other LPS-binding proteins, including the 12-kDa
Limulus LPS-binding protein purified by a procedure utilizing LPS-affinity chromatography (8), except TL-3 and TL-P. TPL-2,
isolated in this study, differs from most other LPS-binding proteins
with a near neutral isoelectric point (pI = 7.65), instead of a
higher pI value. The basic nature of these proteins has been considered
to be an important factor in their interaction with the negatively
charged LPS molecule (27). It could be argued, however, that the proper
positioning of the basic amino acid in the three-dimensional structure
of the protein is more important than the overall basic nature of the
protein for the binding to LPS. In this respect, it is noted that there
are three clusters of basic amino acids in the TPL-2 sequence that
might be critical for its binding to LPS.
Heteromers of TPL-1 and TPL-2--
In addition to forming
homo-oligomers, TPL-1 and TPL-2 appear to form heteromers with each
other with molecular masses of about 76 kDa (Fig. 1E,
lane 2). The absence of this band in the two
control samples (Fig. 1E, lanes 1 and
3) assures that the 76-kDa band observed in Fig.
1E (lane 2) represents the TPL-1-TPL-2 heteromer. The band with molecular mass of about 180 kDa observed in
lanes 1 and 2, but not in
lane 3, most likely originated from the
anti-TPL-1 antibodies attached to the agarose gel. During boiling in
SDS buffer, TPL-1 antibodies were dissociated from the agarose
gel and reacted with anti-TPL-2 antiserum in Western blot. Although the
mechanism by which stable homo- and hetero-oligomers of TPL-1 and TPL-2
are formed remains to be clarified, the physiological function of TPL-1
and TPL-2 could be related to their propensity to form clusters of
interlocking molecules to immobilize and entrap the invading microorganisms.
Biological Function of TPL-1 and TPL-2: Binding to
Bacteria--
TPL-1 and TPL-2 have been shown to bind to three species
of bacteria, S. pneumoniae R36A, V. parahaemolyticus, and
E. coli Bost-12 in a dose-dependent and
saturable manner (Fig. 7A).
The specificity of the binding is demonstrated by inhibition with GlcNAc/GlcNAc plus 2 M KCl, respectively. Although both
pre- and post-affinity-column plasma proteins bind to bacteria,
significantly more pre-column plasma proteins bind to bacteria (Fig.
7B), indicating the contribution of affinity-column purified
TPL-1/TPL-2 in binding to bacteria. One possible binding site of TPL-1
and TPL-2 with these bacteria would be at the NAc moiety of the
GlcNAc-MurNAc cell wall peptidoglycan. Likewise, lectins with affinity
for the N-acetyl-group, tachylectin-2 (23), tachylectin-P
(24), and tachylectin-5A and -5B (14), could act as innate defense
molecules by binding to the peptidoglycans of bacteria.
Conclusion--
In contrast to adaptive immune system in having
repertoires of specific antigen receptors and antibodies, the
phylogenetically ancient innate immune system uses germline-encoded
receptors for recognition of common antigens on the surface of
microbial pathogens, such as proteases (4), polyphenol oxidases (28),
pathogen-specific lectins (29), and antibiotic peptides (5, 6). They
work in concert for the recognition, immobilization, and elimination of
the invading pathogens. The pathogen-specific lectins in the hemolymph
are expected to distinguish between self and nonself and to serve as
the first line of defense upon the entry of pathogens. The interaction
between lectins and pathogens results in the recruitment of other
defense mechanisms, which ultimately are responsible for the
immobilization and elimination of the invading pathogens.
C-reactive protein, identified as a pattern-recognition molecule in the
hemolymph of American horseshoe crabs, Limulus polyphemus, is a polymorphic mixture of closely related proteins (30). Recent study
has further shown that a family of genes (31) encodes three main
classes of the polymorphic CRPs. One (tCRP-1) binds to phosphocholine
(PC)/phosphoethanolamine (PEA) ligand in the presence of
Ca2+ but not to sialic acid-ligand, another (tCRP-2) binds
to both PC/PEA ligand and to the sialic-ligand, and a third (tCRP-3)
binds neither to the PC/PEA ligand nor to the sialic acid ligand (31). Yet, they all share an extensive sequence homology, and a
hexameric structure (30, 31).
In this study, two additional pattern recognition molecules, TPL-1 and
TPL-2 were isolated from T. tridentatus, their cDNA sequence determined, and their biochemical properties investigated. The
results obtained suggest that, in general, lectin-like pattern recognition molecules: 1) consist of small molecular weight subunit of
protein with mass of 15-25 kDa, which are encoded by families of
closely related genes; 2) tend to form homo- or hetero-oligomers; and
3) could assemble to yield a myriad of complex structures with
different binding specificity and affinity for ligands, that mimic the
diversity of the immunoglobulin system. The innate defense of horseshoe
crabs depends on this kind of system to recognize and entrap the varied
and everchanging nature of the invading pathogens. The glycostructures
of TPL-1 and TPL-2 might be responsible for mediating the formation of
stable interlocking cluster of the oligomers, through
protein-carbohydrate interactions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
2-macroglobulin. Hemocyanin functions as an
oxygen-carrying protein. CRPs are lectins that bind to phosphocholine
of the pneumococcus C-polysaccharide (9) and to the chromatin of
damaged cells (10).
2-Macroglobulin exhibits protease
inhibitory activity with a broad specificity that can block the
activities of proteases secreted from invading microorganisms (11). The
Limulus CRPs, along with the C3 homologue,
2-macroglobulin, participate in a complement-like
hemolytic activity in horseshoe crab hemolymph.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
6 mol/ml of drained gel by assuming the
average molecular mass of LPS to be 5,000 daltons.
20 °C. TPL-1/TPL-2-specific
IgGs were affinity-purified from immunized rabbit plasma by
staphylococcal protein A column chromatography. Antibodies recovered
were concentrated by ammonium sulfate precipitation (50% saturation)
and used in the Western blot and immunoassay. The titer of the specific
antibody was assayed by either immunoblotting or immunodiffusion.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-1,6-linked
D-galactose and an unusual
3,6-anhydro-L-galactose. Sepharose CL is prepared from
Sepharose by reacting with 2,3-dibromopropanol under alkaline condition, resulting in cross-linkages between the 6-OH of
D-galactose of one chain and 2-OH of the
3,6-anhydro-L-galactose of the other chain, via
2-hydroxypropyl bridges (22). Evidently, TPL-1 binds to this structure
in a GlcNAc-dissociable manner.
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Fig. 1.
SDS-PAGE and Western blot analyses of TPL-1
and TPL-2. *, purified TPL-1 (5 µg) and TPL-2 (8 µg) were
subjected to SDS-PAGE with (R) or without reduction
(NR) by 2.5% 2-mercaptoethanol. The gel was stained with
Coomassie Blue. The positions of the mass, in kDa, of standard proteins
are indicated at the left. A, lane
1, TPL-1, no reduction; lane 2, TPL-2
no reduction. B, lane 1, TPL-1, with
reduction; lane 2, TPL-2 with reduction. *,
Western blot analysis of pre- and post-column whole plasma (8 µg/µl), purified TPL-1 (0.1 µg/µl), and TPL-2 (0.16 µg/µl)
with and without reduction. C, using antiserum against
TPL-1. Without reduction: lane 1, pre-column
plasma; lane 2, post-column plasma;
lane 3, TPL-1; lane 4,
TPL-2. With reduction: lane 5, pre-column plasma;
lane 6, post-column plasma; lane
7, TPL-1; lane 8, TPL-2. D,
using antiserum against TPL-2. Without reduction: lane
1, pre-column plasma; lane 2,
post-column plasma; lane 3, TPL-1;
lane 4, TPL-2. With reduction: lane
5, pre-column plasma; lane 6,
post-column plasma; lane 7, TPL-1;
lane 8, TPL-2. E, detection of
TPL-1-TPL-2 heteromer in the plasma. Condition of immunoprecipitation
is described under "Materials and Methods." Supernatant (5 µl)
recovered from the boiling of the gel suspension with the nonreducing
SDS buffer were subjected to Western blot using antiserum against
TPL-2. Lane 1, buffer alone incubated with
biotinylated anti-TPL-1 antibodies coupled to streptavidin-agarose;
lane 2, plasma incubated with biotinylated
anti-TPL-1 antibodies coupled to streptavidin-agarose; lane
3, plasma incubated with streptavidin-agarose without
biotinylated anti-TPL-1 antibodies coupled to it.
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Fig. 2.
Nucleotide and deduced amino acid sequences
of TPL-1. Nucleotide and amino acid residues are
numbered on the left. The underlines
represent sequences determined by amino acid sequence analysis of the N
terminus of the intact protein and a tryptic peptide. The residues in
the gray box indicate corresponding nucleotide
sequences used as primers for PCR. The broken
underlines correspond to nucleotide sequences used in
5'-RACE and 3'-RACE. The putative signal sequence and glycosylation
site are printed in italics and bold,
respectively. An asterisk marks the stop codon.
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Fig. 3.
Sequence alignment of TPL-1 with
tachylectin-1 (TL1) and tachylectin-P
(TL-P) of T. tridentatus and
tectonin-1 and tectonin-2 of Myxomyces physarum
polycephalum. The alignment was performed using
CLUSTALW and PILEUP program of GCG package. Identical residues are
shown in gray boxes.
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Fig. 4.
Mass spectrometric analysis of TPL-1 and
TPL-2. The HPLC-purified TPL-1 and TPL-2 eluted from the
affinity-columns were subjected to mass spectrometric analysis as
described under "Materials and Methods." A, mass
spectrometric analysis of TPL-1. B, mass spectrometric
analysis of TPL-2.
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Fig. 5.
Nucleotide and deduced amino acid sequences
of TPL-2. Nucleotide and amino acid residues are
numbered on the left. The underlines
represent sequences determined by amino acid sequence analysis of the N
terminus of the intact protein. The residues in the gray
box indicate corresponding nucleotide sequences used as a
primer for PCR. The broken underlines correspond
to nucleotide sequences used for cDNA cloning and for multiple gene
analysis. The putative signal sequence and glycosylation site are
printed in italics and bold, respectively. An
asterisk marks the stop codon.
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[in a new window]
Fig. 6.
Alignment of deduced amino acid sequences of
TPL-2 related genes found in T. tridentatus. The
alignment was performed using PILEUP program of GCG package. Divergent
residues are shown in white boxes.
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Fig. 7.
Binding of TPL-1/TPL-2and plasma to
bacteria. A, TPL-1/TPL-2 incubated with immobilized
bacteria: S. pneumoniae R36A (1), V. parahaemolyticus (2), and E. coli Bos-12
(3). Open triangle, TPL-1;
open circle, TPL-1; cross, TPL-2 + 0.4 M GlcNAc + 2 M KCl; open
square, TPL-1+ 0.4 M GlcNAc. B, pre-
or post-column plasma incubated with immobilized bacteria: S. pneumoniae R36A (open triangle, pre-column;
closed triangle, post-column), V. parahaemolyticus (open square, pre-column;
closed square, post column), and E. coli
Bos12 (open circle, pre-column;
closed circle, post-column).
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ACKNOWLEDGEMENT |
---|
We thank Dr. Gilbert Jay of the OriGene Technologies Inc., for valuable comments on the biological role of lectins as innate defense molecules and for proofreading the manuscript, and Professor Yuan-Chuan Lee of the Johns Hopkins University for valuable discussion on the possible role of glycostructure in stabilizing the oligomer structure of glycoproteins. We are indebted to Drs. Kay-Hooi Khoo, Po-Huang Liang, Chia-Larn Kwo, and Sheng-Tai Chiou for helpful discussion throughout this study and Bor-Long Huang, Jian-Horng Leu, and Jin-Mei Chen for valuable contributions in the chromatographic procedures and determination of carbohydrate composition.
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FOOTNOTES |
---|
* This work was supported in part by grants from Academia Sinica, and the Chinese Petroleum Corp.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The amino acid sequences of these proteins can be accessed through NCBI Protein Database under NCBI accession numbers AF264067 (Tachypleus plasma lectin 1) and AF264068 (Tachypleus plasma lectin 2).
Portions of this work were submitted in partial fulfillment for
the degree of Master of Science, National Taiwan University, Taipei, Taiwan.
§ To whom correspondence should be addressed. Present address: OriGene Technologies, Inc., 6 Taft Ct., Suite 300, Rockville, MD 20850. Tel.: 301-365-3085; Fax: 301-365-7950; E-mail: dliu@origene.com and darrellliu{at}aol.com.
Published, JBC Papers in Press, December 22, 2000, DOI 10.1074/jbc.M008414200
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ABBREVIATIONS |
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The abbreviations used are: LPS, lipopolysaccharide; CRP, C-reactive protein; HPLC, high performance liquid chromatography; PC, phosphocholine; PEA, phosphoethanolamine; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; PAGE, polyacrylamide gel electrophoresis; GSP, gene-specific primer; TL, tachylectin; TPL, Tachypleus plasma lectin; PTH, phenylthiohydantoin; BSA, bovine serum albumin.
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REFERENCES |
---|
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---|
1. | Fearon, D. T., and Locksley, R. M. (1996) Science 272, 50-54[Abstract] |
2. | Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J. M., and Hoffmann, J. A. (1996) Cell 86, 973-983[Medline] [Order article via Infotrieve] |
3. | Carroll, M. C., and Prodeus, A. P. (1998) Curr. Opin. Immunol. 10, 36-40[CrossRef][Medline] [Order article via Infotrieve] |
4. | Medzhitov, R., and Janeway, C. A., Jr. (1997) Cell 91, 295-298[Medline] [Order article via Infotrieve] |
5. | Muta, T., and Iwanaga, S. (1996) Curr. Opin. Immunol. 8, 41-47[CrossRef][Medline] [Order article via Infotrieve] |
6. | Iwanaga, S., Kawabata, S., and Muta, T. (1998) J. Biochem. (Tokyo) 123, 1-15[Abstract] |
7. | Fortes-Dias, C. L., Minetti, C. A. S., Lin, Y., and Liu, T.-Y. (1993) Comp. Biochem. Physiol. 105, 79-85 |
8. |
Minetti, C. S. A.,
Lin, Y.,
Cislo, T.,
and Liu, T.-Y.
(1991)
J. Biol. Chem.
266,
20773-20780 |
9. | Volanakis, J. E., and Kaplan, M. H. (1971) Proc. Exp. Biol. Med. 136, 612-614 |
10. |
Robey, F. A.,
and Liu, T.-Y.
(1981)
J. Biol. Chem.
256,
969-975 |
11. | Enghild, J. J., Thogersen, I. B., Salvesen, G., Fey, G. H., Figler, N. L., Gonias, S. L., and Pizzo, S. V. (1990) Biochemistry 29, 10070-10080[Medline] [Order article via Infotrieve] |
12. |
Liu, T.,
Lin, Y.,
Cislo, T.,
Minetti, C. A. S. A.,
Baba, J. M. K.,
and Liu, T. Y.
(1991)
J. Biol. Chem.
266,
14813-14821 |
13. | Kawabata, S., and Iwanaga, S. (1999) Dev. Comp. Immunol. 23, 391-400[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Gokudan, S.,
Muta, T.,
Tsuda, R.,
Koori, K.,
Kawahara, T.,
Seki, N.,
Mizunoe, Y.,
Wai, S. N.,
Iwanaga, S.,
and Kawabata, S.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
10086-10091 |
15. |
Chiou, S. T.,
Chen, Y. W.,
Chen, S. C.,
Chao, C. F.,
and Liu, T. Y.
(2000)
J. Biol. Chem.
275,
1630-1634 |
16. | Mürer, E. H., Levin, J., and Holmer, R. (1975) J. Cell. Physiol. 86, 533-542[Medline] [Order article via Infotrieve] |
17. | Kapitany, R. A., and Zebrowski, E. J. (1973) Anal. Biochem. 56, 361-369[Medline] [Order article via Infotrieve] |
18. | Hong, T. H., Chen, S. T., Tang, T. K., Wang, S. C., and Chang, T. H. (1989) J. Immunol. Methods 21, 151-157 |
19. | Freudenberg, M. A., Fomsgaard, A., Mitov, I., and Calanos, C. (1989) Infection 17, 322-328[Medline] [Order article via Infotrieve] |
20. | Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual , p. 280, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY |
21. | Inamori, K., Saito, T., Iwaki, D., Nagira, T., Iwanaga, S., Arisaka, F., and Kawabata, S. (1999) J. Biol. Chem. 274, 3227-3278 |
22. | Porath, J., Janson, J.-C., and Laas, T. (1961) J. Chromatogr. 60, 167-177[CrossRef] |
23. |
Okino, S.,
Kawabata, S.,
Saito, T.,
Hirata, M.,
Takagi, T.,
and Iwanaga, S.
(1995)
J. Biol. Chem.
270,
31008-31015 |
24. |
Nagai, T.,
Kawabata, S.,
Shishikura, F.,
and Sugita, H.
(1999)
J. Biol. Chem.
274,
37673-37678 |
25. |
Saito, T.,
Kawabata, S.,
Hirata, M.,
and Iwanaga, S.
(1995)
J. Biol. Chem.
270,
14493-14499 |
26. |
Huh, C. G.,
Aldrich, J.,
Mottahedeh, J.,
Kwon, H.,
Johnson, C.,
and Marsh, R.
(1998)
J. Biol. Chem.
273,
6565-6574 |
27. | 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) Science 249, 1429-1431[Medline] [Order article via Infotrieve] |
28. | Hoffmann, J. A., Reichhart, J. M., and Hetru, C. (1996) Curr. Opin. Immunol. 8, 8-13[CrossRef][Medline] [Order article via Infotrieve] |
29. | Medzhitov, R., and Janeway, C. A., Jr. (1998) Curr. Opin. Immunol. 10, 12-15[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Nguyen, N. Y.,
Suzuki, A.,
Cheng, S. M.,
Zon, G.,
and Liu, T. Y.
(1986)
J. Biol. Chem.
261,
10450-10455 |
31. |
Iwaki, D.,
Osaki, T.,
Mizunoe, Y.,
Wai, S. N.,
Iwanaga, S.,
and Kawabata, S.
(1999)
Eur. J. Biochem.
264,
314-326 |