(Received for publication, June 9, 1995; and in revised form, July 12, 1995)
From the
G Activator is a low molecular weight protein
cofactor that stimulates the enzymatic conversion of G
into G
by human
-hexosaminidase A and also the
conversion of G
into G
by clostridial
sialidase (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). Among the five known activator proteins for the
enzymatic hydrolysis of glycosphingolipids, only G
activator is effective in stimulating the hydrolysis of G
.
However, the mechanism of action of G
activator is still
not well understood. Using a unique disialosylganglioside,
GalNAc-G
, as the substrate, we were able to show that in
the presence of G
activator, GalNAc-G
was
specifically converted into GalNAc-G
by clostridial
sialidase, while in the presence of saposin B, a nonspecific activator
protein, GalNAc-G
was converted into both
GalNAc-G
and GalNAc-G
. Individual products
generated from GalNAc-G
by clostridial sialidase were
identified by thin layer chromatography, negative secondary ion mass
spectrometry, and immunostaining with a monoclonal IgM that recognizes
the G
epitope. Our results clearly show that
G
activator recognizes the G
epitope in
GalNAc-G
. Thus, G
activator may interact
with the trisaccharide structure of the G
epitope and
render the GalNAc and NeuAc residues accessible to
-hexosaminidase
A and sialidase, respectively.
Sugar chains in glycosphingolipids of higher animals are
catabolized by lysosomal glycosidases, and some of the hydrolytic steps
have been shown to require the assistance of protein cofactors called
activator proteins(1, 2, 3) . Among the five
known activator proteins, four were derived from a common precursor,
prosaposin, by partial proteolysis (4, 5, 6) and sequentially named as saposins
A, B, C, and D, based on their placement from the amino-terminal end of
prosaposin(3) . The gene of prosaposin is located at a single
locus on chromosome 10 (7, 8) . Functionally, both
saposins A and C can stimulate -glucosidase to hydrolyze
glucosylceramide, and saposin C was also reported to stimulate the
hydrolysis of galactosylceramide(9) . Saposin B, previously
called nonspecific activator protein(10) , has been shown to
have a broad specificity toward a wide variety of glycolipid substrates
and enzymes. Saposin D was shown to stimulate the hydrolysis of
sphingomyelin (11) and ceramide in vivo(12) .
However, the true function of saposin A and D remains to be
established.
The fifth activator protein is the product of a
separate gene located on chromosome 5 (13) and has been named
G activator, since this activator protein was found to
stimulate most efficiently the hydrolysis of G
by
-N-acetylhexosaminidase
A(1, 2, 3) . The fact that G
hydrolysis is not efficiently stimulated by any of the four
saposins and that the deficiency of G
activator in type AB
G
gangliosidosis results in massive cerebral accumulation
of G
(14, 15, 16) indicate the
physiological importance of this activator protein in vivo for
the degradation of G
.
It has been postulated that
G activator extracts a single molecule of G
from the micelles and presents the monomeric form of G
to
-N-acetylhexosaminidase A(17) . It has
also been suggested that the GalNAc residue in G
should be
degradable by
-N-acetylhexosaminidase A without the
assistance of G
activator; however, in biological
membranes, the carbohydrate head group of G
is shielded
from the enzyme cleavage by the head groups of other adjacent
glycosphingolipids. For
-N-acetylhexosaminidase A to
reach the GalNAc residue in G
, it requires G
activator to lift the G
molecule a few angstroms out
of the membrane surface(2) . In contrast, we have shown that
the effectiveness of G
activator in stimulating the
hydrolysis of G
may be due to its ability to recognize and
interact with the specific trisaccharide structure of the G
epitope, GalNAc
1
4(NeuAc
2
3)-Gal, and make
the GalNAc residue in G
accessible to
-N-acetylhexosaminidase A(18) . We further found
that the specificity of G
activator is not limited in
stimulating the hydrolysis of G
as previously
reported(1, 2) . This activator also stimulates the
hydrolysis of NeuAc from G
to produce G
and
works synergistically with saposin B for the hydrolysis of GalNAc from
G
by
-N-acetylhexosaminidase A(18) .
Although we have shown that saposin B stimulates the hydrolysis of GM1
by human hepatic
-galactosidase and that G
activator
stimulates the hydrolysis of G
by
-N-acetylhexosaminidase A(19) , the mechanisms of
action of these two activator proteins are still not well understood.
Among the G-related gangliosides, GalNAc-G
is structurally similar to G
by having an additional
G
epitope linked to the C-4 position of the GalNAc in
G
(20) . It has been widely known that the external
NeuAc of G
is readily hydrolyzed by clostridial sialidase
without the assistance of an activator protein(21) . However,
we found that the same NeuAc in GalNAc-G
cannot be
hydrolyzed by clostridial sialidase without the assistance of G
activator. This suggests that the addition of a GalNAc residue
onto G
alters the susceptibility of the NeuAc to
clostridial sialidase. We hypothesized that G
activator
and saposin B might act differently toward the enzymatic hydrolysis of
the two NeuAc residues in GalNAc-G
. We, herewith, present
evidence to show that G
activator clearly recognizes the
specific branched trisaccharide structure in the G
epitope
of GalNAc-G
, while the stimulatory activity of saposin B
does not require a specific sugar chain structure.
The following chemicals and reagents
of the highest grade were purchased from commercial sources: NeuAc,
clostridial sialidase type X,
isopropyl-1-thio--D-galactopyranoside, ampicillin, and
4-chloro-1-naphthol, Sigma; yeast extract and tryptone, DIFCO;
restriction endonucleases and T4 DNA ligase, Life Technologies, Inc.; Taq DNA polymerase, Promega; T7 sequencing kit version 2.0, U.
S. Biochemicals Corp.; E. coli stain BL-21 (DE3), Novagen;
pQE, MI5[pREP4], and the 6XHis/Ni-NTA purification system,
Qiagen; precoated Silica Gel-60 HPTLC plates, Merck (Darmstadt,
Germany); and the peroxidase-conjugated anti-human IgM (µ chain
specific) antibodies, Cappel.
Figure 1:
Hydrolysis of G and
GalNAc-G
by clostridial sialidase. E,
clostridial sialidase. For the detailed incubation conditions, see
``Experimental Procedures.''
When clostridial
sialidase removes the NeuAc associated with the G epitope
from GalNAc-G
, the product will be
GalNAc-G
, which no longer carries the G
epitope. When the sialidase removes the internal NeuAc from
GalNAc-G
, the product will be GalNAc-G
,
which still retains the G
epitope. If the sialidase
removes both NeuAc residues from GalNAc-G
, then the
product will be a neutral glycosphingolipid, GalNAc-G
. Fig. SIillustrates the cleavage of one or two NeuAc residues
from GalNAc-G
.
Figure SI: Scheme I.
Thus, using GalNAc-G, it
should be possible to differentiate the actions of G
activator and saposin B. Fig. 2A shows that in
the presence of G
activator (lane3),
GalNAc-G
was converted into one major band and one very
minor fast moving band, whereas in the presence of saposin B (lane4), GalNAcG-
was converted into two major
and one minor products. The monoclonal IgM that recognizes the G
epitope (26) was used for the initial identification of
these products as shown in Fig. 2B. All lanes in Fig. 2B correspond to that in Fig. 2A. The monoclonal IgM stained the residual
GalNAc-G
as shown in lanes3`, while in lane4`, a band moving faster than GalNAc-G
was also stained. This band corresponds to the second fast moving
band in Fig. 2A, lane4. This
indicates that, in the presence of saposin B, clostridial sialidase
removed the internal NeuAc residue from GalNAc-G
and that
the product retained the G
epitope. The detailed
structural identification of each product in lanes3 and 4 is presented below.
Figure 2:
Hydrolysis of GalNAc-G by
clostridial sialidase in the presence of saposin B (P1) or
G
activator (P2). A, the HPTLC plate was
sprayed with DPA reagent. B, the identical plate as in A was stained with anti-G
antibodies. NA1,
GalNAc-G
; D1a, G
; ND1a,
GalNAc-G
; and E, clostridial sialidase. The
amounts (µg) of the activator used are indicated in parentheses. The numbers on the top are the lanenumbers referred to in the text. For the
detailed incubation conditions, see ``Experimental
Procedures.''
The amounts of G activator (5 µg) and saposin B (20 µg) used in Fig. 2A, lanes3 and 4, were
based on our prior experiences in using them for the hydrolysis of
other glycosphingolipids. Since these amounts represent two different
activator concentrations, we further compared the effect of these two
activator proteins at two levels of concentrations: 2.7 µM (5 µg of G
activator or 2.5 µg of saposin B)
for the low activator concentration and 10.7 µM (20 µg
of G
activator or 10 µg of saposin B) for the high
activator concentration. As shown in Fig. 2A, only in
the presence of saposin B did clostridial sialidase produce the second
fast moving band from GalNAc-G
(Fig. 2A, lanes4 and 8). When saposin B was in a low
concentration (2.7 µM), very little hydrolysis of
GalNAc-G
was observed (Fig. 2A, lane6). Again, the second fast moving band was stained by the
monoclonal IgM that recognizes the G
epitope (Fig. 2B, lanes4` and 8`).
In contrast, this ganglioside was practically not produced from
GalNAc-G
in the presence of G
activator (Fig. 2B, lanes3`, 5`, and 7`). The fastest moving band in Fig. 2A, lane4 or 8, was not stained by the
monoclonal IgM, indicating the absence of the G
epitope in
this product. Furthermore, this band was not stained by the resorcinol
reagent (34) indicating the absence of NeuAc in this product
and was identified to be GalNAc-G
by mass spectrometry.
Identical results as shown in Fig. 2were obtained by using the
native human hepatic G
activator (25) and saposin
B (24) in place of the recombinant activator proteins (results
not shown).
Figure 3:
The HPTLC plate (A) and the
blotted PVDF membrane (B) showing the products after
incubation of GalNAc-G with clostridial sialidase in the
presence of saposin B (P1) or G
activator (P2). A, lane1, the standard
G
(top) and G
; lane2, the standard G
, G
, and
G
(from top to bottom); lane3, the substrate GalNAc-G
; lane4, the products formed in the presence of saposin B (P1); lane5, the products formed in the
presence of G
activator (P2); and lane6, the standard G
. B,
glycosphingolipid bands transferred onto the PVDF membrane from the
HPTLC plate. All lanes in B are identical to that in A. The soliddots indicate the area that was
punched out for the mass spectrometry. For detailed conditions, see
``Experimental Procedures.''
Pl-a and P2-a
were identified to be GalNAc-G. The deprotonated molecule
and fragmentation patterns of these two glycosphingolipids corresponded
to that of GalNAc-G
as shown in Fig. 4, A and B.
Figure 4: The negative SIMS of P1-a and P2-a in Fig. 3B. A, P1-a; B, P2-a. These spectra identify P1-a and P2-a to be HexNAc-Hex-HexNAc-Hex-Hex-Cer.
P1-m, one of the major products in the presence
of saposin B, was identified to be GalNAc-G (Fig. 5). The deprotonated molecule and the fragmentation
profile of P1-m indicated that the NeuAc residue came off first, and
then the other fragment ions were identical to that of
GalNAc-G
. This band was not produced in the presence of
G
activator (P2).
Figure 5: The negative SIMS of P1-m in Fig. 3B. This spectrum identifies P1-m to be HexNAc-(NeuAc)-Hex-HexNAc-Hex-Hex-Cer.
P1-b and P2-b were identified to be
GalNAc-G. Both mass spectra of P1-b (Fig. 6A) and P2-b (Fig. 6B)
corresponded to GalNAc-G
but different from that of
GalNAc-G
, as the characteristic fragment ions
corresponding to that from G
, G
, and G
were detected.
Figure 6: The negative SIMS of P1-b and P2-b in Fig. 3B. A, P1-b; B, P2-b. These spectra identify P1-b and P2-b to be HexNAc-Hex-HexNAc-(NeuAc)-Hex-Hex-Cer.
Pl-s and P2-s were identified to be the
residual parent GalNAc-G. The TLC mobilities of P1-s and
P2-s (Fig. 3A) and their mass spectra (Fig. 7, A and B) were identical to that of the substrate
GalNAc-G
. Also, the mass spectrum of GalNAc-G
shows that the major molecular species of the ceramide moieties
were long chain base 18:1, fatty acid 18:0 (m/z 564)
and long chain base 20:1, fatty acid 18:0 (m/z 592).
These results agree well with the previous data on the lipid
composition of GalNAc-G
(22) .
Figure 7: The negative SIMS of P1-s and P2-s in Fig. 3B. A, P1-s; B, P2-s. These spectra identify P1-s and P2-s to be HexNAc-(NeuAc)-Hex-HexNAc-(NeuAc)-Hex-Hex-Cer, and they are the residual substrate.
P1-c and P2-c
were the very minor products and their exact structures were not
identified. Both showed the deprotonated molecule of m/z 1886 and the fragment ion of m/z 603, which is
characteristic of the ion [NeuAc-NeuAc + Na -
HO - H]
. As this ion was not
detected in the parent GalNAc-G
, P1-c and P2-c might be
the products of the glycosyltransferring action of clostridial
sialidase (the hydrolysis of the external NeuAc residue from
GalNAc-G
and transferring of the NeuAc to the internal
NeuAc to form NeuAc-NeuAc-containing ganglioside).
Quantitative
estimation of the above products was accomplished by scanning the TLC
plate using a Schimadzu CS-930 TLC scanner(35) . In the
presence of saposin B and 6 units of clostridial sialidase (Fig. 2A, lane4), the production of
GalNAc-G, GalNAc-G
, and GalNAc-G
was in a ratio of 1.5:5.6:2.9, whereas in the presence of the
double amounts of the sialidase (Fig. 3A, laneP1), the ratio was 1:1.56:1.86. These results indicate
that saposin B stimulated, without discrimination, the cleavage of the
external and the internal NeuAc residues of GalNAc-G
, and
the higher sialidase concentration promoted the production of
GalNAc-G
. In contrast, in the presence of G
activator and 6 units of the sialidase (Fig. 2A, lane5), the ratio of GalNAc-GMla and GalNAc-G
was 8.8:1, whereas in the presence of the double amounts of the
enzyme (Fig. 3A, laneP2), the ratio
of these two products was 7:3. This indicates that G
activator specifically stimulated the hydrolysis of the external
NeuAc, which is associated with the G
epitope, and
virtually did not stimulate the hydrolysis of the internal NeuAc from
GalNAc-G
to produce GalNAc-G
(only a trace
of GalNAc-G
was detected by immunostaining as shown in Fig. 2B, lane5`). After the removal
of the external NeuAc residue, some GalNAc-G
was
converted into GalNAc-G
.
The differential hydrolysis of
the external and the internal NeuAc residues in GalNAc-G by one sialidase in the presence of G
activator or
saposin B may indicate that these two NeuAc residues are distinct
within their microenvironments, even though in the same molecule.
Recently, Acquotti et al.(22) studied the
conformational properties of GalNAc-G
as a free monomer
in (CD
)
SO or as inserted in a micelle of fully
deuterated dodecyl phosphocholine in D
O. They concluded
from the H6-C6-C7-H7 and H7-C7-C8-H8 dihedral angles that the two NeuAc
conformations for both GalNAc-G
and G
were
very similar to GM1(36) , G
(37) , and
G
(38) . Moreover, the chemical shifts of the
external and the internal Gal, GalNAc, and NeuAc residues were
completely superimposed, and no distinction could be made between the
two sets of trisaccharide structures in GalNAc-G
.
Therefore, the physico-chemical determinations of GalNAc-G
could not distinguish the external and the internal NeuAc
residues. These results, however, do not corroborate with the results
that saposin B and G
activator can discriminate the two
NeuAc residues. The difference may be due to the fact that the
physico-chemical studies were performed in
(CD
)
SO or dodecyl phosphocholine micelles in
which the behavior of GalNAc-G
molecule might be
different from that found in the aqueous system used for the in
vitro enzymatic hydrolysis. The different specificities expressed
by G
activator and saposin B toward the two NeuAc residues
in GalNAc-G
clearly show the distinct functions of these
two activator proteins.
The unique structural feature of
GalNAc-G is the presence of two G
epitopes,
the branched trisaccharide GalNAc-(NeuAc)-Gal, linked in tandem. This
ganglioside provided us with an excellent model to show for the first
time the distinct mode of action of saposin B and G
activator. Our results strongly suggest that G
activator can recognize the external NeuAc residue in
GalNAc-G
, while saposin B does not exhibit this
specificity. Since G
activator can stimulate the
hydrolysis of only one NeuAc residue between the two supposedly
identical NeuAc residues in GalNAc-G
, it is reasonable to
consider that the two trisaccharide units (G
epitopes) in
GalNAc-G
are not completely identical and they are
distinguishable by G
activator protein. Whether this
difference is the result of intra-saccharide interaction or the
influence by the hydrophobic ceramide is still not clear. We have used
ceramide glycanase (33) to prepare the lipid-free
oligosaccharide from GalNAc-G
and found that the two
NeuAc residues in this oligosaccharide became resistant to clostridial
sialidase in the presence or absence of G
activator or
saposin B. This indicates that the ceramide moiety has a profound
effect on the activator-assisted hydrolysis of sialic acids from
GalNAc-G
.