(Received for publication, September 3, 1996, and in revised form, October 29, 1996)
From the Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112
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
-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.
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 -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 -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-
-GlcNAc or
p-nitrophenyl-
-GlcNAc by
-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
-hexosaminidase A. This hypothesis, however, is
not supported by two simple facts: (a) The water-soluble
tetrasaccharide derived from GM2 cannot be hydrolyzed by
-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
-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.
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).
-Hexosaminidase A (2) and
-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).
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 -hexosaminidase A and GM1 by
-galactosidase, respectively, were confirmed by the methods
described previously (8, 12).
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
-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.
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 GM2The fractions that contained
the protein-lipid complex eluted from the Sephacryl S-200 column were
incubated with 0.5 units of -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.
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 ProteinProtein was determined by the method of Lowry et al. (28) using bovine serum albumin as a standard.
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 -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
2
6Gal. 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 (
2
3Gal versus
2
6Gal), 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
-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.
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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).
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
).
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 -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.
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.
|
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.
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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.