Interaction of GM2 Activator Protein with Glycosphingolipids*

(Received for publication, September 3, 1996, and in revised form, October 29, 1996)

Yoichiro Hama , Yu-Teh Li and Su-Chen Li Dagger

From the Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

GM2 activator protein is a protein cofactor that has been shown to stimulate the enzymatic hydrolysis of both GalNAc and NeuAc from GM2 (Wu, Y. Y., Lockyer, J. M., Sugiyama, E., Pavlova, N.V., Li, Y.-T., and Li, S.-C. (1994) J. Biol. Chem. 269, 16276-16283). To understand the mechanism by which GM2 activator stimulates the hydrolysis of GM2, we examined the interaction of this activator protein with GM2 as well as with other glycosphingolipids by TLC overlay and Sephacryl S-200 gel filtration. The TLC overlay analysis unveiled the binding specificity of GM2 activator, which was not previously revealed. Under the conditions optimal for the activator protein to stimulate the hydrolysis of GM2 by beta -hexosaminidase A, GM2 activator was found to bind avidly to acidic glycosphingolipids, including gangliosides and sulfated glycosphingolipids, but not to neutral glycosphingolipids. The gangliosides devoid of sialic acids, such as asialo-GM1 and asialo-GM2, and the GM2 derivatives whose carboxyl function in the NeuAc had been modified by methyl esterification or reduction, were only very weakly bound to GM2 activator. These results indicate that the negatively charged sugar residue or sulfate group in gangliosides is one of the important sites recognized by GM2 activator. For comparison, we also studied in parallel the complex formation between glycosphingolipids and saposin B, a separate activator protein with broad specificity to stimulate the hydrolysis of various glycosphingolipids. We found that saposin B bound to neutral glycosphingolipids and gangliosides equally well, and there was an exceptionally strong binding to sulfatide. In contrast to previous reports, we found that GM2 activator formed complexes with GM2 and other gangliosides in different proportions depending on the ratio between the activator protein and the ganglioside in the incubation mixture prior to gel filtration. We were not able to detect the specific binding of GM2 activator to GM2 when GM2 was mixed with GM1 or GM3. Thus, the specificity or the mode of action of GM2 activator cannot be simply explained by its interaction with glycosphingolipids based on complex formation. The binding of GM2 activator to a wide variety of negatively charged glycosphingolipids may indicate that this activator protein has functions other than assisting the enzymatic hydrolysis of GM2.


INTRODUCTION

In higher animals, the sugar chains of glycosphingolipids are catabolized by the sequential action of lysosomal exoglycosidases (1). It has been shown that, in addition to beta -hexosaminidase A, the conversion of GM21 into GM3 requires the assistance of GM2 activator, a low molecular weight protein cofactor (2-4). The physiological significance of GM2 activator has been demonstrated by the fact that the congenital defect of this activator protein leads to cerebral accumulation of GM2 in type AB Tay-Sachs disease (5, 6).

Human GM2 activator has been isolated from kidney (4), brain (6), and liver (7). This activator has been shown to be very specific in stimulating the hydrolysis of GalNAc from GM2 by beta -hexosaminidase A (1, 4, 7). This activator protein was also shown to assist the hydrolysis of NeuAc from GM2 by clostridial sialidase (8) and to recognize the branched trisaccharide (GM2-epitope) in GM2 (9). This activator, however, is not required for the hydrolysis of water-soluble synthetic substrates such as 4-methylumbelliferyl-beta -GlcNAc or p-nitrophenyl-beta -GlcNAc by beta -hexosaminidase A. The mode of action of GM2 activator is still not well understood. Through the studies of complex formation between GM2 activator and glycosphingolipids using electrophoresis, isoelectric focusing, and ultracentrifugation (4, 10), Conzelmann and Sandhoff (4) postulated that the action of GM2 activator is to extract a single GM2 molecule from its micelles to form a water-soluble protein-lipid complex (1:1 ratio), which serves as the true substrate for beta -hexosaminidase A. This hypothesis, however, is not supported by two simple facts: (a) The water-soluble tetrasaccharide derived from GM2 cannot be hydrolyzed by beta -hexosaminidase A in the presence or absence of the activator (8) and (b) saposin B, another activator protein whose action is to solubilize glycosphingolipids, does not stimulate the hydrolysis of GM2 by beta -hexosaminidase A.

The results of previous studies on the interaction between glycosphingolipids and the activator proteins isolated from human tissues might have been complicated by the possible presence of contaminated proteins. Recently, we have cloned the cDNA encoding human GM2 activator (11) and also expressed the cDNA in Escherichia coli (8). The availability of pure recombinant human GM2 activator in large quantities made the re-examination of the interactions between GM2 activator and glycosphingolipids possible. To understand the mode of action of GM2 activator, we have studied the interaction of GM2 activator with various glycosphingolipids by TLC overlay and Sephacryl S-200 gel filtration. For comparison, we have also studied in parallel the interaction of glycosphingolipids with saposin B, a nonspecific activator protein that has been reported to stimulate the enzymatic hydrolysis of a wide variety of glycosphingolipids (12). We found that in aqueous medium, such as gel filtration, one molecule of GM2 activator was able to associate with multiple molecules of gangliosides. By TLC overlay, GM2 activator was found to bind to various negatively charged glycosphingolipids without showing preference to any particular sugar chain.


EXPERIMENTAL PROCEDURES

Materials

GM2 was prepared from the brain of a Tay-Sachs patient (13). GA1 and GA2 were prepared from GM1 and GM2, respectively, by mild acid hydrolysis (14). The following glycosphingolipids were the generous gifts: GM1, from Drs. G. Kirschner and G. Toffano (Fidia Research Laboratory, Italy); GalNAc-GD1a and NeuGc-GM1 (15), from Dr. S. Sonnino (University of Milan, Milan, Italy); the chemically modified Me-GM2 (the carboxyl group of NeuAc in GM2 was methyl esterified) and HO-GM2 (the carboxyl group of NeuAc in GM2 was reduced to alcohol) (16, 17), from Dr. S. Handa (Tokyo Medical and Dental University, Tokyo); SM3, from Dr. T. Ishizuka (Teikyo University, Tokyo); and the chemically synthesized gangliosides, KDN-GM3, IV6KDNLnOse4Cer, and IV6KDNLcOse4Cer, from Dr. A. Hasegawa (Gifu University, Gifu, Japan). Oligo-GM2 was prepared from GM2 using ceramide glycanase (18). PE-GM2, the neoglycolipid, was prepared by conjugating II3NeuAcGgOse3 that was derived from GM2 to dipalmitoylphosphatidylethanolamine by reductive amination (19). 3H-Labeled GM1 and GM2 were prepared using the galactose oxidase and NaB3H4 reduction procedure as described by Radin (20) with a slight modification (21). The recombinant GM2 activator and saposin B were both produced in E. coli and purified as described previously (8, 9). beta -Hexosaminidase A (2) and beta -galactosidase (22) were isolated from human liver. The following were purchased from commercial sources: GD2, GM4, GM3, and LacCer, Matreya (Pleasant Gap, PA); GalCer, GlcCer, sulfatide, primulin, polyoxyethylenesorbitan monolaurate (Tween 20), and bovine serum albumin, Sigma; [14C]formaldehyde (specific activity, 55 mCi/mmol), American Radiolabeled Chemicals (St. Louis, MO); Polygram SIL G TLC plate, Macherey-Nagel (Duren, Germany); dimethylamine borane complex, ammonium acetate, Aldrich; Sephacryl S-200 (super fine), Pharmacia (Uppsala, Sweden); polyvinylpyrrolidone, Fisher; Bio Gel P-6, Bio-Rad; X-OMAT AR film, Kodak; Silica gel 60 TLC plate, Merck (Darmstadt, Germany); and Universol (scintillation mixture), ICN (Irvine, CA).

Radiolabeling of GM2 Activator and Saposin B

14C-Labeled GM2 activator and saposin B were prepared by reductive methylation of amino groups with 14C-labeled formaldehyde (23, 24). Briefly, 300 µg of GM2 activator or saposin B were dissolved in 85 µl of 0.2 M phosphate buffer, pH 7.0. To this solution, 88.4 µg of dimethylamine borane complex, which had been dissolved in 10 µl of methanol, was added. After addition of 5 µl (2.2 µmol) of aqueous [14C]formaldehyde, the mixture was left at room temperature for 6 h. Then, the resulting 14C-labeled protein was separated from the reagents by gel filtration on a Bio Gel P-6 column (0.9 × 10 cm) using water as an eluant, followed by dialysis against 10 mM ammonium acetate buffer, pH 6.8, and lyophilized. The stimulatory activities of the 14C-labeled GM2 activator and saposin B on the hydrolyses of GM2 by beta -hexosaminidase A and GM1 by beta -galactosidase, respectively, were confirmed by the methods described previously (8, 12).

TLC Overlay

The three buffer solutions used for studying the interactions between the activator proteins and glycosphingolipids on TLC plates were (a) 25 mM ammonium acetate buffer, pH 4.0, a low ionic strength acidic buffer providing the optimal condition for assaying the hydrolysis of GM2 by beta -hexosaminidase A; (b) 25 mM ammonium acetate buffer, pH 6.8, a low ionic strength neutral buffer; and (c) 250 mM ammonium acetate buffer, pH 4.0, a high ionic strength acidic buffer. Each glycosphingolipid sample (10-15 nmol) in chloroform:methanol (2/1 v/v) was first applied onto a Polygram SIL G TLC plate, and the plate was developed with chloroform:methanol:water (60/35/8, v/v/v). The dried plate was then immersed and kept at 37 °C for 30 min in one of the above mentioned buffer solutions, which contained 1% each of polyvinylpyrrolidone and bovine serum albumin. The plate was then incubated in 5 ml of the same buffer solution containing 50 µg of the 14C-labeled activator protein (250,000 cpm) and 3% polyvinylpyrrolidone at 37 °C for 1 h and washed three times with the buffer solution containing 0.05% Tween 20 and then air dried. Finally, the protein-lipid complexes were detected by placing the TLC plate onto an x-ray film to obtain a radioautogram. After obtaining the radioautogram, the same TLC plate was sprayed with diphenylamine reagent (25) and heated at 110-120 °C for 15-20 min to reveal the glycosphingolipids on the plate.

Gel Filtration Chromatography

For studying the complex formation between GM2 activator and the micellar form of glycosphingolipids using Sephacryl S-200 gel filtration, 25 mM ammonium acetate buffer, pH 4.0, was used as the incubation buffer and also to equilibrate and elute the column. GM2 activator (25 µg, 1.34 nmol) in 100 µl of the buffer solution was mixed with a given amount of a ganglioside or oligo-GM2 and incubated at 37 °C for 30 min. The entire mixture was subsequently applied onto a Sephacryl S-200 column (0.6 × 30 cm) connected to an HPLC system (Waters 600E, Millipore). The column was then eluted with the same buffer at a flow rate of 0.25 ml/min, and the effluent was monitored by the absorbance at 280 nm (Waters 490E UV-VIS detector). Fractions of 0.5 ml (2 min) were collected through the entire run, and each fraction was analyzed for the content of the activator protein and the glycosphingolipid.

Hydrolysis of GM2

The fractions that contained the protein-lipid complex eluted from the Sephacryl S-200 column were incubated with 0.5 units of beta -hexosaminidase A at 37 °C for 3 h. Each incubated fraction was evaporated to dryness, dissolved in 20 µl of chloroform:methanol (2/1, v/v), and analyzed by Silica gel 60 TLC plate using chloroform:methanol:water (60/35/8, v/v/v) as the developing solvent. Gangliosides were visualized by spraying the plate with diphenylamine reagent (25) followed by heating at 110-120 °C for 15-20 min.

Analytical Methods

When the activator protein was incubated with only [3H]GM1 or [3H]GM2, the amount of the [3H]GM1 or [3H]GM2 in the protein-lipid complex was determined as follows: a 50-µl aliquot of each fraction obtained from the Sephacryl S-200 column was mixed with 5 ml of Universol, and the radioactivity was measured by a Tri-Carb model 1600 CA liquid scintillation counter (Packard Instrument Co., IL). When the activator protein was incubated with GM3, which was not radiolabeled, the amounts of GM3 in the protein-lipid complexes were determined by TLC analysis using the resorcinol spray (26) and then quantitated by Scan Jett II CX (Hewlett Packard, Boise, ID) and NIH image 1.55. When the activator protein was incubated with both [3H]GM1 and [3H]GM2, the two gangliosides in the protein-lipid complex were first separated from each other by TLC using chloroform:methanol:water (60/35/8, v/v/v) as the developing solvent. The [3H]GM1 and [3H]GM2 on the plate were first revealed by primulin reagent (27) and then individually scraped off the plate and mixed with Universol; the radioactivity was then measured by a scintillation counter. When the activator protein was incubated with both GM2 and GM3, the amounts of GM2 and GM3 in the protein-lipid complex were determined as follows: an aliquot of each fraction was evaporated to dryness, redissolved in 20 µl of chloroform:methanol (2/1, v/v), and applied onto a TLC plate. The plate was developed with the solvent system as described above for separating GM1 and GM2, and the gangliosides were visualized with the diphenylamine reagent (25). The amounts of GM2 and GM3 were quantitated by scanning the TLC plate with a Scan Jet IICX and analyzed by NIH Image 1.55.

Determination of Protein

Protein was determined by the method of Lowry et al. (28) using bovine serum albumin as a standard.


RESULTS

Interaction of GM2 Activator with Glycosphingolipids on a TLC Plate

The interactions between GM2 activator and glycosphingolipids were examined by TLC overlay on which glycosphingolipids were associated with silica gel. Fig. 1A shows the representative common acidic and neutral glycosphingolipids on the plate that were stained by the diphenylamine reagent (25). While the same amount (15 nmol) of each glycosphingolipid was applied on the plate, GlcCer and GalCer showed weaker staining than GM1, since the color intensity produced by the diphenylamine reagent depends on the sugar content of the glycosphingolipids. The same TLC plate prior to the chemical staining was overlaid with the radiolabeled GM2 activator as described under "Experimental Procedures," and the results are shown in Fig. 1B. The conditions for the overlay were first chosen to use the low ionic strength acidic buffer (25 mM ammonium acetate buffer, pH 4.0), which is the optimal condition for GM2 activator to stimulate the hydrolysis of GM2 by beta -hexosaminidase A. Under this condition, GM2 activator protein binds avidly to gangliosides GM1, GM2, and GM3 (lanes 1, 2, and 3, respectively) but very weakly to the neutral glycosphingolipids, LacCer, GalCer, and GlcCer (lanes 4, 5, and 6, respectively). The bindings of GM2 activator to 14 other glycosphingolipids were further examined under the same conditions. As summarized in Table I, GM2 activator binds to several other acidic glycolipids such as GM4, the synthesized PE-GM2 (8) which contains the oligosaccharide of GM2 linked to phosphatidylethanolamine instead of ceramide, NeuGc-GM1, KDN-GM3 and two chemically synthesized gangliosides, IV6 KDNLcOse4Cer and IV6KDNLnOse4Cer, whose KDN residues are linked through alpha 2right-arrow6Gal. These results indicate that the interactions between GM2 activator and glycosphingolipids require the presence of an acidic moiety on the glycosphingolipid, and the binding is not significantly affected by the sugar chain backbones, the position (alpha 2right-arrow3Gal versus alpha 2right-arrow6Gal), and the nature of sialic acid (NeuAc versus NeuGc or KDN) in the glycosphingolipids. Furthermore, GM2 activator also binds to the sulfated glycosphingolipids, such as sulfatide and SM3, but not to the asialogangliosides, such as GA1 and GA2. These results strongly suggest that the recognition sites on the glycosphingolipids for the binding by GM2 activator are the anionic residues. These observations were further supported by studying the bindings between GM2 activator and the two chemically modified GM2. As shown in Fig. 2, conversion of the carboxylic function of NeuAc in GM2 to a methyl ester (Me-GM2) (Fig. 2, lane 3) or to an alcohol (HO-GM2) (Fig. 2, lane 4) abolishes the ability of the two modified GM2 derivatives to interact with GM2 activator. The results of this binding study explain our previous observation that Me-GM2 and HO-GM2 were not hydrolyzed by beta -hexosaminidase A in the presence of GM2 activator but could be hydrolyzed in the presence of sodium taurodeoxycholate (16). Fig. 2 also shows that sulfatide (lane 1) is very weakly stained by diphenylamine (25) due to its highly acidic sulfate residue. However, this acidic glycosphingolipid binds strongly to GM2 activator.


Fig. 1. Detection of the complex formation between GM2 activator and the common glycosphingolipids GM1, GM2, GM3, LacCer, GlcCer, and GalCer by TLC overlay. A, the TLC plate with the indicated glycosphingolipids was stained with the diphenylamine reagent. B, the radioautogram of the same TLC plate that was overlaid with the 3H-labeled GM2 activator prior to the chemical staining as shown in A. Glycosphingolipids (15 nmol each) were applied onto a TLC plate: lane 1, GM1; lane 2, GM2; lane 3, GM3; lane 4, LacCer; lane 5, GalCer; and lane 6, GlcCer. Each lane in A corresponds to that of B. The buffer solution used for TLC overlay was 25 mM ammonium acetate buffer, pH 4.0. The detailed conditions for the experiment are described in the text.
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Table I.

Binding of glycosphinglipids to GM2 activator

Binding of glycosphingolipids to GM2 activator was examined by TLC overlay. The buffer solution used for this experiment was 25 mM ammonium acetate, pH 4.0. Detailed conditions are described in the text.
Positive
  Galbeta 1right-arrow3GalNAcbeta 1right-arrow4(NeuAcalpha 2right-arrow3)Galbeta 1right-arrow4Glcbeta 1right-arrow1'Cer (GM1)
  GalNAcbeta 1right-arrow4(NeuAcalpha 2right-arrow3)Galbeta 1right-arrow4Glcbeta 1right-arrow1'Cer (GM2)
  NeuAcalpha 2right-arrow3Galbeta 1right-arrow4Glcbeta 1right-arrow1'Cer (GM3)
  NeuAcalpha 2right-arrow3Galbeta 1right-arrow1'Cer (GM4)
  GalNAcbeta 1right-arrow4(NeuAcalpha 2right-arrow3)Galbeta 1right-arrow3GalNAcbeta 1right-arrow4(NeuAcalpha 2right-arrow3)   Galbeta 1right-arrow4Glcbeta 1right-arrow1'Cer (GalNAc-GDla)
  GalNAcbeta 1right-arrow4(NeuAcalpha 2right-arrow8NeuAcalpha 2right-arrow3)Galbeta 1right-arrow4Glcbeta 1right-arrow1'Cer (GD2)
  HSO3-3Galbeta 1right-arrow1'Cer (sulfatide)
  HSO3-3Galbeta 1right-arrow4Glcbeta 1right-arrow1'Cer (SM3)
  Galbeta 1right-arrow3GalNAcbeta 1right-arrow4(NeuGcalpha 2right-arrow3)Galbeta 1right-arrow4Glcbeta 1right-arrow1'Cer (NeuGc-GM1)
  KDNalpha 2right-arrow3Galbeta 1right-arrow4Glcbeta 1right-arrow1'Cer (KDN-GM3)
  KDNalpha 2right-arrow6Galbeta 1right-arrow3GlcNAcbeta 1right-arrow4Galbeta 1right-arrow4Glcbeta 1right-arrow1'Cer (IV6KDNLcOse4Cer)
  KDNalpha 2right-arrow6Galbeta 1right-arrow4GlcNAcbeta 1right-arrow4Galbeta 1right-arrow4Glcbeta 1right-arrow1'Cer (IV6KDNLnOse4Cer)
  GalNAcbeta 1right-arrow4(NeuAcalpha 2right-arrow3)Galbeta 1right-arrow4Glc-PE (PE-GM2)
Negative
  Galbeta 1right-arrow3GalNAcbeta 1right-arrow4Galbeta 1right-arrow4Glcbeta 1right-arrow1'Cer (GA1)
  GalNAcbeta 1right-arrow4Galbeta 1right-arrow4Glcbeta 1right-arrow1'Cer (GA2)
  GalNAcbeta 1right-arrow4(NeuAc-olalpha 2right-arrow3)Galbeta 1right-arrow4Glcbeta 1right-arrow1'Cer (HO-GM2)
  GalNAcbeta 1right-arrow4(NeuAc-Mealpha 2right-arrow3)Galbeta 1right-arrow4Glcbeta 1right-arrow1'Cer (Me-GM2)
  Galbeta 1right-arrow1'Cer (GalCer)
  Glcbeta 1right-arrow1'Cer (GlcCer)
  Galbeta 1right-arrow4Glcbeta 1right-arrow1'Cer (LacCer)


Fig. 2. Effect of the modification of carboxyl group of NeuAc in GM2 on the interaction with GM2 activator. A, the TLC plate with the indicated glycosphingolipids (10 nmol) was stained with diphenylamine reagent. B, the radioautogram of the same TLC plate that was overlaid with the 3H-labeled GM2 activator prior to the chemical staining as shown in A. Other conditions for this experiment were identical to that used for Fig. 1. A and B: lanes 1, sulfatide; lanes 2, GA2; lanes 3, Me-GM2; lanes 4, HO-GM2; and lanes 5, GM2.
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The extent of the bindings of GM2 activator to the glycosphingolipids was significantly reduced by raising the pH and the ionic strength of the buffer (Fig. 3). When the binding assay was carried out in a low ionic strength neutral pH buffer (pH 6.8), almost no bindngs between GM2 activator and glycosphingolipids were detected (Fig. 3, A and A'). Even sulfatide, which usually binds strongly to GM2 activator, was only very weakly bound to the activator protein under the neutral pH (lane 5). The binding detected in a high ionic strength acidic buffer (250 mM ammonium acetate, pH 4.0) was also considerably reduced (Fig. 3B'). GA2 (lane 6) showed no bindings with GM2 activator under all conditions tested. These results corroborated our previous observation that the conversion of GA2 to LacCer was not effectively stimulated by GM2 activator (8), and the hydrolysis of the GalNAc from GM2 was greatly inhibited by the high ionic strength of the buffer solution; however, no such effect was observed for the hydrolysis of the GalNAc from GA2 (asialo-GM2).


Fig. 3. Effect of pH (A and A') and ionic strength (B and B') of the buffer solutions on the complex formation between glycosphingolipids and GM2 activator. A and B, the TLC plates with the indicated glycosphingolipids (10 nmol) were stained with diphenylamine reagent. A' and B', the radioautograms of the TLC plates that were overlaid with the 3H-labeled GM2 activator prior to the chemical staining as shown in A and B, respectively. The buffer solutions used for TLC overlay were 25 mM ammonium acetate buffer, pH 6.8 (A'), and 250 mM ammonium acetate buffer, pH 4.0 (B'). Other conditions for this experiment were identical to that used for Fig. 1. Lane 1, GM1; lanes 2, GM2; lanes 3, GM3; lanes 4, LacCer; lanes 5, sulfatide; lanes 6, GA2.
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Interaction between Saposin B and Glycosphingolipids

Saposin B is a nonspecific activator protein that stimulates the enzymatic hydrolysis of a number of glycosphingolipids catalyzed by different glycosidases (12). This activator protein was reported to bind glycosphingolipids to form lipid-protein complexes (29, 30). Therefore, the interactions between saposin B and glycosphingolipids were also examined in the same manner for comparison. As shown in Fig. 4, saposin B was found to bind not only to gangliosides and sulfatide but also to GA2 and LacCer. Compared with GM2 activator, saposin B bound to glycosphingolipids better at the neutral pH (pH 6.8) (Fig. 4B'), and the general behavior of binding was not greatly affected by the acidic pH (Fig. 4A') or the high ionic strength of the buffer solution (Fig. 4C').


Fig. 4. Detection of the complex formation between saposin B and glycosphingolipids by TLC overlay. A-C, the TLC plates with the indicated glycosphingolipids (10 nmol) were stained with diphenylamine reagent. A'-C', the radioautograms of the same TLC plates that were overlaid with the 3H-labeled saposin B prior to the chemical staining as shown in A-C, respectively. The buffer solutions used for TLC overlay were 25 mM ammonium acetate buffer, pH 4.0 (A'), 25 mM ammonium acetate buffer, pH 6.8 (B'), and 250 mM ammonium acetate buffer, pH 4.0 (C'). Other conditions used were identical to that used for Fig. 1. In all panels, lanes 1, GM1; lanes 2, GM2; lanes 3, GM3; lanes 4, LacCer; lanes 5, sulfatide; and lanes 6, GA2.
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Interaction of GM2 Activator with Gangliosides in Micellar Forms

Since the results of the TLC overlay experiment showed the preferential binding of GM2 activator to the anionic glycosphingolipids, we subsequently examined the interactions between the GM2 activator and the the gangliosides in aqueous medium using Sephacryl S-200 gel filtration to separate the protein-lipid complexes. Sephacryl S-200 column offers a special advantage for this analysis because this column adsorbs the free gangliosides but not the protein-lipid complexes. This enabled us to isolate and analyze the content in the complexes. When GM2 activator was applied alone to the column, the protein was not adsorbed and eluted from the column at the retention time of 28 min (Fig. 5A), whereas applying [3H]GM2 alone, the ganglioside was retained by the Sephacryl S-200 gel. When an incubation mixture containing [3H]GM2 and GM2 activator in a molar ratio of 1:1 or 50:1 was applied to the column, a peak containing both GM2 activator and [3H]GM2 was eluted (Fig. 5, B and C). This peak was confirmed to be the protein-lipid complex by two separate analyses: (a) rechromatography of this complex did not result in the separation of the activator protein from [3H]GM2 and (b) incubation of the complex with beta -hexosaminidase A resulted the conversion of [3H]GM2 into [3H]GM3 (Fig. 6). In these experiments, the recoveries of the activator protein and the gangliosides were determined to be in the range of 57-72% and 69-87%, respectively. As shown in Fig. 5, B and C, the complex derived from the incubation mixture that contained [3H]GM2 and GM2 activator in a molar ratio of 50:1 (Fig. 5C) had a slightly shorter retention time and a broader peak area than that derived from the mixture that contained [3H]GM2 and GM2 activator in an equimolar ratio (Fig. 5B). Similar chromatographic profiles were obtained when GM2 activator was incubated with either GM1 or GM3. However, no complex formation was detected when GM2 activator was incubated with oligo-GM2 (data not shown) indicating that, in addition to the negative charge, the lipid moiety of the glycolipid is also essential for binding.


Fig. 5. Detection of the complex formation between GM2 activator and GM2 by HPLC using Sephacryl S-200 gel filtration chromatography. A, the elution profile of GM2 activator (1.34 nmol) without preincubation with [3H]GM2; B, the elution profile of GM2 activator (1.34 nmol) preincubated with an equimolar ratio of [3H]GM2; C, the elution profile of GM2 activator (1.34 nmol) preincubated with 50 molar excess of [3H]GM2. Each mixture was applied onto a Sephacryl S-200 column (0.6 cm in diameter × 30 cm) connected to a HPLC system. The detailed conditions for the chromatography are described in the text. Solid line, absorption at 280 nm; bullet , radioactivity.
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Fig. 6. TLC analysis showing the conversion of GM2 into GM3 by beta -hexosaminidase A in the GM2 ganglioside·GM2 activator complex. GM2 activator (1.34 nmol) was preincubated with 50 molar excess of GM2, and the entire mixture was subjected to gel filtration through a Sephacryl S-200 column. The complex was isolated and incubated with beta -hexosaminidase A and analyzed by TLC as described in the text. Lane l, the complex was incubated without beta -hexosaminidase A; lane 2, the complex was incubated with beta -hexosaminidase A; lane 3, the standard GM2 was incubated with beta -hexosaminidase A but without the activator protein; lane 4, GM2 standard; and lane 5, GM3 standard.
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The complexes formed between GM2 activator and the different molar ratios of gangliosides were individually isolated from the Sephacryl S-200 column and analyzed for the ratio between the ganglioside and the activator protein. As shown in Table II, when GM2 activator was incubated with an equimolar ratio of either GM1 or GM2, the molar ratio between GM2 activator and the respective ganglioside in the complex was found to be approximately 1:1. However, when the activator protein was incubated with 50 molar excess of either GM1 or GM2, the molar ratio between the ganglioside and GM2 activator in the complex was found to be about 50:1 in both cases. When the bindings between GM2 activator and a 50-fold molar excess of GM3 was examined, we found that the ratio of GM3 to the activator protein was about 80:1. It is well documented that in an aqueous medium GM3 exists as vesicles that are larger than micelles (31). Therefore, it is not surprising to find that the ratio of GM3/activator protein to be larger than that of GM2/activator protein or GM1/activator protein. As also shown in Table II, the association of saposin B to GM2 was very similar to that of GM2 activator protein. Thus, the interactions between the activator and the gangliosides detected in aqueous medium are similar for saposin B and GM2 activator. Whereas, the bindings on TLC overlay showed that saposin B bound to all glycosphingolipids, and GM2 activator bound preferentially to the anionic glycosphingolipids.

Table II.

Ratio between the ganglioside and GM2 activator in the lipid-protein complex


Mixture (mol:mol) Ganglioside/ GM2-Acta found in the complex

GM1:GM2-Act
   1:1 1.08
  50:1 48.2
GM2:GM2-Act
   1:1 1.03
  50:1 51.0
GM3:GM2-Act
  50:1 83.9
GM2:Saposin B
  50:1 46.6

a  GM2-Act, GM2 activator protein.

The Bindings of GM2 Activator to the Mixture of GM1 and GM2 or GM2 and GM3

We have reported that GM2 activator was able to recognize the branched trisaccharide epitope of GM2 (8, 9). We, therefore, examined whether GM2 activator can specifically bind only to GM2 when GM2 was mixed with GM1 or GM3. GM2 was first mixed with GM1 or GM3 in chloroform:methanol (2/1, v/v), dried, and redispersed in an aqueous buffer solution. The aqueous ganglioside mixture was then incubated with GM2 activator and subjected to Sephacryl S-200 gel filtration as described under "Experimental Procedures." The complexes were isolated, and the amounts of GM2, GM1, and the activator protein (or GM2, GM3, and the activator protein) were determined. As shown in Table III, GM2 activator did not appear to bind preferentially to GM2 to form the activator protein·GM2 complex in 1:1 ratio. Rather, it associated with the mixture of gangliosides in the proportion similar to that in the original ganglioside mixture. For example, when GM2 activator was incubated with a mixture containing an equimolar ratio of GM1 and GM2, the molar ratio of GM2 activator, GM1, and GM2 in the complex was found to be close to 1:1:1. However, when GM2 activator was incubated with a mixture containing 25-fold excess of GM1 and GM2, the detected ratio of the activator protein to GM1 and GM2 in the complex was 1:17.3:17.0. No preferential extraction of GM2 from the two ganglioside mixtures was observed. A similar result was obtained from the incubation of GM2 activator with a mixture of GM2 and GM3. These results indicate that the composition of the complexes formed under the micellar form of ganglioside was determined by the pre-existing status of the ganglioside micelles.

Table III.

Analysis of the lipid-protein complex formed between GM2 activator and the mixed gangliosides


Mixture (mol:mol) Determined ratio of ganglioside/GM2-Acta in the complex
GM1 GM2 GM3 GM2-Act

mol/mol
GM1:GM2:GM2-Act
   1:1:1 0.81 0.84 1.0
  25:25:1 17.3 17.0 1.0
GM2:GM3:GM2-Act
  25:25:1 23.2 25.8 1.0

a  GM2-Act, GM2 activator protein.


DISCUSSION

Among the five activator proteins that stimulate the enzymatic hydrolysis of glycosphingolipids, saposin B and GM2 activator have been shown to interact and affect the glycosphingolipid substrates (29, 30). Several methods have been used to demonstrate the complex formation between the activator proteins and glycosphingolipids, and in some studies the molar ratios between the protein and the lipid were also determined. For example, Fischer and Jatzkewitz (32) studied the complex formation between saposin B and sulfatide using electrophoresis and reported that the ratio of these two components in the complex was 1:1. Also using electrophoresis, Wenger and Inui (33) reported the ratio of the two compounds in the saposin B·GM1 and saposin B·sulfatide complexes to be 1:4 and 1:2.6, respectively. Vogel et al. (34) studied the binding of saposin B to the individual gangliosides, such as GM1, GM2, GM3, and GD1a, as well as sulfatide by centrifugation and determined the molar ratios between saposin B and each of these gangliosides in the protein-lipid complexes to be almost 1:1. For GM2 activator, Conzelmann et al. (4, 10) concluded from their studies using ultracentrifugation, isoelectric focusing, and electrophoresis that GM2 activator can form the activator protein·GM2 complex in 1:1 ratio. These experiments were carried out under the conditions required for the specific methodology used (for example, high sucrose density for ultracentrifugation and high pH for electrophoresis). Using TLC overlay, we have shown clearly that the high ionic strength or high pH of the buffer solution inhibited the interactions between GM2 activator and the glycosphingolipid substrates. Therefore, we chose to analyze the complex formation between GM2 activator and gangliosides using 25 mM ammonium acetate buffer, pH 4.0, which is optimal for the enzymatic hydrolysis of GM2 in the presence of GM2 activator.

By TLC overlay, GM2 activator was found to bind to various anionic glycosphingolipids without showing preference to any particular sugar chain. Thus, GM2 activator does not behave like lectins, which display the recognition of specific saccharide structure. The involvement of an anionic residue of a glycosphingolipid in the complex formation with an activator protein has been suggested. We have reported that the carboxylic function of the NeuAc in GM2 was important for the action of GM2 activator (16). Also, Mitsuyama et al. (35) have reported the binding of saposin B to the affinity column packed with the immobilized sulfatide or its derivatives as ligands. In the present studies using TLC overlay, we have clearly demonstrated that the anionic group in glycosphingolipids is vital for the complex formation with GM2 activator but not with saposin B. While the bindings of GM2 activator to gangliosides and sulfatides are greatly affected by the assay conditions, such as the pH and the ionic strength of the buffer solutions (Figs. 1, 3, and 4), no such effects were found for saposin B. These results, again, support the importance of the negative charge in a glycosphingolipid to form the glycolipid·GM2 activator complex.

Wynn (36) proposed the triple binding domain theory of a glycosphingolipid to saposin B based on the conformational studies of the glycosphingolipids. He predicted that there are three possible interactions between a glycosphingolipid and the protein: (a) the hydrophobic interaction of the hydrocarbon chains of the ceramide moiety and a complementary hydrophobic domain in the protein molecule; (b) the electrostatic interaction between sialic acid or sulfate group and a positively charged group of the protein; and (c) the hydrophilic interaction between a hydroxyl group in a sugar moiety and a complementary plane of the protein. Wynn (36) also pointed out that the glycolipid which has at least two of these structural features will strongly bind to saposin B. Our results on the binding behavior of both saposin B and GM2 activator toward glycosphingolipids agree well with this model, since we have shown that GM2 activator was not able to distinguish the saccharide backbone, the number of sugar residues, and the position or the nature of sialic acid (Table I). It is evident that GM2 activator is not specific to bind only GM2. As both saposin B and GM2 activator were shown to be able to transport glycosphingolipids from the donor to the acceptor liposomes (10), GM2 activator may have a specific role in vivo to transport the acidic glycosphingolipids.

Our results on the complex formation between GM2 activator and the micellar forms of gangliosides agree well with the studies of Cantu et al. (37). They studied the micelle formation in mixed gangliosides using light scattering and neutron scattering and reported that when GM2 and GT1b were mixed in different molar ratios in aqueous solution, the two gangliosides formed a single family of mixed micelles rather than that of two families of unmixed micelles, and the ratio of each ganglioside in the mixed micelles depended on the molar concentration of each ganglioside (37).

In contrast to previous reports (4, 10), we were not able to explain the mode of action of GM2 activator based on our studies on the complex formation between this activator protein and glycosphingolipids, especially GM2. The fact that GM2 activator interacts with a wide variety of anionic glycosphingolipids indicates that this activator protein may have functions other than assisting the enzymatic hydrolysis of GM2.


FOOTNOTES

*   This research was supported by National Institutes of Health Grant NS 09626. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Biochemistry, SL43, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112. Tel.: 504-584-2459; Fax: 504-584-2739.
1    The abbreviations used are: GM2, II3NeuAcGgOse3Cer; GM4, I3NeuAcGalCer; SM3, II3SO3 LacCer; GM3, II3NeuAcLacCer; GA2, asialo-GM2, GgOse3Cer; GD2, II3(NeuAc)2GgOse3Cer; Oligo-GM2, II3NeuAcGgOse3; GM1, II3NeuAcGgOse4Cer; GA1, asialo-GM1,GgOse4Cer; KDN, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid; IV6KDNLcOse4Cer, KDNalpha 2,6Galbeta 1,3GlcNAcbeta 1,3Galbeta 1,4GlcCer; IV6KDNLnOse4Cer, KDN alpha 2,6Galbeta 1,4GlcNAcbeta 1,3Galbeta 1,4GlcCer.

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