From the
A new type of O-linked oligosaccharide has been
discovered on several proteins secreted by the Gram-negative bacterium
Flavobacterium meningosepticum, including Endo F
Post-translational glycosylation is now widely recognized as an
important process for modifying the structure/function of eukaryotic
proteins. Glycosylation additions in bacterial systems, however, are
much less well understood and thus far restricted mainly to
oligosaccharide polymers and lipopolysaccharides and to complex cell
wall and cell surface components. Mescher and Strominger in 1976
(1) first reported the presence of a single N-linked
and multiple O-linked carbohydrates in the envelope protein of
Halobacterium salinarium. More recent studies have reported
the presence of N-linked carbohydrate in bacterial cell wall
proteins
(2, 3) and O-linked glycan in
Thr/Pro-rich regions of cellulase complexes of the cellulolytic
bacteria Clostridium thermocellum and Bacteriodes
cellulosolvens(4) .
In the course of our studies on the
hydrolases produced by Flavobacterium meningosepticum, we
observed anomalies which suggested a type of glycosylation different
from what had been previously observed in prokaryotes. This
Gram-negative bacterium secretes into the culture medium at least eight
major proteins that can be purified to homogeneity by hydrophobic
interaction chromatography in conjunction with high-resolution
ion-exchange chromatography
(5, 6) . These proteins
include two proteases, termed P27 and P40
Prokaryotic extracellular glycoproteins like Endo
F
The supernatant from the thermolytic digestion of Endo F
Compositional analyses were also accomplished by preparing alditol
acetate derivatives of glycosyl components followed by gas
chromatography and combined gas chromatography/mass spectrometry
(GC/MS) analysis. Trimethylsilyl derivatives of the methyl glycosides
were prepared to detect any amino sugars
(17, 18) and
analyzed by GC and GC/MS (compositional analyses were performed by Dr.
Roberta K. Merkle at the University of Georgia, Complex Carbohydrate
Research Center).
The secreted proteins of Flavobacterium meningosepticum are separated initially by hydrophobic interaction chromatography
on columns of either Toyopearl TSK-butyl
(5) or Toyopearl
TSK-phenyl (7). A typical chromatogram of 12 liters of culture medium
on a TSK-phenyl column was surveyed for carbohydrate content by the
phenol-H
Mass
analysis of the free oligosaccharide demonstrated a molecular weight of
1264 Da for the major component. An intense signal for the M +
H
Evidence has also been obtained that Endo F
The gene for Endo F
represents a synopsis of sequences of
F. meningosepticum secreted proteins and their sites of
glycosylation. In every instance where there is a DS* sequence, the
starred serine is fully substituted by addition of the common
Flavobacterium oligosaccharide. We have found only one
instance of a D-T*-T sequence (Endo F
The function
of this novel oligosaccharide will require further study. The only
other reported O-linked glycan on prokaryotes are those found
in the extracellular cellulase complexes of Clostridium
thermocellum and Bacteriodes cellulosolvens(4) .
These glycans are linked mainly to threonine via a galactosyl group,
and the attachment sites appear to be restricted to threonine/proline
enriched areas somewhat reminiscent of O-linked carbohydrates
of eukaryotic systems. In contrast, the Flavobacterium oligosaccharide has a completely different glycan structure that
is attached randomly at very specific consensus sites. The
oligosaccharide moiety may be involved in protein folding/stability as
has been demonstrated in mammalian systems. We have successfully used
the pMAL amplification system for overproduction of PNGase
F
We anticipate the production of antibody probes to
ascertain whether this oligosaccharide and its linkage sequence is
unique to Flavobacterium, or importantly, is an unreported but
more common glycoform in other secreted bacterial glycoproteins.
We thank Arthur W. Phelan and William Heffernan for
excellent technical assistance and Dr. Li-Ming Changchien for Edman
sequence analysis.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(three sites), Endo F
(one site), and a P40 protease
(one site). The oligosaccharide moiety is covalently attached via a
mannose residue to a serine or threonine at consensus sites
corresponding to Asp-Ser* or Asp-Thr*-Thr. Preliminary characterization
by mass spectroscopy revealed an oligosaccharide of 1244 Da at each of
the proposed glycosylation sites. Collision-associated dissociation
analysis showed a characteristic daughter ion series of m/z 218, 394, and 556, indicative of a common Flavobacterium oligosaccharide. Compositional analysis demonstrated an unusual
profile of monosaccharides, including hexoses, methylated hexoses, and
uronic acid derivatives.
(
)(7) and four oligosaccharide chain-cleaving enzymes, including
one amidase, termed PNGase
(
)
F, and three
endoglycosidases designated as Endo F
, F
, and
F
(5). The genes for all four oligosaccharide
chain-cleaving enzymes were cloned and their nucleotide sequences
determined
(8, 9, 10) . The genes for Endo
F
and Endo F
were expressed in Escherichia
coli, and the molecular weights of the cloned enzymes were
compared with those of the native enzymes on SDS-polyacrylamide gel
electrophoresis. Native Endo F
ran slightly larger and
native Endo F
significantly larger than their cloned enzyme
counterparts. Mass spectrometry confirmed that Endo F
and
Endo F
were modified and contained approximately 3731 and
1244 daltons of mass, respectively, not accounted for in the gene
structure. Re-examination of Edman sequence data led to the suggestion
that Endo F
and Endo F
were
post-translationally modified with carbohydrate during secretion by
possible linkage to the hydroxyl of serine
(10) . A problem
existed in characterizing the oligosaccharide moieties of Endo F
and Endo F
, since isolation of these enzymes from 12
liters of cultural filtrate yielded only 9 and 15 nmol of protein,
respectively. We will demonstrate that the P40 protease is also a
glycoprotein containing a carbohydrate moiety O-linked to
serine at one unique well defined aspartyl-serine consensus site. This
oligosaccharide is easily obtained in amounts of 1 µmol and serves
as a model for Endo F
and Endo F
. In fact, mass
spectrometry data strongly suggests that the O-linked
carbohydrate on P40, Endo F
, and Endo F
are
identical.
, Endo F
, and P40 that have O-linked
oligosaccharides at specific consensus sites have never been described
before. This report outlines the isolation of glycopeptides containing
these consensus sites and demonstrates the existence of a new type of
bacterial glycan that is distinguished by having an unusual array of
uronic acids and methylated neutral sugars linked via mannose to
serine/threonine. The following paper
(11) presents a detailed
characterization of this novel glycan.
Proteins
Endo F and Endo F
were isolated essentially as described
(5) . The final
purification step of Endo F
and Endo F
was
achieved on a high resolution sulfopropyl (Protein-Pak SP8HR, Waters
Chromatography Division) column at pH 4.5
(6) . P40 was
chromatographed on TSK-phenyl-Toyopearl 650 M(3) and
then rechromatographed on TSK-butyl-Toyopearl 650S using the conditions
outlined for TSK-butyl-Toyopearl 650 M column
chromatography
(5) . Pooled fractions were dialyzed at 4 °C
against two changes of 10 mM EDTA and one change of 5
mM EDTA followed by one change of distilled water for 4 h
before lyophilization.
Tryptic Digestion
Lyophilized P40 (42 mg) was
warmed at 37 °C for 45 min in 1.6 ml of 0.4 M Hepes, pH
8.25, containing 8 M urea. The solution was diluted 3-fold
with 4.8 ml of 0.4 M Hepes and 315 µl of
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin (4.0 mg/ml, 3% w/w) in 2 mM HCl, 100 mM
CaCl was added. After 6 h, a second identical aliquot of
trypsin was added. The digestion was incubated at 37 °C for 24 h
and was terminated by addition of 0.4 ml of 8 M
H
PO
. A precipitate was removed by
centrifugation.
Thermolytic Digestion
Lyophilized Endo F (1.23 mg) was suspended in 0.4 ml of 0.2 M Hepes, pH
8.24, containing 5 mM CaCl
; and 50 µg of
thermolysin, 3% w/w, (Calbiochem) was added. After 6 h, a second
identical aliquot of thermolysin was added. The digestion was incubated
at 50 °C for 24 h and was terminated by addition of 50 µl of
4.0 M H
PO
.
Cyanogen Bromide Digestion
To approximately 500
pmol of Endo F in a 500-µl Eppendorf tube was added 100
µl of 70% trifluoroacetic acid and a crystal of CNBr. The sample
was flushed with N
, capped, and allowed to sit for 24 h at
room temperature. The solution was lyophilized to near dryness,
redissolved in 500 µl of water, lyophilized again, and then
redissolved in 200 µl of water prior to separation on a 1
50-mm ABI RP-300 column.
Glycopeptide Purification
Supernatant (7.5 ml)
from the tryptic digestion of P40 was applied to a column of Toyopearl
HW-40S (1.5 234 cm, Supelco) equilibrated in 0.1 N
acetic acid containing 1% butanol. The flow rate was 11.2 ml/h, and
fractions of 2.8 ml were collected. Peptides were detected by
absorbance at 230 nm. Carbohydrate was monitored by the
phenol-H
SO
assay
(12) . The glycopeptide
was shown to be exclusively in the nonretarded position on the column.
These fractions were pooled, concentrated by rotary evaporation at 40
°C, and rechromatographed on a column of Bio-Gel P-4 (0.9
91 cm, 200-400 mesh, Bio-Rad) equilibrated in 0.1%
trifluoroacetic acid. The flow rate was 12 ml/h, and fractions of 0.8
ml were collected. Glycopeptide was detected by absorbance at 215 nm
and by reacting aliquots with fluorescamine
(13) . Carbohydrate
was monitored as before and pertinent fractions were lyophilized.
was handled identically to that of P40 except on a smaller scale.
The initial separation was on a TSK HW40S column (0.6
93 cm)
equilibrated in 0.1% trifluoroacetic acid. The flow rate was 6.0 ml/h,
and fractions of 0.5 ml were collected. Peptides were monitored at 215
nm. The glycopeptide-containing fractions (tubes 28-30) were
lyophilized. Ten percent was applied directly to a Bio-Gel P-4 column
(0.6
63 cm) and 90% was subjected to alkaline-borohydride
oligosaccharide release (see below) prior to chromatography on Bio-Gel
P-4.
Oligosaccharide Release
Lyophilized P40
glycopeptide (1 µmol) was dissolved in 450 µl of freshly
prepared 1 M NaBH in 50 mM NaOH
(14) and incubated at 45 °C. After 16 h, the reaction was
terminated by the addition of glacial acetic acid to pH 4.0, and the
sample was applied to the aforementioned larger Bio-Gel P-4 column.
Uncleaved peptide, released oligosaccharide, and released peptide were
detected as before. Endo F
glycopeptide was handled
similarly, and released oligosaccharide was separated from reduced
peptide on a Bio-Gel P-4 column (0.6
63 cm).
Amino Acid Analyses
Peptides were hydrolyzed in
constant boiling HCl at 110 °C for 22-24 h and analyzed for
amino acids with a Beckman System Gold amino acid analyzer or a
Pickering amino acid resolution column and buffers on a Waters 625 LC
system.
Edman Degradation
Automated Edman degradation was
performed with a model 477A Applied Biosystem pulsed liquid sequenator
equipped with a model 120A amino acid analyzer. Samples of 250-2000
pmol were applied in 100-200 µl of 25% acetic or formic acid.
Peptide composition determined by amino acid analysis was used to
predict the number of Edman cycles to be completed for each peptide.
Carbohydrate Analysis
Suitable aliquots were
hydrolyzed either with 2 N trifluoroacetic acid or with 2
N hydrochloric acid in N-flushed, evacuated tubes
for varying time intervals. Acids were removed by lyophilization (2
N trifluoroacetic acid) or under vacuum at 40 °C (2
N HCl). Monosaccharide samples were taken up in water (115
µl), made alkaline with 2 N NaOH (10 µl), and aliquots
(100 µl) were analyzed by HPAEC/PAD on a CarboPac PA-I column,
using the chromatography conditions of Clarke et
al.(15) , or on a MA-1 column using conditions recommended
by the manufacturer (Dionex) for sugar alcohols. Released, reduced P40
oligosaccharide was chromatographed on a Carbo-Pac PA-100 column using
the general procedure of Townsend et al.(16) . Eluant 1
was 500 mM sodium acetate, eluant 2 was water, and eluant 3
was 1 M NaOH. The column was equilibrated in 10 mM
sodium acetate, 200 mM sodium hydroxide at a flow rate of 1
ml/min. After sample addition, the concentration of sodium acetate was
increased to 400 mM over 60 min using a linear gradient.
Mass Spectrometry
Data were acquired with a
Finnigan-MAT (San Jose, CA) TSQ-700 triple quadruple mass spectrometer
equipped with a Finnigan electrospray ionization (ESI) source.
Salt-free samples were infused into the ESI source at a flow rate of 2
µl/min. Collisional activated dissociation (CAD) spectra were
obtained on doubly and/or singly charged ions; argon was used as the
collision gas. Carboxylic acid groups were converted to the
corresponding methyl esters as described previously
(19) .
Peptide amine terminal NH groups were acetylated with a 3:1
mixture of methanol/acetic anhydride.
SO
assay. The only protein peak that
was coincident with carbohydrate was that of the protease, P40
(peak C, Ref. 7). The fractions containing Endo F
and F
, although glycosylated, showed no carbohydrate
owing to the lack of sensitivity of this technique at their
concentration levels. P40 was rechromatographed on a column of
TSK-butyl (not shown), desalted by dialysis against dilute EDTA, and
then subjected to total tryptic hydrolysis. The soluble peptides,
containing 80% of the original carbohydrate, were subjected to gel
filtration on a column of TSK HW-40S (Fig. 1). All of the
carbohydrate was recovered in a glycopeptide eluting in the nonretarded
fraction of the column. Amino acid analysis of the glycopeptide
indicated the presence of six amino acids, with 2 mol of serine/mol of
aspartic acid and no apparent amino sugars. The glycopeptide was then
subjected to alkaline borohydride to release the oligosaccharide
moiety. After overnight incubation at 45 °C excess borohydride was
decomposed with glacial acetic acid, and the reaction mixture was
separated on a Bio-Gel P-4 column (Fig. 2). All components were
monitored by absorbance at 215 nm; the carbohydrate- and
peptide-containing fractions were determined with
phenol-H
SO
and fluorescamine, respectively. The
peak corresponding to the intact glycopeptide (Fig. 2, solid
bar) was greatly decreased following alkaline borohydride
treatment and replaced by two lower molecular weight fractions; a
moderate-sized peak containing the majority of the carbohydrate
(Fig. 2, CHO), and a large fluorescamine-positive,
carbohydrate-negative peak containing the peptide. Molecular weight
analysis by ESI-MS indicated a mass of 746.6 Da for the deglycosylated
peptide. The CAD spectra of the native, acetylated, and methyl
ester-converted peptide demonstrated a sequence of
S-X-X-D-A-T-K, where X is either leucine or
isoleucine. Edman degradation established that the sequence was
S-I-L-D-A-T-K. The presence of 1 mol of alanine corresponds to the
complete reduction of the dehydroalanine formed by
-elimination of
the oligosaccharide moiety at the serine residue under the conditions
employed. Thus, the P40 glycopeptide corresponds to the sequence
S-I-L-D-S*-T-K (), where the starred serine
represents the site of attachment to the oligosaccharide.
Figure 1:
Gel filtration
of tryptic digest of P40 on Toyopearl HW-40S. The column (1.5
234 cm) was equilibrated in 0.1 N acetic acid at a flow rate
of 11.2 ml/h, and fractions of 2.8 ml were collected. Peptides were
detected by absorbance at 230 nm (
) and carbohydrate was
monitored by the phenol-H
SO
assay (
).
The bar marks fractions pooled for further
study.
Figure 2:
Gel
filtration of P40 glycopeptide mixture after release of oligosaccharide
chain by 1 M NaBH in 50 mM NaOH at 45
°C for 16 h. The column (0.9
91 cm) was equilibrated in
0.1% trifluoroacetic acid at a flow rate of 9 ml/h, and fractions of
0.8 ml were collected. Peptides were detected by absorbance at 215 nm
(
) and by reaction of aliquots with Fluram (
); carbohydrate
was monitored by the phenol-H
SO
assay (
).
Filled rectangle, position of original glycopeptide before
carbohydrate release; open rectangle, fractions pooled for
further study.
Preliminary information concerning the Flavobacterium oligosaccharide moiety was obtained as follows. Mild acid
hydrolysis of the free oligosaccharide followed by HPAEC chromatography
on a PA-1 column indicated the presence of glucose, mannose, and
glucuronic acid, plus at least two unknown components in significant
amount. Sugar composition determined by GC and GC/MS analysis of the
TMS and alditol acetate derivatives indicated the presence of mannitol,
presumably the linking sugar of the oligosaccharide, and nearly
equimolar amounts of a 2-O-methyl-deoxyhexose (probably
rhamnose), 2-O-methyl mannose, glucose, and glucuronic acid,
as well as lower amounts of mannose. The presence of mannitol was
verified by HPAEC/PAD chromatography using a MA-1 column. No amino
sugars were apparent using any of the above techniques.
ion, compared with Na
and
K
chelated species, indicated the presence of a basic
moiety in the oligosaccharide, in spite of the absence of amino sugars
in the compositional analysis. The CAD spectrum of this oligosaccharide
(Fig. 3A) exhibited a daughter ion series with prominent
signals at m/z 218, 394, and 556; this daughter ion pattern
was also observed in the CAD spectra of glycopeptides with the 1244-Da
pendant group. Also, the m/z 218 signal indicated the possible
presence of an N-acetylhexuronic acid, and the m/z 218 - m/z 394 difference of 176 indicated the
possible presence of a hexuronic acid or a methyl hexose. To test for
the presence of hexuronic acids the released oligosaccharide was
treated with methanolic HCl, to convert any carboxylate groups into
methyl esters. Molecular mass analysis of the resultant derivative
indicated a mass of 1306 Da. This shift of 42 Da corresponds to the
presence of three carboxylic acid groups in the P40 oligosaccharide. In
Fig. 3B, the CAD spectrum of the methyl ester converted
oligosaccharide, the shift of the m/z 218 ion to m/z 232 supports an assignment of N-acetylhexuronic acid,
whereas the constant difference of 176 between the m/z 232 and
m/z 408 ions argues for the presence of a methyl hexose as an
adjacent sugar group.
Figure 3:
Comparison of the CAD spectra of
(A) the free oligosaccharide from P40 and (B) the
corresponding methyl ester derivative.
The reduced P40 oligosaccharide was
chromatographed directly on a PA-100 column. Because of the three
acidic groups in this oligosaccharide, it was necessary to increase the
standard gradient sodium acetate concentration 2-fold in order to elute
the oligosaccharide in the middle of the profile. Under these
conditions the oligosaccharide was resolved into three glycoforms; a
main component representing 83% of the PAD response, with minor
components accounting for 14 and 3%, respectively. These findings are
consistent with MS analyses that indicated a main component with a mass
of 1264 Da, a minor component with a mass of 1104 Da (-160 Da),
and a trace component with a mass of 1441 Da (+176 Da).
and Endo
F
are O-glycosylated like the P40 protease
described above. A glycopeptide was isolated from Endo F
using the same procedures developed for the P40 protease, albeit
on a smaller scale. Intact enzyme was digested at 50 °C by
thermolysin, and the glycopeptide was isolated by gel filtration in the
nonretarded fractions of a TSK-HW40S column. The glycopeptide was
subjected to oligosaccharide release by alkaline-borohydride followed
by gel filtration on Bio-Gel P-4 to separate the components.
Trifluoroacetic acid hydrolysis of intact glycopeptide followed by
HPAEC chromatography on a PA-1 column gave a pattern of unknown and
known sugars very similar to that of the P40 oligosaccharide. Edman
degradation of the carbohydrate-free peptide established the following
sequence for the 10 cycles programmed: L-Q-D-X-T-K-L-P-G-G.
The X represents a blank cycle which should contain
phenylthiohydantoin-derivative amino-butyric acid, for which there was
no standard. The sequence L-Q-D-T-T-K corresponds to residues
46-51 of the Endo F
molecule
(10) . Mass
spectrometry determined a molecular mass of 2431 Da for the
glycopeptide. The CAD spectrum of this species was consistent with the
peptide L-Q-D-T*-T-K-L-P-G-G-T-G, modified, like the P40 protease, with
a pendant moiety of mass 1244 Da. This MS/MS spectrum exhibited the
characteristic daughter ion pattern of m/z 218, 394, 556
associated with the putative common Flavobacterium glycoform.
The methyl ester converted sample exhibited a mass of 2501 Da,
corresponding to the presence of five carboxylic acids (three on the
oligosaccharide and two on the peptide chain), i.e. the
expected value for this glycopeptide.
has been isolated and cloned in E. coli(10) . A
comparison of the molecular weights of native versus cloned
Endo F
showed a molecular weight difference of +3731
for the native enzyme, suggesting potential multiple glycosylation
sites. Edman data of two tryptic peptides containing Asp-Ser sequences
revealed no phenylthiohydantoin-derivatives for these serines. To
account for the mass difference, we proposed
(10) that native
Endo F
was post-translationally modified during secretion
by glycosylation with an oligosaccharide moiety at three specific
serine residues in an Asp-Ser diad, namely at positions 27-28,
43-44, and 97-98. This proposal was confirmed by the
following mass analysis. Cleavage of Endo F
with cyanogen
bromide followed by reverse phase high performance liquid
chromatography separation on a RP-300 (C8) column yielded four distinct
fractions. Infusion ESI mass analysis of one of these fractions
revealed two nonglycosylated peptides, with respective molecular masses
of 9482 (major product) and 9711 Da (minor product). These values
coincide with the calculated masses for the projected cleavage products
(as homoserine) at amino acid residues 206-288 and 206-290
of Endo F
. The three other CNBr fractions were glycosylated
peptides with masses of 2327, 5369, and 18,460 Da, respectively. These
values each correlate to predicted cleavage residue sites 39-47,
1-38, and 51-205 with the addition of a 1244-Da
oligosaccharide moiety (). For example, the CAD spectrum of
the 2327-Da peptide is consistent with the glycopeptide sequence,
R-W-L-P-D-S*-L-D-Hse. Moreover, each glycopeptide fraction showed the
characteristic oligosaccharide daughter ion series of m/z 218,
394 and 556.
) and it too is fully
substituted. A DT sequence alone, however, is not sufficient to allow
for glycosylation, as evidenced by the finding that the single DT
sequences in Endo F
, Endo F
, Endo
F
, and P40 () do not contain covalently bound
carbohydrate. These substitutions should be evident by MS analysis even
if on less than 10% of the protein. It is of interest to note that all
five nonsubstituted DT sequences are followed by asparagine, aspartic
acid, or glutamine. Further sequence studies will reveal whether these
residues could be a negative signal for glycosylation.
(
)
and glycosylasparaginase
(20) in
E. coli. Neither of these proteins is glycosylated by F.
meningosepticum. In contrast, Endo F
which is heavily
O-glycosylated, is unable to be amplified in E. coli with the pMAL system despite being properly engineered into the
vector. Endo F
appears to be unstable without its normal
carbohydrate complement in this system and is recovered in trace
amounts.
Table:
Consensus sites for glycosylation of F.
meningosepticum proteins
-(N-acetyl-
-D-glucosaminyl)asparagine
amidase; Endo, endo-
-N-acetylglucosaminidase; HPAEC/PAD,
high performance anion-exchange chromatography/pulsed amperometric
detection; GC/MC, gas chromatography/mass spectrometry; ESI,
electrospray ionization; CAD, collisional activated dissociation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.