From the Department of Microbiology, Stockholm University, S-106 91 Stockholm, Sweden
Received for publication, August 30, 2002, and in revised form, November 15, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent studies of peptidoglycan recognition
protein (PGRP) have shown that 2 of the 13 Drosophila PGRP
genes encode proteins that function as receptors mediating immune
responses to bacteria. We show here that another member, PGRP-SC1B, has
a totally different function because it has enzymatic activity and
thereby can degrade peptidoglycan. A mass spectrometric analysis of the
cleavage products demonstrates that the enzyme hydrolyzes the
lactylamide bond between the glycan strand and the cross-linking
peptides. This result assigns the protein as an
N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), and the corresponding gene is thus the first of this class
to be described from a eukaryotic organism. Mutant forms of PGRP-SC1B
lacking a potential zinc ligand are enzymatically inactive but
retain their peptidoglycan affinity. The immunostimulatory properties
of PGRP-SC1B-degraded peptidoglycan are much reduced. This is in
striking contrast to lysozyme-digested peptidoglycan, which retains
most of its elicitor activity. This points toward a scavenger function
for PGRP-SC1B. Furthermore, a sequence homology comparison with phage
T7 lysozyme, also an N-acetylmuramoyl-L-alanine amidase, shows that as many as six of the Drosophila PGRPs
could belong to this class of proteins.
A prominent feature of the innate immune system is the rapid and
massive response to intruding microorganisms. Signal transduction pathways are immediately activated to induce genes for antimicrobial peptides or signaling molecules such as tumor necrosis factor- In the late 1980s, Janeway (5) formulated the concept of pattern
recognition. This concept implies that the innate immune system is set
to recognize molecular patterns that are common to most microbes and
that are essential structural parts of the microbial cell. Such
pathogen-associated molecular patterns
(PAMPs),1 e.g.
lipopolysaccharide, peptidoglycan (PGN), and The differential recognition of different pathogens enables insects to
respond with a somewhat adapted response, depending on the nature of
the infecting pathogen (8). In Drosophila melanogaster there
are 13 genes for peptidoglycan recognition proteins (PGRP) (9). A gene
knock out of one of these, PGRP-SA, was shown to be defective in the
response to Micrococcus luteus in adult flies via the
Toll/Dif pathway (10). In similar genetic screens, one of the
membrane-bound forms, PGRP-LCx, was shown to be required for activation
of the Relish pathway (11-13). It had been demonstrated earlier (14)
that PGRP is needed to trigger the prophenol oxidase cascade with PGN
in Bombyx mori. Because these three PGRP proteins have been
shown to have affinity for peptidoglycan, they are all true PRRs (9,
14).2
A homology comparison of PGRP and bacteriophage T7 lysozyme identified
PGRP as part of the N-acetylmuramoyl-L-alanine
amidase superfamily of proteins (15). This enzyme hydrolyzes the bond between the N-acetylmuramyl group in the glycan strand and
the L-alanine in the stem peptide in peptidoglycan. None of
the two receptor PGRPs (SA and LCx) has such amidase
activity.2 However,
N-acetylmuramoyl-L-alanine amidase activity has
been found in mammalian sera, and the enzymatic properties of a human enzyme have been well characterized (16, 17), although the gene for
this enzyme is not known. This is in contrast to the many known genes
of bacterial N-acetylmuramoyl-L-alanine
amidases, which take part in the degradation of the bacterial cell wall (18).
In the present study we report that one member of the PGRP family, the
Drosophila SC1B, is an
N-acetylmuramoyl-L-alanine amidase, and we
examine the immunogenicity of peptidoglycan being digested with this enzyme.
Protein Expression and Purification--
The inserts of the
cDNA clones pBacPAK9/PGRP-SC1B-His and pBacPAK9/PGRP-SA-His (9)
were excised from the baculovirus vector pBacPAK9 with EcoRI
and XhoI and ligated into the pMT/V5-His expression vector
(Invitrogen). Because the insert contains six histidine codons followed
by a stop codon, neither the V5 epitope nor the vector His tag was
utilized. The vector contains a copper-inducible metallothionein promoter.
The Drosophila Expression System (Invitrogen) was employed
to produce Schneider 2 (S2) cell lines expressing the following His-tagged proteins: PGRP-SA( Peptidoglycan Purification--
Insoluble peptidoglycan from
Staphylococcus aureus Cowan 1, M. luteus Ml 11, and Bacillus megaterium Bm 11 was prepared as described
(20). In short, bacterial lawns were grown overnight at 37 °C on
nutrient agar plates. Cells were collected and suspended in saline and
boiled for 20 min. Bacteria were pelleted, washed with saline, water,
and acetone, and then dried at 37 °C. Bacteria were resuspended in
cold water with an equal volume of 0.1-mm glass beads and homogenized
using a Bead Beater (Biospec Products) 10 times for 2 min and filtered
through a glass filter. Unbroken cells were sedimented by
centrifugation at 1,500 × g for 10 min at 4 °C, and
cell walls were collected at 6,500 × g for 30 min at
4 °C. Cell walls were then treated with 100 µg/ml RNase A (Sigma), 50 µg/ml DNase I (Sigma), and 0.25% toluene. The suspension was incubated with slow shaking for 18 h in 37 °C. Trypsin (200 µg/ml) (Sigma) was added and incubated for another 18 h at
37 °C. Cell walls were collected, washed, and lyophilized. For
teichoic acid removal, S. aureus and B. megaterium PGN were incubated with 5% trichloroacetic acid at
22 °C for 18 h. The insoluble walls were heated at 90 °C for
15 min, washed three times with water and three times with acetone, and dried.
Enzymatic Digestion of Peptidoglycan--
Insoluble
trichloroacetic acid-treated PGN from S. aureus was
suspended in PBS (pH 7.2 at 4 mg/ml) and briefly sonicated using an
XL2020 Sonicator (Misonix). PGN (1 mg/ml) was then incubated with
PGRP-SC1B or hen egg white lysozyme (EC 3.2.1.17) (Sigma) at different
concentrations. Clearance of the turbid solutions was monitored as
decrease in absorbance at 540 nm on a Titertec iEMS Reader MF
(Labsystems) for 10 h at 25 °C with occasional shaking. To
measure substrate specificity and to compare the activities of
PGRP-SC1B and the mutants PGRP-SC1B[C168S] and PGRP-SC1B[C168A], the assay was slightly modified. Tris buffer (60 mM Tris,
pH 8.0, 100 mM NaCl) was used, and the final concentrations
of PGRP-SC1B and PGN were 10 and 330 µg/ml, respectively.
Peptidoglycans used were from S. aureus and B. megaterium (with and without teichoic acids) and M. luteus (lacking teichoic acid). A540
was measured every 5 min for 3 h. The initial rate was calculated
as Antibacterial Assay--
The antibacterial activity of PGRP-SC1B
against S. aureus, B. megaterium, Bacillus
subtilis, Bacillus thuringiensis, Micrococcus luteus, Escherichia coli D22, E. coli D31,
and Enterobacter cloacae Immune Stimulation of mbn-2 Cells--
Drosophila
mbn-2 cells were grown in 35-mm culture dishes in Schneider's
Drosophila medium (PAN Biotech) supplemented with 10% fetal calf serum (Invitrogen). Cells were seeded at 1 × 106 cells/ml and grown for 24 h at 25 °C. S. aureus trichloroacetic acid-treated PGN (1 mg/ml) was pretreated
by incubation for 10 h at 25 °C with hen egg white lysozyme (20 µg/ml), PGRP-SC1B (20 µg/ml), or with a mixture of both enzymes.
Control PGN was incubated in PBS under the same conditions. Digested or
control PGN was added to the mbn-2 cells at a concentration of 5 µg/ml. As additional control, mbn-2 cells were treated with PGRP-SC1B
(20 µg/ml), hen egg white lysozyme (20 µg/ml), and PBS. Total RNA
was isolated from the mbn-2 cells at 2, 6, and 24 h after induction.
Isolation of RNA and Northern Analysis--
Total RNA was
isolated using Trizol (Invitrogen), essentially following the
manufacturer's instructions. RNA (15 µg per lane) was separated on a
denaturing 1% agarose gel and subsequently capillary-blotted onto a
Hybond-XL nylon filter (Amersham Biosciences). The filter was probed
with [32P]dCTP random prime-labeled (Amersham Biosciences
Rediprime II kit) cDNAs for cecropin A1, attacin, diptericin, and
ribosomal protein 49. High stringency hybridization at 42 °C was
performed and a PhosphorImaging screen was exposed to the filter.
Scanning was done using a FLA-3000 scanner (Fuji film), and data were
analyzed with Image Gauge 3.45 software (Fuji film).
HPLC Analysis of Digested Peptidoglycan--
Peptidoglycan from
S. aureus (1.8 mg/ml) was cleaved with PGRP-SCIB (6 µg/ml)
in 50 mM ammonium bicarbonate buffer, pH 7.0, at 22 °C
for 18 h with rocking. After incubation, a sample was hydrolyzed
with lysostaphin (EC 3.4.24.75) (12.5 µg/ml) (Sigma) for 18 h at
22 °C with rocking.
Reverse phase HPLC was carried out using a Varian 5000 Liquid
Chromatograph with a 5-µm Brownlee 4.6 × 30-mm RP-18 column. The sample was centrifuged for 4 min at 13,000 rpm, and a 200-µl aliquot of the supernatant was applied to the column. The initial solvent was 0.1% trifluoroacetic acid, and the sample was eluted with
a gradient of acetonitrile containing 0.08% trifluoroacetic acid at a
flow rate of 0.3 ml/min. The gradient profile was as follows: 0%, 0 min; 15%, 20 min; 25%, 25 min; and 60%, 30 min. All separations were
monitored at 214 nm. The fractions were collected manually and
subsequently subjected to MALDI-TOF mass spectral analysis.
MALDI-TOF Mass Spectrometry--
The masses of the samples were
measured by MALDI-TOF MS using a Voyager DE STR (Applied Biosystems)
operating in the reflector, positive ion mode. The accelerating voltage
was set to 20 kV with an extraction delay time of 125 ns. For each
spectrum, 250 laser shots were averaged. Mass spectra were calibrated
externally by Calibration Mixtures 1 (SequazymeTM Peptide
Mass Standard Kit, Perspective Biosystems). The sample (0.5 µl) was
applied and mixed with 0.5 µl of 2,5-dihydroxybenzoic acid in 50%
acetonitrile on the sample plate.
Mutagenesis--
Site-directed mutagenesis was performed to
produce two mutants of PGRP-SC1B-His (C168S and C168A) using a two-step
PCR strategy. First, two PCRs were done with pMT/PGRP-SC1B-His as
template. One reaction used 5'-CATCTCAGTGCAACTAA-3', a complementary
sequence to the pMT/V5-His expression vector upstream of PGRP-SC1B, as the sense primer plus a mutagenic primer complementary to bases 483-519 in the coding sequence of PGRP-SC1B. The other reaction was
with the complementary mutagenic primer plus
5'-TAGAAGGCACAGTCGAGG-3', a sequence downstream of PGRP-SC1B in
the vector. The mutagenic primers substitute the Cys-168 TGC codon for
a TCC serine codon or a GCC alanine codon. After separation on a 1%
agarose gel, the PCR products were purified using the Mini-elute gel
extraction kit (Qiagen). In the second PCR step, the purified fragments
from the first PCR step were used as templates with the same flanking vector primers as above.
The resulting PCR product was purified using gel extraction, cleaved
with restriction enzymes EcoRI and XhoI
(Invitrogen), and inserted into pMT/V5-His. The mutations were
confirmed by DNA sequencing.
PCR and Plasmid DNA Sequencing--
PCRs were run using a
PC-960G Gradient Thermal Cycler (Corbett Research) and Deep Vent
polymerase (New England Biolabs). Oligonucleotide primers were
purchased from DNA Technology A/S (Aarhus, Denmark). Sequencing of
plasmid constructs was performed using DYEnamic terminator ET cycle
sequencing kit (Amersham Biosciences).
Peptidoglycan Binding Assay--
Insoluble trichloroacetic
acid-treated peptidoglycan (1 mg/ml) from S. aureus was
incubated with PGRP-SC1B, PGRP-SC1B[C168A], PGRP-SC1B[C168S], or
PGRP-SA in Tris buffer (60 mM Tris, pH 8.0, 100 mM NaCl) for 30 min at 4 °C. The samples were
centrifuged at 13,000 × g for 10 min, and the
supernatants were collected. The PGN pellet fraction was washed with
Tris buffer, centrifuged, and dissolved in SDS loading buffer.
Supernatant and pellet fractions were analyzed on a 15%
SDS-polyacrylamide gel followed by staining with Coomassie Brilliant Blue.
PGRP-SC1B Degrades Bacterial Cell Walls--
A recombinant
PGRP-SC1B protein was synthesized using the Schneider expression system
and S2 insect cells. Codons coding for 6 histidine residues were added
to the C terminus by PCR to facilitate purification of recombinant
protein. After metal chelate affinity column purification, the protein
was essentially pure (Fig.
1C). We tested the
peptidoglycan degrading activity of the purified protein. The activity
is compared with that of egg white lysozyme in PBS at pH 7.2 (Fig. 1).
The activity is dose-dependent, and the kinetic curves with
2 µg/ml lysozyme and 5 µg/ml PGRP-SC1B are similar. The kinetics of
the enzymatic digestion of PGN thus shows that PGRP-SC1B is almost as
efficient as hen egg white lysozyme for degradation of PGN from
S. aureus.
Enzyme Activity against Different Peptidoglycans--
Table
I shows the activity of PGRP-SC1B against
purified peptidoglycans from different bacteria. The protein is active
against all peptidoglycans tested; however, it has by far the highest activity against trichloroacetic acid-treated S. aureus
peptidoglycan that lacks teichoic acid. We also noted an increased
activity against PGN from B. megaterium after
trichloroacetic acid treatment.
The Enzyme Is an N-Acetylmuramoyl-L-alanine
Amidase--
To find out which bond is cleaved by PGRP-SC1B, we
compared the HPLC profiles of undigested S. aureus
peptidoglycan and peptidoglycan cleaved with PGRP-SC1B (Fig.
2). The profile of undigested control peptidoglycan shows no peaks. In contrast, there is a complex pattern
of peaks in the PGRP-SC1B-cleaved sample. Because S. aureus peptidoglycan has a high degree of cross-linking (22), the size and
structure of the PGRP-SC1B-cleaved peptides vary greatly, which
explains the profile.
To simplify the pattern, PGRP-SC1B-treated peptidoglycan was digested
with the endopeptidase lysostaphin, which hydrolyzes the pentaglycine
bridges that cross-link the stem peptides (1). The number of peaks in
the chromatogram is dramatically reduced to one major and one minor
peak (Fig. 2C). To determine the masses of the peptidoglycan
fragments, the material in the major peak was analyzed using MALDI-TOF
mass spectrometry.
The spectrum (Fig. 3) is dominated by
peaks with masses corresponding to those of the fragments that can be
derived from the known structure of S. aureus peptidoglycan
(Fig. 3, inset). The pattern is compatible with PGRP-SC1B
cleaving between N-acetylmuramic acid and
L-alanine in peptidoglycan. The multitude of peaks in the
spectrum is fully explained by (i) lysostaphin cleavage at different
positions in the penta-glycine bridges and by (ii) two parallel series
of peaks generated by sodium replacement of a hydrogen ion. For
example, the peak at 702.4 Da is in good agreement with a protonated
stem peptide plus five glycine residues with a calculated mass of 703.3 (Table II).
Analysis of PGRP-SC1B Mutants--
In bacteriophage T7 lysozyme,
Cys-130 is a zinc ligand essential for
N-acetylmuramoyl-L-alanine amidase activity. To
examine the impact of the homologous Cys-168 in PGRP-SC1B on amidase
activity and PGN-binding properties, we constructed two mutants using
site-directed mutagenesis. In the receptor type PGRPs (PGRP-SA and
PGRP-LCx), a serine residue is found in this position. Therefore, we
made the substitution C168S as well as C168A as a control. Fig.
4A shows that both mutants
have lost enzymatic activity. This suggests that cysteine in position
168 is an active-site residue, most likely a zinc ligand, also
in PGRP and thereby required for amidase activity. However, the
capacity to bind peptidoglycan (Fig. 4B) is retained in both
mutants in consonance with the fact that receptor function is not
dependent on this residue being a metal ligand.
The Enzyme Does Not Show Antibacterial Activity--
The finding
that PGRP-SC1B degrades peptidoglycan prompted us to test if PGRP-SC1B
is bacteriolytic. PGRP-SC1B did not exhibit any antibacterial activity
against any of the five Gram-positive and three Gram-negative bacterial
strains employed in the plate assay (data not shown). Thus the PGN
layer of living bacteria is not susceptible to degradation by
PGPR-SC1B. As indicated in Table I, this inertness is partly due to the
presence of teichoic acid. We cannot, however, exclude the possibility
that PGRP-SC1B has antibacterial effects in synergy with other immune proteins.
The Digested Peptidoglycan Has Lost Immunogenicity--
As PGN is
a strong elicitor of immune responses in insects, we assayed the
influence of PGRP-SC1B on this elicitor activity. Challenge of
Drosophila mbn-2 cells with intact peptidoglycan (Fig.
5) results in a typical immune induction
pattern, namely an early response of cecropin A1 that persists over
time and a more delayed response of attacin and diptericin.
Interestingly, the inducibility of the antibacterial peptide genes was
drastically reduced when PGRP-SC1B-degraded peptidoglycan was added to
the cells. In contrast, the lysozyme-digested PGN remained
immunostimulatory to the mbn-2 cells because the immune genes tested
were induced to almost the same levels as when exposed to intact PGN.
None of the control treatments with protein alone had immune
stimulatory effect. We also tested conditions more similar to the
physiological situation in the fly in which both lysozymes and
PGRP-SC1B are constitutively expressed. The cells were practically
unable to respond to the double-digested peptidoglycan, implying an
efficient scavenging effect abolishing immunogenic PGN concentrations
in the insect.
What is the physiological role for PGRP-SC1B? We have shown
clearly that it degrades peptidoglycan and that the degradation products are less immunostimulatory, but not all peptidoglycans are
equally well degraded. Those from S. aureus and M. luteus contain L-Lys and a peptide cross-linking
bridge of a slightly different structure. B. megaterium and
most Gram-negative bacteria have PGN with
meso-diaminopimelic acid and direct cross-linking between
stem peptides. This PGN is the most resistant of those tested, but the
differences are small. We observed the largest difference between PGN
with and without teichoic acid. In nature other enzymes may initially
remove teichoic acid from the cell wall. In the physiological
situation, PGRP-SC1B also works in concert with lysozyme to make the
peptidoglycan structure more accessible for PGRP amidases to abolish
PGN immunogenicity. With lysozyme we also saw a difference in activity
against PGN with and without teichoic acid (not shown), confirming
earlier studies on lysozyme specificity (23).
The ancient connection to phage T7 lysozyme was noticed when the first
PGRPs were cloned (15), and these proteins were extensively tested for
amidase activity but were found to be negative. From the known crystal
structure of bacteriophage T7 lysozyme and a mutational analysis, five
amino acid residues have been shown to be required for enzymatic
activity (24). A sequence homology comparison between the
Drosophila PGRPs and T7 lysozyme with respect to these
residues shows that PGRP-SC1B has four of these five residues conserved
(Fig. 6). The fifth amino acid residue,
Lys-128 in T7 lysozyme, is replaced by a threonine in PGRP-SC1B;
however, in T7 lysozyme it was shown that a threonine can be
substituted for the lysine residue with retained but reduced activity.
If one allows for a threonine in this position one observes that five
other members of the Drosophila PGRP family have these five amino acid residues in common. These potentially enzymatically active
proteins are PGRP-SB1, PGRP-SB2, PGRP-SC1A, PGRP-SC2, and PGRP-LB. If
our hypothesis is correct, the expression pattern of PGRP-SB1 (9),
i.e. high inducibility in the fat body and secretion, makes
it a good candidate for being a modulator of immunity reactions in fat
body and hemocytes. The PGRP-SC1B gene is expressed
predominantly in gut cells; the localization of the protein is not
known, but a luminal export is likely. With such a localization, one
should not exclude a digestive function for this particular form.
PGRP-SC2 has a similar expression pattern as
PGRP-SC1B, but in addition it is induced in the fat
body.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(1).
Pre-formed humoral protein cascades can also be triggered by a single
microbe through strong binding to cell wall fragments (2). It is
therefore important that organisms have efficient mechanisms to remove
such immunogenic substances. This is necessary to be able to respond to
a second infection and to minimize overreaction to foreign material and
the risk of septic shock. Usually microbes are internalized for further
processing after being bound to cellular scavenger receptors (3,
4).
-glucan, were postulated to be recognized by pattern recognition receptors (PRRs). In
mammals, the Toll-like receptors (TLR) are involved in the response to
a variety of PAMP molecules, and in at least one case, a TLR has been
shown to be a true PRR (6, 7).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
His), PGRP-SC1B(
His), and the mutants PGRP-SC1B[C168S](
His) and PGRP-SC1B[C168A](
His). The vector pCoHYGRO encoding a hygromycin-B resistance gene was used for selection. Transfection was performed using the calcium phosphate method (19). Stable clones were selected in Schneider's
Drosophila medium (PAN Biotech) supplemented with
10% fetal calf serum and hygromycin-B (300 µg/ml). Transformed cells
were adapted to serum-free medium (HyClone Hy-Q-CCM3) and grown in
500-ml suspension cultures at 21 °C. At a concentration of 3 × 106 cells/ml, the cells were induced with CuSO4
(500 µM) and grown for 5 days. Cells were centrifuged
(3,000 × g for 20 min at 4 °C), and the medium was
assayed for protein. Proteins in the medium were precipitated with 70%
saturated ammonium sulfate overnight at 4 °C. The precipitate was
spun down (8,000 × g, 20 min, 4 °C), and the pellet
was dissolved in 30 ml of water. The suspension was dialyzed against 2 liters of binding buffer (5 mM imidazole, 600 mM NaCl, 20 mM Tris, pH 7.9) with two changes
of buffer over an 18-h period. Proteins were applied to the superloop
in a fast protein liquid chromatography system (Amersham Biosciences
AB) and passed through a nickel-charged HiTrap chelating HP column (Amersham Biosciences) at 1 ml/min. The column was washed with 60 mM imidazole, 600 mM NaCl, 20 mM
Tris, pH 7.9, and elution was performed with a linear gradient to 600 mM imidazole. The proteins eluted at ~20% elution buffer
and were essentially free from other proteins, which was confirmed with
a Coomassie Brilliant Blue-stained 15% SDS-polyacrylamide gel.
A540/(
time × [enzyme]).
12 was tested in a zone
inhibition assay on thin agar plates (21). Bacteria were grown to
mid-log phase, and 2 µl from a 100× bacterial dilution was mixed
with 8 ml of 47 °C yeast tryptone agar and spread on a Petri dish.
Samples (2 µl) of PGRP-SC1B (2-50 µg/ml) were loaded to 2-mm
diameter holes in the agar plate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 1.
Kinetics of S. aureus
peptidoglycan degradation. Insoluble peptidoglycan (1 mg/ml)
was incubated with PGRP-SC1B (A) or hen egg white lysozyme
(B) in PBS, pH 7.2. Protein concentrations were 0 ( ), 2 (
), 5 (
), 10 (
), and 20 (
) µg/ml. Enzymatic activity was
recorded as the optical clearance of the solution at 540 nm. Other
incubation conditions are described under "Experimental
Procedures." C, Coomassie-stained SDS-15%
polyacrylamide gel showing the purity of the affinity-purified
PGRP-SC1B protein used. Molecular mass marker proteins are shown
on the left.
Enzymatic activity of PGRP-SCIB to peptidoglycan from different
species
View larger version (11K):
[in a new window]
Fig. 2.
HPLC separation of S. aureus peptidoglycan fragments. Samples (360 µg) of insoluble peptidoglycan were incubated in ammonium bicarbonate
buffer (A), with PGRP-SC1B (B), and with
PGRP-SC1B plus lysostaphin (C). The samples were
centrifuged, and the supernatants were fractionated using a Brownlee
Spheri-5 column and an acetonitrile gradient (shown on the
right-hand side).
View larger version (24K):
[in a new window]
Fig. 3.
MALDI-TOF mass spectrum of peptidoglycan
cleavage products. Fraction I (in Fig. 2C) from the
HPLC separation of peptidoglycan cleaved with PGRP-SC1B plus
lysostaphin was analyzed using MALDI-TOF mass spectrometry. The
indicated masses at the peaks correspond to those of an S. aureus peptidoglycan stem peptide plus two to six glycine
residues. A schematic structure of S. aureus peptidoglycan
with PGRP-SC1B cleavage sites indicated is shown in the
inset. NAG, N-acetylglucosamine;
NAM, N-acetylmuramic acid.
Summary of peptidoglycan fragment mass mapping analysis
View larger version (37K):
[in a new window]
Fig. 4.
PGRP-SC1B mutants have lost the amidase
activity but bind peptidoglycan. A, insoluble
trichloroacetic acid-treated PGN from S. aureus (0.33 mg/ml)
was incubated with PGRP-SC1B ( ) or the mutants PGRP-SC1B[C168A]
(
) and PGRP-SC1B[C168S] (
) in 60 mM Tris, pH 8.0, 100 mM NaCl (
). Protein concentration was 10 µg/ml.
B, PGN (1 mg/ml) was incubated with PGRP-SC1B, wild
type (wt), and mutants, and PGRP-SA at 4 °C for 30 min.
Free PGRP (f) was separated from bound (b) by
centrifugation and analyzed on a 15% SDS-PAGE gel with Coomassie
staining. Marker (M) proteins are shown on the
left.
View larger version (57K):
[in a new window]
Fig. 5.
PGRP-SC1B-degraded peptidoglycan shows low
elicitor activity. Drosophila mbn-2 cells were
challenged with staphylococcal peptidoglycan (5 µg/ml). PGN (1 mg/ml)
had been preincubated at 25 °C for 10 h in PBS with PGRP-SC1B
(20 µg/ml), hen egg white lysozyme (20 µg/ml), or with a mixture of
PGRP-SC1B (20 µg/ml) and lysozyme (20 µg/ml). Total RNA was
isolated at 2, 6, or 24 h after challenge with PGN (times are
indicated above the lanes). Control cells were stimulated
with PBS, PGRP-SC1B, or lysozyme. RNA was isolated after 6 h and
subjected to Northern analysis. The filter was probed with
32P-labeled cDNA for cecropin A1, attacin, and
diptericin. The ribosomal protein 49 (rp49) was used as a loading
control showing that similar amounts of RNA were loaded in each
lane.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
[in a new window]
Fig. 6.
Multiple alignment of active-site residues of
PGRP family members. Protein sequences were aligned using the
multiple alignment function in the GeneJockey II program (Biosoft, UK).
Proteins with four active-site residues conserved from T7 lysozyme plus
a threonine residue in position 128 are shown in boldface
type. The GenBankTM or Swissprot accession
numbers for the sequences used are as follows: bacteriophage T7
lysozyme (P00806); PGRP from Trichoplusia ni (O76537),
B. mori (Q9XTN0), Calpodes ethlius (AF035445),
D. melanogaster (SA, Q9VYX7; SB1, Q9VV97; SB2, Q9VV96; SC1A,
Q9V3B7; SC1B, Q95SQ9; SC2, Q9V4X; SD, Q9VS97; LA, Q9VSV8; LB, Q9VGN3;
LCw, AAL89928; LCx, Q8T5Q2; LD, Q9GN97; LE, Q9VXN9), Mus
musculus (S, AF076482; L, AF149837), Bos taurus
(AAL87002), and Homo sapiens (S, AF076483; I , AY035376;
I
, AY035377; L, AF384856).
Interestingly, the two PGRPs with receptor functions, PGRP-SA and PGRP-LCx, both have a serine substitution in the position corresponding to the Cys-130 zinc ligand in T7 lysozyme. This modification removes one of the three potential zinc ligands and would make these proteins inactive enzymes. This substitution can also be found in other Drosophila PGRPs as well as in the B. mori PGRP being a receptor for the prophenol oxidase cascade (14). Also some of the mammalian forms have this substitution, making them candidates for being signaling PGRPs rather than enzymes. Furthermore, our analysis of PGRP-SC1B mutants shows Cys-130 to be essential for amidase activity but not for binding PGN. A cysteine residue in this position can thus serve as a marker for an effector type PGRP, and a non-cysteine residue could be an indication of a PGRP receptor function.
The concept of receptors and scavengers belonging to the same family of proteins contains an apparent logic. If PGRP-SC1B binds to the same motif in PGN as PGRP-SA and LCx, an obvious explanation for the scavenger effect of PGRP-SC1B is that it cleaves in the middle of the binding motif for the receptor PGRPs. This effect is not obtained with lysozyme because a different bond is cleaved. It has been suggested that the minimal PGN structure required to elicit an immune response in insects contains the two sugar moieties N-actetylglucosamine and N-acetylmuramic acid bound to the stem peptide Lys-Glu-DAP-Ala (25). This is consistent with our results, as such an active product is obtained by lysozyme cleaving the glycan strand but not by PGRP-SC1B, which instead hydrolyzes the bond between the peptide and the glycan strand. PGRP-SC1B is in this way efficiently destroying the PAMP properties of peptidoglycan.
In the mammalian system, recognition of peptidoglycan is thought to be mediated by TLR2 directly binding to PGN (6, 26). A receptor function for PGRP as in insects has not yet been demonstrated in mammals, but the PGN structure recognized seems to be similar if not identical in the two systems. Drosophila PGRP-SC1B is the first eukaryotic N-acetylmuramoyl-L-alanine amidase gene to be described. However, a human N-acetylmuramoyl-L-alanine amidase has been purified from serum and shown to reduce the immunostimulatory effect of peptidoglycan (27). It will be interesting to find out if the amidase activity in vertebrate sera also can be ascribed to PGRP proteins.
Studies of scavenger functions in Drosophila have
concentrated on cell-mediated responses performed by macrophage-like
cells, expressing scavenger receptors that engulf bacteria and
bacterial fragments (28). One of these cellular receptors is a member of the PGRP family (13). Our study now shows that a secreted PGRP
protein also can accomplish a scavenging task in innate immunity.
![]() |
FOOTNOTES |
---|
* This work was supported by the Swedish Natural Science Research Council Grant BU 4027-308 and European Union Grant QLK2-CT-2000-00336.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.
To whom correspondence should be addressed: Dept. of Microbiology,
Stockholm University, S-196 91 Stockholm, Sweden. Tel.: 46-8- 164160;
Fax: 46-8-6129552; E-mail: hakans@mibi.su.se.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M208900200
2 P. Mellroth and H. Steiner, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PAMP, pathogen-associated molecular pattern; PGRP, peptidoglycan recognition protein; PGN, peptidoglycan; TLR, Toll-like receptor; PRR, pattern-recognition receptor; PBS, phosphate-buffered saline; MALDI-TOF, matrix-assisted laser desorption ionization time of flight; HPLC, high pressure liquid chromatography.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Silverman, N.,
and Maniatis, T.
(2001)
Gene Dev.
15,
2321-2342 |
2. |
Takaki, Y.,
Seki, N.,
Kawabata Si, S.,
Iwanaga, S.,
and Muta, T.
(2002)
J. Biol. Chem.
277,
14281-14287 |
3. | Gough, P. J., and Gordon, S. (2000) Microbes Infect. 2, 305-311[CrossRef][Medline] [Order article via Infotrieve] |
4. | Aderem, A., and Underhill, D. M. (1999) Annu. Rev. Immunol. 17, 593-623[CrossRef][Medline] [Order article via Infotrieve] |
5. | Janeway, C. A. J. (1989) Cold Spring Harbor Symp. Quant. Biol. 54, 1-13[Medline] [Order article via Infotrieve] |
6. |
Iwaki, D.,
Mitsuzawa, H.,
Murakami, S.,
Sano, H.,
Konishi, M.,
Akino, T.,
and Kuroki, Y.
(2002)
J. Biol. Chem.
277,
24315-24320 |
7. | Sieling, P. A., and Modlin, R. L. (2002) Curr. Opin. Microbiol. 5, 70-75[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Lemaitre, B.,
Reichhart, J. M.,
and Hoffmann, J. A.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
14614-14619 |
9. |
Werner, T.,
Liu, G.,
Kang, D.,
Ekengren, S.,
Steiner, H.,
and Hultmark, D.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
13772-13777 |
10. | Michel, T., Reichhart, J. M., Hoffmann, J. A., and Royet, J. (2001) Nature 414, 756-759[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Choe, K. M.,
Werner, T.,
Stöven, S.,
Hultmark, D.,
and Anderson, K. V.
(2002)
Science
296,
359-362 |
12. | Gottar, M., Gobert, V., Michel, T., Belvin, M., Duyk, G., Hoffmann, J. A., Ferrandon, D., and Royet, J. (2002) Nature 416, 640-644[CrossRef][Medline] [Order article via Infotrieve] |
13. | Rämet, M., Manfruelli, P., Pearson, A., MatheyPrevot, B., and Ezekowitz, R. A. B. (2002) Nature 416, 644-648[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Yoshida, H.,
Kinoshita, K.,
and Ashida, M.
(1996)
J. Biol. Chem.
271,
13854-13860 |
15. |
Kang, D. W.,
Liu, G.,
Lundström, A.,
Gelius, E.,
and Steiner, H.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
10078-10082 |
16. | Valinger, Z., Ladesic, B., and Tomasic, J. (1982) Biochim. Biophys. Acta 701, 63-71[Medline] [Order article via Infotrieve] |
17. | Vanderwinkel, E., de Pauw, P., Philipp, D., Ten Have, J. P., and Bainter, K. (1995) Biochem. Mol. Med. 54, 26-32[CrossRef][Medline] [Order article via Infotrieve] |
18. | Shockman, G. D., Daneo-Moore, L., Kariyama, R., and Massidda, O. (1996) Microb. Drug Resist. 2, 95-98[Medline] [Order article via Infotrieve] |
19. | Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual , pp. 16.33-16.36, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
20. | Rosenthal, R. S., and Dziarski, R. (1994) Methods Enzymol. 235, 253-285[Medline] [Order article via Infotrieve] |
21. | Hultmark, D., Engström, A., Bennich, H., Kapur, R., and Boman, H. G. (1982) Eur. J. Biochem. 127, 207-217[Abstract] |
22. | Ghuysen, J.-M., Strominger, J. L., and Tipper, D. J. (1968) Compr. Biochem. 26, 53-104 |
23. | Ohta, K., Komatsuzawa, H., Sugai, M., and Suginaka, H. (1998) Microbiol. Immunol. 42, 231-235[Medline] [Order article via Infotrieve] |
24. | Cheng, X., Zhang, X., Pflugrath, J. W., and Studier, F. W. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4034-4038[Abstract] |
25. | Iketani, M., Nishimura, H., Akayama, K., Yamano, Y., and Morishima, I. (1999) Insect. Biochem. Mol. Biol. 29, 19-24[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Schwandner, R.,
Dziarski, R.,
Wesche, H.,
Rothe, M.,
and Kirschning, C. J.
(1999)
J. Biol. Chem.
274,
17406-17409 |
27. | Hoijer, M. A., Melief, M. J., Debets, R., and Hazenberg, M. P. (1997) Eur. Cytokine Netw. 8, 375-381[Medline] [Order article via Infotrieve] |
28. | Rämet, M., Pearson, A., Manfruelli, P., Li, X. H., Koziel, H., Gobel, V., Chung, E., Krieger, M., and Ezekowitz, R. A. B. (2001) Immunity 15, 1027-1038[Medline] [Order article via Infotrieve] |