From the
Glycosphingolipids were isolated from kidneys, urethers, and
bladders (including urethrae) of C3H/HeN mice. Binding was studied of a
clinical isolate and recombinant strains of uropathogenic P-fimbriated
Escherichia coli to these glycolipids. A series of
receptor-active glycolipids with Gal
The binding pattern for mouse kidney glycolipids differed from that
for kidney glycolipids of man and monkey. In particular, the dominant
8-sugar glycolipid in the mouse was not detected in the primates. A
second difference was found in the binding of E. coli to
kidney glycoproteins on blots after electrophoresis; the mouse showed
distinct receptor-active bands while human and monkey did not. These
differences may be of relevance when using the mouse as a model for
clinical urinary tract infection of man.
An important factor in the pathogenesis of bacterial infections
is the ability of the bacteria to adhere to host tissues
(1) ,
often by means of specific binding of bacterial adhesins to host cell
carbohydrates
(2, 3) . P-fimbriated Escherichia coli are important in human urinary tract infections and are known to
adhere to the epithelial cells
(4, 5, 6) . The
adhesion has been shown to depend on Gal
The mouse is
currently used as a model of human urinary tract infection
The animals were anesthetized with
diethyl ether and tissues were removed using sterile instruments.
Kidneys from 60 animals, and urethers, bladders, and urethrae (in one
piece) from 10 animals were obtained. The tissues were immediately
frozen using cryostat spray and stored at -70 °C until
analysis was performed. Before lipid extraction the urethers were
separated from bladders plus urethrae. Lipids were extracted with a
Soxhlet apparatus using chloroform/methanol (2:1 for 24 h, and 1:9 for
24 h). The combined extracts were subjected to mild-alkaline hydrolysis
(0.2
M KOH in methanol, 3 h), dialyzed against water, and
purified on a silica straight-phase column. No further purification of
lipids from urethers and bladders plus urethrae, 2.1 and 2.7 mg,
respectively, was carried out, while the purification of the mouse
kidney preparation continued as described by Karlsson
(19) . The
kidney glycosphingolipids were separated into two main fractions, a
neutral one containing uncharged lipids, 8.5 mg, and an acid fraction
containing gangliosides and other charged lipids.
Fractionation of
neutral glycolipids was achieved by HPLC chromatography on a Spherisorb
S10W silica column, 250
Reference
glycosphingolipids were obtained as follows: globotriaosylceramide,
globoside, and P1 glycolipid from human erythrocytes
(20) ,
Le
For analysis of
glycolipid mixtures using bacteria and antibodies, the glycolipids were
separated on TLC with chloroform/methanol/water, 60:35:8, on aluminum
backed silica nano plates of 0.2-mm phase thickness (Merck, Germany).
Chemical detection was done with anisaldehyde
(25) or
resorcinol staining
(26) . The procedure used for incubation of
TLC plates with labeled biological reagents has been described
(27) . Radioactivity was detected by autoradiography (Kodak
XAR-5 film, Eastman Kodak).
SDS-PAGE (gradient gel, 8-25%) and Coomassie R-350
(PhastGel
For
incubation with bacteria, the nitrocellulose membrane with
electroblotted proteins was preincubated in blocking solution (3%
bovine serum albumin, 50 m
M Tris-HCl, 200 m
M NaCl,
0.1% NaN
The acid glycolipids obtained from the mouse
kidneys showed two distinct binding bands (Fig. 1 B)
which had the same mobility as GL-II and GL-III. No chemical
identification was made of these glycolipids.
HPLC fractions 8
(GL-II), 11 (GL-III), and 15 (GL-IV) were subjected to negative-ion
FAB-MS analysis (Fig. 4, A-C, respectively). The glycolipid in
fraction 8 was composed of five hexoses (of which at least one was
terminal) and two internal N-acetylhexosamines. One of the
N-acetylhexosamines was located next to a terminal hexose. The
FAB-MS obtained from fraction 11 (Fig. 4 B) showed the
presence of five hexoses, two N-acetylhexosamines, and one
Fuc. The Fuc and at least one hexose were terminally placed. This
glycolipid had two dominating ceramide species which differed by two
carbon atoms. The mass spectrum of fraction 15 closely resembled that
of fraction 11. However, fraction 15 contained one dominating ceramide
species with six carbons less than the light ceramide species of
fraction 11. This conclusion was also consistent with the difference in
mobility on the TLC plate. Because of the high intensity of m/z 1736.8 and 1387.8 relative to m/z 1590.8 and 1225.7, it
is probable that the molecule contained two branching points. This
irregular intensity of ions is also seen for fraction 11
(Fig. 4 B).
Gas chromatography-mass
spectroscopic analysis of partially methylated alditol acetates was
performed on the pooled fractions 13-15 in parallel to analysis
of the reference compounds gangliotetraosylceramide and Le
The anomeric region of the
Testing with an anti-Le
The glycolipid mixtures from the mouse were
tested for binding with a human clinical isolate of uropathogenic
E. coli (DS17) as shown in Fig. 6 D. Binding was
seen with DS17 to all three mouse organs, giving the same pattern as
the class II bacteria (Figs. 1 B and 6 B).
DS17-8, an isogenic strain with a frameshift mutation in the
adhesin gene, did not bind to any glycolipid (not shown).
Identification of galabiosylceramide, globotriaosylceramide,
and the Forssman glycolipid in mouse kidneys was achieved using
monoclonal antibodies in combination with TLC mobility ().
The specificity of the antibody against Gal
The MS and NMR data confirm
the suggested structure for GL-III, as being
Gal
The glycolipid GL-II was suggested by Sekine et al. (44) to be defucosylated GL-III, a suggestion that was
confirmed both with FAB-MS analysis and by
Sekine et al. (45, 50) have
shown that DBA mice lack a dominant gene that BALB mice carry. This
means that DBA mice cannot synthesize GL-III but accumulate
Gal
The mixture of
acid glycolipids from C3H/HeN mouse kidneys contained two TLC bands
that were bound by P-fimbriated E. coli. The observation that
anti-Le
In mouse bladder plus urethra
and urether the dominating E. coli-binding activity was found
in the 4-5-sugar region, suggesting the presence of globoside and
the Forssman glycolipid. The presence of the latter was confirmed by
antibody binding. These organs contain one late eluting band, each of
which bound E. coli carrying the class II adhesin (shown in
Fig. 1
). The mobilities of these two bands coincided with those
of GL-II and GL-III in the mouse kidney and also with two acid
glycolipids from mouse kidney.
The binding of P-fimbriated E.
coli to glycoproteins extracted from mouse kidneys was
demonstrated, although it is not clear if this binding is to
carbohydrate or peptide
(53) . The presence of receptor-active
glycoproteins could be of relevance for the use of mice as model
animals for urinary tract infection
(16, 17, 18) . Such proteins, if secreted into
the urine
(54) or added in experimental situations
(18) , may act as competitive inhibitors for E. coli adherence to epithelial cells of the urinary tract. In this
respect, it was shown that Gal
Differences in glycolipid
composition between epithelial and subepithelial cells have been
demonstrated in several cases, e.g. for human urethers
(61) , human intestine (Ref. 21 and references therein), mouse
intestine
(62) , and rat intestine
(56) . The difference
in degree of ceramide hydroxylation as found for mouse kidney
glycolipids (higher for lipids with 4 or less sugars and lower for
lipids with more than 4 sugars) are analogous to data previously
obtained for human kidney
(63, 64) . This may mean that
the short-chain glycolipids predominate in kidney epithelial membranes
rather than in the subepithelial tissue.
E. coli carrying
class II adhesin showed a good binding to GL-II, GL-III, and GL-IV
(I). The class III adhesin, however, behaved differently.
The bacteria with this adhesin bound strongly to the Forssman
glycolipid
(12) but did not bind to GL-II, GL-III, and GL-IV
even though the latter are also elongated globoside structures. A
comparison between the minimum energy conformers of GL-III, the
Forssman glycolipid, and Globo-A reveals the reasons behind these
observations (Fig. 8). Optimal binding of the class III adhesin
is to the Forssman glycolipid (Fig. 8 B) involving the
terminal trisaccharide GalNAc
Chemical shifts, in ppm relative to TMS, of anomeric
protons in the eight-sugar fraction (GL-III) and the non-fucosylated
7-sugar fraction (GL-II). Spectra were run at 30 °C.
The method of
identification of each compound and tissue is indicated with
subscripts.
Data are based on the present
report and published results (12, 65, 67). -, denotes weak or no
binding; +, intermediate binding; and ++, strong
binding.
4Gal in common, previously
shown to be recognized by these bacteria, was identified by use of
specific monoclonal antibodies, fast-atom bombardment and
electron-impact mass spectrometry, and proton nuclear magnetic
resonance spectroscopy: galabiosylceramide (Gal
4Gal
Cer),
globotriaosylceramide (Gal
4Gal
4Glc
Cer), globoside
(GalNAc
3Gal
4Gal
4Glc
Cer), the Forssman glycolipid
(GalNAc
3GalNAc
3Gal
4Gal
4Glc
Cer),
Gal
4GlcNAc
6(Gal
3)GalNAc
3Gal
4Gal
4Glc
Cer,
and
Gal
4(Fuc
3)GlcNAc
6(Gal
3)GalNAc
3Gal
4Gal
4Glc
Cer.
4Gal-containing
glycolipids on the host cells
(5, 7) and a number of
glycolipid isoreceptors with Gal
4Gal in terminal or internal
positions have been identified
(8) . Recently, it was
demonstrated for human red blood cells that Gal
4Gal was absent
from glycoproteins and exclusively present in the glycolipid form
(9) . The molecular genetics and biogenesis of P-fimbriae have
been extensively studied, including the sequence of the three classes
of adhesin, I, II, and III
(10, 11) , which have
slightly different affinities for various isoreceptors of glycolipids
(12, 13, 14, 15) . With respect to human
and dog urinary tract infection, clinical isolates of E. coli differ in adhesin class, which is related to different glycolipid
patterns of the two species. In isolates of human origin, the class II
adhesin predominates. This adhesin preferentially binds to
globoside,
(
)
a glycolipid that dominates in human
urinary tract. E. coli isolates from dog infections, however,
express mostly the class III adhesin, which binds more strongly to the
Forssman glycolipid, a major glycolipid in dog kidney.
(
)(16, 17, 18) . It is therefore of
interest to investigate the carbohydrate basis for E. coli adhesion in this animal. Thus, model C3H/HeN mice were analyzed
for the presence of receptor-active glycoconjugates in various parts of
the urinary tract using both clinical isolates and recombinant strains
of uropathogenic E. coli. It was found that the isoreceptor
pattern differed significantly from that found in human and monkey
urinary tract with respect to both glycolipids and glycoproteins.
Isolation of Glycolipids
Female C3H/HeN mice
(original breeding stock, Charles River Laboratories, Margate, Kent,
UK) were kept at the animal facilities at the Department of Infection
and Immunology, Astra Arcus, S-181 85 Södertälje, Sweden,
where the model infections were performed.The mice were
used at 8-10 weeks of age.
8-mm inner diameter, particle size 10
µm (Phase Separation Ltd., Queensferry, UK). The glycolipid
mixture, 6 mg, was applied and eluted with a gradient starting with
chloroform/methanol/water, 60:35:8 (by volume unless otherwise stated),
and ending with 10:10:3, 2 ml/min. Mild hydrolysis of acid
glycosphingolipids to remove sialic acid was achieved in 1 ml of acetic
acid/water, 1:100 (300 µg of lipid), 100 °C, 1 h.
and Le
from human small intestine
(21) , the Forssman glycolipid
(22) and Le
(23) from dog intestine, and gangliotetraosylceramide from feces
of axenic mouse
(24) . Reference mixtures of glycolipids were
obtained from dog intestine, calf brain, human kidneys, human
erythrocytes (blood group A), and human primary liver cancer, which
contains Le
structures
(20) .
Isolation and Analysis of Mouse and Monkey Kidney
Proteins
Dried residues (20 mg) remaining after the lipid
extraction of C3H/HeN mice and cynomolgus monkey ( Macaca
fascicularis) kidneys were mixed with 1 ml of 50 m
M
Tris-HCl buffer (pH 8.0) containing 2.5% SDS. The mixture was incubated
at room temperature overnight (gentle shaking), heated to 95 °C for
10 min, and then centrifuged at 10,000 g for 10 min.
To extract residual lipids from the SDS-solubilized material, this was
treated with 1-butanol/water, 9:1, three times. Prior to SDS-PAGE of
the supernatant, the samples were diluted to 2-4 mg of protein/ml
(determined by BCA Protein Assay, Pierce), and 2-mercaptoethanol (5%)
was added.
Blue R, Pharmacia) staining were carried out
with a Pharmacia Phast System
(Pharmacia, Sweden)
according to the protocols of the manufacturer. Briefly, samples were
heated to 95 °C for 5 min and centrifuged at 10,000
g for 2 min before electrophoresis to remove unsolubilized material.
After electrophoresis the gel was either stained for protein or sugar
(Glycan detection kit, Boehringer, Germany), or electroblotted onto a
nitrocellulose membrane (0.45 µm) in 20% methanol containing 192
m
M glycine and 25 m
M Tris at pH 8.3.
, pH 8.0) for 1.5 h. The membrane was then
incubated with
S-labeled E. coli in
phosphate-buffered saline (0.14
M phosphate buffer, pH 7.2,
0.14
M NaCl, 5 m
M KCl). After 1.5-2 h the
membrane was washed in 50 m
M Tris-HCl, 0.2
M NaCl,
0.05% Tween 20 (pH 8.0), dried at room temperature, and exposed to
x-ray film overnight.
Chemical Analysis
Mass spectra were obtained with
a ZAB-2F/HF (VG Analytical, Manchester, UK) and a Jeol SX 102A (Jeol,
Tokyo, Japan), both sector instruments, either in the positive-EI or
negative-FAB mode (Xe atom bombardment, 8 kV). Triethanolamine was used
as matrix. Methylation was performed according to Ref. 28 (and
references therein) and gas chromatography-mass spectroscopic analysis
of partially methylated alditol acetates was done according to Refs. 29
and 30. For analysis of partially methylated alditol acetates, a
quadropole MS (Trio-II, VG Masslab, Altricham, UK) was used. The
capillary column was a DB-1, 15 m 0.25 mm, inner diameter,
0.2-µm film thickness (J& Scientific). FAB-MS analysis of
glycolipids was performed directly from the TLC plate as described
(31) with the first MS of a Jeol HX/HX110A instrument. The
conditions for the aquisition of EI spectra were: 70 eV electron
ionization potential, 10 kV accelerating voltage, 300 µA trap
current, ion source temperature 375 °C, and scan time 26 s.
H NMR spectra on deuterium-exchanged glycolipid
fractions were acquired at 11.74 telsa on a JEOL ALPHA-500 (Jeol,
Tokyo, Japan). Samples were dissolved in 0.5 ml of
Me
SO/D
O, 98:2, and spectra recorded at 30
°C with a digital resolution of 0.4 Hz. Chemical shifts are given
relative to tetramethylsilane using the internal solvent peak. Nuclear
Overhauser enhancements were measured using the standard software of
the instrument. The recycle time was at least five times the
longitudinal relaxation time ( T
).
Growth and Labeling of Bacteria
Four different
P-fimbriated E. coli strains were used, HB101/pPIL291-15
(a gift from Dr. I. van Die, Vrije Universiteit, Amsterdam, The
Netherlands, and Dr. B. Westerlund, University of Helsinki, Finland
(32) ), HB101/pPAP5
(33) , HB101/pDC1
(34) ,
HB101/pPAP601
(35) , and DS-17 (a gift from Dr. K. Tullus, S:t
Görans barnsjukhus, Stockholm, Sweden
(36) ). The bacteria
were cultivated on colonization factor agar plates supplemented with
[S]methionine (400 µCi/10 ml, Amersham
International, UK) at 37 °C overnight. They were collected by
centrifugation, washed twice with phosphate-buffered saline, and
resuspended in phosphate-buffered saline to approximately 1
10
colony-forming units/ml. The bacteria were diluted to an
activity of approximately 1
10
cpm/ml.
Antibodies
The following mouse monoclonal
antibodies were used: anti-Le(BL-G15, Monosan, Bio-Zak,
Järfälla, Sweden), three antibodies binding to terminal
Gal
4Gal sequences, pk002, P001, and MC2102 = 87:5 (all
obtained from Accurate Chemical & Science Corp., New York); and
anti-Forssman (MAS033b, Seralab, Göteborgs Termometerfabrik,
Sweden). The secondary antibodies used (rabbit anti-mouse
immunoglobulins, Z109, DAKO A/S, Denmark) were labeled with
I
(37) .
Molecular Modeling
Minimum energy conformers of
the GL-III glycolipid, identified below as
Gal4(Fuc
3)GlcNAc
6(Gal
3)GalNAc
3Gal
4Gal
4Glc
Cer,
were calculated within the Biograf molecular modeling program
(Molecular Simulations Inc.) using the Dreiding-II force field
(38) on a Silicon Graphics 4D/35TG workstation. Partial atomic
charges were generated using the charge equilibration method
(39) , and a distance dependent dielectric constant
= 3.5r was used for the Coulomb interactions. In addition a
special hydrogen bonding term was used in which D
was set
to 4 kcal/mol.
Comparison of Glycolipid Pattern of Mouse Urinary Tract
Organs
The acid and neutral glycosphingolipids of C3H/HeN mouse
kidneys were isolated and purified separately. TLC separations of the
neutral glycosphingolipids visualized chemically with anisaldehyde are
shown in Fig. 1 A. For comparison, glycolipids from human
kidneys, as well as purified globoside, were included in the
chromatogram. The neutral glycolipids from mouse kidneys are dominated
by a very slow-moving band with more than 6 sugars (GL-III,
Fig. 1
) although several more weakly-staining bands, particularly
in the 3-4-sugar region, are also seen.
Figure 1:
Binding of P-fimbriated E.
coli to glycosphingolipids isolated from mouse urinary tract
organs. Plate A, anisaldehyde staining of neutral glycolipids
from human kidney, mouse kidney, mouse urether, mouse bladder plus
urethra, and globoside. Plate B, binding of E. coli (HB101/pPIL291-15) to purified glycolipids, mixtures of
neutral ( n), and mixtures of acid ( a) glycolipids as
indicated. The amount of each glycolipid mixture is shown as
µg/lane in parentheses. Numbers in italics indicate the approximate position of glycolipids with different
numbers of sugars. The arrow shows the direction of
elution.
The mixture of acid
glycolipids from mouse kidneys and the crude preparations of lipids
from urethers and bladders plus urethrae were used for bacterial
binding studies. The latter preparations were not purified sufficiently
for chemical staining (the bands seen in Fig. 1of urethers and
bladders plus urethrae do not have the green color characteristic of
glycoconjugates stained with anisaldehyde).
Mapping of Receptors for P-fimbriated E.
coli
Initially P-fimbriated E. coli, HB101/pPIL291-15, which binds very strongly to
Gal4Gal-containing glycolipids, was used to screen the glycolipid
preparations as shown in Fig. 1 B. Globotriaosylceramide,
globoside, and the Forssman glycolipid were applied in the first three
lanes as references. The bacteria bound strongly to neutral glycolipids
from mouse kidneys in the 4-5-sugar region. In addition, four
slower moving bands were strongly bound by the bacteria (GL-I, GL-II,
GL-III, and GL-IV). The urethers and bladders plus urethrae both
contained binding glycolipids in the 4- and 5-sugar regions, while in
the preparation of bladders plus urethrae weak binding is also seen to
a band in the 3-sugar region and also to a band comigrating with GL-II.
For the urether a weak binding band was obtained which had the same
mobility as GL-III.
Identification of Binding
Glycosphingolipids
Antibodies were used to reveal the presence
of terminally placed Gal4Gal structures in glycolipids from mouse
kidneys. In the 2-sugar region, the binding of the antibody MC2001
showed that Gal
4Gal
Cer was present in mouse kidneys (Fig.
2 A). The three bands probably differed in ceramide
composition. Dog intestine was included in the analysis as a positive
reference.
(
)
In the 3-sugar region, the antibody
pk002 bound weakly, Fig. 2 B, but strongly to the
reference globotriaosylceramide. The lack of a clear binding by
monoclonal antibody P001 indicated that the P1 antigen is absent from
mouse kidneys (Fig. 2 C). Further investigation with an
antibody specific for the Forssman glycolipid (Fig. 2 D),
showed that this glycolipid was present in the mouse but not in human
kidneys. A weak band was also detected in the preparation of mouse
bladders plus urethrae but not in the urethers.
Figure 2:
TLC
separation of glycolipids from various sources and analysis with
monoclonal antibodies. The following antibodies were used: A,
MC2102, which detects Gal4Glc
Cer; B, pk002, which
binds globotriaosylceramide, and more weakly the P1 antigen;
C, P001, which binds the P1 antigen; and D, MAS033b,
which binds the Forssman glycolipid. The amount of each glycolipid
mixture is shown as µg/lane in parentheses. Numbers in italics indicate the approximate position of
glycolipids with different numbers of
sugars.
To elucidate the
structure of GL-II, GL-III, and GL-IV in the mouse kidneys, the neutral
glycolipid mixture was subjected to HPLC fractionation on a silica
column. The separation conditions were chosen to optimize separation of
slow-moving compounds. Analysis by TLC of the fractions obtained is
shown in Fig. 3. Fractions 4 and 5 were analyzed by FAB-MS in two ways
after separation by TLC. First, the 3- and 4-sugar regions were scraped
off the plates separately, extracted in chloroform/methanol, 2:1, and
their mass spectra were collected. Second, they were analyzed by direct
TLC FAB-MS. The 2-sugar region gave spectra consistent with
(Hex)Cer, the 3-sugar region with (Hex)
Cer, and
the 4-sugar region gave spectra showing that the glycolipid was
HexNAcHexHexHexCer. All three compounds had similar ratios of
hydroxylated to unhydroxylated ceramide (1:1).
Figure 4:
MS analysis of mouse kidney glycolipids.
Negative-ion FAB-MS of native glycolipids are shown in A-C,
and the positive-ion EI-MS of a methylated glycolipid in D.
The HPLC fractions analyzed were: A, 8; B, 11;
C, 15; and D, 11-12. Peaks labeled with an
asterisk originated from the matrix, triethanolamine.
Tentative theoretical fragmentation patterns and molecular weight are
shown above each spectrum.
Fractions 11 and 12 were pooled and
analyzed by EI-MS as the methylated derivative
(Fig. 4 D). Molecular ions of m/z 2347.1 and
2375.2 were obtained which correspond to ceramides with sphingosine and
22- and 24-carbon fatty acids, respectively. These ceramides are also
seen at m/z 632.6 and 660.6. Large peaks appear at 638.3,
1087.5, and 1291.5, which correspond to the following oxonium ions:
HexHexNAcFuc, (Hex)(HexNAc)
Fuc, and
(Hex)
(HexNAc)
Fuc, respectively. These three
oxonium ions, [Ox]
, lose either Fuc
[Ox-Fuc+H]
or Hex
[Ox-Hex+H]
giving rise to the
following sets of ions: m/z 450.2 and 420.2 from 638.3,
m/z 899.4 and 869.4 from 1087.5, and m/z 1073.5 and
1103.5 from 1291.5. Ions from terminal Hex (219.1 and 219.1-32
= 187.1) and Fuc (189.1 and 189.1-32 = 157.1) were
also obtained. The presence of a set of m/z at 692.6 (Cer),
2173.4 (M-Hex), and 2203.1 (M-Fuc), indicates that minor amounts of the
glycolipid might carry phytosphingosine.
.
The following monosaccharides were identified; terminal Gal and Fuc,
4-substituted Gal and Glc, 3-substituted Gal, and 3,4-substituted
GlcNAc. A second di-substituted HexNAc, with a longer retention time,
but similar MS, was obtained.
H NMR spectrum (Fig. 5 C, Table I) of GL-III (HPLC
fraction 10) shows two
-signals ( J
4 Hz) at 4.82 and
4.80 ppm ( a and b in Fig. 5 C)
corresponding to Fuc
3 and Gal
4, respectively. These signals
can be conclusively assigned from the nuclear Overhauser enhancement
(NOE, magnetic dipole coupling through space) experiments. The NOE
between the 4.80 ppm signal and a typical Gal H4 signal at 3.82 ppm
(40, 41) , as well as a quartet at 3.76 ppm (H2 of
Gal
), are shown in Fig. 5 B. The Fuc H5 gave rise to a
quartet of 4.67 ppm ( c in Fig. 5 C). Of the two
HexNAc
signals at 4.61 and 4.49 ppm ( d and e in
Fig. 5C) the former showed NOEs (Fig. 5 A)
to Gal
H5 (4.19 ppm) and Gal
H4 (3.98 ppm) and also a large
NOE to a signal at 3.59 ppm. This corresponds well with published
values for the Gal
H3
(40, 41) , and thus confirms
the 4.61 ppm signal as arising from the GalNAc
3 of a globo-core
structure. The signal at 4.49 ppm is within the region for GlcNAc
shifts, see for example, Ref. 42. Two well separated Gal
signals
were seen at 4.29 and 4.26 ppm ( f and g in
Fig. 5C), respectively, the latter arises from the
internal Gal
4 linked to Glc, which in turn was seen at 4.19 ppm,
overlapping two other signals. The former Gal is consistent with a
terminal Gal
4 of a Le
terminal
(43) . The other
signals at 4.19 ppm (overlapping the Glc anomer) were triplets from the
H5 of Gal
and a terminal Gal
3
(41) . NOEs were
measured from all anomeric protons (not shown), and they confirmed the
saccharide sequence as determined above. Since most non-anomeric
signals are unassigned, the NOE analysis was based on the multiplicity
of signals and their chemical shifts compared to model structures. For
most glycosidic linkages there was no uncertainty. Only the linkage
between the terminal Le
fragment and the globo-core was
unclear. There were NOEs (not shown) into the region of H6 protons but
also an NOE to a typical Gal H4 signal, all of moderate strength. Thus,
the results from the NMR indicate that the structure is
Gal
4(Fuc
3)GlcNAc
6/4(Gal
3)GalNAc
3Gal
4Gal
4Glc
Cer.
This spectrum corresponds well with that published by Sekine et al. (44) . However, molecular modeling indicates that a
4-linked Le
determinant most likely can be excluded since
the strong NOE expected between the GlcNAc
H1 and H4 of
GalNAc
is in this case not observed, whereas in the 6-linked form
several conformers are compatible with the NOE effects.
Figure 5:
Proton-NMR spectra of glycolipid fraction
10. C, the normal spectrum; B, NOE difference
spectrum after irradiation at the 4.80 ppm signal; and A, NOE
difference spectrum after irradiation at the 4.61 ppm signal. In C the following letters are used to assign signals: a,
Fuc3 H1; b, Gal
4 H1; c, Fuc
3 H5;
d, GalNAc
3 H1; e, GlcNAc
4/6 H1; f,
Gal
4 H1; g, Gal
4 (internal) H1; h-j,
Gal
3 H1, Glc
1 H1, and Gal
4 H5, and k, Gal
4
H4. All spectra were recorded at 30 °C, and in the NOE difference
spectra the dispersion-like remains of the residual water peak have
been baseline corrected.
GL-II (HPLC
fraction 8) was available in very small amounts and therefore only a
single H NMR spectrum could be acquired. The spectrum
obtained (not shown) contained the same globo-core signals
() as GL-III, but the Fuc signals (H1, H5, and methyl) were
missing. In accordance with this, the GlcNAc (to which the Fuc was
linked in fraction 10) anomer signal had shifted upfield to 4.39 ppm,
whereas the terminal Gal
4 had shifted upfield to 4.21 ppm
(45) . An additional component was present in fraction 8, which
had a HexNAc signal at 4.57 ppm. Also the EI-MS of methylated fraction
8 (not shown) contained an ion ( m/z = 260, typical for
terminal HexNAc) that supports the presence of a second component.
antibody showed strong staining
of one band in fractions 10 and 15 (Fig. 3 C). A second
weak band was also seen in fraction 15 and two weak bands in fraction
8. Several binding bands were present in the Le
-containing
reference, human liver tumor. The antibody did not cross-react with
Lc
, nLc
, Le
, Le
, or
Le
(not shown). When testing the mouse kidney gangliosides,
neither the mixture of acid glycolipids as such, nor the desialylated
mixture, were bound by the anti-Le
antibody (data not
shown).
Figure 3:
HPLC
separation of mouse kidney glycolipids. A, TLC separation of
glycolipids before HPLC; B and C after HPLC
fractionation. In A and B chemical detection was used
(anisaldehyde), and in C binding by anti-Leantibody.
Binding of E. coli with Class I, II, and III Adhesins to
Mouse Glycolipids
E. coli strains expressing the three
different classes of P-adhesins were tested for binding to mouse
urinary tract glycolipids together with human kidney glycolipids and
purified globotriaosylceramide, globoside, and the Forssman glycolipid
(Fig. 6, A-C, and Tables II and III). E. coli carrying the class I adhesin bound strongly to
globotriaosylceramide of human kidney and to globoside of human and
mouse kidney (Fig. 6 A). It also bound to a TLC band in
the 4-sugar region of the mouse bladders plus urethrae preparation.
Weaker binding to GL-I and GL-III in mouse kidney was also found. The
class II bacteria, which bound equally strongly to
globotriaosylceramide, globoside, and the Forssman glycolipid on TLC,
bound to the same structures as the class I carrying bacteria. In
addition, it bound to bands in the mouse kidney and in the bladders
plus urethrae that had slightly lower mobility than globoside. The
binding to the GL-I, GL-II, GL-III, and GL-IV glycolipids seen for
HB101/pPIL291-15 was also observed for this class II E.
coli.
Figure 6:
Binding of P-fimbriated E. coli to glycolipid mixtures from urinary tract organs of C3H/HeN mice.
The glycolipids were separated by TLC before incubation with
P-fimbriated bacteria. The following E. coli isolates were
used: A, class I, HB101/pPAP5; B, class II,
HB101/pDC1; C, class III, HB101/pPAP601; and D, the
clinical isolate DS-17. The amount of each glycolipid mixture is shown
as µg/lane in parentheses. Numbers in italics indicate the approximate position for glycolipids with different
numbers of sugars.
The class III adhesin carrying E. coli, however,
showed a very different binding pattern (Fig. 6 C). It
clearly preferred the Forssman glycolipid to the shorter
Gal4Gal-containing glycolipids. In addition to the Forssman
glycolipid of the mouse kidneys, it also bound to bands with the same
mobility in both the urether and the bladder plus urethrae
preparations. However, GL-II, GL-III, and GL-IV of mouse kidneys were
only very weakly bound.
Binding of E. coli to Proteins of Mouse
Kidneys
Proteins extracted from mouse and monkey kidneys were
resolved by SDS-PAGE and stained with Coomassie Blue (Fig.
7 A). Specific staining of sugars indicated that all protein
bands were glycosylated (not shown). Incubation of the blotting
membrane with E. coli pDC1, which carries a class II adhesin
(Fig. 7 B), showed that bacterial binding to components in both
the monkey and the mouse preparations occurred. However, when the SDS
extracts were further treated with butanol to extract remaining lipids,
only binding to the mouse kidney proteins was seen.
Molecular Modeling
The glycosidic dihedral angles
for the globoside part of GL-III were found to be very similar to
earlier published values
(46) as were those for the terminal
Ledeterminant
(47) . In the case of the
GlcNAc
6(Gal
3)GalNAc
structural element, energy minima
were located by varying the dihedral angles of GlcNAc
6GalNAc
while in turn keeping constant one of the three staggered conformations
of the C5-C6 bond of GalNAc
as well as dihedral angles of
Gal
3GalNAc
. The global minimum energy structure was found to
be backfolded with the Le
determinant pointing toward the
ceramide. However, this conformer may be excluded on the grounds that
the Le
determinant would be unavailable for antibody
binding as well as being too close to the membrane surface in a
membrane-bound environment. The next energetically favored conformer
(0.8 kcal/mol above the global minimum and 2.6 kcal/mol below
subsequent conformers) shows an extended conformation (see Fig.
8 A) consistent with both binding and
H NMR data
given above.
4Gal
Cer (MC2102)
and globotriaosylceramide (pk002) has been studied
(
)
and the anti-Forssman antibody has a strict specificity
(48) . Gal4GalCer has earlier been identified (the anomeric
configuration, however, was not determined) in kidneys of some mouse
strains
(49) . The presence of globotriaosylceramide and
globoside was confirmed by FAB-MS analysis of the 3- and 4-sugar
region. In the 2-sugar region, lactosylceramide was probably present in
addition to the galabiosylceramide. Due to the low abundance of GL-I,
no chemical identification was performed. The anti-Le
antibody bound to GL-II, GL-III, and GL-IV. Since both MS and NMR
data showed that GL-II lacked Fuc, this antibody seems to be able to
cross-react with Gal
4GlcNAc
- when present in
Gal
4GlcNAc
6(Gal
3)GalNAc
3Gal
4Gal
4Glc
Cer
but not in nLc
. The minor, unidentified, component that was
present in this sample could also be responsible for this antibody
binding, although being devoid of Fuc.
4(Fuc
3)GlcNAc
6(Gal
3)GalNAc
3Gal
4Gal
4Glc
Cer.
FAB analysis of native GL-III showed uneven appearance of fragment ions
that indicate the presence of two branches. Further evidence for such
branching was obtained from EI-MS, where fragment ions from terminal
Hex, HexHexNAcFuc, and (Hex)
(HexNAc)
Fuc were
obtained while ions from HexHexNAc and Hex(HexNAc)
Fuc or
(Hex)
HexNAcFuc were absent. The binding position x in -GlcNAc
x(Gal
3)GalNAc
3- was, however, more
difficult to identify. But the combined
H NMR and molecular
modeling results point rather strongly to a
6-linked branch in
agreement with the suggestion of Sekine et al. (44) ,
who based their conclusion on data from partially methylated alditol
acetates.
H NMR in the
present study.
3Gb
Cer. Apparently the mice used here, C3H/HeN,
resemble BALB mice in their expression of GL-III.
antibodies did not bind to these compounds excludes
the possibility that the separation of the kidney lipids into neutral
and acid components was incomplete. Mono- and disialylated
Gal
3Gb
Cer have been identified both in DBA mouse
kidneys
(51) and in human vagina
(52) . However, Sekine
et al. (51) found a correlation between the presence
of sialylated Gal
3Gb
Cer and absence of
Le
-elongated Gal
3Gb
Cer. Therefore,
sialylated Gal
3Gb
Cer would not be expected to exist in
C3H/HeN mice. In addition, it has been shown that gangliosides that are
bound by P-fimbriated E. coli are present in monkey urinary
tract tissues.
(
)
4Gal structures were apparently
absent from secreted material in human urine
(54) , indicating
that E. coli may have selected for this specificity to assure
adhesion to cell membranes. Based on the results of Fig. 7, it is
not unlikely that receptor-active glycoproteins are secreted into mouse
urine.
Figure 7:
Binding of P-fimbriated E. coli to protein extracts from mouse and monkey kidneys. In A,
the SDS-PAGE gel was stained with Coomassie Brilliant Blue, and in
B, the autoradiogram after binding of S-labeled
bacteria (HB101/pDC1) to an electrophoretic blot on nitrocellulose is
shown. 2-4 µg of protein was applied per lane. The
numbers to the left denote apparent molecular weights
(kDa).
When using animal models for experiments with human pathogens
it is important to know if binding-active glycoconjugates are present
in the target tissue. Differences in glycoconjugate composition of the
same organ from different species
(20, 55, 56) and also between strains of the same species are well known
(49, 57) . It may be more difficult to demonstrate the
presence of receptors on individual target cells with chemical
techniques. However, alternative methods exist, using tissue sections
and binding with antibodies, bacteria, or lectins
(58, 59, 60) .
3GalNAc
3Gal
4
(12, 65) . The strong binding of the class III adhesin
(12) to Globo-A (I) is due to the fact that the
terminal blood group A determinant does not occlude the binding
epitope. Furthermore, the Fuc
2 residue of Globo-A occupies
approximately the same position as the GalNAc
3, including the
acetamido moiety, in the Forssman glycolipid, thus giving rise to only
minor steric interferences. For the GL-III glycolipid it is seen that
the Gal
3 residue is not expected to be the major
(66) cause for the loss of adhesin binding since it occupies a
position close to that of the GalNAc
3 residue in the Forssman
glycolipid, a conclusion which is supported by the relatively small
reduction in binding affinity seen for Gal
3-substituted globoside
(12) . The Le
determinant of GL-III
(Fig. 8 A), however, interferes directly with adhesin
accessibility due to its attachment to the central residue of the
trisaccharide epitope and thus explains the complete loss of binding in
this case. The most probable conformation of GL-III, as found here, is
also compatible with retained binding of the class II adhesin whose
optimal binding epitope includes the Gal
4Gal
4 segment of
globoside with some involvement of the terminal GalNAc
3
(12) .
Figure 8:
Minimum energy conformers of
Gal4(Fuc
3)GlcNAc
6(Gal
3)GalNAc
3Gal
4Gal
4Glc
Cer
( A), GalNAc
3GalNAc
3Gal
4Gal
4Glc
Cer
( B), and
GalNAc
3(Fuc
2)Gal
3GalNAc
3Gal
4Gal
4Glc
Cer
( C). Both side and top views are shown in which the methyl
carbons of Fuc, GalNAc, and GlcNAc are black for orientation.
A comparison of the terminal trisaccharide
(GalNAc
3GalNAc
3Gal
4) of the optimal receptor for the
class III adhesin (the Forssman glycolipid, B), which also
constitutes the binding epitope, with the corresponding part of Globo-A
( C) shows that the fucose residue assumes a position very
close to that of the terminal GalNAc
3 in the Forssman glycolipid,
resulting in only a minor steric hindrance, thus explaining the strong
binding found for this isoreceptor. In GL-III ( A), however,
the 6-linked Le
determinant interferes with the central
part of the binding epitope causing complete loss of class III binding.
The class II binding epitope (GalNAc
3Gal
4Gal
4) is not
affected, on the other hand, since an extended conformation of the
6-linked branch leaves the epitope essentially
unobstructed.
Table: Proton-NMR data of GL-II, GL-III, and
references
Table: Glycosphingolipids
in C3H/HeN mouse kidneys, urethers, and bladders plus urethra that are
bound by P-fimbriated E. coli of class II
Table:
Comparison of class I, II, and III
adhesins in their relative affinities for Gal4Gal-containing
glycosphingolipids on TLC plates
, GalNAc
3Gal
4Gal
4Glc
Cer;
Cer, ceramide; d18:1-24:0, ceramide with sphingosine and a
saturated fatty acid chain; galabiosylceramide, Gal
4Gal
Cer;
globotriaosylceramide, Gb
, Gal
4Gal
4Glc
Cer;
Forssman glycolipid,
GalNAc
3GalNAc
3Gal
4Gal
4Glc
Cer; P1 antigen,
Gal
4Gal
4GlcNAc
3Gal
4Glc
Cer; Globo-A,
GalNAc
3(Fuc
2)Gal
3GalNAc
3Gal
4Gal
4Glc
Cer;
gangliotetraosylceramide, GgO
,
Gal
3GalNAc
4Gal
4Glc
Cer; lactotetraosylceramide,
Lc
, Gal
3GlcNAc
3Gal
4Glc
Cer;
lactoneotetraosylceramide, nLc
,
Gal
4GlcNAc
3Gal
4Glc
Cer; Le
,
Gal
4(Fuc
3)GlcNAc
3Gal
4Glc
Cer; Le
,
Fuc
2Gal
4(Fuc
3)GlcNAc
3Gal
4Glc
Cer;
Le
, Gal
3(Fuc
4)GlcNAc
3Gal
4Glc
Cer;
Le
,
Fuc
2Gal
3(Fuc
4)GlcNAc
3Gal
4Glc
Cer; HPLC,
high-pressure liquid chromatography; NMR, nuclear magnetic resonance;
NOE, nuclear Overhauser enhancement; MS, mass spectrometry; FAB,
fast-atom bombardment; EI, electron impact; GC, gas chromatography;
PAGE, polyacrylamide gel electrophoresis.
1-4Gal Adhesins of Uropathogenic Escherichia coli, Ph.D. thesis, University of Umeå, Sweden
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.