| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 275, Issue 2, 937-941, January 14, 2000
,§, and
From the University Department of Paediatrics, John
Radcliffe Hospital, Oxford OX3 9DU, United Kingdom
,,, and
From ¶ M-Scan, Silwood Park, Ascot SL5 7PZ, United Kingdom and
the Department of Biochemistry, Imperial College,
London SW7 2AZ, United Kingdom
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
Louse-borne relapsing fever, caused by
Borrelia recurrentis, provides one of the best documented
examples of the causative role of tumor necrosis factor (TNF) in the
pathology of severe infection in humans. We have identified the
principal TNF-inducing factor of B. recurrentis as a
variable major lipoprotein (Vmp). Here we report the complete gene
sequence of Vmp, including its lipoprotein leader sequence. Using
metabolically labeled forms of the native Vmp we confirm that the TNF
inducing properties are associated with the lipid portion of the
molecule. Quadrupole orthogonal time of flight mass spectrometry
unequivocally locates the lipidic moiety at the
NH2-terminal cysteine of the native polypeptide, and
indicates the existence of three forms which are consistent with the
structures C16:0, C16:0, C16:0 glyceryl cysteine; C18:1, C16:0, C16:0
glyceryl cysteine; and C18:0, C16:0, C16:0 glyceryl cysteine. These
data provide the first direct evidence that the TNF inducing lipid
modification of native Borrelia lipoproteins is a
structural homologue of the murein lipoprotein of Escherichia coli.
Lipoproteins have been implicated as the principal mediators of
the inflammatory response that occurs in the human host during a
spirochetal infection. In vitro, it has been clearly
established that outer surface lipoproteins of spirochetes causing Lyme
disease and syphilis induce the production of inflammatory mediators
from a variety of cell types (1-11). Recently, we identified a
variable major lipoprotein
(Vmp)1 as the principal TNF
inducing stimulus of Borrelia recurrentis, the causative
agent of louse borne relapsing fever (12). The TNF-inducing stimulus of
B. recurrentis is of particular interest since this is the
only human infectious disease in which anti-TNF therapy has been
conclusively demonstrated to have a beneficial role (13).
Several studies have demonstrated that lipid modification is essential
for the potent biological activities of spirochetal lipoproteins (3,
12, 14, 15). The structure of the lipid modification of a native
spirochetal lipoprotein has not been elucidated but features of
lipoproteins from Borrelia burgdorferi provide
insight into a possible structure. Metabolic labeling studies with
palmitic acid and the presence of a consensus lipoprotein leader
sequence indicate a lipid modification similar to that of
Escherichia coli murein lipoprotein (16). However, indirect evidence from functional comparisons suggests that the lipid
modifications of B. burgdorferi and E. coli
lipoproteins may differ. First, B. burgdorferi lipoproteins
have greater potency than E. coli murein lipoprotein (2, 3,
5, 17, 18); and second, B. burgdorferi lipoproteins are
considerably more potent than corresponding lipopeptides synthesized
with the E. coli type of lipid modification having three
palmitic acid residues (Pam3Cys) (3).
The lack of direct evidence concerning the native lipid modifications
of spirochetal lipoproteins, and the established clinical importance of
TNF induction in louse borne relapsing fever, led us to investigate the
biochemical structure of the Vmp of B. recurrentis.
Cloning and Sequencing of vmp A1--
DNA isolated from B. recurrentis (19) was digested with HindIII and cloned
into pBluescript. The resulting library was screened with a partial vmp
A1 gene PCR product (12) that was radiolabeled using a random priming
kit (Megaprime kit, Amersham Pharmacia Biotech). Positive clones were
sequenced using an ABI 377 sequencer using BigDye terminator chemistry
(Perkin-Elmer). DNA sequences were assembled and analyzed using a
software package from the Wisconsin Genetics Computer Group, Madison, WI.
Purification of Native Vmp A1--
B. recurrentis
isolate A1 was grown in BSK II medium plus 6% rabbit serum. Cells from
a 1-liter culture were harvested at late logarithmic phase and washed
twice with HEPES-buffered saline (HBS; 25 mM HEPES, pH 7.5, 150 mM NaCl) and sonicated in 1 ml of HBS. The lysate was
centrifuged for 15 min at 10,000 × g, and the pellet
was extracted twice with HBS and once with methanol:chloroform:water (8:4:3). The pellet was solubilized with 0.1% SDS in HBS, and further
purified and desalted by reverse phase (RP)-HPLC (12). This preparation
was of greater than 95% purity.
Assay of TNF Induction--
To assay TNF induction we used the
human monocyte cell line MonoMac6 (MM6, German Collection of
Micro-organisms and Cell cultures) (20). Cells were grown in RPMI 1640 medium containing 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium
pyruvate, 1 × non-essential amino acids, 9 µg/ml insulin, 1 mM oxaloacetic acid, and 10% fetal calf serum. Prior to
assay cells were plated out in 96-well flat bottom plates at 2 × 105 cells/ml (100 µl/well) in the presence of 100 ng/ml
phorbol 12-myristate 13-acetate and incubated for 1 h. Test
stimuli were then added to the cells for 16 h and supernatants
were assayed for TNF by an enzyme-linked immunosorbent assay as
described previously (21).
Purification of Native TNF Inducing Lipopeptide Derived from Vmp
A1--
A 15-ml culture of B. recurrentis isolate A1 was
grown from early to late logarithmic phase in the presence of 4 mCi of
[9,10-3H]palmitic acid. The cells were harvested and
washed three times with HBS, sonicated, and extracted with HBS and
methanol:chloroform:water as above and then extracted with 0.1% SDS in
HBS. The SDS extract was further purified by preparative
electrophoresis (12). TNF inducing fractions containing Vmp were
concentrated by ultrafiltration and precipitated with 10%
trichloroacetic acid. The precipitated material was resuspended in 2 M urea, 0.1 M ammonium bicarbonate buffer, and
digested for 16 h at 37 °C with 2% (w/w) trypsin. The digest
was dried under vacuum, and redissolved in 0.2 ml of 0.1%
trifluoroacetic acid in water to which 1.2 ml of chloroform:methanol (2:1) was added. The chloroform:methanol phase was removed, dried under
vacuum, and redissolved in hexane, 2-propanol, 0.1% trifluoroacetic acid in water (3:4:0.25, solvent A). The recovery of radioactivity at
each stage was determined to be greater than 60% by scintillation counting. The sample was applied to a Nova-Pak silica column (3.9 × 150 mm, Waters, normal phase (NP)-HPLC) equilibrated with solvent A
at a flow rate of 0.5 ml/min. After washing for 15 min with solvent A
the column was developed with a 1% per min gradient of solvent B
(hexane, 2-propanol, 0.1% trifluoroacetic acid in water; 3:4:0.85) to
50%, and then to 100% solvent B over 15 min. Prior to assay for TNF
inducing activity 2% of each fraction was dried under vacuum and
redissolved in 0.1% SDS in HBS. To generate larger amounts of
lipopeptide for mass spectrometry the process was repeated for a
1-liter culture without palmitic acid metabolic labeling and the
preparative gel electrophoresis step was omitted. The estimated yield
of lipopeptide by amino acid and fatty acid analysis was in the low
picomole range.
Mass Spectrometry--
Matrix-assisted laser desorption mass
spectrometry was carried out on a Voyager STR mass spectrometer (PE
Biosystems) operating in reflectron mode with delayed extraction of
ions, using 2,5-dihydroxybenzoic acid as matrix.
Electrospray mass spectrometry was carried out on a quadrupole
orthogonal time of flight mass spectrometer (Q-TOF) instrument (Micromass), using direct flow injection for the intact protein data
acquisition, and nanospray on a Z-source for acquiring the lipopeptide
MS and collisionally activated decomposition MS/MS data. MS/MS data
were obtained using argon as collision gas and collision energies
between 40 and 60 eV (22-24).
Characterization of Vmp A1 Lipoprotein--
The protein component
of the lipoprotein was investigated by cloning and sequencing the gene
encoding Vmp A1. A genomic library was screened using a 715-base pair
polymerase chain reaction product that partially encodes vmp
A1 (12). An open reading frame encoding a protein of 363 amino
acids was identified in positive clones (Fig.
1A). This sequence contains a
consensus prokaryotic membrane lipoprotein leader sequence from residue
1 to 26. Thus, the putative mature protein contains 338 amino acids and
has a predicted molecular mass of 34,287 daltons. The complete
structure and organization of vmp A1 is not relevant to this
study and will be presented elsewhere.2
The mass of the native biologically active lipoprotein purified by
RP-HPLC was determined by mass spectrometry using a Q-TOF instrument
(Fig. 1B). The data transform to give a minimum molecular mass of 35,077 daltons for Vmp A1, that is 790 daltons higher than that
predicted from the gene sequence, thus confirming the presence of one
or more post-translational modifications on Vmp A1. Two other proteins
of masses 35,101 and 35,120 daltons were detected; these may be sodium
adducts or other forms of Vmp A1 having modifications of greater
molecular mass. The peak located at about 35,025 daltons may be a
cone-voltage induced loss from the molecular ions or a minor impurity.
Isolation of TNF Inducing Tryptic Peptide--
We have
demonstrated previously that digestion of Vmp A1 with proteinase K had
no effect on the TNF inducing activity (12). We also discovered that in
the process of generating tryptic peptides for NH2-terminal
sequencing the TNF inducing peptide was lost after RP-HPLC, despite the
digest retaining TNF inducing activity. Since a lipid is essential for
activity we reasoned that the active peptide should carry the lipid
modification and hence would be extremely hydrophobic. Using a palmitic
acid labeled preparation of purified Vmp A1 we established a
methodology to isolate the active tryptic peptide (Fig.
2A). After trypsin treatment
the radioactivity and TNF inducing activity could be partitioned into chloroform:methanol, 2:1, and fractionated by normal phase HPLC using
hexane, 2-propanol, 0.1% trifluoroacetic acid in water gradient. There
was a single peak of TNF inducing activity eluting at 45 to 47 min that
was coincident with the principal peak of radioactivity. Thirty-five
percent of total activity was recovered in fractions 44 to 48. This
result conclusively links the TNF inducing activity of the Vmp A1
lipoprotein with its lipid moiety. The absorbance profile at 214 nm for
this peak was biphasic in nature indicating that it contained at least
two components. For this reason fractions 44 to 48 from an unlabeled
preparation were screened by MALDI-TOF. The spectra of the most active
fractions 45 and 46 (Fig. 2B) showed a group of signals in
the 1500 dalton mass range that were absent from inactive flanking
fractions. Interpretation of these spectra revealed that both these TNF
inducing fractions contained three molecules that ionized to give
signals at m/z 1524, 1550, and a weaker 1552. The
other signals present in these spectra correspond to the +1 and +2
sodium adducts of these molecules. Interestingly, the relative
abundance of the three molecules differs in these fractions.
Characterization of TNF Inducing Lipopeptide by Mass
Spectrometry--
To determine the structure of the TNF inducing
molecules of masses 1524, 1550, and 1552 daltons tandem mass
spectrometry was performed on the Q-TOF instrument. First, it was
necessary to discover whether the anticipated lipopeptide would ionize
by electrospray, and the nanospray ES-MS spectrum of fraction 45 is
shown in Fig. 3A. Because of
the acidic (0.1% trifluoroacetic acid) solvent used, sodium adduct
formation is minimized, and as expected two groups of signals are
observed at m/z 1524 and 1550. The group of
signals at 1550 includes a weak 1552 signal. Collisionally activated
decomposition spectra were then derived in the MS/MS mode for the
signal at m/z 1524, producing the MS/MS spectrum shown in Fig. 3B. This spectrum is interpreted as follows:
clear COOH-terminal (y") ions are observed for the peptide
fragmentation at m/z 632, 518, 431, 374, 317, 260, and 147 identifying a sequence, XNSGGGIK for the
tryptic peptide, where X has an NH2-terminal (b-ion) mass of 893 daltons. From the vmp A1 sequence this
peptide occurs at the amino terminus of the protein, and the deduced
amino acid on the NH2-terminal side of the asparagine is
cysteine. Thus the 893 signal observed in Fig. 3B
corresponds in mass to a N-palmitoyl S-dipalmitoyl glyceryl cysteine residue. All of the
COOH-terminal ions in Fig. 3B, as well as the
NH2-terminal fragment ions at m/z
893, 1007, and 1094 give proof of localization of the mass increment on
the NH2-terminal cysteine residue. One palmitoyl residue is
on the amino-terminal group itself since a doubly charged ion, rather
than the singly charged 1524, would have been observed in this analysis
if the NH2 terminus had been free.
MS/MS analysis of the m/z 1550 group of signals
in Fig. 3A showed identical COOH-terminal (y") fragment
ions, together with signals equivalent to the m/z
893 ion at m/z 919 and 921 (data not shown).
These data are entirely consistent with the following lipid
modifications for these peptides C16:0, C16:0, C18:1 glyceryl cysteine,
and C16:0, C16:0, C18:0 glyceryl cysteine. Similar results were
obtained in the MS/MS analysis of fraction 46.
The discovery that the lipid moiety is essential for the potent
cytokine inducing and mitogenic properties of bacterial lipoproteins has renewed interest in their structure. Since the seminal paper of
Hantke and Braun (25) describing the structure of the lipid modification of E. coli murein lipoprotein several other
lipoproteins have been characterized (26-28). These recent
investigations have revealed significant differences in the structure
of the lipid modification of bacterial lipoproteins. Moreover, the
biological potency of bacterial lipoproteins varies considerably and
this variation results from the type of lipid modification present on
the lipoprotein (28, 29). It has been widely assumed that spirochetal
lipoproteins possess a lipid modification similar to E. coli
murein lipoprotein having three palmitic acid residues (Pam3Cys). Given
the importance ascribed to spirochetal lipoproteins in the pathogenesis
of disease we undertook a detailed structural characterization of a
native spirochetal lipoprotein. Our results clearly demonstrate that
the TNF inducing activity of B. recurrentis Vmp A1 resides
in the NH2-terminal lipid modification that is indeed
similar in structure to that of E. coli murein lipoprotein.
Analysis by mass spectrometry revealed that Vmp A1 is not a single
entity, but consists of three lipoproteins having identical peptide
sequence but differing lipid moieties. The first hint of heterogeneity
in Vmp A1 came from the Q-TOF analysis of the intact protein when
molecules of masses 35,077, 35,101, and 35,120 daltons were observed.
These molecules are of greater molecular weight than predicted from the
gene sequence, confirming the presence of one or more
post-translational modifications on Vmp A1. To elucidate the structure
of the post-translational lipid modification of Vmp A1 that confers
bioactivity, a methodology to isolate the TNF inducing active
lipopeptide was established utilizing a normal phase HPLC system. Three
lipopeptides of masses 1524, 1550, and 1552 daltons were present in the
active fractions. Characterization of these lipopeptides by MS/MS
showed unequivocally that the lipid moieties are located on the
NH2-terminal peptide. Comparison of the actual mass data on
the isolated lipopeptide with the gene sequence revealed the presence
of three types of lipid attachment of masses 789, 815, and 817 daltons.
These masses are consistent with the following fatty acid substitutions
to a NH2-terminal glyceryl cysteine: 1) C16:0, C16:0,
C16:0; 2) C16:0, C16:0, C18:1; 3) C16:0, C16:0, C18:0. Structures 1 and
2 have been identified previously in recombinant B. burgdorferi Osp A (rOsp A) expressed in E. coli
(30), and structure 2 also has been found in E. coli murein
lipoprotein (31). Structure 3 has not been reported previously. It is
noteworthy that C18:0 was detected in the fatty acid analysis of
lipoprotein extracts from other spirochetes but was not found in
E. coli murein lipoprotein (25, 32). In conclusion, analysis of the intact lipoprotein and active lipopeptide has revealed that Vmp
A1 has only one post-translational lipid modification occurring at the
NH2 terminus that confers bioactivity. Moreover our data
provide the first direct proof of localization of the triacyl moiety at
the NH2-terminal cysteine residue of a lipoprotein, with
one of the fatty acyl groups being on the amino-terminal group itself.
Clinical investigations of the Jarisch-Herxheimer reaction of louse
borne relapsing fever have provided perhaps the clearest example to
date of the causal role of TNF in the pathogenesis of severe infectious
disease in humans (13, 33). We have extended this investigation by
providing a comprehensive structural description of the native
TNF-inducing moiety of B. recurrentis isolate A1. A key
issue raised by this structural study is the mode of presentation of
the TNF-inducing lipid component of Vmp A1 to the responsive host cell.
Purified spirochetal lipoproteins and synthetic lipopeptides stimulate
cells through toll-like receptor 2 (34, 35) and signaling is
facilitated by CD14 (36-38). However, when Vmps are released upon
lysis of the spirochetes they are presumably still associated with
other membrane components, possibly forming micelles or larger
aggregates. Thus the active lipid component may remain embedded within
a hydrophobic structure and apparently unable to interact directly with
protein receptors on the responsive cell surface. Because of the
importance of B. recurrentis Vmp and other spirochetal
lipoproteins in disease pathogenesis a fuller understanding of how this
class of lipid toxin interacts with host cells is required.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 1.
Characterization of Vmp A1 lipoprotein.
A, the complete deduced amino acid sequence of Vmp A1 gives
a predicted molecular mass of 34,287 Da. The consensus prokaryotic
lipoprotein leader sequence from residue 1 to 26 is
underlined. These sequence data are available from
EMBL/GenBank/DDBJ under accession number AJ237608. B, the
actual mass of the intact lipoprotein was determined by electrospray
mass spectrometry using a Q-TOF instrument. The transformed data
presented gives a minimum molecular mass of 35,077 daltons for Vmp A1,
some 790 daltons greater than predicted. Two other proteins of masses
35,101 and 35,120 daltons were detected; these may be sodium adducts or
other forms of Vmp A1 having modifications of greater molecular
mass.
View larger version (26K):
[in a new window]
Fig. 2.
Isolation of TNF inducing tryptic peptide by
normal phase HPLC. A, fractions from the normal phase HPLC
separation of a tryptic digest of palmitic acid labeled Vmp A1 were
assayed for TNF inducing activity on MM6 cells at a dilution of 1/5000.
The coincidence of peaks of radioactivity (dpm) and UV
absorbance (A214) with bioactivity confirm that the TNF
inducing component of trypsin-digested Vmp A1 is a lipopeptide.
B, analysis of Fractions 45 (left panel) and 46 (right panel) from the normal phase HPLC separation of a
tryptic digest by matrix-assisted laser desorption ionization mass
spectrometry. Interpretation of these spectra reveals three TNF
inducing lipopeptides in these fractions of m/z
1524, 1550, and 1552. Other signals correspond to the +1 and +2 sodium
adducts of these ions. Numbers 1-8 indicate ions having
m/z of 1524, 1546, 1550, 1552, 1568, 1572, 1574, and 1594.
View larger version (15K):
[in a new window]
Fig. 3.
Characterization of TNF inducing lipopeptide
by mass spectrometry. A, analysis of Fraction 45 from normal
phase HPLC separation of tryptic digest by electrospray mass
spectrometry using a Q-TOF instrument. The spectrum contains two groups
of signals at m/z of 1524 and 1550. B,
collisionally activated decomposition spectra were derived in the MS/MS
mode for the ion of m/z 1524 present in Fraction
45. Interpretation of the spectrum reveals clear COOH-terminal
(y'') ions for the peptide fragmentation at
m/z 632, 518, 431, 374, 317, 260, and 147 identifying a sequence: XNSGGGIK, for the tryptic peptide
where X has an NH2-terminal mass of 893 daltons.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
FOOTNOTES |
|---|
* This work was supported by the Medical Research Council (United Kingdom) and the quadrupole orthogonal time of flight research was funded by the Wellcome Trust (to H. R. M. and A. D.).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 Paediatrics, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom. Tel.: 44-1865-221071; Fax: 44-1865-220479; E-mail: dominic.kwiatkowski@paediatrics.ox.ac.uk.
2 V. Vidal, I. G. Scragg, S. J. Cutler, and D. Kwiatkowski, submitted for publication.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: Vmp, variable major lipoprotein; Q-TOF, quadrupole orthogonal time of flight; MS, mass spectrometry; m/z, mass/charge; HPLC, high performance liquid chromatography; TNF, tumor necrosis factor.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Radolf, J. D., Norgard, M. V., Brandt, M. E., Isaacs, R. D., Thompson, P. A., and Beutler, B. (1991) J. Immunol. 147, 1968-1974[Abstract] |
| 2. |
Ma, Y.,
and Weis, J. J.
(1993)
Infect. Immun.
61,
3843-3853 |
| 3. | Radolf, J. D., Arndt, L. L., Akins, D. R., Curetty, L. L., Levi, M. E., Shen, Y., Davis, L. S., and Norgard, M. V. (1995) J. Immunol. 154, 2866-2877[Abstract] |
| 4. |
Ma, Y.,
Seiler, K. P.,
Tai, K. F.,
Yang, L.,
Woods, M.,
and Weis, J. J.
(1994)
Infect. Immun.
62,
3663-3671 |
| 5. |
Tai, K. F.,
Ma, Y.,
and Weis, J. J.
(1994)
Infect. Immun.
62,
520-528 |
| 6. | Morrison, T. B., Weis, J. H., and Weis, J. J. (1997) J. Immunol. 158, 4838-4845[Abstract] |
| 7. | Wooten, R. M., Modur, V. R., McIntyre, T. M., and Weis, J. J. (1996) J. Immunol. 157, 4584-4590[Abstract] |
| 8. | Sellati, T. J., Abrescia, L. D., Radolf, J. D., and Furie, M. B. (1996) Infect. Immun. 64, 3180-3187[Abstract] |
| 9. | Burns, M. J., Sellati, T. J., Teng, E. I., and Furie, M. B. (1997) Infect. Immun. 65, 1217-1222[Abstract] |
| 10. | Ebnet, K., Brown, K. D., Siebenlist, U. K., Simon, M. M., and Shaw, S. (1997) J. Immunol. 158, 3285-3292[Abstract] |
| 11. | Marietta, E. V., Weis, J. J., and Weis, J. H. (1997) J. Immunol. 159, 2840-2848[Abstract] |
| 12. | Vidal, V., Scragg, I. G., Cutler, S. J., Rockett, K. A., Fekade, D., Warrell, D. A., Wright, D. J., and Kwiatkowski, D. (1998) Nat. Med. 4, 1416-1420[CrossRef][Medline] [Order article via Infotrieve] |
| 13. |
Fekade, D.,
Knox, K.,
Hussein, K.,
Melka, A.,
Lalloo, D. G.,
Coxon, R. E.,
and Warrell, D. A.
(1996)
N. Engl. J. Med.
335,
311-315 |
| 14. |
Akins, D. R.,
Purcell, B. K.,
Mitra, M. M.,
Norgard, M. V.,
and Radolf, J. D.
(1993)
Infect. Immun.
61,
1202-1210 |
| 15. |
Weis, J. J.,
Ma, Y.,
and Erdile, L. F.
(1994)
Infect. Immun.
62,
4632-4636 |
| 16. |
Brandt, M. E.,
Riley, B. S.,
Radolf, J. D.,
and Norgard, M. V.
(1990)
Infect. Immun.
58,
983-991 |
| 17. | Bessler, W. G., Cox, M., Lex, A., Suhr, B., Wiesmuller, K. H., and Jung, G. (1985) J. Immunol. 135, 1900-1905[Abstract] |
| 18. | Lex, A., Wiesmuller, K. H., Jung, G., and Bessler, W. G. (1986) J. Immunol. 137, 2676-2681[Abstract] |
| 19. |
Barbour, A. G.,
and Garon, C. F.
(1987)
Science
237,
409-411 |
| 20. | Ziegler-Heitbrock, H. W., Thiel, E., Futterer, A., Herzog, V., Wirtz, A., and Riethmuller, G. (1988) Int. J. Cancer 41, 456-461[Medline] [Order article via Infotrieve] |
| 21. |
Allan, R. J.,
Rowe, A.,
and Kwiatkowski, D.
(1993)
Infect. Immun.
61,
4772-4776 |
| 22. | Morris, H. R., Paxton, T., Dell, A., Langhorne, J., Berg, M., Bordoli, R. S., Hoyes, J., and Bateman, R. H. (1996) Rapid Commun. Mass Spectrom. 10, 889-896[CrossRef][Medline] [Order article via Infotrieve] |
| 23. | Morris, H. R., Paxton, T., Panico, M., McDowell, R., and Dell, A. (1998) in Mass Spectrometry of Biological Materials (Larsen, B. A. , and McEwen, C., eds) , Marcel Dekker, New York |
| 24. | Billker, O., Lindo, V., Panico, M., Etienne, A. E., Paxton, T., Dell, A., Rogers, M., Sinden, R. E., and Morris, H. R. (1998) Nature 392, 289-292[CrossRef][Medline] [Order article via Infotrieve] |
| 25. | Hantke, K., and Braun, V. (1973) Eur. J. Biochem. 34, 284-296[Medline] [Order article via Infotrieve] |
| 26. | Weyer, K. A., Schafer, W., Lottspeich, F., and Michel, H. (1987) Biochemistry 26, 2909-2914[CrossRef] |
| 27. |
Muhlradt, P. F.,
Kiess, M.,
Meyer, H.,
Sussmuth, R.,
and Jung, G.
(1997)
J. Exp. Med.
185,
1951-1958 |
| 28. |
Muhlradt, P. F.,
Kiess, M.,
Meyer, H.,
Sussmuth, R.,
and Jung, G.
(1998)
Infect. Immun.
66,
4804-4810 |
| 29. | Metzger, J. W., Beck-Sickinger, A. G., Loleit, M., Eckert, M., Bessler, W. G., and Jung, G. (1995) J. Pept. Sci. 1, 184-190[CrossRef][Medline] [Order article via Infotrieve] |
| 30. | Bouchon, B., Klein, M., Bischoff, R., Van Dorsselaer, A., and Roitsch, C. (1997) Anal. Biochem. 246, 52-61[CrossRef][Medline] [Order article via Infotrieve] |
| 31. | Pittenauer, E., Quintela, J. C., Schmid, E. R., Allmaier, G., Paulus, G., and de Pedro, M. A. (1995) J. Am. Soc. Mass Spectrom. 6, 892-905[CrossRef] |
| 32. |
Belisle, J. T.,
Brandt, M. E.,
Radolf, J. D.,
and Norgard, M. V.
(1994)
J. Bacteriol.
176,
2151-2157 |
| 33. |
Negussie, Y.,
Remick, D. G.,
DeForge, L. E.,
Kunkel, S. L.,
Eynon, A.,
and Griffin, G. E.
(1992)
J. Exp. Med.
175,
1207-1212 |
| 34. |
Brightbill, H. D.,
Libraty, D. H.,
Krutzik, S. R.,
Yang, R. B.,
Belisle, J. T.,
Bleharski, J. R.,
Maitland, M.,
Norgard, M. V.,
Plevy, S. E.,
Smale, S. T.,
Brennan, P. J.,
Bloom, B. R.,
Godowski, P. J.,
and Modlin, R. L.
(1999)
Science
285,
732-736 |
| 35. |
Hirschfeld, M.,
Kirschning, C. J.,
Schwandner, R.,
Wesche, H.,
Weis, J. H.,
Wooten, R. M.,
and Weis, J. J.
(1999)
J. Immunol.
163,
2382-2386 |
| 36. |
Wooten, R. M.,
Morrison, T. B.,
Weis, J. H.,
Wright, S. D.,
Thieringer, R.,
and Weis, J. J.
(1998)
J. Immunol.
160,
5485-5492 |
| 37. |
Sellati, T. J.,
Bouis, D. A.,
Kitchens, R. L.,
Darveau, R. P.,
Pugin, J.,
Ulevitch, R. J.,
Gangloff, S. C.,
Goyert, S. M.,
Norgard, M. V.,
and Radolf, J. D.
(1998)
J. Immunol.
160,
5455-5464 |
| 38. |
Giambartolomei, G. H.,
Dennis, V. A.,
Lasater, B. L.,
and Philipp, M. T.
(1999)
Infect. Immun.
67,
140-147 |
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. Asai, M. Hashimoto, H. M. Fletcher, K. Miyake, S. Akira, and T. Ogawa Lipopolysaccharide Preparation Extracted from Porphyromonas gingivalis Lipoprotein-Deficient Mutant Shows a Marked Decrease in Toll-Like Receptor 2-Mediated Signaling Infect. Immun., April 1, 2005; 73(4): 2157 - 2163. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Venema, H. Tjalsma, J. M. van Dijl, A. de Jong, K. Leenhouts, G. Buist, and G. Venema Active Lipoprotein Precursors in the Gram-positive Eubacterium Lactococcus lactis J. Biol. Chem., April 18, 2003; 278(17): 14739 - 14746. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Masuda, S.-i. Matsuyama, and H. Tokuda Elucidation of the function of lipoprotein-sorting signals that determine membrane localization PNAS, May 28, 2002; 99(11): 7390 - 7395. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. A. Udalova, V. Vidal, I. G. Scragg, and D. Kwiatkowski Direct Evidence for Involvement of NF-kappa B in Transcriptional Activation of Tumor Necrosis Factor by a Spirochetal Lipoprotein Infect. Immun., September 1, 2000; 68(9): 5447 - 5449. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
