Glycobiology Advance Access originally published online on September 13, 2007
Glycobiology 2007 17(11):1156-1166; doi:10.1093/glycob/cwm095
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Sulfatide binding properties of murine and human antiganglioside antibodies
2 Division of Clinical Neurosciences, Glasgow Biomedical Research Centre, University of Glasgow, G12 8TA Scotland
3 Institute of Physiological Chemistry, University of Bonn, Nussallee 11, D-53115 Bonn, Germany
4 Laboratory of Carbohydrate Chemistry, Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia
5 CERMAV-CNRS, BP53, F-38041, GRENOBLE, Cedex 9, France
6 Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
1 To whom correspondence should be addressed: Tel: +44-141-330-8388; Fax: +44-141-330-4297; e-mail: k.townson{at}clinmed.gla.ac.uk
Received on May 10, 2007; revised on August 28, 2007; accepted on September 3, 2007
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Abstract |
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Antiganglioside antibodies form an important component of the innate and adaptive B cell repertoire, where they provide antimicrobial activity through binding encapsulated bacterial glycans. In an aberrant role, they target peripheral nerve gangliosides to induce autoimmune nerve injury. An important characteristic of antiganglioside antibodies is their ability to selectively recognize highly defined glycan structures. Since sialylated and sulfated glycans often share lectin recognition patterns, we here explored the possibility that certain antiganglioside antibodies might also bind 3-O-sulfo-β-D-galactosylceramide (sulfatide), an abundant constituent of plasma and peripheral nerve myelin, that could thereby influence any immunoregulatory or autoimmune properties. Out of 25 antiganglioside antibodies screened in solid phase assays, 20 also bound sulfatide (10–5 to 10–6 M range) in addition to their favored ganglioside glycan epitope (
10–7 M range). Solution inhibition studies demonstrated competition between ganglioside and sulfatide, indicating close proximity or sharing of the antigen binding variable region domain. Sulfatide and 3-O-sulfo-β-D-galactose were unique in having this property amongst a wide range of sulfated glycans screened, including 4- and 6-O-sulfo-β-D-galactose analogues. Antiganglioside antibody binding to 3-O-sulfo-β-D-galactose was highly dependent upon the spatial presentation of the ligand, being completely inhibited by conjugation to protein or polyacrylamide (PAA) matrices. Binding was also absent when sulfatide was incorporated into plasma membranes, including myelin, under conditions in which antibody binding to ganglioside was retained. These data demonstrate that sulfatide binding is a common property of antiganglioside antibodies that may provide functional insights into, and consequences for this component of the innate immune repertoire. Key words: antibody / ganglioside / neuropathy / sulfatide
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Introduction |
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Sulfated glycolipids frequently act as ligands for a diverse range of human and microbial glycan binding proteins including CD1 restricted T cell receptors (De Libero and Mori 2005
), selectins (Aruffo et al. 1991), chemokines (Sandhoff et al. 2005), laminins (Talts et al. 1999), and toxins (Rousset et al. 1998; Wang et al. 2006). In particular, a wide range of studies have shown that immunglobulins react with sulfatide (3-O-sulfo-β-D-galactosylceramide) in both normal and disease states (Fredman et al. 1991; Avila et al. 1993; Dabby et al. 2000; Ilyas et al. 2003; Lopate et al. 2005) and many sulfatide-binding antisera and monoclonal antibodies (mAbs) have been isolated and studied (Hakomori 1974; Fredman et al. 1988; Kirschning et al. 1997; Wang et al. 2006).
Sulfatides are widely distributed throughout mammalian tissues, including serum, and are particularly prevalent in both neuronal and myelin membranes within the nervous system (Ishizuka 1997). As such, they have long been considered potential targets for autoimmune neurological disease pathogenesis. This cause and effect relationship has been studied in many model systems without a clear consensus, at least in part due to the likely diversity of the different antisulfatide antibodies studied. Nevertheless, it seems clear that antisulfatide antibodies are able to induce pathophysiological changes in the nervous system under certain experimental conditions (Petratos and Gonzales 2000; Rosenbluth and Moon 2003; Rosenbluth et al. 2003; Kanter et al. 2006; Wang et al. 2006).
One intriguing feature of certain classes of sulfatide-reactive antibodies is that in addition to binding sulfatide, they may more promiscuously bind other structurally similar (Eurelings et al. 2001) or even dissimilar ligands (Aotsuka et al. 1992). Conversely, antibodies raised against, and apparently specific for, a particular class of antigens may also bind sulfatide (Merten et al. 2003). Certain proteins, notably C-type lectins that bind sialyl oligosaccharides also bind sulfated structures (Galustian et al. 1997, 2004), both of which present a negative charge, and sulfation can strongly influence the binding of lectins, including siglecs, to their sialyl-glycans (Campanero-Rhodes et al. 2006). Since gangliosides are also sialylated, we have here analyzed the sulfated glycan binding properties of human and mouse antiganglioside antibodies to determine whether any dual specificity exists that may potentially contribute to their functional or pathophysiological properties.
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Binding of antiganglioside antibodies to sulfatide
Twenty mouse and five human antiganglioside mAbs with diverse specificities for neuropathy-associated ganglioside targets including GM1, GD1a, GD1b, GD3, and GQ1b were assessed in ELISA for binding to sulfatide. At a concentration of 10 µg/mL, 20/25 mAbs bound sulfatide in ELISA (Table I). No correlation was observed between the ganglioside binding profile of a mAb and its ability or inability to also bind sulfatide. Binding profiles for six mouse mAbs – 3 IgGs and 3 IgMs – representative of different ganglioside specificities (TBG3, GD1a; EG1, GD3/GQ1b; DG2, GM1; EM4, GD1a; EM1, GQ1b/GD3; MOM3, GM1/GD1b) and human anti-GM1 mAbs (SM1, BR1, WO1) were also assessed by titration analyses (Figure 1 and 2) and exhibited the sigmoid shaped curves typically seen for antibody–antigen interactions. In order to determine and compare the affinities of the mAb–sulfatide interaction with the mAb–ganglioside interaction, surface plasmon resonance was conducted using Fab fragments derived from the three representative mouse IgG mAbs, TBG3, EG1, and DG2, and the data are shown in Figure 3. The KDs for TBG3 and GD1a (9.5 x 10–7 M) (Boffey et al. 2005
) and DG2 and GA1 (3 x 10–7 M) (Townson et al. 2007) have been previously reported. Whereas affinities for the preferred ganglioside were in the 10–7 M range for these three antibodies, affinities for sulfatide were on average 1 to 2 log lower – in the 10–5 to 10–6 M range.
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Dependence of antibody binding on the sulfate and its position in the galactose ring
We next determined whether the antibody–sulfatide interaction depended upon the presence of sulfate, and its position in the galactose ring. Firstly, we established that galactocerebroside did not bind these antiganglioside antibodies (data shown for mAbs DG2, EM1, and BR1 – Figure 4B, D and F, lane 2). Secondly, we observed that cleavage of the sulfate from 3-O-sulfo-β-D-galactosylceramide by mild acid hydrolysis abolished binding, shown here by thin layer chromatography (TLC) overlay (Figure 4B, D and F, lanes 3, 4) and also by ELISA (Figure 4C, E and G). Thirdly, we determined the requirement for the sulfate to be positioned at the third carbon of the galactose ring by comparing the binding of DG2 to 3-sulfated galactose and to galactose with substitutions in the 4 and 6 positions. In solution inhibition studies, 3-sulfated galactose (in the form of ozonolyzed 3-O-sulfo-β-D-galactosylceramide, (sulph-OS)) was an effective inhibitor of solid phase mAb–sulfatide interaction, whereas D-Galactose-4-O-sulfate (Gal-4-sulfate) and D-Galactose-6-O-sulfate (Gal-6-sulfate) had no inhibitory activity (Figure 5, top panels). These data thus demonstrate both dependence of antibody binding on the sulfate and its location on the third carbon, as is found on sulfatide.
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Occupancy of overlapping antigen-binding sites for ganglioside and sulfatide
The binding studies described above did not address whether sulfatide and ganglioside occupy the same position in the antigen-combining site or whether they bind to separate and distinct regions of the antibody molecule. To address this, we conducted solution inhibition studies to identify any competition for binding sites between ganglioside and sulfatide. Either ganglioside (GM1, GD1a, and GD3 for DG2, TBG3, and EG1, respectively) or sulfatide were immobilized, and the competing oligosaccharides introduced in solution. All antibodies were inhibited from binding solid phase ganglioside by the presence of solution phase sulfatide, and vice-versa, indicating that the antigen binding sites were competitively overlapping. Effective inhibition was thus observed irrespective of whether ganglioside or sulfatide were in the solid or solution phases (Figure 5).
Binding of antiganglioside mAbs to sulfated oligosaccharide conjugates
In order to investigate whether sulfatide recognition of these antiganglioside mAbs was specific for 3-sulfated galactose, or was actually a more promiscuous interaction with a range of sulfated oligosaccharides, we examined binding by ELISA of a selection of mAbs (CGG1, DG1, DG2, EG1, TBG3, MOG35, DO1, and BR1) to a panel of sulfated oligosaccharide–polyacrylamide (PAA) conjugates (Supplementary data, Table S
). The only antibody–ligand pairing was DG2 and Galβ1-3GalNAcβ (Tββ), which is the terminal disaccharide of GM1, GA1, and GD1b. The other anti-GM1 mAbs require sialic acid for binding, except for DO1. The lack of binding of DO1 to Tββ may be due to the more complex binding interactions of IgMs in comparison with IgGs. No binding was observed to any of the sulfated structures, including 3-O-Su-Galβ. It was therefore, not possible to establish the recognition patterns of the mAbs, as it appeared that the epitope may not have been presented on PAA in a way that could be recognized. Similarly, we observed that presentation of the epitope on bovine serum albumin (BSA) was also critical, as none of the sulfatide-binding mAbs were able to significantly bind 3-O-Su-Galβ-BSA (data not shown). Alternatively, this also raised the question as to whether the lipid component of sulfatide was necessary for binding.
Interaction of antiganglioside antibodies with sulfatide lacking one or both acyl chains
To examine in more detail the requirement for the lipid component of sulfatide for binding of these mAbs to the 3-O-Su-Galβ epitope, solution inhibition ELISAs were performed with sulph-OS, which lacks one acyl chain, and enzymatically cleaved sulfatide (ceramide glycanase treated 3-O-sulfo-β-D-galactosylceramide (sulph-CG)), which has both acyl chains removed. Binding of DG2 to solid phase GM1 and sulfatide was measured after mixing with the soluble sulfatide variants. DG2 interacted with both sulph-OS and sulph-CG, inhibiting binding to GM1 and sulfatide (Figure 6). Although not essential for binding, inhibition was greater when one of the acyl chains was present. This was most striking with GM1 binding – 100% inhibition at 1 mM compared with 45% for sulph-CG.
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Binding of antiganglioside antibodies to sulfatide in biological membranes
The binding of mAbs to sulfatide liposomes observed during the Biacore affinity measurements showed that antiganglioside mAbs are able to bind sulfatide effectively when incorporated into a membrane environment as well as when immobilized in pure form on ELISA polystyrene plates. We then determined whether antiganglioside mAbs were able to bind sulfatide in a more biologically relevant neural membrane environment. To achieve this, PC12 cells that do not naturally contain sulfatide (at least in the clone used for this study) were incubated with sulfatide, which become incorporated into the plasma membrane, and then GM1 epitopes on the cells were blocked by incubating with cholera toxin B subunit (CTB). The cells were then assessed for antiganglioside mAb binding by immunofluorescence microscopy and by FACS. The widely used mouse IgM mAb, O4, which binds sulfatide in cells and tissues, was used as the indicator of membrane-associated sulfatide binding. EG3, which did not bind sulfatide in ELISA or Biacore, was used as a negative control mAb. PC12 cells incubated with sulfatide became primed to bind O4 (Figure 7, top panels, and Figure 8C), thereby demonstrating effective sulfatide incorporation into the plasma membrane. For antiganglioside antibody binding, PC12 cells remained negative or unchanged from baseline (Figure 7, lower panels, and Figure 8).
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To address whether sulfatide-binding antiganglioside mAbs are able to bind sulfatide in peripheral nerve membranes, frozen sections from cerebroside sulfotransferase overexpressing, sulfatide accumulating (CSThigh) and sulfatide deficient mice (CGT–/–) were assessed for binding of O4, and the antiganglioside mAb, DG2, that binds both GM1 and sulfatide. As expected, O4 did not bind sulfatide deficient nerve, but bound sulfatide overexpressing nerve strongly (Figure 9, top panels). DG2 bound both sulfatide deficient and sulfatide overexpressing nerve strongly and equally well, by virtue of binding GM1 ganglioside (Figure 9, middle panels). When binding of DG2 to GM1 was blocked by preincubation of sections with CTB, DG2 binding was abolished in both sulfatide deficient and sulfatide overexpressing nerve sections (Figure 9, lower panels), indicating that DG2 cannot bind sulfatide in sulfatide rich nerve membranes. O4 staining intensity was unaffected by CTB preincubation, indicating that CTB is not masking sulfatide in nerve sections (not shown).
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Discussion |
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These data demonstrate that a high proportion of both mouse and human antiganglioside antibodies used in this study are also able to bind sulfatide, independent of their specificity for the very highly defined glycan epitopes on the gangliosides or bacterial lipooligosaccharides against which they were raised and cloned. Thus antiganglioside antibodies that are specific for terminal Gal(β1-3)GalNAc disaccharides (e.g., DG2), terminal Neu5Ac(
2-3)Gal(β1-3)GalNAc trisaccharides (e.g., TBG3) or internal Neu5Ac(2-8)Neu5Ac epitopes (e.g., EG1) may also bind 3-O-sulfo-β-D-galactosylceramide (i.e., sulfatide). The promiscuous binding of this class of antiglycan antibodies to sulfatide is therefore, not due to a general inability to discriminate between different glycan structures, as the ganglioside specificity is very discrete, and the sulfatide binding is entirely dependent on the sulfate being in the 3-position of the galactose. The antibodies may, however, be binding ganglioside and sulfatide at the same binding site, or at least at binding sites in close proximity, as binding to one ligand could inhibit binding to the other. Sulfatide binding is also not a universal feature of antiganglioside antibodies, since some antibodies of similar specificity to those identified above only bind sulfatide weakly or not at all, despite binding well to their ganglio-series glycan structure. We have previously shown that some antiganglioside antibodies are polyreactive, as defined by binding actin, thyroglobulin, tubulin or DNA (Boffey et al. 2004); however, this does not correlate closely with the ability to also bind sulfatide. For example, EG1 binds sulfatide strongly, but showed no binding to the above polyreactive antigens. By reviewing previously published mAb sequencing data, we have also examined the variable region gene usage of sulfatide binding antiganglioside antibodies in comparison with those that do not bind sulfatide and did not observe any distinctive patterns (data not shown). It appears as though the interaction of these antiganglioside mAbs with sulfatide depends on the scaffold to which the monosaccharide is attached. Although the lipid moiety is not essential for binding, as mAbs interact well with sulph-CG that lacks this component, the presentation of the monosaccharide epitope in solid phase is important. This element of complexity is demonstrated by the lack of binding of the mAbs to 3-O-Su-Galβ presented on both PAA and BSA.
In general, the binding to sulfatide is at least a log lower affinity than binding to the dominant ganglioside glycan epitope, as assessed by both the affinity determinations in liposomal membranes using Biacore and the half-maximal binding data derived from ELISA. Nevertheless, this does not indicate insignificance of the sulfatide binding properties of these antibodies, since at appropriate concentrations of sulfatide presented in the appropriate format (in micelles or a simple liposome membrane environment), interaction with sulfatide is observed.
The immunofluorescence evidence suggests that at least for the antiganglioside mAbs studied here, binding to sulfatide in normally organized myelin and neural membranes may be insignificant, and thus direct pathogenic effects of these antibody–sulfatide interactions are unlikely. Other antisulfatide antibodies, such as O4, that bind sulfatide in neural membranes may have the capability to drive direct pathogenic effects. However, there are other potentially important immunological, physiological or pathological actions for antibody–sulfatide interactions. Sulfatide is present in human plasma at a concentration of 0.6–0.7 nmol/mL and this concentration may vary in disease states (Buschard et al. 2005). In plasma, sulfatide may have antiatherosclerotic and anticoagulant activity (Kyogashima 2004) and it is possible that antisulfatide antibody might engage free or protein- bound circulating sulfatide and thereby modulate its biological effects. Antisulfatide antibodies may be able to bind the sulfatide saccharide presented by CD1 molecules and thereby modulate CD1-restricted sulfatide specific T cell interactions. Similarly, ganglioside and sulfatide binding B cell receptors may sense circulating sulfatide and thereby modulate B cell activation.
Apart from their association with disease states, many normal individuals have significant titres of "antisulfatide" antibodies. There is clearly considerable diversity amongst antisulfatide antibodies in terms of binding properties and functional effects, as demonstrated for modulation of insulin metabolism (Buschard et al. 2005). It is clear from this and other studies that these antibodies may have other more significant carbohydrate antigen recognition domains than sulfatide binding. Conversely, sulfatide reactivity could be viewed as a more general property of a wide range of carbohydrate binding antibodies and this characteristic may comprise a significant proportion of the innate antibody repertoire. The functional consequences of this are currently unknown. This study also demonstrates that identifying sulfatide reactivity in polyclonal sera, or in mAbs using solid phase assays, says little about sulfatide binding capacity in biological membranes. It also acts in a cautionary way to remind us that having identified sulfatide as a ligand for an antibody, this may represent one of the many possible ligands that could confound interpretation of subsequent studies unless it is identified. The advent of glycoarray screening would be one way through which such multiple ligand interactions could be easily identified.
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Purification and characterization of antiganglioside mAbs
Murine antiganglioside mAbs were cloned following immunization of HeN or GalNAc transferase knockout mice with Campylobacter jejuni lipooligosaccharides or gangliosides in adjuvant as previously described (Goodyear et al. 1999
; Bowes et al. 2002; Boffey et al. 2004, 2005). The anti-GD3 mAb, R24 (that also binds GQ1b) was obtained from American Type Culture Collection (Manassas, VA) (Dippold et al. 1980). Human mAbs were cloned from peripheral blood mononuclear cells isolated from patients with Guillian-Barré syndrome (DO1), multifocal motor neuropathy (WO1, BR1, and SM1) (Willison et al. 1994; Paterson et al. 1995; Goodyear et al. 1999) or ataxic
neuropathy (HA1) (Willison et al. 1996). Murine IgGs were purified from tissue culture supernatants using HiTrap Protein A (IgG3) or Protein G (IgG1, IgG2b) affinity columns (Amersham Pharmacia Biotech, Buckinghamshire, UK). Human IgMs were purified by gel filtration using a Sepharose 6B column (Amersham Pharmacia Biotech).
Immunoassays and affinity determinations
Reactivity of mAbs with glycolipids was assessed by a standard ELISA using Immulon 2 microtitre plates (Dynatech Laboratories, Sussex, UK) coated with 200 ng of glycolipid per well and assays conducted at 4°C (Willison et al. 1999). Affinity analysis was performed on Fab fragments using surface plasmon resonance (Biacore 2000, Biacore AB, Uppsala, Sweden) as previously reported (Boffey et al. 2005). Briefly, ganglioside- or sulfatide-containing liposomes (0.2 mg/mL in phosphate buffered saline [PBS]) were prepared from 1,2-dimyristoyl-rac-glycero-3-phosphocholine (Calbiochem, Nottingham, UK) and ganglioside/sulfatide (Sigma, Poole, Dorset, UK) in a (w/w) ratio of 100:1, and resized to 50 nm. Approximately 5000 response units (RU) of liposomes were immobilized on an L1 pioneer sensor chip (Biacore AB) by injection at 2 µL/min. Fab fragments of Protein A/G purified mAbs were prepared by papain digestion (ImmunoPure Fab Preparation Kit, Pierce, Northumberland, UK) The liposome layer was regenerated with 100 mM NaOH. Measurements were carried out in PBS at 25°C. Kinetic analyses were performed using the BIA evaluation 3.1 package (Biacore AB).
Binding of antiganglioside mAb to desulfated sulfatide
The sulfate group of sulfatide was hydrolyzed using mild acid. Briefly, 5 mL of 50 mM HCl in methanol was incubated with 5 mg lyophilized sulfatide (Sigma) for 16 h at room temperature. Following this, 15 mL chloroform, 2.5 mL methanol, and 5.5 mL 0.2% Na2CO3 were added and the solution sonicated and centrifuged for 5 min at 1000 rpm. The upper phase was removed and the lower phase, containing the desulfated sulfatide, was washed with chloroform:methanol:KCl (3:48:47 v/v) and then chloroform:methanol:distilled H2O (3:48:47 v/v). The extent of desulfation was assessed by TLC on silica gel high performance TLC plates (Merck, Nottingham, UK) with chloroform:methanol (2:1 v/v) running solvent and orcinol staining. Binding of murine IgG (DG2), IgM (EM1), and human IgM (BR1) mAbs to desulfated sulfatide and other glycolipids was assessed by TLC overlay as previously described (Willison et al. 1994). The glycolipids were separated by TLC, as above, and the plate blocked overnight with 2% (w/v) BSA. MAb, diluted to 10 µg/mL in 1% (w/v) BSA in PBS, was then applied to the plate and incubated for 1 h. Binding was detected using horse radish peroxidase (HRP)-conjugated anti-mouse IgG or IgM antibody (Sigma) or anti-human IgM antibody (DakoCytomation, Glostrup, Denmark), and developed using Super Signal West Pico enhanced chemiluminescence (Pierce).
Natural and synthetic oligosaccharides
Gangliosides, sulfatides, and the oligosaccharide of GD3 (disialyllactose, DSL) were purchased from Sigma. Gal-4-sulfate and Gal-6-sulfate were from Dextra Laboratories (Reading, UK). The oligosaccharide of GM1 (EGM1-OS) was produced in Escherichia coli (Antoine et al. 2003; Townson et al. 2007). GD1a oligosaccharide (GD1a-OS) and sulph-OS were prepared by ozonolysis of GD1a and sulfatide respectively, which cleaves one of the acyl chains of ceramide and renders the oligosaccharides soluble. Briefly, GD1a and sulfatide were dissolved to 1 mg/mL in methanol and cooled in a dry ice/ethanol bath. Ozone was generated from oxygen using a bench top generator (Model OL80W, Ozone Services, Burton, BC, Canada). Ozone application time was optimized at 17 min by TLC monitoring of cleaved and uncleaved products. Ozone was then dispersed by passing oxygen through the sample, and dimethylsulfoxide (500 µL) added, stirred for 30 min on dry ice, then 90 min at room temperature. After drying under a stream of nitrogen, the cleaved long chain aldehyde was separated by adding 10 mL n-hexane, sonicating for 5 min and then centrifuging at 1400 rpm for 10 min. The n-hexane was then drawn off and the oligosaccharides dried under nitrogen.
Sulph-CG was prepared by enzymatic cleavage of both acyl chains by ceramide glycanase (Calbiochem). Sulfatide (5 mg) was dissolved by sonication in 40 mL 50 mM sodium acetate, pH 5.0, containing 1 mg/mL sodium cholate, and then 5 U of ceramide glycanase added and incubated, shaking for 64 h at 37°C. The sulph-CG was purified from the digestion mixture using Oasis HLB cartridges (Waters Ltd, Milford, MA) and then lyophilized. Purity was assessed by TLC.
Oligosaccharide inhibition ELISA
Inhibition of mAbs binding to solid phase gangliosides and sulfatide by their soluble counterparts was investigated by inhibition ELISA. MAbs were diluted in 0.1% (w/v) BSA in PBS and used at concentrations equivalent to half maximal binding, as determined by titration in ELISA. Stock solutions of oligosaccharides were prepared in distilled water to 5–10 mg/mL and further diluted in 0.1% (w/v) BSA in PBS. Equal volumes of mAb and oligosaccharide were mixed prior to application to a standard ganglioside ELISA (50 µL/well). Percentage inhibition was calculated by comparison to the binding of mAb/antisera alone (100%). All assays were performed in duplicate and repeated at least three times.
Binding of mAbs to a panel of sulfated oligosaccharide conjugates
Binding by ELISA of a selection of the mAbs in Table to a panel of sulfated oligosaccharide–PAA conjugates (Supplementary data, Table S) was measured. The ELISA protocol was optimized for oligosaccharide–PAA conjugates, rather than gangliosides, and consequently was slightly different from the ELISAs described above. ELISA 96-well plates (NUNC Maxisorp, Denmark) were coated with sugar–PAA conjugates (Lectinity Holding, Inc, Moscow, Russia) 10 µg/mL in 0.05 M Na-carbonate buffer, pH 9.6, for 1 h at 37°C. Plates were then blocked with 3% BSA in PBS (w/v) for 2 h at 4°C and washed three times with PBS containing 0.1% Tween-20 (washing buffer). MAbs were diluted to 50 µg/mL in 0.3% BSA/PBS and 100 µL added per well. The plates were then incubated for 2 h at 4°C. Plates were washed three times with washing buffer and incubated with anti-mouse Ig-HRP (Sigma) (1:1000 in 0.3% BSA/PBS) or anti-human Ig-HRP for 2 h at 4°C. After washing, the plates were developed as for the ganglioside ELISAs described above. Binding of DG2 to 3-O-Su-Galβ-BSA was also tested, following the ganglioside ELISA protocol, but coating with 10 µg/mL PBS overnight at 4°C and then washing prior to blocking.
Analysis of antiganglioside mAb binding to sulfatide in cell membranes
PC12 cells were cultured in poly-L-lysine (Sigma) coated tissue culture flasks with DMEM containing 7.5% foetal calf serum (FCS) and 7.5% horse serum (Sigma). After harvesting, the cells were coated onto coverslips overnight at 37°C (1.5 x 104 cells/coverslip). Sulfatide was reconstituted in a small volume of distilled H2O by incubating at 37°C for 1 h with frequent vortexing and sonication. Serum-free DMEM was added to give a sulfatide concentration of 20 µg/mL. To allow sulfatide to insert into PC12 cell plasma membranes, coverslips were rinsed in serum-free DMEM and then incubated at 37°C for 18 h in serum-free DMEM with or without sulfatide. Coverslips were then washed in serum-free DMEM, and any GM1 on the cell surface was blocked by incubating with CTB (Sigma) (4 µg/mL in serum-free DMEM) for 1 h at 4°C. After washing, as before, antiganglioside mAb (DG1, DG2 or EG3) or antisulfatide positive control mAb O4 were diluted in serum-free DMEM (10 µg/mL) and added for 1 h at room temperature. All further washing steps and dilutions were performed in DMEM containing 7.5% FCS and 7.5% horse serum. After washing, FITC-labeled goat anti-mouse IgG (for DG1, DG2, and EG3) or IgM (for O4) (Southern Biotech, Birmingham, AL) diluted 1/300 (v/v) was added for 1 h, in the dark, at room temperature. Coverslips were then washed and fixed in methanol for 30 min at –20°C. Coverslips were mounted with vectashield containing DAPI (Vector Laboratories, Peterborough, UK). To check for nonspecific secondary antibody binding, sulfatide treated and untreated cells were stained as above, but without mAb.
Flow cytometry was performed on PC12 cells grown overnight in poly-L-lysine coated tissue culture dishes with DMEM containing 7.5% FCS and 7.5% horse serum. After washing with chilled, sterile PBS, sulfatide was added, reconstituted in serum-free medium as described above, and the cells incubated at 37°C for 18 h. All washes and dilutions were performed in PBS containing 2% FCS and all incubations were for 1 h at 4°C. Sulfatide-treated or untreated cells were harvested and aliquots of 1 x 105 cells incubated with CTB (4 µg/mL). The cells were then washed and incubated with antiganglioside mAb or antisulfatide control mAb (10 µg/mL), followed by FITC-labeled goat anti-mouse IgG or IgM. Binding was analyzed using a FACScan (Becton Dickinson, Oxford, UK). Only very low levels of nonspecific secondary antibody binding were observed, and these were subtracted.
Analysis of antiganglioside mAb binding to sulfatide in peripheral nerve sections
Sciatic nerves were removed from sulfatide accumulating and cerebroside sulfotransferase (CST) overexpressing (Arylsulfatase A-deficient/PLP-CST transgenic mice, 7 months) (H Ramakrishnan et al., in preparation) and sulfatide deficient (UDP-galactose:ceramide galactosyltransferase knock-out mouse (CGT–/–), 3 weeks) mice (Coetzee et al. 1996), embedded immediately in semi-frozen OCT Embedding Matrix (CellPath, Hemel Hempstead, UK) and mounted onto a cryostat chuck. Nerves were cryosectioned at 15 µm in the transverse plane onto L-lysine-coated (Sigma) glass slides. Sections were stained unfixed or fixed in ice-cold methanol or 4% paraformaldehyde. Slides were incubated with DG2 or O4 (10 µg/mL PBS) for 1 h at 4°C, followed by three rinses in PBS and incubation at 4°C for 2 h in FITC conjugated anti-mouse IgG and IgM, respectively (1/300 (v/v) PBS). In order to block DG2 binding to GM1, the nerves were pretreated with CTB (4 µg/mL PBS) for 1 h at 4°C, before rinsing and application of DG2. Substitution of primary antibody with PBS confirmed that in both control and CTB treated sections, there was no nonspecific binding of the secondary antibody. Nerves were mounted in Citifluor (Citifluor, Canterbury, UK) for imaging under identical settings with a Zeiss (Oberkochen, Germany) Pascal confocal microscope.
Supplementary data for this article is available online at http://www.glycob.oxfordjournals.org.
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This work was supported by grants to HJW from the Wellcome Trust (GR060349 and GR077041) and to DRB from the Canadian Institute of Health Research and Alberta Ingenuity.
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsFundingConflict of interest statementReferences |
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None declared.
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Abbreviations |
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BSA, bovine serum albumin; CGT–/–, sulfatide deficient mice; CSThigh, cerebroside sulfotransferase overexpressing, sulfatide accumulating mice; CTB, cholera toxin B subunit; DSL, disialyllactose; EGM1-OS, oligosaccharide of GM1; GD1a-OS, GD1a oligosaccharide; FCS, foetal calf serum; Gal-4-sulfate, D-galactose-4-O-sulfate; Gal-6-sulfate, D-galactose-6-O-sulfate; MAbs, monoclonal antibodies; PAA, polyacrylamide; PBS, phosphate buffered saline; Sulph-OS, ozonolyzed 3-O-sulfo-β-D-galactosylceramide; Sulph-CG, ceramide glycanase treated 3-O-sulfo-β-D-galactosylceramide; TLC, thin layer chromatography
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