From the TB Research Group, Department of Bacterial
Diseases, Veterinary Laboratories Agency (Weybridge), New Haw,
Addlestone, Surrey KT15 3NB, United Kingdom, the ¶ Department of
Biological Sciences, Imperial College of Science Technology and
Medicine, London SW7 2AY, United Kingdom, ** M-Scan Mass
Spectrometry Research and Training Centre, Silwood Park, Ascot SL5 7PZ,
United Kingdom, the
Service de
Microbiologie, Hopital Saint Louis, 1 Avenue Claude Vellefaux, 75475 Paris, Cedex 10, France, and the §§ Centre for
Molecular Microbiology and Infection, Imperial College of Science
Technology and Medicine, London SW7 2AY, United Kingdom
Received for publication, August 5, 2002, and in revised form, January 6, 2003
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mycobacterium tuberculosis and
Mycobacterium bovis, the causative agents of human and
bovine tuberculosis, have been reported to express a range of surface
and secreted glycoproteins, although only one of these has been
subjected to detailed structural analysis. We describe the use of a
genetic system, in conjunction with lectin binding, to characterize the
points of attachment of carbohydrate moieties to the polypeptide
backbone of a second mycobacterial glycoprotein, antigen MPB83 from
M. bovis. Biochemical and structural analysis of the native
MPB83 protein and derived peptides demonstrated the presence of 3 mannose units attached to two threonine residues. Mannose residues were
joined by a (1 Protein glycosylation is ubiquitous in eukaryotes and the diverse
array of carbohydrate moieties that can be fashioned to a polypeptide
backbone makes glycoproteins ideal molecules to allow specific
interactions with other molecules. In contrast, the number of reports
of this post-translational modification by prokaryotes is comparatively
low. Examples of eubacterial glycoproteins identified to date include
cell surface or secreted proteins of pathogens that have antigenic
properties and play a role in host pathogen interaction (1-6).
Glycosylation of pilin has been postulated to enhance the adherence of
Neisseria meningitidis to endothelial cells (2), whereas
N-glycosylation of the platelet aggregation-associated protein of Staphylococcus sanguis may promote colonization
of the endocardium (7, 8). Removal of a sero-specific glycosyl moiety
from the flagellin of Campylobacter jejuni has been reported to alter the O antigenicity of the organism (9, 10). Similarly, Romain
et al. (11) demonstrated that removal of covalently bound mannose from the Mycobacterium tuberculosis antigen MPT32
reduced by 10-fold its ability to elicit a delayed-type
hypersensitivity reaction in guinea pigs immunized with
Mycobacterium bovis BCG.
M. tuberculosis and M. bovis are closely related
members of the "M. tuberculosis complex" (MTb complex)
that secrete a series of immunodominant antigens that have been
reported to be glycosylated, on the basis of their ability to bind
lectins such as concanavalin A
(ConA)1 (12-15). Structural
confirmation of protein glycosylation by mycobacteria was provided by
detailed chemical compositional analysis of MPT32, a 45/47-kDa secreted
antigen of M. tuberculosis (15, 16). Mannose, mannobiose,
and mannotriose substituents were found O-linked to four
threonine residues on the protein backbone. Also, site-directed mutagenesis of the 19-kDa lipoprotein antigen of M. tuberculosis implicated threonine residues in ConA binding,
consistent with the observed O-linked glycosylation
(17).
On the basis of monoclonal antibody-binding studies, Fifis
et al. (13) proposed that the 25/23-kDa secreted
antigen of M. bovis was a glycosylated form of the
major M. bovis antigen MPB70. They suggested that the
increases in molecular weight, relative to the 22,000 MPB70,
were attributable to glycosylation with mannose, as treatment of these
antigens with M. bovis expresses high levels of both MPB70 and MPB83 and
these proteins are strongly recognized by the immune system during M. bovis infection in cattle and badgers (19, 20). Although the proteins are expressed only at low levels in M. tuberculosis when grown in vitro, they are highly
immunogenic during infection with live bacteria in mice (18) and man
(21). In addition, vaccination of mice with a plasmid encoding MPB83
has been shown to confer significant protection against challenge with
M. bovis (22). Thus, given its strong recognition by the
immune system, further characterization of MPB83 is required. Open
reading frames with homology to MPB70 and MPB83 can be
identified in genome sequences from Streptomyces coelicolor
and other microbes, but a physiological function has yet to be ascribed
to these proteins.
The aims of this study were to explore the glycosylation status of
MPB83 and MPB70, to map specific sites for glycosylation within
the molecules, and to elucidate the chemical structure of the
carbohydrate moiety. Using a combination of genetic and biochemical
approaches we have demonstrated that MPB83 but not MPB70 is
glycosylated, and that glycosylation occurs via attachment of short
(1 Bacterial Strains and Culture Conditions--
Escherichia
coli DH5 Oligonucleotides and PCR Amplifications--
The genes encoding
MPB70, MPB83, and those regions of MPB83 used for deletion mapping,
were amplified by PCR using the oligonucleotides described in Table
I. Oligonucleotides were supplied by
Oswel, Southampton, United Kingdom. PCR amplifications were carried out in a DNA thermal cycler (Biometra) using 0.5 units of cloned
Pfu DNA polymerase (Stratagene). PCR reactions were
performed in 50-µl volumes containing: 50 mM-KCl, 10 mM Tris-HCl (pH 8.8), 0.01% (w/v) gelatin, 5 mM MgCl2, 200 µM of each
dinucleotide triphosphate (dNTP), and 20 pmol of each oligonucleotide
primer overlaid with mineral oil. The parameters for amplification were
one denaturation cycle at 96 °C for 2 min followed by 30 cycles of
denaturation at 96 °C for 1.5 min, hybridization at 56 °C for 1.5 min, and elongation at 72 °C for 1.5 min. A final extension at
72 °C for 5 min was also performed.
Construction of PhoA Hybrids--
mpb70 and
mpb83 were amplified from the cosmid pA3, described
previously (18), using the oligonucleotide pairs 1,2 and 3,4, respectively (Table I). Amplified fragments were purified using Wizard
PCR Prep columns (Promega) and digested with the restriction endonucleases BamHI and KpnI (Promega). Digested
fragments were cloned into BamHI/KpnI-digested
p19pro-PhoA (Fig. 1), a modified version of pSMT3(19-PhoA) (17)
containing a leaderless E. coli alkaline phosphatase
(phoA) gene, using standard procedures (Sambrook et
al. (23)). p19proSP-PhoA contains the promoter and signal peptide
of the 19 kDa of M. tuberculosis in-frame with the PhoA gene
in 19pro-PhoA and is used as a positive control for anti-alkaline phosphatase antibody activity and as a negative control for ConA binding. Truncated versions of mpb83 were amplified using
oligonucleotide 83PF with the corresponding reverse complementary
oligonucleotide and cloned into p19pro-PhoA as described above.
Recombinants with functional alkaline phosphatase activity were
selected after transformation into competent E. coli DH5 Site-directed Mutagenesis--
Complementary forward and reverse
oligonucleotide primers 9 and 10 (Table I) were used in conjunction
with the reverse complementary and forward oligonucleotide primers, 4 and 3 (Table I), for mpb83, respectively. Following
purification, the two products, having an overlap of 30 bp, were
combined and amplified by PCR using oligonucleotide primers 3 and 8 to
give a fragment of mpb83 incorporating the desired mutation.
All cloned DNA fragments were DNA sequenced using the TaqFS
dideoxy terminator cycle sequencing kit in conjunction with a 373A
Automated DNA Sequencer (Applied Biosystems Inc.). Each nucleotide was
sequenced a minimum of three times from both strands. DNA sequence data
analysis and protein sequence comparison were performed using the
DNASTAR software package (DNASTAR Inc.).
SDS-PAGE and Western Blotting--
For SDS-PAGE, recombinant
M. smegmatis was grown in 40 ml of MADC-Tween 80 at
37 °C, 225 rpm, for 48 h. The mycobacteria were pelleted by
centrifugation (4,000 rpm for 10 min, Sigma 3K10, rotor 11133), washed
twice with 40 ml of PBS (pH 7.0), and resuspended in 3 ml of PBS (pH
7.0). The resulting suspensions were sonicated on ice 10 times for
60 s with 90-s intervals, using a Soniprep 150 (MSE Ltd) equipped
with a 1-cm probe. Sonicated extracts were centrifuged at 14,000 rpm
for 30 min at 4 °C, the supernatant was isolated and filtered
through a 0.22-µm filter. Total protein concentrations of sonicated
extracts were determined using the bicinchoninic acid (BCA) protein
assay (Pierce and Warriner). 10 µg of extracts were solubilized by
heating at 100 °C for 3 min in an equal volume of sample loading
buffer {5 mM Tris-HCl, pH 6.8, 5% (v/v)
2-mercaptoethanol, 2% (w/v) sodium dodecyl sulfate, 10% (v/v)
glycerol, 0.002% (w/v) bromphenol blue). Samples were fractionated by
electrophoresis through 12.5% acrylamide (w/v) SDS-polyacrylamide gels
using a discontinuous Tris-HCl buffer system as described by Laemmli
(25) and transferred to nitrocellulose by electroblotting as described
previously (26). Rainbow protein markers (Amersham Biosciences)
were run as molecular mass standards.
ConA Analysis and Antibody Detection--
ConA binding was
determined by first blocking the nitrocellulose membranes with 20 ml of
5% bovine serum albumin (Sigma) in PBS with 0.1% Tween 20 (Bio-Rad)
for 1 h at room temperature. After washing the membranes three
times for 10 min with PBS containing 0.5% Tween 20, they were
incubated with 20 ml of peroxidase-conjugated ConA at 1 mg/ml in PBS,
0.5% Tween 20 without bovine serum albumin for 1 h at room
temperature. The membranes were then washed twice for 10 min with PBS
containing 0.5% Tween 20 and finally for 10 min with PBS only. The
substrate solution for visualizing peroxidase activity was prepared by
dissolving 30 mg of 4-chloronaphthol (Sigma) in 12 ml of methanol
followed by addition of 96 ml of PBS and 17 µl of 30% (v/v) hydrogen
peroxide. For detecting PhoA protein expression or MPB83, the membranes
were blocked with 0.01 M Tris-buffered saline (TBS)
containing 3% (w/v) skimmed milk powder (TBSM), and then incubated for
1 h at room temperature. Following three 10-min washes with TBS
containing 0.05% (v/v) Tween 20 (TBST) the nitrocellulose membranes
were incubated for 2 h at 37 °C with monoclonal antibody raised
against bacterial alkaline phosphatase (Sigma) or MBS43 (VLA
Weybridge), both at a dilution of 1 in 8000 in 30 ml of TBST containing
3% (w/v) skimmed milk (TBSTM). Membranes were then washed in TBST as
described previously and incubated for 2 h at 37 °C with
alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma) diluted 1 in 8000 in TBSTM. After three further 10-min washes at room temperature
with TBS bound alkaline phosphatase-conjugated antibody was detected
with SigmaFast tablets (Sigma) dissolved in 15 ml of water.
Purification of Native MPB83--
Native MPB83 was purified from
a M. bovis culture supernatant. M. bovis AN5 was
grown as a surface pellicle on Bureau of Animal Industry medium (14 g
of L-asparagine, 1.5 g of dipotassium hydrogen phosphate, 0.74 g of sodium citrate, 1.5 g of magnesium
sulfate, 0.3 g of ferric sulfate, 0.08 g of zinc sulfate,
0.008 g of manganese chloride, 0.00138 g of cobaltous chloride, 10 g of glucose, 100 g of glycerol/liter) for 10 weeks at 37 °C.
The culture was filtered through a stainless steel mesh strainer and
then passed through a 0.2-µm VacuCap bottle top filter (Gelman
Sciences). The culture supernatant was filtered a second time though a
0.2-µm VacuCap bottle top filter to ensure sterility.
The culture supernatant was diluted 3-fold in preparative anion
exchange chromatography (AEC) loading buffer (20 mM Tris, pH 8, 0.05% (v/v) Igepal CA-630) and applied to a XK50/20 column (Amersham Biosciences) containing 200 ml of Fast Flow DEAE anion exchange medium (Amersham Biosciences) using a fast protein liquid chromatography instrument. Adsorbed protein was eluted from the column
by applying a linear increasing gradient of 0-250 mM
sodium chloride over a 1-liter volume. Fractions containing MPB83 were identified by separation on SDS-PAGE gels, transfer to nitrocellulose, and probing with the MPB83-specific antibody MBS43 using a Western blotting procedure. The purity of each MPB83 containing fraction was
assessed by total protein analysis of SDS-PAGE gels using a silver
stain procedure. Fractions containing MPB83 were pooled and
concentrated using a 50-ml stirred cell concentrator containing a YM10
membrane (10,000 molecular weight cut-off) (Millipore UK).
The enriched MPB83 containing material was further purified by high
resolution AEC using a MonoQ HR10/10 column. The sample was diluted
5-fold in AEC loading buffer (20 mM Tris, pH 8) and applied
to a MonoQ HR10/10 column using a fast protein liquid chromatography
instrument. The adsorbed material was eluted from the column by
applying a linear increasing gradient of 0-250 mM sodium
chloride over a 100-ml volume. Fractions containing MPB83 were
identified as described above. The pooled fractions were concentrated
by ultrafiltration using a CentriPrep 10 (10,000 molecular weight
cut-off) concentrator unit (Millipore UK) centrifuged at 2500 × g (MSE Mistrial 2000 centrifuge, Sanyo Gallenkamp).
The AEC purified material was diluted 5-fold in hydrophobic interaction
chromatography loading buffer (20 mM sodium phosphate, pH
7, 1 M ammonium sulfate) and applied to a HR5/5
phenyl-Superose hydrophobic interaction chromatography column (Amersham
Biosciences) using a fast protein liquid chromatography instrument.
Adsorbed material was eluted by applying a linear, decreasing gradient, of 1-0 M ammonium sulfate over a volume of 80 ml.
Fractions containing MPB83 were identified as described above. The
pooled fractions were concentrated by ultrafiltration using Centricon
10 (10,000 Mr cut-off) concentrator units
(Millipore UK) centrifuged at 4000 × g (MSE Mistral
2000 centrifuge, Sanyo Gallenkamp). The concentrated material was
dialyzed against 4× 2 liters of PBS using a 3,500 Mr cut-off dialysis cassette (Perbio Science).
Dialysis was performed at 4 °C and a minimum of 2 h was allowed
between each change of dialysis buffer. The dialyzed material was
filtered through a 0.2-µm syringe filter and the protein
concentration was determined using the BCA protein estimation assay.
Chemical Deglycosylation of Native MPB83--
Purified native
MPB83 was subjected to a chemical deglycosylation procedure using a
GlycoFree deglycosylation kit (Glyko Inc.). This method employs
the use of anhydrous trifluoromethanesulfonic acid, which cleaves
N- and O-linked glycans nonselectively from glycoproteins while leaving the primary structure of the protein intact
(27). Purified native MPB83 was initially desalted by dialysis against
4× 2-liter volumes of water using a 3,500 Mr cut-off dialysis cassette (Perbio Science). The desalted sample was
then frozen at
Briefly, a reaction vial, containing 0.5 mg of lyophilized material,
was placed in a dry ice/ethanol bath and 50 µl of
trifluoromethanesulfonic acid was slowly added. The vial was then
incubated at Nanoelectrospray Mass Spectrometry--
Intact MPB83 and
products of tryptic digestion were analyzed by ES-MS using a nanospray
ion source on a hybrid quadrupole orthogonal acceleration time of
flight (Q-TOF) mass spectrometer (Micromass UK) (28, 29). Signals
attributable to glycopeptides were passed separately for collisionally
activated decomposition tandem MS experiments (CAD MS/MS) into a
collision cell filled with argon gas, using collision energies from 10 to 40 eV as appropriate (28, 29).
Carbohydrate Analysis--
The modifying sugars of purified
MPB83 were identified by first hydrolyzing the protein (50 µg) with 4 M trifluoroacetic acid at 95 °C for 4 h to release
the sugars. The hydrolysis reaction was dried under vacuum at 40 °C
in a SCV100H SpeedVac apparatus (Stratech Scientific) and then
resuspended in 100 µl of water. A sample of 10 µl was then analyzed
on a CarboPak PA1 anion exchange column (Dionex Corp.) using a 625LC
high performance liquid chromatography instrument (Waters) equipped
with a pulsed amperometric detector (Dionex Corp.).
Sugars were eluted from the column with 16 mM sodium
hydroxide at a flow rate of 1 ml/min. Sugars were identified by
comparing the column retention time with that of sugar standards
prepared at known concentrations run under identical conditions (all
sugar standards were supplied by Sigma).
FAB-MS and GC-MS Linkage Analysis--
Reductive elimination,
permethylation, preparation of partially methylated alditol acetates,
and acquisition of FAB-MS and GC-MS data were performed as described
previously (30, 31).
Recombinant MPB83 but Not MPB70 Is Glycosylated by
M. smegmatis--
A recombinant mycobacterial expression system
(17, 32) was used to determine whether MPB70 and MPB83 were
glycosylated by mycobacteria. The genes encoding these proteins were
cloned in-frame with E. coli alkaline phosphatase (PhoA)
lacking its own signal sequence in the mycobacterial shuttle vector
19pro-PhoA (Fig. 1) as described under
"Experimental Procedures." PhoA was used as a hybrid partner to
allow the level of expression of the different constructs to be
monitored and to verify that the fusion proteins were being exported.
The fusion proteins encoded by these constructs expressed functionally
active alkaline phosphatase in both E. coli DH5
Glycosylation of the fusion proteins was monitored by their ability to
bind ConA following fractionation of recombinant cell extracts by
denaturing SDS-PAGE and electrotransfer to nitrocellulose membranes. In
these experiments only MPB83-PhoA expressed by M. smegmatis
mc2155 was recognized by ConA (Fig.
2A, lane 5). ConA
did not bind MPB83-PhoA expressed by E. coli DH5
Immunoblotting with antibodies against PhoA also revealed an increase
in the apparent molecular weight of MPB83-PhoA expressed by M. smegmatis mc2155 compared with that of the protein
expressed by E. coli DH5 Identification of a Glycosylation Motif in MPB83 Using PhoA Hybrid
Proteins and Site-directed Mutagenesis--
To identify regions of
MPB83 involved in ConA binding, in-frame hybrid proteins were produced
in which increasing amino-terminal regions of MPB83 were fused to PhoA
as described under "Experimental Procedures." This approach was
used to generate the set of in-frame fusion proteins listed in Table
II.
Analysis of ConA binding to these recombinant fusion proteins expressed
in M. smegmatis mc2155 demonstrated that
proteins containing the first 36 amino acid residues (aa) of MPB83
failed to bind ConA (Fig. 3A,
lane 3). However, when the number of amino acids of MPB83
fused to PhoA was increased to 56 or 63, ConA binding was restored
(Fig. 3A, lanes 4 and 5). Probing the
corresponding blot with antibody to PhoA again demonstrated a similar
amount of hybrid protein in each of the extracts (Fig. 3B),
indicating that differences in ConA binding were not a consequence of
different expression levels of the hybrid proteins. ConA did not bind
any of the fusion proteins when expressed by E. coli (data
not shown).
The amino acid sequence of MPB83 between positions 36 and 56 (37PKPATSPAAPVTTAAMADPA56) contains four
potential O-mannosylation sites (Thr41,
Ser42, Thr48, and Thr49). Alignment
of the amino acid sequences of MPB83 and MPB70 revealed the presence of
a cluster of 10 amino acids (residues 46-55) present in MPB83 but
absent from MPB70 (highlighted in Fig.
4). This 10-amino acid region showed an
overall similarity with the four chemically characterized glycopeptides
of MPT32 (16) in having a threonine cluster within a region flanked by
proline residues. In light of this similarity the effect of mutagenesis
of the two residues (Thr48, Thr49) on ConA
binding of p63aaPhoA was investigated. The substitution of threonine to
valine was chosen, as the resultant modification, exchange of a methyl
group for a hydroxyl group, was the most conservative change with
respect to molecular weight.
Sonicated extracts of M. smegmatis mc2155
expressing the mutated and native 63aaPhoA fusion protein were resolved
by SDS-PAGE and transferred to nitrocellulose membranes. Incubation of
the membrane with peroxidase-labeled ConA revealed that substitution of
the Thr48 and Thr49 residues of MPB83 with
valine resulted in the complete ablation of ConA binding (Fig.
3C, lane 2). The mutated 63aaPhoA fusion also had
a lower apparent molecular weight than that of the native fusion
protein (Fig. 3D, lanes 1 and 2). In
contrast, the threonine substitutions had little effect on the apparent
molecular weight of the fusion construct expressed in E. coli (data not shown).
Purification of MPB83 from M. bovis AN5 Culture
Supernatant--
25- and 23-kDa forms of MPB83 were recognized by the
monoclonal antibody MBS43 following the separation of the M. bovis culture supernatant by SDS-PAGE, as shown in Fig.
5B, lane 1. Both
molecular weight forms of MPB83 were purified using a chromatography
strategy comprising two AEC steps followed by a hydrophobic
interaction chromatography step, as described under "Experimental
Procedures." The yield of the 23- and 25-kDa forms of MPB83 from a
2-liter culture supernatant were 5 mg and 200 µg, respectively,
demonstrating the predominance of the 23-kDa form of MPB83 in the
culture supernatant. The purity of the 23-kDa form is shown in Fig. 5.
Amino-terminal sequencing data of the 25 kDa showed multiple calling on
the NH2-terminal residue with the predominant form shown in
Fig. 6, and its low yield is consistent
with previous observations that the 25-kDa form is cell-associated
(18). It was not possible to make a positive amino acid assignment for
the first two residues of the 23-kDa form of MPB83 (Fig. 6); the
subsequent residues corresponded to the sequence from Ala50
onwards. Inability to assign amino acids at sites of carbohydrate attachment is a characteristic property of O-glycosylation
(33), and it is interesting to note that the unassigned residues
correspond to the O-glycosylation sites predicted from the
mutagenesis experiments.
Definition of MPB83 Glycosylation by Mass
Spectrometry--
Nanospray ES-MS of the 23-kDa form of MBP83 on the
Q-TOF (28, 29) identified two partially resolved components with masses of 18,048 and 18,066 Da (data not shown). The mass difference of these
two components is consistent with partial oxidation of a methionine
residue in the sequence to methionine sulfoxide, (a theoretical
increase of 16 Da), suggesting that the mass of the 23-kDa form of
native MPB83 is 18,048 (± 2) Da. This experimentally calculated mass
is 484 (±2) Da greater than that of the theoretical mass of 17,564 calculated from the gene sequence having taken into account the amino
acid truncation identified by amino-terminal sequence analysis (Fig.
6). The difference between the experimentally determined and
theoretical calculated mass of this form of MPB83 corresponds well with
the mass of 3 hexose units (486 Da). In confirmation that MPB83 is
modified by the addition of 3 hexose units, chemical deglycosylation of
this material by trifluoromethanesulfonic acid produced an unoxidized
form of MPB83 with a mass of 17,560 (±2) Da (data not shown). The
reduction in mass of MPB83 following the deglycosylation procedure
(488 ± 2 Da) confirms the loss of the 3 hexose units. These data
demonstrate the effectiveness of the trifluoromethanesulfonic acid
deglycosylation procedure in removing glycans while leaving the primary
sequence of the protein intact.
To identify the site of glycosylation, a tryptic digest of the 23-kDa
form of MPB83 was purified by C18 reverse phase high performance liquid
chromatography and fractions were screened for the presence of
glycopeptides by ESI-MS on the Q-TOF. The molecule was mapped in its
entirety with the exception of TDAK (residues 145-148) and Asp
(residue 187). Fraction 36 of the LC trace produced a mass spectrum
indicative of the presence of a glycopeptide, with a doubly charged ion
at m/z 988. This mass does not map onto the MPB83
molecule, but corresponds to the peptide TTAAMADPAADLIGR (residues
48-62) present as the Met sulfoxide plus 3 hexose units.
The Q-TOF nanospray MS-MS spectrum of the 988 ion is shown in Fig.
7. The spectrum clearly shows the full
correct sequence assignment via y" ions (28) at
m/z 1388, 1287, 1216, 1145, 998, 927, 812, 715, 644, 573, 458, 345, 232, and 175 plus a number of confirmatory b ions
(28), consistent with its identification as a glycosylated derivative
of peptide 48-62. Because sugars are lost at relatively low collision
energies compared with peptide bond fragmentation, coupled with the
poor ion yields at the first position (b1) in a sequence,
the spectrum does not allow assignment of which threonine(s) carries
the Hex3 mass increment. An experiment was therefore
designed to move the first threonine residue of the glycopeptide to a
pseudo b2 position by the NH2-terminal reaction with phenylisothiocyanate to form the phenylthiocarbamyl glycopeptide. Q-TOF MS analysis of the phenylisothiocyanate-treated sample confirmed a shift of the m/z 988 signal to a new doubly
charged ion at m/z 1056. The MS-MS spectrum of
m/z 1056 is shown in Fig.
8. This spectrum is a summation of data
acquired at a variety of low collision energies in an attempt to
capture peptide fragment ions still retaining the hexose substitutions.
A detailed analysis of three of these variable collision energy spectra
(Fig. 9), in particular the CAD MS-MS
spectrum acquired at 10 eV, reveals the presence of sugar-containing
peptide fragment ions corresponding to cleavage between
NH2-terminal threonine residues Thr1 and
Thr2. These are present as b1 ions carrying the
phenylthiocarbamyl group at m/z 237 (Thr1Hex0), 399 (Thr1Hex1), 561 (Thr1Hex2), and 723 (Thr1Hex3), and y14" ions at
m/z 1388 (Thr2Hex0), 1550 (Thr2Hex1), 1712 (Thr2Hex2), and 1874 (Thr2Hex3). A confirmatory b13
fragment ion set is also present at m/z 1393, 1555, 1717, and 1879 corresponding to the peptide fragment 48-60 carrying 0-3 hexose residues, respectively. A first-order analysis of
peak intensities suggests that a major molecular component of the
m/z 1056 [M + 2H]2+ ion is a
peptide with
Thr1Hex1 Identification of the Modifying Sugars and Linkage
Analysis--
The identities of the modifying sugars of MPB83 were
determined by Dionex ion chromatography following their release by acid hydrolysis as described under "Experimental Procedures."
Comparative analysis of the MPB83-hydrolyzed material with standard
sugars separated under the same conditions identified a component of MPB83 with an elution time corresponding to mannose (Fig.
10, chromatographs A and
B). To confirm that the MPB83 modifying sugar was mannose, the hydrolysis sample was spiked with mannose. Co-elution of the component peaks corresponding to the mannose standard and the sugar
detected from MPB83 confirmed the identity of the sugar as mannose
(Fig. 10, chromatograph C). Furthermore, the absence of any
material with an elution time corresponding to arabinose in the MPB83
sample indicates the absence of sample contamination by the
lipopolysaccaride antigen common to many strains of mycobacteria, lipoarabinomannan (LAM). Similar to the observations of Dobos et
al. (16) during their characterization of the 45/47-kDa
glycoprotein of M. tuberculosis, the minor peaks observed in
the MPB83 chromatographs are most likely attributable to components of
the chromatographic supports used in its purification.
The m/z 988 [M + 2H]2+ MPB83
tryptic glycopeptide was reductively eliminated to release the
O-glycans, which were permethylated and examined by FAB-MS
on a ZAB 2SE 2FPD mass spectrometer. The FAB spectrum (not shown)
exhibited a major signal at m/z 493 corresponding to HexHexitol with a minor signal at m/z 697 corresponding to Hex2Hexitol. GC-MS linkage analysis of the
partially methylated alditol acetates, derived from the permethylated
products of reductive elimination, gave a peak corresponding to
terminal mannose (diagnostic fragment ions at m/z
205, 161, 145, 129, and 101) together with a peak that gave a mass
spectrum consistent with a linked hexitol. The latter was observed at
the elution position corresponding to 3- or 4-linked mannitol. These
partially methylated alditol acetates co-elute under the conditions
employed in this experiment. Authentic 2-linked mannitol elutes
approximately 0.1 min earlier. The fragment ions, which have been shown
by examination of authentic standards to be diagnostic of 2-, 3-, and
4-linked hexitols, are shown in Fig.
11A. Significantly, the mass
spectrum obtained from the sample hexitol peak was identical to the 3- or 4-mannitol standards and lacked signals at m/z
161 and 129, which are major ions in the spectra of 2-linked hexitols
(see Fig. 11A for origin of these ions). This result was
surprising because 2-linked mannose is the usual linkage observed in
mycobacterial glycopolymers. To confirm our initial observation, and to
resolve the ambiguity of 3- and 4-linked mannitol, which give identical
mass spectra, the reductive elimination procedure was carried out in
duplicate using sodium borodeuteride as the reducing reagent. The
deuterium incorporated during the reduction "tags" carbon-1 and
therefore allows 3- and 4-mannitol to be differentiated via the shift
in mass of the respective fragment ions containing carbon-1. The duplicate experiments gave comparable data and the results from one set
of experiments are reproduced. Fig. 11C shows the spectrum of the partially methylated alditol acetates of authentic 3-linked mannitol and Fig. 11B shows the mass spectrum of the peak
from the MPB83 sample with the same retention time. All diagnostic fragment ions in the 3-mannitol standard are shared by the sample. Importantly the presence of m/z 206 and the
absence of m/z 205 rules out 4-linked mannitol
(see Fig. 11A for m/z assignments). Additionally, the absence of m/z 162 and 130, which are the deuterated counterparts of m/z 161 and 129 (see above) and the presence of m/z 90 provides unequivocal evidence that the reducing end mannose is 3-linked
and not 2-linked.
Several of the immunodominant antigens of M. tuberculosis and M. bovis have been reported to be
glycosylated (13, 16, 32, 34). However, to date the unambiguous
demonstration of mycobacterial glycosylation has been proved only for
the M. tuberculosis antigen MPT32 (16). In this paper we
have demonstrated that the M. bovis antigen MPB83 is
O-glycosylated with mannose via threonine residues.
Furthermore, linkage analysis of the carbohydrate reveals a
Man(1 ConA binding activity of mycobacteria-expressed PhoA fusion proteins
provides evidence for glycosylation of MPB83 but not of the closely
related MPB70 antigen. Further deletion analysis and site-directed
mutagenesis identified an essential role in glycosylation for the
residues at positions 48 and 49 of MPB83. These residues are not
present in the corresponding MPB70 sequence. Direct evidence of
glycosylation of Thr48 and Thr49 was obtained
by nanospray MS-MS analysis of a tryptic peptide corresponding to MPB83
residues 48-62. The amino acid sequence of the MPB83 glycosylation
domain resembles glycosylation sites in MPT32 in the presence of
threonine flanked by alanine and proline residues (16).
Whereas there is no clearly defined motif that allows accurate
prediction of O-glycosylation sites, the presence of a
proline residue In this study Edman degradation amino-terminal sequencing was performed
on both the 23- and 25-kDa forms of MPB83. The data shows that the
23-kDa form of MPB83 is generated by proteolytic cleavage immediately
before Thr48 from the mature acylated 25-kDa form (18).
This raises the possibility that glycosylation may function either as a
signal for cleavage or as a means of preventing amino-terminal
degradation of the protein following cleavage from its acylated anchor.
In support of this latter idea, amino-terminal glycosylation of the cholecystokinin octapeptide (CCK-8) and of glucagon-like
peptide-1-(7-36) amide (Tglp-1) leads to their increased resistance to
serum aminopeptidase degradation (36, 37). In their study of the
M. tuberculosis 19-kDa antigen, Herrmann and colleagues
(17) also described a truncated form of the protein in which the
glycosylation motif formed the new NH2 terminus. This
product was observed when glycosylation had been prevented by threonine
to valine substitutions, leading these authors to conclude that
glycosylation inhibited the initial proteolytic cleavage from the
acylated form of this antigen. This model is not consistent with the
findings of this study, where the NH2-terminal threonine of
the truncated 23-kDa form is glycosylated. It is interesting to
note that such a 23-kDa form is not observed when the gene
mpb83 is expressed by M. smegmatis, suggesting
that M. bovis may possess peptidases not present in the
nonpathogenic mycobacteria.
The dominant glycoform identified for MPB83 was Thr48
substituted with a single mannose residue and Thr49
substituted with a Man(1 Although similar to the M. tuberculosis antigen MPT32 in its
carbohydrate composition, there is a fundamental difference in the
sugar linkages present in the MPB83 glycoprotein. Whereas MPT32
contains (1 The detailed structural characterization of MPB83 described in this
paper provides an essential platform for further exploration of the
biological significance of antigen glycosylation by mycobacterial pathogens. Expression of native and site-directed variants of glycoproteins in recombinant hosts will allow assessment of the role of
post-translational modification in recognition by the host immune
response. In parallel, tools for genetic manipulation of mycobacteria
can be used to construct strains deleted for genes encoding individual
glycoproteins or enzymes involved in glycosylation, allowing assessment
of their contribution to the pathogenesis of tuberculosis.
3) linkage, in contrast to the (1
2) linkage
previously observed in antigen MPT32 from M. tuberculosis
and the (1
2) and (1
6) linkages in other mycobacterial
glycolipids and polysaccharides. The identification of glycosylated
antigens within the M. tuberculosis complex raises the
possibility that the carbohydrate moiety of these glycoproteins might be involved in pathogenesis, either by interaction with mannose
receptors on host cells, or as targets or modulators of the
cell-mediated immune response. Given such a possibility
characterization of mycobacterial glycoproteins is a step toward
understanding their functional role and elucidating the mechanisms of
mycobacterial glycosylation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mannosidase resulted in a reduction in their relative
molecular weight. Subsequently, we demonstrated the presence of a gene,
mpb83, which encodes a protein with 61% identity at the
amino acid level to MPB70 (18). In the same study, the protein encoded
for by this gene, MPB83, was shown to bind a monoclonal antibody
specific for the 25/23-kDa M. bovis antigen. Thus,
the 25/23-kDa antigen characterized by Fifis et al. (13) is
MPB83, rather than a glycosylated form of MPB70.
3)-linked mannose chains to two threonine residues.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(Invitrogen) and Mycobacterium
smegmatis mc2155 expressing PhoA hybrid proteins were
grown in Luria broth, 50 µg/ml hygromycin, or in Middlebrook 7H9
supplemented with albumin, dextrose, catalase (MADC), and 0.05% Tween
80, 200 µg/ml hygromycin, respectively. Alkaline phosphatase activity
of E. coli and mycobacterial PhoA recombinants was
determined by the presence of blue colonies when plated on L agar
(containing appropriate hygromycin concentrations) supplemented with 40 µg/ml 5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine (Sigma).
Oligonucleotides used for fragment amplification
(Invitrogen), by the presence of blue colonies when grown on
L-agar supplemented with 40 µg/ml
5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine. Plasmid
DNA was isolated (Qiagen) and subsequently transformed into M. smegmatis mc2155 according to the method of Snapper
et al. (24).
80 °C and lyophilized overnight in a model EF03
freeze dryer (Edwards High Vacuum International). The
deglycosylation procedure was then performed in accordance with
the GlycoFree kit recommendations.
20 °C for 4 h and then returned to a dry
ice/ethanol bath, to which 150 µl of pyridine was slowly added. The
vial was then transferred to dry ice for a further 5 min and then to
wet ice for a further 15 min. The reaction mixture was neutralized by
addition of 400 µl of 0.5% (w/v) ammonium bicarbonate. The
deglycosylated protein was then isolated from the reaction products and
reagents by dialysis against 4× 2 liters of PBS using a 3,500 Mr cut-off dialysis cassette (Perbio Science).
The protein concentration of the dialyzed material was determined using
the BCA protein estimation assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
M. smegmatis mc2155, as determined by the
formation of blue colonies in the presence of
5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine.
View larger version (16K):
[in a new window]
Fig. 1.
Maps of plasmids used in this
study.
(Fig.
2A, lane 4), demonstrating that
post-translational modification of MPB83 is restricted to the
mycobacterial host. Neither the MPB70-PhoA fusion protein nor the PhoA
protein alone expressed by M. smegmatis mc2155
bound ConA (Fig. 2A, lanes 3 and 6,
respectively). Probing a duplicate blot with antibody directed against
PhoA (Fig. 2B) demonstrated a similar amount of hybrid
protein in each of the extracts, indicating that differences in ConA
binding were not a consequence of differences in expression levels of
the recombinant fusion proteins. There was evidence of partial
proteolysis of fusion proteins expressed in M. smegmatis
mc2155 as two bands reacting with anti-PhoA antibody were
observed.
View larger version (76K):
[in a new window]
Fig. 2.
Binding of ConA to MPB83 when expressed by
mycobacteria. Nitrocellulose membranes incubated with
(A) peroxidase-conjugated ConA and (B) rabbit
polyclonal antibody raised against bacterial alkaline phosphatase.
Lanes: 1, purified PhoA protein;
2, E. coli DH5 (p70PhoA);
3, M. smegmatis mc2155
(p70PhoA); 4, E. coli DH5
(p83PhoA);
5, M. smegmatis mc2155
(p83PhoA); 6, M. smegmatis
mc2155 (19proSP-PhoA).
(Fig. 2B, lanes
4 and 5). In contrast, no size difference was observed
between MPB70-PhoA expressed by M. smegmatis
mc2155 or E. coli DH5
(Fig. 2B,
lanes 2 and 3). The difference in apparent
molecular weights of fusion proteins expressed by different bacterial
hosts, coupled with the ConA recognition of MPB83-PhoA expressed by
M. smegmatis mc2155, supports the conclusion
that MPB83 but not MPB70 is glycosylated by mycobacteria.
MPB83 amino terminus PhoA fusions
View larger version (61K):
[in a new window]
Fig. 3.
Deletion mapping and site-directed
mutagenesis of MPB83 to identify regions responsible for ConA
binding. Nitrocellulose membranes incubated with
peroxidase-conjugated ConA (A and C) and rabbit
polyclonal antibody raised against bacterial alkaline phosphatase
(B and D). M. smegmatis
mc2155 expressing MPB83 amino terminus PhoA fusions from
constructs: panels A and B: lanes
1, purified PhoA protein; 2, p28aaPhoA;
3, p35aaPhoA; 4, p56aaPhoA; 5,
p63aaPhoA; 6, p83PhoA; and 7, p19proSP-PhoA.
Panels C and D: lanes
1, p63aaPhoA; 2, p63aa (TT VV)PhoA;
3, p83 PhoA; 4, p19proSP-PhoA.
View larger version (24K):
[in a new window]
Fig. 4.
Comparison of the NH2-terminal
amino acid sequence of MPB83 and MPB70. Differences are
boxed and the putative O-glycosylation sites are
highlighted.
View larger version (73K):
[in a new window]
Fig. 5.
Purification of the 23-kDa native form of
MPB83 from a M. bovis culture supernatant. A
23-kDa form of MPB83 was purified from a M. bovis AN5
culture supernatant by a 3 column chromatography procedure. The purity
of the material following each stage of the purification was determined
by silver stain analysis of SDS-PAGE separated samples (panel
A). The identity of MPB83 was confirmed by probing the material at
each stage in the procedure with a MPB83 specific monoclonal antibody
MBS43 (panel B). Lanes 1, M. bovis AN5
culture supernatant (5 µg); 2, purity following DEAE-Fast
Flow AEC (2.5 µg); 3, purity following Mono-Q AEC (2.5 µg); 4, purity following hydrophobic interaction
chromatography (0.5 µg).
View larger version (5K):
[in a new window]
Fig. 6.
Amino-terminal sequencing of purified forms
of MPB83. The predicted amino-terminal sequence of mature MPB83 is
shown on line 1. The experimentally determined sequences for
the 25- (2) and 23-kDa (3) forms of MPB83
purified from a M. bovis culture supernatant have been
aligned against their corresponding positions in the predicted MPB83
sequence.
Thr2Hex2
glycosylation, although there is certainly evidence from the other
signals noted for the presence of less abundant structures in the
sample, ranging from Thr1Hex3
Thr2Hex0
through
Thr1Hex2
Thr2Hex1 to
Thr1Hex0
Thr2Hex3. A
further point of interest is that whatever the substitution patterns,
only 3 hexose units are attached overall in the principal glycopeptide
observed.
View larger version (16K):
[in a new window]
Fig. 7.
ESI (Q-TOF) CAD MS-MS analysis of the [M + 2H]2+ ion at m/z
988. The major fragment ions correspond to the y" series of
peptide fragments all of which are lacking sugar attachments (see
text).
View larger version (10K):
[in a new window]
Fig. 8.
ESI (Q-TOF) CAD MS-MS analysis of the [M + 2H]2+ ion at m/z 1056, which is the phenylthiocarbamyl derivative of the
m/z 988 component. This spectrum
is a summation of data obtained over a range of collision energies. For
an explanation of the data see discussion of Fig. 9.
View larger version (15K):
[in a new window]
Fig. 9.
ESI (Q-TOF) CAD MS-MS spectra of
m/z 1056 obtained at three different
collision energies. The spectra are presented in narrow mass
ranges for ease of interpretation. Panels A-C, span
m/z 150 to 750; panels D-F, span
m/z 650 to 1050; and panels G-I, span
m/z 1100 to 2200. Panels A,
D, and G correspond to 29 eV collision energy,
panels B, E, and H correspond to 20 eV
collision energy and panels C, F, and
I correspond to 10 eV collision energy. For an explanation
of signals that were used to assign sugar attachment points and site
occupancy see the text.
View larger version (88K):
[in a new window]
Fig. 10.
Analysis of the sugar component of
MPB83. MPB83 was subjected to acid hydrolysis and the released
sugar present in this material (25 µg) was identified by Dionex ion
chromatography (chromatograph B) by comparison with sugar
standards run under the same conditions. Chromatograph A
demonstrates the elution times of the standard sugars arabinose (0.05 µg), glucose (0.05 µg), and mannose (0.05 µg). Chromatograph
C shows the profile of the MPB83 hydrolysis material that
was spiked with 0.03 µg of mannose.
View larger version (26K):
[in a new window]
Fig. 11.
Linkage analysis of reductively eliminated
glycans from MPB83. A, fragmentation pathways for 2-, 3-, and 4-linked hexitols with and without deuterium at C-1, showing the
major fragment ions observed in spectra from authentic standards.
B, mass spectrum of the partially methylated alditol
acetates of the deutero-reduced hexitol from MPB83, which eluted at the
same retention time as authentic deutero-reduced 3-mannitol whose
spectrum is shown in C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3)Man linkage not previously observed in mycobacterial glycoproteins.
1 or +3 relative to the position of a threonine
residue has been found to be useful as a predictor of such sites
in eukaryotic mucin-like proteins (35)
(www.cbs.dtu.dk/services/ NetOGlyc/). The glycosylation site
predicted by site-directed mutagenesis of the 19-kDa antigen of
M. tuberculosis is similarly located in a threonine-rich
region flanked by proline residues (17). Analysis of MPB83 using this
O-glycosylation prediction model identifies nine residues
within MPB83 that are potential sites for O-glycosylation.
These sites include the threonine doublet at positions 48 and 49. Mass
spectrometry analysis demonstrates that this is in fact the only site
of glycosylation, at least in the truncated 23-kDa form of MPB83
included in the present study.
3)Man linkage. However, evidence for a
heterogeneous array of glycoforms from
Thr48(Man3) Thr49(Man0)
through to
Thr48(Man0)Thr49(Man3)
was also observed. A diversity of mannosylated threonine residues was
also seen in the study of the 45/47-kDa antigen of M. tuberculosis, the only other mycobacterial glycoprotein for which
the modifying glycans have been fully characterized (16). The exclusive
use of mannose in mycobacterial glycoproteins characterized to date
differs from the O-linked sugars identified on the
glycosylated pilin of N. meningitidis and the glycosylated
flagellin of C. jejuni. The predominant modification found
in N. meningitidis is a terminal
(1
4)-linked digalactose moiety covalently linked to a
2,4-diacetamido-2,4,6-trideoxyhexose sugar that is directly O-linked to the pilin (2), whereas the C. jejuni flagellin was O-glycosylated with
5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid, Pse5Ac7Ac) (38). The simple
O-mannosylation observed for mycobacteria, reminiscent
of that seen in yeast, enables us to suggest that the mechanism of
mycobacterial glycosylation may also be similar, and may be catalyzed
by a family of protein mannosyltransferases as is the case for yeast
(39). Protein mannosyltransferase activity has been demonstrated in
cell-free extracts from M. smegmatis using peptides from
MPT32 and MPB83 as acceptor molecules (40), and experiments in S. coelicolor identify an M. tuberculosis open
reading frame, Rv1002c, as a likely candidate for this activity
(41).
2)-linked mannobiose and
(1
2),(1
2)-linked mannotriose, the terminal mannose in
MPB83 is (1
3)-linked. The Man(1
2)Man linkage seen in
MPT32 is also seen as a branching linkage in the mannan backbone of LAM
(42), in the di- and trimannosyl units of the Man cap of LAM (43) and
as one of the linkages for the mannose present in the
phosphatidylinositol mannoside (PIM) family of mycoabacterial
phospholipids such as PIM5 and PIM6 (44, 45).
In contrast the (1
3) linkage of the mannobiose glycan of MPB83
is not a configuration previously identified within the M. tuberculosis complex. However, a recent report has shown that LAM
from the fast growing Mycobacterium chelonae, designated CheLAM, contains a mannan core similar to LAM of M. tuberculosis, but differs in its branching, with LAM having
(1
2)-mannose branches as opposed to CheLAM which has
(1
3)-mannose branches (46). The genome sequence of M. tuberculosis contains several open reading frames encoding
potential mannosyltransferases. Two, pimB and
pimC, have been characterized biochemically and shown to
encode (1
6)-transferases involved in PIM biosynthesis (47, 48).
It will be of interest to identify the (1
3)-transferase, and to
determine the factors that influence the preferential (1
2)
versus (1
3) modification of the different glycoproteins.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Jeff Keen for help in NH2-terminal sequencing and Dr. Stephen Gordon for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Department for the Environment Food and Rural Affairs (DEFRA) UK, the Biotechnology and Biological Sciences Research Council, and the Wellcome Trust (to A. D. and H. R. M.).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. Current address: DSTL, Porton Down, Salisbury, Wiltshire SP4 0JQ, United Kingdom. Tel.: 44-1980614950; E-mail: slmichell@dstl.gov.uk.
Biotechnology and Biological Sciences Research Council
Professorial fellow.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M207959200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ConA, concanavalin A; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; AEC, anion exchange chromatography; Q-TOF, orthogonal acceleration time of flight; CAD MS/MS, collisionally activated decomposition tandem mass spectrometry; aa, amino acid(s); LAM, lipoarabinomannan; PIM, phosphatidylinositol mannoside.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Virji, M., Saunders, J. R., Sims, G., Makepeace, K., Maskell, D., and Ferguson, D. J. (1993) Mol. Microbiol. 10, 1013-1028[Medline] [Order article via Infotrieve] |
2. | Stimson, E., Virji, M., Makepeace, K., Dell, A., Morris, H. R., Payne, G., Saunders, J. R., Jennings, M. P., Barker, S., Panico, M., Blench, I., and Moxon, E. R. (1995) Mol. Microbiol. 17, 1201-1214[Medline] [Order article via Infotrieve] |
3. | Wu, H., Mintz, K. P., Ladha, M., and Fives-Taylor, P. M. (1998) Mol. Microbiol. 28, 487-500[CrossRef][Medline] [Order article via Infotrieve] |
4. | Popov, V. L., Yu, X., and Walker, D. H. (2000) Microb. Pathog. 28, 71-80[CrossRef][Medline] [Order article via Infotrieve] |
5. |
McBride, J. W., Yu, X. J.,
and Walker, D. H.
(2000)
Infect. Immun.
68,
13-18 |
6. |
Chia, J. S.,
Chang, L. Y.,
Shun, C. T.,
Chang, Y. Y.,
and Chen, J. Y.
(2001)
Infect. Immun.
69,
6987-6998 |
7. |
Erickson, P. R.,
and Herzberg, M. C.
(1993)
J. Biol. Chem.
268,
23780-23783 |
8. |
Erickson, P. R.,
and Herzberg, M. C.
(1993)
J. Biol. Chem.
268,
1646-1649 |
9. | Doig, P., Kinsella, N., Guerry, P., and Trust, T. J. (1996) Mol. Microbiol. 19, 379-387[CrossRef][Medline] [Order article via Infotrieve] |
10. | Szymanski, C. M., Yao, R., Ewing, C. P., Trust, T. J., and Guerry, P. (1999) Mol. Microbiol. 32, 1022-1030[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Romain, F.,
Horn, C.,
Pescher, P.,
Namane, A.,
Riviere, M.,
Puzo, G.,
Barzu, O.,
and Marchal, G.
(1999)
Infect. Immun.
67,
5567-5572 |
12. | Espitia, C., and Mancilla, R. (1989) Clin. Exp. Immunol. 77, 378-383[Medline] [Order article via Infotrieve] |
13. | Fifis, T., Costopoulos, C., Radford, A. J., Bacic, A., and Wood, P. R. (1991) Infect. Immun. 59, 800-807[Medline] [Order article via Infotrieve] |
14. | Garbe, T., Harris, D., Vordermeier, M., Lathigra, R., Ivanyi, J., and Young, D. (1993) Infect. Immun. 61, 260-267[Abstract] |
15. | Dobos, K. M., Swiderek, K., Khoo, K. H., Brennan, P. J., and Belisle, J. T. (1995) Infect. Immun. 63, 2846-2853[Abstract] |
16. | Dobos, K. M., Khoo, K. H., Swiderek, K. M., Brennan, P. J., and Belisle, J. T. (1996) J. Bacteriol. 178, 2498-2506[Abstract] |
17. | Herrmann, J. L., O'Gaora, P., Gallagher, A., Thole, J. E., and Young, D. B. (1996) EMBO J. 15, 3547-3554[Abstract] |
18. | Hewinson, R. G., Michell, S. L., Russell, W. P., McAdam, R. A., and Jacobs, W. J. (1996) Scand. J. Immunol. 43, 490-499[Medline] [Order article via Infotrieve] |
19. | O'Loan, C. J., Pollock, J. M., Hanna, J., and Neill, S. D. (1994) Clin. Diagn. Lab. Immunol. 1, 608-611[Abstract] |
20. | Goodger, J., Nolan, A., Russell, W. P., Dalley, D. J., Thorns, C. J., Croston, P., and Newell, D. G. (1994) Vet. Rec. 135, 82-85[Medline] [Order article via Infotrieve] |
21. | Roche, P. W., Triccas, J. A., Avery, D. T., Fifis, T., Billman-Jacobe, H., and Britton, W. J. (1994) J. Infect. Dis. 170, 1326-1330[Medline] [Order article via Infotrieve] |
22. | Chambers, M. A., Vordermeier, H., Whelan, A., Commander, N., Tascon, R., Lowrie, D., and Hewinson, R. G. (2000) Clin. Infect. Dis. 30, S283-S287[CrossRef][Medline] [Order article via Infotrieve] |
23. | Sambrook, J., Fitsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY |
24. | Snapper, S. B., Melton, R. E., Mustafa, S., Kieser, T., and Jacobs, W. R., Jr. (1990) Mol. Microbiol. 4, 1911-1919[Medline] [Order article via Infotrieve] |
25. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
26. |
Matsudaira, P.
(1987)
J. Biol. Chem.
262,
10035-10038 |
27. | Sojar, H. T., and Bahl, O. P. (1987) Arch. Biochem. Biophys. 259, 52-57[Medline] [Order article via Infotrieve] |
28. | 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] |
29. | Morris, H. R., Panico, M., Dell, A., and McDowell, R. A. (1998) in Mass Spectrometry of Biological Materials (Larsen, B. S. , and McEwen, C. N., eds), 2nd Ed. , pp. 53-80, Marcel Dekker, New York |
30. | Dell, A., Reason, A. J., Khoo, K. H., Panico, M., McDowell, R. A., and Morris, H. R. (1994) Methods Enzymol. 230, 108-132[Medline] [Order article via Infotrieve] |
31. |
Khoo, K. H.,
Sarda, S.,
Xu, X.,
Caulfield, J. P.,
McNeil, M. R.,
Homans, S. W.,
Morris, H. R.,
and Dell, A.
(1995)
J. Biol. Chem.
270,
17114-17123 |
32. | Herrmann, J. L., Delahay, R., Gallagher, A., Robertson, B., and Young, D. (2000) FEBS Lett. 473, 358-362[CrossRef][Medline] [Order article via Infotrieve] |
33. | Abernathy, J. L., Wang, Y., Eckhardt, A. E., and Hill, R. L. (1992) in Techniques in Protein Chemistry (Angelleti, R. H., ed), Vol. III , pp. 277-286, Academic Press, New York |
34. | Torres, A., Juarez, M. D., Cervantes, R., and Espitia, C. (2001) Microb. Pathog. 30, 289-297[CrossRef][Medline] [Order article via Infotrieve] |
35. | Gooley, A. A., Classon, B. J., Marschalek, R., and Williams, K. L. (1991) Biochem. Biophys. Res. Commun. 178, 1194-1201[Medline] [Order article via Infotrieve] |
36. | O'Harte, F. P., Mooney, M. H., Kelly, C. M., and Flatt, P. R. (1998) Diabetes 47, 1619-1624[Abstract] |
37. | O'Harte, F. P., Mooney, M. H., Lawlor, A., and Flatt, P. R. (2000) Biochim. Biophys. Acta 1474, 13-22[Medline] [Order article via Infotrieve] |
38. |
Thibault, P.,
Logan, S. M.,
Kelly, J. F.,
Brisson, J. R.,
Ewing, C. P.,
Trust, T. J.,
and Guerry, P.
(2001)
J. Biol. Chem.
276,
34862-34870 |
39. | Immervoll, T., Gentzsch, M., and Tanner, W. (1995) Yeast 11, 1345-1351[Medline] [Order article via Infotrieve] |
40. |
Cooper, H. N.,
Gurcha, S. S.,
Nigou, J.,
Brennan, P. J.,
Belisle, J. T.,
Besra, G. S.,
and Young, D.
(2002)
Glycobiology
12,
1-8 |
41. | Cowlishaw, D. A., and Smith, M. C. (2001) Mol. Microbiol. 41, 601-610[CrossRef][Medline] [Order article via Infotrieve] |
42. | Misaki, A., Azuma, I., and Yamamura, Y. (1977) J. Biochem. (Tokyo) 82, 1759-1770[Abstract] |
43. |
Venisse, A.,
Riviere, M.,
Vercauteren, J.,
and Puzo, G.
(1995)
J. Biol. Chem.
270,
15012-15021 |
44. | Lee, Y. C., and Ballou, C. E. (1965) Biochemistry 4, 1395-1404[Medline] [Order article via Infotrieve] |
45. |
Chatterjee, D.,
Hunter, S. W.,
McNeil, M.,
and Brennan, P. J.
(1992)
J. Biol. Chem.
267,
6228-6233 |
46. |
Guerardel, Y.,
Maes, E.,
Elass, E.,
Leroy, Y.,
Timmerman, P.,
Besra, G. S.,
Locht, C.,
Strecker, G.,
and Kremer, L.
(2002)
J. Biol. Chem.
277,
30635-30668 |
47. |
Schaeffer, M. L.,
Khoo, K. H.,
Besra, G. S.,
Chatterjee, D.,
Brennan, P. J.,
Belisle, J. T.,
and Inamine, J. M.
(1999)
J. Biol. Chem.
274,
31625-31631 |
48. | Kremer, L., Gurcha, S. S., Bifani, P., Hitchen, P. G., Baulard, A., Morris, H. R., Dell, A., Brennan, P. J., and Besra, G. S. (2002) Biochem. J. 363, 437-447[CrossRef][Medline] [Order article via Infotrieve] |