Báo cáo khóa học: Differential carbohydrate epitope recognition of globotriaosyl ceramide by verotoxins and a monoclonal antibody pdf

Eur. J. Biochem. 271, 405–417 (2004) Ó FEBS 2004

doi:10.1046/j.1432-1033.2003.03941.x

Differential carbohydrate epitope recognition of globotriaosyl
ceramide by verotoxins and a monoclonal antibody
Role in human renal glomerular binding
Davin Chark1,2, Anita Nutikka1, Natasha Trusevych1,3, Julia Kuzmina1,3 and Clifford Lingwood1,2,3
1

Research Institute, Division of Infection, Immunity, Injury and Repair, The Hospital for Sick Children, Ontario, Canada;
Department of Laboratory Medicine & Pathobiology and 3Department of Biochemistry, University of Toronto, Canada

2

The role of renal expression of the glycosphingolipid
verotoxin receptor, globotriaosylceramide, in susceptibility
to verotoxin-induced hemolytic uremic syndrome is unclear.
We show that a single glycosphingolipid can discriminate
multiple specific ligands. Antibody detection of globotriaosylceramide in renal sections does not necessarily predict verotoxin binding. The deoxyglobotriaosylceramide
binding profile for verotoxin 1, verotoxin 2 and monoclonal
anti-globotriaosylceramide are distinct. Anti-globotriaosylceramide had greater dependency on the intact a-galactose
and reducing glucose of globotriaosylceramide than verotoxin 1, while verotoxin 2 was intermediate. These ligands
differentially stained human kidney sections. Glomerulopathy is the primary verotoxin-associated pathology in
hemolytic uremic syndrome. For most samples, verotoxin 1
immunostaining within adult glomeruli was observed (type
A). Some samples, however, lacked glomerular binding
(type B). Anti-globotriaosylceramide (and less effectively,
verotoxin 2) stained all glomeruli. Verotoxin 1/anti-globotriaosylceramide tubular staining was comparable. Type B
glomerular/tubular globotriaosylceramide showed minor,
but significant, fatty acid compositional differences. Verotoxin 1 type B glomerular binding became evident following

pretreatment with cold acetone, or methyl-b-cyclodextrin,
used to deplete cholesterol. Direct visualization, using fluorescein isothiocyanate-verotoxin 1B, showed paediatric,
but no adult glomerular staining; this was confirmed by
anti-fluorescein isothiocyanate immunostaining. Acetone
induced fluorescein isothiocyanate-verotoxin 1B glomerular
staining in type A, but poorly in type B samples. Comparison of fluorescein isothiocyanate-verotoxin 1B and native
verotoxin 1B deoxyglobotriaosylceramide analogue binding
showed an alteration in subspecificity. These studies indicate
a marked heterogeneity of globotriaosylceramide expression
within renal glomeruli and differential binding of verotoxin 1/verotoxin 2/anti-globotriaosylceramide to the same
glycosphingolipid. Verotoxin 1 derivatization can induce
subtle changes in globotriaosylceramide binding to significantly affect tissue binding. Heterogeneity in glomerular
globotriaosylceramide expression may play a significant
(cholesterol-dependent?) role in determining renal pathology
following verotoxemia.

The glycosphingolipid (GSL) globotriaosylceramide
(Gala1–4Galb1–4glucosyl ceramide, Gb3), is the functional
receptor for the verotoxins (VTs, also termed Shiga toxins,
or Stx’s) produced by Escherichia coli [1]. Gastrointestinal
infection with E. coli producing such toxins can result in
hemorrhagic colitis which may progress to hemolytic uremic
syndrome (HUS), particularly in young children [2]. Gb3
is also CD77, a differentiation marker of human germinal
centre B cells [3], the Pk blood group antigen [4] and a

marker of certain tumour cells [5–8], such that VT1 can be
used as an antineoplastic agent [9,10]. A fraction of Gb3
is found in cell surface cholesterol-enriched lipid microdomains – ÔraftsÕ [11,12]. This organization appears to

regulate the intracellular routing of the verotoxin–Gb3
complex [13], such that protein synthesis can be induced,
rather than inhibited, by VT for cells in which Gb3 is not
raft-associated. As for all GSLs, heterogeneity of fatty acid,
and to a lesser extent, of sphingoid base, generates a
spectrum of lipid isoforms of Gb3. Both the lipid structure
[14–17] and the local membrane phospholipid microenvironment [18] impinge upon verotoxin–Gb3 binding. In
addition, there are variants of verotoxin, primarily VT1,
VT2 and VT2c [19], which differentially bind to the
carbohydrate moiety of Gb3, as determined by differential
binding to deoxyGb3 analogues [20] and Gb3 lipid isoforms
[14,16]. These variants are also differentially involved in
disease [21,22].
Gb3 is also involved in the signal transduction of CD19
[23] and a2-interferon [24,25], due to N-terminal sequence
similarity between the verotoxin B subunit and the

Correspondence to C. Lingwood, Research Institute, Division of
Infection, Immunity, Injury and Repair, The Hospital for Sick
Children, Ontario M5G 1X8, Canada.
Fax: + 1 416 813 5993, Tel.: + 1 416 813 5998,
E-mail:
Abbreviations: AP, alkaline phosphatase; FITC, fluorescein isothiocyanate; Gb3, globotriaosylceramide; GSL, glycosphingolipid; NGS,
normal goat serum; SGC, sulfogalactosyl ceramide; TBS, Trisbuffered saline; VT, verotoxin; VTEC, verotoxigenic E. coli.
(Received 15 October 2003, accepted 24 November 2003)

Keywords: Membrane glycosphingolipid receptor; lipid
isoforms; carbohydrate presentation; hemolytic uremic
syndrome; cholesterol.

Ó FEBS 2004

406 D. Chark et al. (Eur. J. Biochem. 271)

N-terminus of CD19 [26] and the a2-interferon receptor [27].
Verotoxin B subunit and monoclonal anti-Gb3 can induce
apoptosis [28,29], particularly in lymphoid cells [30]. Tissue
screening with monoclonal anti-Gb3 indicates a wider Gb3
distribution [31,32] than inferred from pathogenesis of HUS
or VT tissue targeting and pathology in animal models [33].
We have shown previously the expression of Gb3 within
the renal glomerulus, as monitored by the binding of
fluorescein isothiocyanate (FITC)-labelled VT1, correlates
with the age-related incidence of HUS following verotoxigenic E. coli (VTEC) infection [34]. Ninety per cent of HUS
cases occur in children under 3 years of age. We showed VT
binding in paediatric glomeruli, whereas, little or no binding
was seen in the glomeruli of adult human renal sections
[34]. The distribution of Gb3 was thus implicated in the
epidemiology and hence, etiology, of VT-induced disease
[35]. However, this differential renal glomerular Gb3
expression has recently been questioned. At 4 °C, VT1
binds similarly to both adult and paediatric glomeruli [36].
Anti-Pk is used to type red cells, yet the binding of VT
to human red cells is only observed at 4 °C and is not
significant under physiological temperature conditions [37].
We therefore questioned whether anti-Gb3 and VT bind
Gb3 in the same manner and whether any differences could
shed light on the Gb3 expression in the human kidney in
relation to VT-induced disease. Our present studies validate

our previous age-related binding [34], but show a marked
difference in Gb3 recognition according to ligand. This work
adds a new, clinically relevant, dimension to the lipidmediated heterogeneity of Gb3 recognition, which may
provide a precedent for other GSL receptor functions.

Materials and methods
VT1, VT1B and VT2 were purified as described [38–40]. Rat
mAb anti-Gb3 (38.13) hybridoma was a generous gift from
J. Wiels (Institut Gustave Roussy, Villejuif, France).
Culture supernatant was affinity purified using Gb3-celite
[41]. SULF1 mAb anti-sulfogalactosyl ceramide (SGC)
culture supernatant [42] was kindly provided by P. Fredman
(Department Clinical Neuroscience, University of Goteborg, Sweden). Rabbit anti-VT2e was supplied by C. Gyles
(Department Microbiology, University of Guelph, Ontario).
Polyclonal rabbit anti-VT1B 6869 Ig was prepared in our
laboratory. Biotin-conjugated goat anti-(rat IgM) serum
and biotin-conjugated goat anti-rabbit Ig were from Jackson Immunoresearch Laboratories (West Grove, PA,
USA). Horseradish peroxidase (HRP)-conjugated goat
anti-rabbit and HRP-conjugated goat anti-mouse Igs were
purchased from Sigma. StreptABComplex/alkaline phosphatase (AP) were from Dako (Carpinteria, CA, USA).
Avidin/Biotin blocker was from Vector (Burlingame, CA,
USA) True Blue Peroxidase substrate and HistoMark
Red were from Kirkegaard & Perry Laboratories (KPL,
Gaithersburg, MD, USA).
Receptor enzyme linked immunosorbant assay (RELISA)
The wells of a 96-well Evergreen microtitre plate (DiaMed
Laboratory Supplies Inc. Mississauga, ON, CA) were
incubated with 150 lL aliquots of 5% (w/v) BSA in
10 mM NaCl/Pi (pH 7.2) for 2 h at room temperature,

washed three times with ddH2O and allowed to dry
completely. Ten microliters of Gb3 (100 lgỈmL)1) in an
85% ethanolic solution was then added in duplicate to each
well and dried overnight. After blocking with 1% BSA/
NaCl/Pi (150 lL per well) for 1 h, wells were successively
treated with 50 lL each of serially diluted VT1, VT2, or
mAb 38.13 in 1% BSA/NaCl/Pi; corresponding antibodies
(rabbit anti-VT1B 6869, rabbit anti-VT2e, or biotin-conjugated goat anti-rat IgM, respectively, all at 1 : 2000 in 1%
BSA/NaCl/Pi); and finally the corresponding horseradish
peroxidase-conjugated goat anti-rabbit or HRP-conjugated
streptavidin, both at 1 : 2000 in 1% BSA/NaCl/Pi). Each
step consisted of a 1 h incubation (100 lL per well)
followed by two washings with 1% BSA/NaCl/Pi. After
one final wash with NaCl/Pi, freshly prepared 2,2Â-azino-bis3-ethylbenzthiazoline-6-sulfonic acid solution (ABTS;
Sigma 0.5 mgặmL)1 ABTS, 0.3 lLặmL)1 30% hydrogen
peroxide in 0.08 M citrate/0.1 M phosphate buffer, pH 4.0)
was added (100 lL per well) to the wells. After a 40 min
incubation at room temperature, absorbence at 405 nm in
each well was measured with an ELISA plate reader
(Dynatech Laboratories). The assay was also performed
using VT1, VT2 or mAb 38.13 (each at 1 lgỈmL)1) on
serially diluted concentrations of Gb3.
Tissue preparation
Human renal cortical tissue was harvested 24 h post
mortem. Grossly normal appearing tissue was excised,
embedded in Tissue Tek OCT Compound (Sakura Finetek,
Torrence, CA, USA), and snap-frozen in liquid nitrogen.
Frozen tissue was then sectioned (6 lm) and stored at
)70 °C. Sections to be stained were dried overnight at room
temperature and all subsequent steps were performed in a

humid chamber.
Staining with FITC-conjugated VT1B
Sections were blocked with 1% normal goat serum (NGS)
(Jackson Immunoresearch Laboratories, Westgrove, PA,
USA) in 50 mM Tris-buffered saline (TBS) pH 7.4 for
20 min at room temperature. NGS (1% in TBS) was used as
the diluent for all toxins and antibodies. Sections were
stained with FITC-labelled VT1B, prepared as described
[43] at 5 lgỈmL)1 for 1 h at room temperature, extensively
washed with TBS and fixed for 20 min in 4% paraformaldehyde/NaCl/Pi. After washing, sections were treated with
50 mM ammonium chloride for 10 min, washed, mounted
with fluorescent mounting media (Dako, Carpinteria, CA,
USA), and observed under a Polyvar fluorescent microscope under incident UV illumination.
Immunoperoxidase detection of FITC-VT1B
Sections were blocked with endogenous peroxidase blocker
(1 mM sodium azide, 1 mL)1 glucose oxidase, 10 mM
glucose) at 37 °C for 1 h. After extensive rinses with TBS,
sections were blocked with 1% NGS/TBS for 20 min at
room temperature. Serial sections were stained with FITC–
VT1B (1 lgỈmL)1) for 30 min, washed with TBS and
incubated with either biotin-conjugated rabbit anti-FITC
(1 : 1000) or rabbit anti-VT1B 6869 (1 : 1000) for 30 min.


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Globotriaosyl ceramide in human renal glomerular binding (Eur. J. Biochem. 271) 407

After washing and a 30 min incubation with HRPconjugated streptavidin (1 : 500) or HRP-conjugated goat
anti-rabbit Ig (1 : 500), respectively, FITC–VT1B binding

was chromagenically visualized with True Blue Peroxidase
substrate incubated at room temperature for 5 min.
Sections were immersed in water for 5 min, dehydrated
through an ethanolic series, cleared with xylene, and
mounted with Permount. Negative control slides were treated without FITC–VT1B. The sensitivity of the immunoperoxidase detection allowed for the use of 1 lgỈmL)1
FITC–VT1B instead of the 5 lgỈmL)1 used for visualization
by fluorescence.
Immunostaining with VT1, VT1B or VT2
The immunoperoxidase staining procedure for VT1 (or
VT1B) was performed as previously [44] but with modifications. Sections were first blocked for endogenous peroxidase as above, and blocked for 20 min with 1% (v/v)
NGS/TBS. Sections were then successively incubated with
1 lgỈmL)1 VT1 (or VT1B), rabbit anti-VT1B 6869
(1 : 1000), and HRP-conjugated goat anti-rabbit Ig
(1 : 500). Each step consisted of a 30 min incubation
followed by extensive washing with TBS. VT1 (or VT1B)
binding was visualized with True Blue then washed,
dehydrated cleared, and mounted as above. Temperaturedependent binding of VT1 was performed in the same
manner, except all binding steps were performed at either
4 °C or 37 °C. Sections to be stained with VT2 were blocked
with Avidin/Biotin blocker followed by 1% (v/v) NGS/TBS
as above, and successively treated with 10 lgỈmL)1 VT2,
rabbit anti-VT2e (1 : 1000), biotin-conjugated goat antirabbit (1 : 1000), and StreptABComplex/AP. Each step
consisted of a 30 min incubation followed by extensive
washing with TBS. VT2 binding was visualized with
HistoMark Red AP substrate. Sections were washed,
dehydrated, cleared, and mounted as above. Control slides
were treated identically but not exposed to the toxins.
Double immunostaining with VT1 and mAb 38.13
Renal sections to be double immunostained were treated
with endogenous peroxidase blocker, Avidin/Biotin blocker

and 1% (v/v) NGS/TBS as above, with TBS washing
between steps. After a 30 min incubation with VT1 and
mAb 38.13 together (1 lgỈmL)1 and 5 lgỈmL)1, respectively), sections were successively treated with biotin-conjugated goat anti-rat IgM (1 : 1000), and StreptABComplex/
AP, in the same manner as above, and mAb 38.13 binding
was visualized with HistoMark Red substrate. After washing with water and 20 min blocking with 1% (v/v) NGS/
TBS, sections were incubated with rabbit anti-VT1B 6869
(1 : 1000) and HRP-conjugated goat anti-rabbit Ig
(1 : 500), each step consisting of 30 min followed by TBS
washing. VT1 binding was visualized with True Blue
Peroxidase substrate and slides were washed, dehydrated,
cleared, and mounted as above. Serial tissue sections were
also stained with VT1 and mAb 38.13 independently.
Negative control slides were treated without VT1 or with
a rat IgM isotype control for mAb 38.13. Using this
combination of substrates, True Blue Peroxidase must be
developed last as the blue product is soluble in TBS.

Epitope unmasking treatments
Frozen sections were either incubated with 1.8 mL)1
neuraminidase from Clostridium perfringens (Sigma) in
0.1 M acetate buffer (pH 4.7) for 3 h at 37 °C, 0.125%
trypsin (Sigma) in NaCl/Pi for 30 min at room temperature,
acetone for 5 min at 4 °C or 10 mM methyl-b-cyclodextrin
for 45 min at 37 °C. After extensive washing with TBS,
sections were blocked with endogenous peroxidase blocker
and 1% (v/v) NGS/TBS as above, and successively treated
with 1 lgỈmL)1 VT1, rabbit anti-VTB 6869, and HRPconjugated goat anti-rabbit Ig in the same manner as
described. VT1 binding was visualized with True Blue
Peroxidase substrate. Direct binding of FITC–VT1B was
also assayed as above after acetone treatment.

Renal sulfatide staining
Renal sections were treated with endogenous peroxidase
blocker and 1% (v/v) NGS/TBS as above, and stained with
mouse anti-SGC SULF1 (2 lgỈmL)1) followed by incubation with HRP-conjugated goat anti-mouse Ig (1 : 500).
Each step consisted of a 30 min incubation followed by TBS
washing. SULF1 binding was visualized with True Blue
Peroxidase substrate.
Thin layer chromatography overlay of deoxyGb3
analogues
Deoxy derivatives of a synthetic Gb3 analogue containing
globotriaose in anomeric linkage to a bis-C16-alkyl sulfone
aglycone were synthesized as described [45,46]. Glycolipids
(5 lg) were resolved on Sil G UV plastic-backed silica TLC
plates (Machery-Nagel) with chloroform/methanol/water
(65 : 25 : 4, v/v/v), dried, and one plate was stained with
0.5% (w/v) orcinol in 3 M H2SO4. The remaining plates
were blocked with 0.6% gelatin in water at 37 °C overnight.
After washing extensively with water, plates were incubated
with VT1 (0.3 lgỈmL)1), VT1B (0.3 lgỈmL)1), VT2
(3 lgỈmL)1), undiluted mAb 38.13 culture supernatant, or
FITC–VT1B (0.3 lgỈmL)1) for 2 h at room temperature (all
dilutions in TBS). After three washings with TBS, plates
were incubated with the corresponding antibody (rabbit
anti-VTB 6869, rabbit anti-VT2e, biotin-conjugated goat
anti-rat IgM, or biotin-conjugated rabbit anti-FITC) at
1 : 1000 for 1 h, followed, after washing, by HRP-conjugated goat anti-rabbit or HRP-conjugated streptavidin as
appropriate at 1 : 1000 for 1 h. Finally, plates were washed
extensively with TBS and developed with 4-chloro-1-naphthol peroxidase substrate.
Renal glomeruli purification
Renal cortical tissue was obtained at autopsy from a 68 yearold. Type B phenotype kidney was dissected and stored at

)20 °C. Tissue was then thawed, minced with a razor blade
into a paste-like consistency, and pushed through a 50 mesh/
230 lm stainless steel tissue sieve screen (Bellco Glass, Inc.
Vineland, NJ, USA). The filtrate containing intact glomeruli
was washed through a 150 mesh/94 lm screen with NaCl/
Pi. Glomerular cores were washed off the screen and
collected separately from the final filtrate (tubular fraction).


408 D. Chark et al. (Eur. J. Biochem. 271)

Ó FEBS 2004

Purity of glomerular and tubular fractions was verified
microscopically. Tissue retained by the 50 mesh sieve was
designated the glomeruli-depleted fraction. Each fraction
was centrifuged at 2000 g for 3 min and pellets were
extracted in chloroform:methanol (2 : 1) overnight. The
lower phase of a Folch partition was dried down and
saponified in 0.1 M NaOH/100% methanol overnight. After
neutralizing with HCl and desalting, lower phase lipids were
separated on TLC plates. VT1 and mAb 38.13 overlays were
performed as described above.
Mass spectrometry
The glomerular and tubular glycolipid fractions purified
from the type B kidney above were subject to mass
spectrometry to determine the ceramide heterogeneity.
The lower phase GSL extracts (above) were saponified
with 1 M NaOH in methanol, neutralized with HCl,
partitioned against water and washed twice. GSLs were

isolated from the lower phase by elution from a silica
column in acetone/methanol (9 : 1, v/v). This fraction was
then passed through a DEAE column in methanol to
remove acidic GSLs. Approximately 10 pmole Gb3 was
analyzed without further separation in the Mass Spectrometry Laboratory at the University of Toronto. Saturated
2,5-dihydroxybenzoic acid in methanol was used as the
matrix solution. The sample was dissolved in 20 lL
methanol and 1 lL was spotted on the sample target, and
then 1 lL of the saturated matrix solution loaded into the
mass spectrometer. The MALDI MS was acquired in
DE-reflection, positive mode on an Applied Biosystems
Voyager-DE STR MALDI-TOF mass spectrometer
equipped with a 377 nm laser. The accelerating voltage
was set at 20 KV, grid voltage at 94%, guide wire at 0.05%,
extraction delay time of 175 nsec and low mass gate at
800 Da. The mass spectra were externally calibrated with
the molecular mass of a mixture of standard peptides.

Results
Comparison of ligand/Gb3 binding by RELISA
The binding of VT1, VT2, and mAb anti-Gb3 (38.13) to
Gb3 was assessed using a RELISA where binding was
determined as a function of the ligand concentration
(Fig. 1A) and the immobilized Gb3 concentration (Fig. 1B).
While binding parameters cannot be calculated using this
assay, all three ligands showed similar dose-dependent,
saturable binding to Gb3.
Binding to deoxyGb3 analogues shows different hydroxyl
dependence for VT1and anti-Gb3
To examine the carbohydrate recognition epitopes of Gb3

for VT1, VT2, and mAb anti-Gb3, binding to a series of
synthetic monodeoxyGb3 analogues was assayed by TLC
overlay (Fig. 2). The hydroxyl groups of the trisaccharide
moiety were each removed in turn. A marked difference
between the binding profile of the antibody and VT1
was clearly seen, while VT2, although more weakly,
bound many of the same deoxyGb3 analogues as the
mAb anti-Gb3. Deoxy substitutions within the terminal

Fig. 1. Binding of VT1, mAb anti-Gb3 and VT2 to Gb3. The binding of
VT1 (d), mAb 38.13 (j), and VT2 (m) to immobilized Gb3 as a
function of (A) ligand concentration (at 1 lg Gb3 per well) (B) Gb3
concentration (at 1 lg ligand per mL), as determined by RELISA.
Similar saturable Gb3 binding is seen for all three ligands.

a-galactose were less tolerated by mAb anti-Gb3 than VT1,
most notably at the 3¢ and 4¢ deoxy positions. Similarly,
deoxy substitutions in the b-glucose, proximal to the
ceramide lipid, were more adverse for mAb anti-Gb3.
Substitutions at the 2 or 6, but not 3-deoxy positions, were
well tolerated by VT1, whereas 3-deoxyglucose but not 2 or
6 substitution allowed mAb anti-Gb3 binding. VT2 binding
was sensitive to any glucose substitution and, like mAb
anti-Gb3, little residual binding after any a-galactose
hydroxyl substitution was seen. With exception of the 3
position, hydroxyl substitutions within the b-galactose were
not tolerated by all three ligands. In general, all hydroxyls
within the trisaccharide were required for full VT2 binding,
as binding to the deoxy analogues were much weaker than
to the parent Gb3 bisalkyl analogue. Also of note, VT2, but

not VT1, required the presence of the hydroxyl at the
6-glucose position.
Renal frozen section binding of VT1, VT2 and antiGb3
are not equivalent
The binding of these three ligands to adult human renal
tissue was then compared by overlay of frozen serial
sections. VT1, VT2 and mAb anti-Gb3 staining were


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Globotriaosyl ceramide in human renal glomerular binding (Eur. J. Biochem. 271) 409

binding within the glomerulus was observed (Fig. 3Aa), as
seen at room temperature. At 4 °C, VT1 glomerular binding
was detected in a serial section (Fig. 3Bb). Tubular VT1
staining was also more distinct and discriminatory at 37 °C
(and at room temperature) compared with 4 °C.
Type B glomerular and tubular Gb3 are similar
and bound by VT1

Fig. 2. Comparison of VT1, mAb anti-Gb3 and VT2 binding to
monodeoxyGb3 analogues by TLC overlay. DeoxyGb3 analogues were
separated by TLC and visualized by (A) orcinol chemical detection.
Immunodetection of ligand binding was detected by TLC overlay for
(B) VT1, (C) mAb anti-Gb3 or (D) VT2. Gb3 analogues tested
for binding: lanes (1) parent Gb3 bisalkyl analogue; (2) 2¢¢-deoxy;
(3) 3¢¢-deoxy; (4) 4¢¢-deoxy; (5) 6¢¢-deoxy; (6) 2¢-deoxy; (7) 3¢-deoxy;
(8) 6¢-deoxy; (9) 2-deoxy; (10) 3-deoxy and (11) 6-deoxy analogues.
A distinct hydroxyl requirement is seen for each Gb3 ligand.

compared using double-label immunohistochemistry, where
HRP, staining blue, was used to detect VT1 binding, and
AP, staining red, was used to detect either VT2 or anti-Gb3
binding. Immunolabelling resulted in a purple stain where
VT1 and anti-Gb3 colocalized. In the majority of samples,
the staining of anti-Gb3 and VT1 corresponded. In some
cases, however, tubular staining by anti-Gb3 was distinct
from that of VT1 and this varied from sample-to-sample.
More significantly, renal glomerular staining by anti-Gb3 vs.
VT1 could be quite distinct. In all adult samples studied
(renal tissue from four autopsies ages 38, 46, 68 and 73 years
with no renal pathology), mAb anti-Gb3 staining within the
glomerulus was observed (Fig. 3Ac,g), while VT1 staining
within the glomerulus was present in three samples both in
single (Fig. 3Aa) and double (Fig. 3Ab) staining with antiGb3 (designated type A phenotype). In the other sample
(from a 68 year-old; designated type B phenotype), VT1
staining within the glomerulus was clearly absent both by
single immunostaining (Fig. 3Ae) and double immunostaining with VT1 and anti-Gb3 (Fig. 3Af) of serial sections.
Tubular staining was similar to that observed in type A
samples. MAb anti-Gb3 and VT1 staining were, for the
most part, coincident. However, some tubules stained by
anti-Gb3 were not bound by VT1 (e.g. some pink tubules
are seen in Fig. 3Af). VT2 glomerular staining was seen in
all samples, although glomerular staining in the type B
sample was somewhat less (compare Fig. 3Ad with h).
Ligand binding was examined routinely at room temperature. However, for type B sections, the effect of temperature
on VT1 glomerular binding was determined. At 37 °C, no

Renal glomeruli were isolated from this sample to determine

whether the Gb3 content was in any way unusual. GSL was
isolated from the residual tubular fraction also. The
glycolipid fraction from the glomeruli and tubules were
subjected to VT1 and mAb anti-Gb3 TLC overlay
(Fig. 4A). No differences in the Gb3 species detected by
such binding, as compared to the renal glomerular and
glomerular-depleted fractions, were observed. Mass spectrometric analysis (Fig. 4B) showed both the glomerular and
tubular Gb3 to be comprised primarily of C16, C22, C24
and C24:1 fatty acids. C18 and C20 fatty acid Gb3 were
detected in the tubular but not the glomerular fraction.
Hydroxylated C20 fatty acid was detected in glomerular but
not tubular Gb3.
Differential FITC–VT1B and VT1B renal section staining
A major fraction of the adult renal samples used in this
study were positive for VT1 glomerular staining (type A)
using the indirect immunoperoxidase staining procedure.
This contrasts with our previous report in which no adult
glomeruli were labelled by direct binding of FITC-conjugated VT1 [34]. We therefore compared the renal staining
of FITC-labelled VT1B with unmodified VT1B staining
detected by the immunoperoxidase procedure (Fig. 5).
Adult renal medulla showed coincident FITC–VT1B
(Fig. 5A) and immunoperoxidase VT1B labelling (Fig. 5B)
of the same tubules. Some tubules were more reactive with
VT1B than FITC–VT1B. FITC–VT1B labelling within the
renal cortex (Fig. 5C,E) validated our previous results [34]
that FITC-labelled toxin does not stain any adult renal
glomeruli. In a paediatric sample, FITC–VT1B staining of
glomeruli (and some tubules) was evident (Fig. 5G) as we
had reported previously [34]. Pediatric glomerular and
tubular staining by VT1 by immunoperoxidase detection

was also clear (Fig. 5H). In type B adult renal sections
(Figs 5E,F), immunoperoxidase anti-VT1 labelling of
VT1B-treated serial sections confirmed the lack of glomerular staining. However, in type A samples, while FITC–
VT1B glomerular staining was negative (Fig. 5C), the
immunoperoxidase staining of VT1B-treated serial sections
showed renal glomeruli bound native VT1B (Fig. 5D). To
verify the distribution of FITC–VT1B in these renal frozen
sections, we used an indirect peroxidase system using antiFITC as the primary antibody (Fig. 5I,K). This indirect
immunoperoxidase assay, confirmed that the FITC-labelled
toxin was not found within any adult renal glomeruli but
restricted to the tubules (Fig. 5I,K), as detected previously
by monitoring tissue section fluorescence directly (Fig.
5A,C,E). However, when FITC-conjugated VT1B-treated
type A serial sections were visualized using the anti-VT1
peroxidase system, additional glomerular VT1B staining
could be observed (Fig. 5J), indicating the presence of


410 D. Chark et al. (Eur. J. Biochem. 271)

Ó FEBS 2004

Fig. 3. Comparison of VT1, mAb anti-Gb3 and VT2 staining of adult renal frozen sections. (A) Serial renal sections from the sample from the 46 yearold (a–d) and the 68 year-old (e–h) were stained with VT1 alone (a,e), VT1 and mAb 38.13 together (b,f), mAb 38.13 alone (c,g), or VT2 alone (d,h)
at room temperature. In single immunostained sections: (a,e) VT1 (blue) (c,g) mAb anti-Gb3 (red) (d,h) VT2 (red); colocalization of ligands stains
purple in VT1/38.13 double immunostained sections (b,f). The sample from the 46 year-old is representative of type A phenotype (VT1 positive
glomeruli) and that from the 68 year-old of type B phenotype (VT1-negative glomeruli). MAb anti-Gb3 stains all glomeruli. (B) VT1 staining of a
type B section at 37 °C (a) is compared with staining of a serial section stained at 4 °C (b). VT1 glomerular staining is seen at 4 °C but is absent at
37 °C. Tubular staining is more distinct and discriminatory at 37 °C. Magnification, ·22.

residual unlabelled VT1B within the FITC–VT1B preparation, binding to the type A glomeruli. Such anti-VT1

glomerular labelling was not seen for FITC–VT1B treated
type B sections (Fig. 5L), and glomeruli were similarly antiFITC negative (Fig. 5K).

affects the deoxyGb3 analogue binding profile as monitored
with anti-FITC (Fig. 6C) when compared to the native
VT1B-subunit (Fig. 6A). Specifically, binding to the
6-deoxyglucose analogue is much reduced. The binding of
residual unconjugated toxin within the FITC–VT1B preparation was detected with anti-VT1 Fig. 6B.

FITC-conjugation alters VT1B deoxyGb3 binding
Based on these results, we suspected that modification of
the VT1B by FITC-conjugation might have subtly altered
the ability of VT1B to bind Gb3. We therefore compared the
binding of FITC-labelled VT1B and native VT1B to the
series of deoxyGb3 analogues by TLC overlay. Separate
binding assays were visualized using either the anti-FITC
peroxidase or the anti-VT peroxidase detection system
(Fig. 6). Conjugation of VT1B with FITC significantly

Basis for lack of VT1B renal type B glomerular staining
The basis of the differential VT1 and anti-Gb3 binding of
the type B renal sample was examined. Cell surface GSLs
may be cryptic [47] via masking by adjacent proteins or
sialated glycoconjugates [48]. Pretreatment of type B renal
sections with either trypsin or neuraminidase/sialidase had
no effect on VT1 glomerular binding (Fig. 7A,B); however,
strong VT1 binding was observed after cold acetone


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Globotriaosyl ceramide in human renal glomerular binding (Eur. J. Biochem. 271) 411

inhibit VT1 binding to Gb3/cholesterol lipid domains
in vitro [51]. We therefore stained serial sections with VT1
and mAb anti-SGC (Fig. 7I–L). No glomerular anti-SGC
staining was seen, but much of the anti-SGC staining was
found for structures not stained by VT1. Similarly, VT1
staining was largely exclusive of anti-SGC binding. This
was apparent for all sections but was particular clear in
type B samples.

Discussion

Fig. 4. Characterization of type B glomerular Gb3. (A) Glycolipids
from purified renal fractions from a type B (VT1 glomerular-negative)
sample were separated by TLC and visualized by: (a) orcinol chemical
detection; (b) VT1 overlay; (c) mAb anti-Gb3 overlay; lanes: (1) glycolipid standards at 1 lg each; (2) tubular fraction; (3) glomerular
fraction and (4) glomerular-depleted fraction. Each lane represents
neutral lipids standardized for Gb3 content. Glycolipid standards are,
from top of the plate, glucosylceramide, galactosylceramide, lactosylceramide, Gb3, Gb4, Forssman glycolipid. (B) MALDITOF MS. (a)
Tubular Gb3, (b) glomerular Gb3 from type B kidney. Only the Gb3
mass range is shown. The masses highlighted are the sodium adducts of
Gb3 containing the indicated fatty acid.

treatment (Fig. 7C). As cold acetone may extract steroids,
we pretreated kidney sections with methyl-b-cyclodextrin to
deplete cholesterol more selectively [49]. This induced
punctate VT1 glomerular staining (Fig. 7D) in the type B
sample. As cold acetone pretreatment was able to induce

renal glomerular VT1 binding in type B samples, the same
procedure was performed to examine whether the binding
of FITC-VT1B, which did not bind to any adult glomeruli,
could be similarly induced. FITC–VT1B glomerular binding was induced by acetone treatment of type A samples but
less significantly in type B (Fig. 7E–H).
SGC is highly expressed in the kidney [50] and we have
described SGC as a ÔdeceptorÕ which when present, can

Cell membrane GSL receptor recognition is a more complex
process than the recognition of protein or glycoprotein
receptors by their appropriate ligands. Glycolipids are
dynamic structures, both in terms of their lateral mobility
and their organization within the plasma membrane. The
identification of cholesterol and sphingolipid enriched,
detergent resistant plasma membrane lipid microdomains,
which serve as foci for transmembrane signal transduction
[52], has provided a strong impetus to consider GSLs as
more than structural bilayer components [53]. The heterogeneity within the lipid moiety of GSLs generates a series of
isoforms for each carbohydrate and this heterogeneity may
determine or modulate their organization within rafts [54]
and subsequent intracellular trafficking pathways [13,55]. In
the case of VT1–Gb3 binding, although the binding
specificity is defined solely by amino acid/carbohydrate
contacts [20,56] the lipid-free carbohydrate has a barely
detectable binding affinity [15,17]. Indeed, the lipid-free
globotriaose oligosaccharide and Gb3 glycolipid bind in
separate sites within the VT1B subunit pentamer [57]. In
addition, the fatty acid content of Gb3 can markedly affect
VT binding and this effect is different for the different forms
of VT [14,16]. Hydroxylation of specific Gb3 fatty acid

isoforms for example, preferentially enhances VT2 binding,
correlating the high renal hydroxylated fatty acid-Gb3
content [58] and VT2 susceptibility of mice [59]. Gb3 fatty
acid heterogeneity also promotes VT binding [60]. In a lipid
bilayer, Gb3 containing fatty acids shorter than C16 were
not bound. Increasing the chain length up to C22 increased
VT1 binding while that of VT2c was preferential for C18
[14]. Such effects can be modulated by the phospholipid acyl
chain length within the GSL containing membrane microenvironment [18]. Thus, the hydrophobic component and
membrane play a central role in VT/Gb3 recognition. We
have proposed that this effect is mediated by an H-bond
network within the interface region at the ÔserineÕ moiety of
the GSL [61] to restrict sugar conformation and solvation.
Molecular modelling predicted two Gb3 binding sites
within each monomer of the B subunit pentamer [20] with
Ôsite 1Õ being predominant, while cocrystallization studies
identified three Gb3 sites, with Ôsite 2Õ dominating [56]. Our
present studies are the first to identify substitutions within
the glucose moiety of Gb3 that affect VT binding, and as
such, are more consistent with Ôsite 1Õ, rather than Ôsite 2Õ,
Gb3 occupancy. Moreover, the major difference between
VT1 and VT2 binding was the lack of VT2 binding to the
6-deoxyglucose analogue, which is consistent with a predicted H-bond from this hydroxyl to VT2, but not VT1, when
Gb3 is docked in Ôsite 1Õ [20]. The presence of multiple
binding sites predicts that receptor multivalency plays a
significant role in determining Gb3 binding avidity [62] but


412 D. Chark et al. (Eur. J. Biochem. 271)

Ó FEBS 2004

Fig. 5. Comparison of VT1B and FITC–VT1B staining of renal frozen sections. Medullar staining by (A) FITC–VT1B visualized by incident UV
illumination and (B) immunoperoxidase detection of VT1B binding to the corresponding serial frozen section. Arrows highlight tubules labelled by
both procedures. Arrowheads indicate tubules preferentially labelled by native VT1B. Sections are from a type A sample. Type A and B phenotype
renal cortical staining by FITC–VT1B (C,E), respectively, and immunoperoxidase detection of VT1B binding to the corresponding serial frozen
section (D,F). Type A and B are both negative for FITC–VT1B glomerular binding. Only Type A exhibits glomerular staining by immunoperoxidase detection of VT1B. FITC–VT1B (G) and VT1 immunoperoxidase (H) staining of paediatric glomeruli. Anti-FITC staining of an FITC–
VT1B treated Type A section (I) and anti-VT1 staining of the FITC–VT1B treated corresponding serial section (J). Anti-FITC immunoperoxidase
staining of an FITC–VT1B treated of Type B section (K) and anti-VT1 staining of the FITC–VT1B treated corresponding serial section (L). No
glomerular anti-FITC staining is seen but anti-VT1 detects VT1B glomerular binding (due to residual unlabelled VT1B in the FITC–VT1B sample)
but only in type A sections. Magnification, ·11.

Fig. 6. Comparison of FITC–VT1B and VT1B binding to deoxyGb3 analogues. The effect of FITC-conjugation on VT1B/Gb3 binding was assessed
by TLC overlay. Lane (1) parent Gb3 bisalkyl analogue; (2) 2¢-deoxy; (3) 3¢-deoxy; (4) 4¢-deoxy; (5) 6¢-deoxy; (6) 2¢-deoxy; (7) 3¢-deoxy; (8) 6¢-deoxy;
(9) 2-deoxy; (10) 3-deoxy and (11) 6-deoxy analogues. VT1B binding detected by anti-VT1 (A); FITC–VT1B binding detected by anti-VT1B (B) or
anti-FITC (C). FITC-labelling of VT1B reduced binding to the 6 deoxyglucosyl analogue (lane 11).

relating this to membrane Gb3 glycolipid binding remains
unclear [57].
In the present study, we have shown five important new
findings: (i) VT1 binds deoxyGb3 analogues in a distinct
manner from VT2 and mAb anti-Gb3; (ii) all adult renal
glomeruli contain Gb3 as monitored by mAb anti-Gb3
binding; (iii) FITC-conjugation of VT1B alters the Gb3
binding such that adult renal glomerular Gb3 is not bound;
(iv) unmodified VT1 (and VT1B) bind renal glomerular Gb3
within a significant fraction of adult samples and (v) VT1

(and VT1B) unreactive renal glomerular Gb3 can be made
accessible to FITC-VT1B/VT1 binding by cold acetone or

methyl-b-cyclodextrin pretreatment. Our studies also validate our previous work [34] that FITC-VT1 glomerular
binding is restricted to paediatric renal samples.
Based on our present tissue staining results, we now
propose two categories for Gb3 expression within the adult
renal glomerulus: (type A) Gb3 present and reactive with
VT1, VT2, and mAb anti-Gb3; (type B) Gb3 present but
only reactive with mAb anti-Gb3, and to a lesser degree,


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Globotriaosyl ceramide in human renal glomerular binding (Eur. J. Biochem. 271) 413

Fig. 7. Modulation of glomerular VT1 staining. Frozen renal sections of type B phenotype were treated by the following procedures prior toVT1/
peroxidase immunostaining: (A) trypsin; (B) sialidase; (C) acetone and (D) methyl-b-cyclodextrin. FITC–VT1B staining of type A (E,F) and type B
(G,H) renal sections without (E,G) and after (F,H) acetone treatment. Serial frozen sections of type A (I,J) and type B (K,L) phenotype were stained
with VT1 (I,K) and mAb anti-SGC (J,L). Only VT1 glomerular staining was seen for type A samples. In type B, neither VT1 nor anti-SGC
glomerular staining was observed. VT1 and anti-SGC staining are largely exclusive (Arrows show structures stained with VT1 but not anti-SGC,
and arrowheads show those stained with anti-SGC but not VT1). Magnification, ·11.

VT2. We have reported this VT1 binding phenotype
previously [44]. The relative frequency of these categories
and their relationship to the incidence of HUS following
gastrointestinal VTEC infection remains to be determined.
Studies along similar lines may also shed light on the
incidence of HUS in children.
Our use of serial frozen section staining allows the direct
comparison of ligand binding and the effect of tissue
modification. Our findings greatly expand the physiological
significance of lipid-based heterogeneity of Gb3 receptor

function, which now may impinge on the epidemiology of
HUS following gastrointestinal VTEC infection. Our renal
section sample size is very small and therefore the variation
in renal glomerular Gb3 expression in relation to HUS
cannot be addressed in this study. Nevertheless our studies
identify the phenotypic prototypes we would expect to
influence the incidence of VT-induced renal disease and
suggest the mechanism separating them. The binding of
VT2 to adult renal glomeruli, not bound by VT1, is
consistent with the more common association of this variant
with renal disease [63–65].
Binding studies of VT1, VT2, mAb anti-Gb3, and FITC–
VT1B to deoxyGb3 analogues show differential binding to
the sugar moiety and support our contention that these
ligands can preferentially bind different isoforms of Gb3.
However, our TLC overlay binding data does not completely rationalize the differences in renal tissue staining
observed. This binding assay, though demonstrating distinct
Gb3 binding subspecificity, is an ineffective mimic of cell

membrane Gb3 presentation [15]. It is clear that the
deoxyGb3 binding of mAb anti-Gb3 and VT2 is more
restricted than that of VT1. While this is consistent with the
similar glomerular staining by mAb anti-Gb3 and VT2 but
not VT1 in type B samples, overall, mAb anti-Gb3 renal
glomerular staining is more, rather than less, widespread
than VT1. Deoxy substitution at Glcb2OH or Glcb6OH is
not tolerated by mAb anti-Gb3. Access to these hydroxyls
could be restricted due to the close apposition of the
ceramide moiety [66] maintained by intramolecular
H-bonding of the ceramide N-H with the anomeric oxygen

[67,68]. The Glcb6OH may form an H-bond with 2-hydroxy
fatty acid containing GSLs [69] within the interface of the
membrane bilayer. Thus, particularly when such H-bonds
are formed, mAb anti-Gb3 (and VT2) binding may be
restricted; in contrast VT1 binding could be unaffected. In
contrast, the Glcb3OH is involved in an intramolecular
H-bond with the b-galactose [20] and the lack of VT1, as
opposed to mAb anti-Gb3 binding to this analogue may
indicate that restriction of rotation around this anomeric
linkage is more important for VT1, as compared to mAb
anti-Gb3 and VT2 binding. Such considerations could be
used to explain the differential VT1, as opposed to mAb
anti-Gb3, renal glomerular staining by proposing that excess
membrane cholesterol may restrict the formation of the
Glcb3OH/Galb5O intramolecular H-bond. In terms of
FITC–VT1B binding, the major difference is reduced
binding to the 6-deoxyglucose analogue as compared to
VT1B (or VT1). The glucose residue of Gb3 is closely


414 D. Chark et al. (Eur. J. Biochem. 271)

apposed to the interface between hydrophilic and lipophilic
components of the GSL. As such, changes in the lipid
moiety or its membrane environment may more severely
impinge on this region to restrict FITC–VT1B binding to
explain the lack of adult renal glomerular binding.
Our earlier studies showed that FITC-coupling at Lys53
of the VT1B subunit inhibited FITC-VT1B/Gb3 binding
[43] and that brief coupling times were necessary to avoid

this. The modified Gb3 binding subspecificity we now
observe may be due to FITC-coupling to other lysine
residues within, or adjacent to, the Gb3 binding site (e.g.
Lys28). Brief FITC-coupling to VT1 has no effect on
cytotoxicity in vitro [43] and FITC–VT1B can compete with
native VT1 for Gb3 binding [51].
These studies reveal a surprising complexity of tissue Gb3
receptor function. Our finding that the same GSL can be
available for one specific ligand but not another, demonstrates a new aspect to GSL receptor function. This is
relevant to the recent report that VT1 and anti-Gb3 induce
apoptosis via different signalling pathways, despite binding
to the same GSL [29]. There are several other examples
where a single GSL is recognized by several ligands [61,69–
75] and differential recognition may also occur.
The lack of VT1 (and VT1B) binding in glomeruli that
were mAb anti-Gb3 reactive (type B) is likely to be a
property of the membrane Gb3 lipid environment as both
ligands were able to effectively bind purified glomerular Gb3
(from the same sample) by TLC overlay. Mass spectrometry
of these samples showed a very similar fatty acid composition. However there were differences in the minor species
[C18 and C20 for tubular and C20(OH) for glomerular Gb3]
which could have a bearing on VT1 reactivity. Fatty acid
heterogeneity promotes VT1 binding [60] and hydroxylation
can preferentially increase VT2 binding [16]. Our finding
that mAb anti-Gb3 reactive glomerular structures become
VT1 reactive after cold acetone treatment is consistent with
a role of the membrane environment in Gb3 receptor
function. While glycolipids and membrane phospholipids
are poorly, if at all, soluble in cold acetone, immunolocalization of GSL after acetone treatment should nevertheless,
be interpreted with caution. Solubility may be sufficient for

GSL diffusion. However, as anti-Gb3 reveals the presence of
Gb3 in the type B glomeruli, we favour an explanation
whereby Gb3 presentation is altered by acetone to promote
VT1 binding. Cholesterol is acetone soluble and might be
extracted from the glomerular membranes to achieve such
an effect. This is the most likely interpretation, since
cholesterol depletion with methyl-b-cyclodextrin had a
similar effect to induce VT1 recognition, although this is
the first time methyl-b-cyclodextrin has been used to extract
tissue section cholesterol. However, the selectivity of
methyl-b-cyclodextrin to deplete cholesterol has recently
been more rigorously investigated [76]. The conditions used
in our study, while preferential for cholesterol, may also
extract some sphingomyelin, glycolipids and phosphatidyl
choline from cells. Thus a role for cholesterol cannot be
inferred conclusively. Cholesterol intercalates with sphingolipids and cholesterol/Gb3 enriched lipid microdomains play
a central role in determining VT1 cell sensitivity [13,77].
Although addition of cholesterol promotes VT1/Gb3 binding in an in vitro lipid microdomain binding assay we have
developed [51,78], excess cholesterol is inhibitory for VT1

Ó FEBS 2004

(but not VT2) binding (C. Lingwood & A. Nutikka,
unpublished observation). If glomerular membrane cholesterol was high, limited cholesterol extraction might therefore
selectively increase VT1 binding membrane Gb3 rafts to
explain our observation. The more aggregated VT1 staining
seen after methyl-b-cyclodextrin treatment would be consistent with optimization of VT1/Gb3 raft binding. Thus,
potentially, the glomerular cholesterol content may be a risk
factor for VT1 binding and hence, for the development of
HUS. This may also provide the basis of the age-related

FITC–VT1B glomerular binding we have found.
The lack of VT1 glomerular binding in type B samples is
not due to SGC inhibition of VT1/Gb3 binding as these
glomeruli lack SGC. SGC is a major component of the renal
GSL fraction [50] and may be involved in ion transport [79].
We have found the addition of SGC inhibits the binding of
VT1 to Gb3/cholesterol microdomains prepared in vitro [51].
The extensive expression of SGC in distal tubules could
relate to their relative resistance to VT1 [80]. VT1 and antiSGC renal binding were found to be, for the most part,
mutually exclusive. This gross separation of Gb3 and SGC
implies functional distinction.
A recent report [36] has contested that there is no agerelated difference in human glomerular Gb3 expression
and reported that Gb3 can be stained by VT1 or anti-Gb3
in all renal glomeruli. In these studies, Gb3 was quantitated by VT1/TLC overlay. While suitable for comparison, this does not quantitate Gb3 in absolute terms.
Chemical detection or mass spectrometry is necessary to
avoid bias due to preferential binding of VT1 to select
Gb3 isoforms. Moreover, in this study, VT1 binding to
renal sections at 4 °C was monitored, and 90% of the
binding was reported lost at room temperature [36]. The
relevance of binding of VT1 to glomeruli (and to red
blood cells [37]) at 4 °C only, to the pathophysiology of
VT-induced disease is questionable. It is possible that at
4 °C, the heterogenous Gb3 binding phenotypes we
observe, are reduced to a common lower affinity mechanism, universally present. At 4 °C, the lateral mobility of
Gb3 will be less, reducing the entropic penalty on ligand
binding. Indeed, we found VT1 glomerular binding in type
B samples at 4 °C. In addition, membrane lipid organization will be altered below their phase transition
temperatures [18]. The lack of VT1 glomerular binding
in type B samples at room temperature, was also seen at
37 °C, validating our binding studies at room temperature

as physiologically relevant. Tubular binding was also more
restricted but more distinct at 37 °C as compared to 4 °C.
This suggests heterogeneity of tubular VT1 sensitivity
under physiological conditions.
While the role of Gb3 in mediating VT cytotoxicity
in vitro [13,55,81] and in animal models [33,82,83] has been
established, the role in human disease has yet to be defined.
Renal glomerulus-bound VT1 was found in paediatric, but
not in adult cases of HUS [35]. This is consistent with our
original report [34] and present studies using FITC-VT1B.
The VT-dependent etiology of HUS in the elderly and the
young may be different. The reduced susceptibility of adults
might be ascribed to a type B Gb3 expression, but, though
our numbers are small, type A appears to predominate,
implicating other factors. Possibly renal Gb3 expression can
be modulated. Certainly renal Gb3 expression and VT1


Ó FEBS 2004

Globotriaosyl ceramide in human renal glomerular binding (Eur. J. Biochem. 271) 415

sensitivity in the baboon model can be up-regulated by LPS
[84].
Our studies show that ÔpresentationÕ of glomerular Gb3,
rather than the quantity of Gb3, varies as a function of age.
FITC–VT1B still shows an age-related binding. While
acetone treatment induced FITC–VT1B glomerular binding
in type A samples, binding in type B samples was only
slightly induced, indicating a robust distinction in terms

of the Gb3 parameters necessary for FITC–VT1B as
opposed to VT1B binding. We would expect individuals
with type B phenotype to be more resistant to VT-induced
renal disease.
Our studies suggest VT/glomerular Gb3 binding could be
related to the glomerular cholesterol content. Cholesterol
homeostasis is important in renal glomerular function
which is a primary lesion in hypercholestrolemia [85]. In
addition, detergent-resistant, renal cortical plasma membrane cholesterol is elevated under stress conditions [86],
suggesting potential variability in this parameter. While
FITC-VT1 renal glomerular binding may provide a (fortuitous) marker of the age-related susceptibility to VT-induced
HUS, it is worth considering whether the toxin might be
modified in vivo in some way during infection to mirror the
FITC-VT1 binding phenotype.

Acknowledgements
This work was supported by CIHR grant #MT13073 and student
stipend support (to D. C.) from HSC. The technical assistance of Beth
Binnington in preparation of samples for mass spectrometry is
gratefully acknowledged. We thank Ling Xu, of the Molecular
Medicine Research Centre, University of Toronto for mass spectrometry and the Department Pathology, Toronto General Hospital for
renal autopsy samples.

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