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 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 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 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 was intermediate These ligands differentially stained human kidney sections Glomerulopathy is the primary verotoxin-associated pathology in hemolytic uremic syndrome For most samples, verotoxin 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 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 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: + 416 813 5993, Tel.: + 416 813 5998, E-mail: cling@sickkids.ca 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 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 °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 °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 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 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 : 2000 in 1% BSA/NaCl/Pi); and finally the corresponding horseradish peroxidase-conjugated goat anti-rabbit or HRP-conjugated streptavidin, both at : 2000 in 1% BSA/NaCl/Pi) Each step consisted of a h incubation (100 lL per well) followed by two washings with 1% BSA/NaCl/Pi After one final wash with NaCl/Pi, freshly prepared 2,2¢-azino-bis3-ethylbenzthiazoline-6-sulfonic acid solution (ABTS; Sigma – 0.5 mgỈmL)1 ABTS, 0.3 lLỈmL)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 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 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 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 lgỈmL)1 for h at room temperature, extensively washed with TBS and fixed for 20 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, mL)1 glucose oxidase, 10 mM glucose) at 37 °C for h After extensive rinses with TBS, sections were blocked with 1% NGS/TBS for 20 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 Ó FEBS 2004 Globotriaosyl ceramide in human renal glomerular binding (Eur J Biochem 271) 407 After washing and a 30 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 Sections were immersed in water for 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 lgỈmL)1 FITC–VT1B instead of the 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 with 1% (v/v) NGS/TBS Sections were then successively incubated with 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 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 °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 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 incubation with VT1 and mAb 38.13 together (1 lgỈmL)1 and 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 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 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 h at 37 °C, 0.125% trypsin (Sigma) in NaCl/Pi for 30 at room temperature, acetone for at °C or 10 mM methyl-b-cyclodextrin for 45 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 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 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 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 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 : 1000 for h, followed, after washing, by HRP-conjugated goat anti-rabbit or HRP-conjugated streptavidin as appropriate at : 1000 for 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 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 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 lL was spotted on the sample target, and then 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 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 lg Gb3 per well) (B) Gb3 concentration (at 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 or 6, but not 3-deoxy positions, were well tolerated by VT1, whereas 3-deoxyglucose but not or 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 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 Ó FEBS 2004 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 °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 °C Type B glomerular and tubular Gb3 are similar and bound by VT1 Fig 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 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 °C (b) VT1 glomerular staining is seen at °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 Ó FEBS 2004 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 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 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 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 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 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, Ó FEBS 2004 Globotriaosyl ceramide in human renal glomerular binding (Eur J Biochem 271) 413 Fig 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 °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 °C only, to the pathophysiology of VT-induced disease is questionable It is possible that at °C, the heterogenous Gb3 binding phenotypes we observe, are reduced to a common lower affinity mechanism, universally present At °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 °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 °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 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Tubular VT1 staining was also more distinct and discriminatory at 37 °C (and at room temperature) compared with °C Type B glomerular and tubular Gb3 are similar and bound by VT1 Fig Comparison of. .. for VT 1and 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