Monoclonal Antibody A2B5, Which Detects Cell Surface Antigens, Binds to Ganglioside GT3 (II3 (NeuAc)3LacCer) and to Its 9-O-Acetylated Derivative*

The monoclonal antibody A2B5 recognizes antigens at the surface of neuronal and glial cells but also at the surface of thymus epithelia and pancreatic islet cells. Although these antigens have been characterized as polysialogangliosides, A2B5 also reacts with other un- identified gangliosides. In order to characterize fur- ther the epitope of A2B5, two new ganglioside anti- gens isolated from chicken brain are identified in this study. One is the and and the

The monoclonal antibody A2B5 recognizes antigens at the surface of neuronal and glial cells but also at the surface of thymus epithelia and pancreatic islet cells. Although these antigens have been characterized as polysialogangliosides, A2B5 also reacts with other unidentified gangliosides.
In order to characterize further the epitope of A2B5, two new ganglioside antigens isolated from chicken brain are identified in this study.
One is the ganglioside NeuAca2-8NeuAca2-8NeuAca2-3GalBl-4Glcfil-lceramide (G-& and the other is a 9-0-acetylated derivative of G&. This derivative was purified from lo-day embryonic chicken brain. Acetyl groups substituted on sialic acid were removed either by alkali treatment or by incubation with influenza virus C, which contains receptor-destroying enzyme (a neuraminidate 9-0-acetyl esterase). The product of alkali treatment or viral action was detected by the antibody 18B8 which is specific for GT3. The deacetylated product still reacts with A2B5.
These data and the results of mild oxidation of the antigen with sodium periodate suggest that the epitope recognized by antibody A2B5 contains the trisialyl structure found in GT3 but does not include the polyalcohol chain of the terminal sialic acid which can be oxidized by periodate or acetylated without modifying the affinity for the antibody. The epitope recognized by A2B5 is different from the epitope recognized by the antibody 18BS in that 18B8 requires the three sialic acids with an intact and unsubstituted polyalco-ho1 chain. Antibody 18BS does not bind to 9-O-acetylated GT~ or GT3 oxidized by sodium periodate.
Monoclonal antibody A2B5 was originally prepared against chicken embryo retina cells (1). The antigens recognized by the antibody are localized on the plasma membrane of retina neurons, and antigen expression depends on the stage of neuronal differentiation. The antigens are detected on bipotential rat glial progenitor cells in optic nerve which differentiates into either oligodendrocytes which loose the antigen or type 2 astrocytes which maintain the antigen (2). The neuronal differentiation of an embryonal carcinoma cell line (3)  acid modulates the expression of cell surface A2B5 antigens. The antibody labels Alzheimer's neurofibrillary tangles and fetal human neurons (5) and astrocytes (6). However, the A2B5 antigens are not restricted to nervous tissues as they also occur in human pancreatic islet tumor cells and other tumors derived from cells of the amine precursor uptake and decarboxylation series (7) and in rat insulinoma cells (8,9). They are also expressed by thymic epithelial cells, which contain thymopoietin and thymosin cul (10, 11). The antibody A2B5 detects polysialogangliosides on cell surfaces, and its immunoreactivity is lost by treatment of cells or tissues with neuraminidase (1). However, its specificity is still controversial. Even though one of its ganglioside antigens has been characterized as GsI,' (12) and a recent report provided evidence of specific reactivity of A2B5 with GT~ and GQ1, (13), other reports showed reactivity with other gangliosides such as GBlb and disialogangliosides (14,15) or with many gangliosides in neuronal tissue which do not correspond to the major gangliosides of this tissue (15). Since monoclonal antibody A2B5 should be used to obtain new fundamental insight into the tissue distribution and the metabolism of the gangliosides it recognizes, the specificity of this antibody has been reexamined. Two major ganglioside antigens were isolated from embryonic chicken brain and identified in this study; one is the trisialoganglioside GT3 and the other is its 9-0-acetylated derivative. Thus, antibody A2B5 differs from antibody 18B8 which detects synapse-associated antigens (16) and which has been previously described as specific for GT3 (17). The chemical or enzymatic modifications of the gangliosides allowed us to improve the definition of the structure of the epitope recognized by A2B5 and to compare its epitope with that recognized by 18B8. was washed as before in cold phosphate-buffered saline (PBS), dried, and exposed to XAR-5 x-ray film (Eastman Kodak).

Solid Phase Radioimmunoassay
The binding of antibody to glycolipid was measured by solid phase radioimmunoassay as previously described (17,19); glycolipid in 20 ~1 of ethanol was added to wells of a round bottom polyvinyl chloride microtiter plate (Linbro), and the solutions were dried by evaporation. The wells were then filled with buffer A. After 30 min, the wells were emptied and to each was added 30 ~1 of monoclonal antibody solution diluted 1:20 with buffer A. The wells were covered with Parafilm, incubated for 1 h at room temperature, washed three times with buffer A, and then to each was added about 60,000 cpm of I*?-labeled goat anti-mouse IgM antibody in 30 ~1 of buffer A. After 1 h, the wells were washed six times with cold phosphate-buffered saline, cut from the plate, and assayed for 125I in a y scintillation spectrometer.

Isolation of the Ganglioside Antigens
Lipids were extracted from lo-12-day chick embryonic brains (115 g wet weight) with chloroform:methanol:H20 (4:8:3 by volume as previously described (17,20  GDs -matographed 0; HPTLC plates, and the ganglioside antigens were visualized by immunostaining with antibodies A2B5 and 18B8 as described under "Experimental Procedures." 17, and 20-day chick embryonic brain, respectively. The migration of standard glycolipids is indicated at the margins. body 18B8 specific for GT3 (17), binds to purified GT3. Other purified gangliosides tested for their ability to bind antibody A2B5 included GM3, Gr.,n, Gm,, Gnib, Gns, GT,b, GM1 lactone, and Gnlb lactone. None of these gangliosides binds to antibody A2B5 (Fig. 1, panel B). When the antibody A2B5 binding was tested by immunostaining of thin layer chromatograms, no reactivity of the antibody with these gangliosides was observed (data not shown). Antibody A2B5 binds to other gangliosides isolated from chicken brain, and their accumulation in the brain changes during embryonic development (Fig. 2). One major antigen (antigen I) with a chromatographic mobility similar to that of Goa is recognized by A2B5 but not by 18B8. In an attempt to identify antigen I, 8-and lo-day embryonic chicken brains which contain a relatively high concentration of the antigen were used for its isolation. In order to protect possible ester linkages in the structure of ganglioside, the purification procedure omitted the alkaline hydrolysis step normally used to eliminate phospholipids. The ganglioside antigen was purified on a anion exchange column and eluted by a 0.12-0.16 M ammonium acetate gradient. The ganglioside was then purified by silicic acid chromatography, and the column fractions were analyzed by immunostaining thin layer chromatograms with the antibody A2B5. Fig. 3, panel A, shows the elution of antigen I in fractions 70-110 of the silicic acid column. The doublets, which probably differ in their ceramides, could be separated. The antigen eluted in fractions 102-118 was identified as GT3 by using antibody 18B8 (data not shown). When the fractions were analyzed by chemical staining with resorcinol reagent (Fig. 3, panel B), only one ganglioside, which was coincident with the antigen I, was detected in fractions 84. However, immunostaining of chromatograms of the pooled 9-0-Acetylated Ganglioside Antigens Gangliosides from lo-day embryonic chicken brain were fractionated by anion exchange chromatography. The fractions containing antigen I were subjected to silicic acid chromatography as described under "Experimental Procedures." Panel A, aliquots of the fractions eluted from the column of Bio-Sil were chromatographed on HPTLC plates, and the ganglioside antigens were visualized by immunostaining of chromatograms with antibody A2B5. Panel B, gangliosides of fractions containing antigens were visualized with resorcinol reagent. Lane G, unfractionated glycolipids from 20 mg (wet weight) of tissue. The migration of standard glycolipids is indicated at the margins. fractions with antibodies A2B5 and 18B8 detected a weak contamination of antigen I with GrS. Antigen I Is an Alkali-labile Ganglioside-The alkali lability of gangliosides is due to the presence of ester linkages which are present in 0-acetylated gangliosides or in lactones of gangliosides isolated from different tissues (22,23,(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). The 0-acetylated gangliosides are usually more resistant to alkali treatment than the lactones of gangliosides. Purified antigen I was treated with aqueous ammonia as described under "Experimental Procedures." Treated and nontreated gangliosides were then analyzed in parallel by thin layer chromatography, and the chromatogram was immunostained with the antibody A2B5. As seen in Fig. 4, lane 2, antibody A2B5 did not detect antigen I treated with concentrated ammonia.
This result suggests that antigen I is an alkalilabile ganglioside and probably contains 0-acetylated sialic acid residues.
Identification of Alkali-labile Antigen I-In order to identify the alkali-labile antigen, the product of its alkali treatment was analyzed. Purified antigen I was chromatographed on HPTLC plates, and the chromatograms were exposed to ammonia vapors (22). The reactivity of antibodies A2B5 and 18B8 against the treated ganglioside was tested by immunostaining of chromatograms (Fig. 5, panel A). Antibody A2B5 still binds to the product of hydrolysis of the alkali-labile antigen. Antibody 18B8, which did not bind to this antigen, now binds to the hydrolyzed molecule identified as Gr.+ To confirm this data, which suggest that antigen I is the alkalilabile GTJ, antigen I was analyzed by two-dimensional thin layer chromatography with treatment with ammonia between runs (23). This method allows identification of one ganglioside and its alkali-labile derivative by comparing their chromatographic mobilities in each dimension. As is shown in Fig. 5, panel B, two additional gangliosides (spots II and X) generated by alkali treatment are recognized by both antibodies. The antigen II generated by treatment with ammonia after the first dimension has the same chromatographic mobility as GT1 in the second dimension.
These results suggest that antigen II is probably GrJ. Moreover, the new antigen II has the same chromatographic mobility in the first dimension as antigen I, suggesting that antigen II derives from alkali-labile antigen I. These results provide strong evidence that the alkali-labile antigen I can be identified as alkali-labile GT.?. The antigen X generated by exposure to ammonia is still not identified. Antigen I Is a Substrate for the Receptor-destroying Enzyme of ZNF-C-To test the possibility that alkali-labile GT3 is Oacetylated, the purified ganglioside was treated with influenza C virus whose surface glycoprotein (hemagglutinin) recognizes N-acetyl-9-0-acetylneuraminidic acid (25) and contains the receptor-destroying enzyme identified as a neuraminidate Oacetyl esterase which cleaves preferentially 9-0-acetyl groups from N-acetyl-9-0-acetylneuraminic acid (26-28). This enzyme is inactivated by DFP, a serine esterase inhibitor (29). Gangliosides were chromatographed on HPTLC plates, and the chromatograms were incubated with INF-C virus or DFPinactivated INF-C virus as described under "Experimental Procedures." Immunostaining of chromatograms with A2B5 did not detect any modification of the antigen catalyzed by the viral enzyme (Fig. 6, A-C). A new antigen identified as CT1 by antibody 18B8 is produced by treatment of ganglioside with INF-C virus (Fig. 6, panel B). The new GTB has the same chromatographic mobility as antigen I. These results suggest that antigen I is the substrate of influenza C-neuraminidate-9-0-acetyl esterase, and it is identified as 9-0-acetylated Gr3. GTZI was not formed if the virus was preincubated with DFP ( Fig. 6, panel C). No Gr:% was produced in the control (Fig. 6,  panel A) showing the absence of esterase activity in the amniotic fluid.
Effect of Mild Oxidation of the Antigens by Sodium Periodate-The role of the polyalcohol chain of GTZ1 in the epitope recognized by antibody A2B5 was studied. The ganglioside I. Panel A, after thin layer chromatography of gangliosides, the plates were not submitted (-) or submitted (+) to ammonia vapors as described under "Experimental Procedures." Ganglioside antigens were visualized by immunostaining with antibodies A2B5 and 18B8. Lane 1, antigen 1; lane 2, 10 ng of purified Gr3. Panel B, gangliosides isolated from 15 mg (wet weight) of lo-day embryonic chicken brain were submitted to two-dimensional thin layer chromatography using solvent A in both dimensions. The direction of each run is shown by the arrows I and 2. Plates (-) were not treated. Plates (+) were exposed to ammonia vapors after the first run, as described under "Experimental Procedures." Ganglioside antigens were visualized by immunostaining with antibodies A2B5 and 18B8. I, refers to the antigen I. II and X, refer to the antigens generated by exposure to ammonia.
antigens were submitted to mild oxidation by sodium periodate, which cleaves, under defined conditions, the polyalcohol exopyranosyl chain of the sialyl residues (30). If the chain is substituted (0-acetylation or glycosidic linkages) oxidation does not occur. In the case of Gr3 only one unsubstituted polyalcohol chain is on the external sialic acid and can be oxidized. Mild oxidation experiments were carried out after chromatography of gangliosides as described under "Experimental Procedures." As 0-acetylation on C9 or C8 will prevent oxidation of the chain, the chromatograms were submitted to ammonia vapors prior to being treated by sodium periodate. As is shown by immunostaining of chromatograms, the binding of antibody A2B5 to ganglioside antigens and to Gr, was not affected by a treatment with as much as 5 mM sodium periodate (Fig. 7, panel B). Identical results were obtained when the antigens were deacetylated prior to being treated with periodate (Fig. 7, panel C). In contrast, the binding of antibody 18B8 to purified Gr3 and to other gangliosides was completely abolished after their oxidation with 1 mM periodate (Fig. 7, panels E and F).
These results show the role of the polyalcohol chain, present in antigen Gr3, in the structure of the epitope recognized by the antibodies. The binding of antibody A2B5 to oxidized Gr3 suggests that the binding of the antibody to Gr3 does not require carbon 9 of the polyclonal chain. This carbon can be also esterified as in 9-0-acetylated Gr3 the ganglioside is still recognized by the antibody. On the other hand, the binding of antibody 18B8 depends on the presence of carbon 9 which must be unsubstituted as the 18B8 antibody does not bind to oxidized Gr3 or to 9-0-acetylated Gr3.

DISCUSSION
Monoclonal antibody A2B5 detects polysialogangliosides on the surface of many different tissues from various species (l-9). However, the exact epitope recognized by this antibody is not well defined. Previous reports showed the reactivity of the antibody with Gr3 (13) and Gale (12), which is a derivative of Gr3 (41), while other studies reported the reactivity of the antibody with gangliosides which are not derivative of Gr3 (14) or with many unidentified gangliosides in neuronal tissue (15). The specificity of the antibody A2B5 was reexamined, and this work characterizes the major antigens found in chicken brain. One of these antigens was characterized as Gr3. The specificity of binding of antibody A2B5 to Gr3 was revealed by immunostaining of thin layer chromatograms and solid phase radioimmunoassay using purified Gr3. This result agrees with the earlier observations (13). Monoclonal antibody 18B8 which is directed against Gr3 (17) was used as a control. The binding of both antibodies to the lipid was compared and found to be similar. A second major antigen detected by A2B5 migrates faster than Gr3 in chromatography. It was isolated from lo-day embryonic chicken brain and characterized as 9-0-acetylated GTR by the following criteria. (i) During purification, which did not include the alkali treatment normally used to hydrolyze the glycerophospholipids, the antigen was eluted from an anion exchange column in the trisialoganglioside fraction by 0.12-0.16 M ammonium acetate. (ii) This antigen is alkali-labile, and the product of the alkali treatment was identified as GT3 by its chromatographic mobility and the binding of antibody 18D8. (iii) Its chromatographic mobility on thin layer chromatography is higher than that of Gr3 and could be explained by an increased hydrophobicity because of the presence of an 0-acetyl group substitution at the hydroxyl position of sialic acid residues. (iv) Finally, the ganglioside is a substrate of receptor-destroying enzyme of influenza C virus which is a 9-0-acetyl neuraminidate esterase (25-28). The transformation of the antigen by influenza C virus is inhibited by preincubation of the virus with the esterase inhibitor diisopropyl fluorophosphate. The product of the enzymatic reaction binds antibody 18B8 and . . is identified as GT3.
The antibody A2B5 does not bind to mono-and disialogangliosides or to GTlb and Gplb. These results agree with previous reports (12, 13). These data suggest that the antibody A2B5 binding requires the trisialyl structure with (~2-8 linkages, found in Gr3 and its derivative Gale. The antibody 18B8, like the antibody A2B5, binds to the trisialyl structure found in GT3. However, these antibodies are different since antibody A2B5 binds to the alkali-labile 9-0-acetylated Gr3 whereas 18B8 does not. After mild oxidation of Gr3 with sodium periodate which cleaves the polyalcohol exopyranosyl chain of external sialic acid, A2B5 still binds to the ganglioside, but the binding of antibody 18B8 is abolished. These results suggest that the epitope recognized by antibody A2B5 does 9-0-Acetylated Gun&side Antigens