(Received for publication, December 29, 1995; and in revised form, February 1, 1996)
From the
G activator protein is a protein cofactor which
stimulates the enzymatic hydrolysis of both GalNAc and NeuAc from
G
. We have previously isolated two cDNA clones, G
activator cDNA and G
cDNA, for human G
activator protein (Nagarajan, S., Chen, H.-C., Li, S.-C., Li,
Y.-T., and Lockyer, J. M.(1992) Biochem. J. 282,
807-813). G
mRNA is an RNA alternative splicing
product that contains exons 1, 2, 3, and intron 3 of the genomic DNA
sequence of G
activator protein (Klima, H., Tanaka, A.,
Schnabel, D., Nakano, T., Schröder, M., Suzuki, K.,
and Sandhoff, K.(1991) FEBS Lett. 289, 260-264).
G
cDNA encodes a protein (G
protein)
containing 1-109 of the 160 amino acids of human G
activator protein, plus a tripeptide (VST) encoded by intron 3 at
the COOH terminus. Thus, G
protein can be regarded as a
form (truncated version) of G
activator protein. We have
expressed G
cDNA in Escherichia coli using pT7-7
as the vector. The recombinant G
protein was purified to
an electrophoretically homogeneous form and was found to stimulate the
hydrolysis of NeuAc from G
by clostridial sialidase, but
not the hydrolysis of GalNAc from G
by
-hexosaminidase A. Like G
activator protein,
G
protein also specifically recognized the terminal
G
epitope in GalNAc-GD1a and stimulated the hydrolysis of
only the external NeuAc from this ganglioside by clostridial sialidase.
These results enabled us to discern the enzymatic hydrolyses of GalNAc
and NeuAc from the G
epitope and established that the
NeuAc recognition domain of G
activator protein is located
within amino acids 1-109. The presence of G
mRNA in
human tissues and the selective stimulation of NeuAc hydrolysis by
G
protein indicate that this activator protein may be
involved in the catabolism of G
through the
asialo-G
pathway.
It has been well established that the catabolism of ganglioside
G(
)requires the assistance of a protein
cofactor called G
activator
protein(1, 2) . The physiological importance of
G
activator protein has been shown by the presence of an
autosomal recessive genetic disease, the AB variant of Tay-Sachs
disease, which is caused by the deficiency or the defect of G
activator protein(3, 4, 5) . In
addition to G
activator protein, four other activator
proteins for the catabolism of glycosphingolipids have been reported.
These four activator proteins are derived from the proteolytic
processing of a single precursor protein,
prosaposin(6, 7, 8) , and have been named
saposin A, B, C, and D according to their placements from the amino
terminus of the prosaposin(9) . Saposin B is also known as a
nonspecific activator protein and has been found to have a
detergent-like activity which stimulates the hydrolyses of various
glycolipids by different glycosidases(10) . Among the five
activator proteins, only G
activator protein is derived
from a separate gene(11) . We have isolated two distinct cDNA
clones for human G
activator protein(12) . One of
them, G
activator cDNA, which has also been isolated by
others(13, 14) , encodes almost the entire amino acid
sequence of the native G
activator protein isolated from
human kidney (15) . The other clone, G
cDNA,
which was reported only by us, has an identical 5`-terminal sequence as
that of G
activator cDNA from nucleotides 1 to 302, but
different for the next 346 nucleotides toward the 3` end. Klima et
al.(16) isolated the genomic DNA which covered 94% of
G
activator cDNA, and identified the presence of three
introns and four exons. The last exon, exon 4, spanned the segment
coding for the carboxyl terminus of G
activator protein
and the entire 3`-untranslated region of the G
activator
cDNA. Comparing the sequence of G
cDNA with this genomic
DNA, we found that the last 346 nucleotides of G
cDNA
were identical to the sequence of 5` end of intron 3 (the exons and the
introns are defined based on G
activator mRNA). Thus,
G
mRNA is an alternative splicing product of G
activator RNA in which the potential 5` splicing site between
exon 3 and intron 3 is not subjected to the splicing process. As shown
in Fig. 1, the coding region of G
activator cDNA
contains the end portion of exon 1, all of exons 2 and 3, and the front
portion of exon 4. While the coding region of G
cDNA
contains the identical exon 1, 2, and 3 as in G
activator
cDNA, and a stretch of 9 nucleotides encoding a tripeptide, VST, at the
COOH terminus which is derived from intron 3. This 9-nucleotide
sequence is immediately followed by a stop codon.
Figure 1:
Gene structure of
G activator protein and G
protein. E1,
E2, E3, and E4 represent exon 1, exon 2, exon 3, and exon
4, respectively. I1, I2, and I3 represent intron 1,
intron 2, and intron 3, respectively. The word
``stop'' means stop codon. VST is the last
three amino acids encoded by intron 3. The sizes of the gene fragments
are not in the exact proportion.
It has been
postulated that the function of G activator protein is to
extract a single G
molecule from the micelles and to
present the substrate-activator complex to
-hexosaminidase
A(17) , or to lift G
from biological membranes
where the sugar chain of G
molecules may be shielded by
other complex lipids with larger headgroups(18, 2) .
In contrast, we have shown that the action of G
activator
protein in stimulating the hydrolysis of G
by
-hexosaminidase A may be due to its ability to recognize and
interact with the branched trisaccharide of G
, the
G
epitope(19) . This view is further supported by
the finding that G
activator protein also stimulates the
hydrolysis of NeuAc from G
by clostridial
sialidase(19) . Based on the selective hydrolysis of only the
terminal NeuAc residue in GalNAc-GD1a, we postulated that G
activator protein can specifically recognize the G
epitope in this ganglioside (20) .
Since the amino
acid sequence encoded by G cDNA consists of the segment
of G
activator protein from amino acid residues 1 to 109
and an additional tripeptide sequence VST at the COOH terminus, the
protein encoded by G
cDNA (G
protein) can
be regarded as a truncated form of G
activator protein. It
is, therefore, important to compare the specificity of G
protein with that of G
activator protein. We have
produced the recombinant G
protein and found that this
activator protein possesses only one of the two known activities of
G
activator protein.
Figure 2:
SDS-PAGE analysis of the purified
recombinant G protein and G
activator
proteins. A high density gel of Phamacia Phast system was used. Lane 1, the prestained protein standards: from the top,
phosphorylase b, 112 kDa; bovine serum albumin, 84 kDa;
ovalbumin, 53.2 kDa; carbonic anhydrase, 34.9 kDa; soybean trypsin
inhibitor, 28.7 kDa; and lysozyme, 20.5 kDa. Lane 2, the
recombinant G
protein (0.2 µg). Lane 3, the
recombinant G
activator protein (0.2 µg). The gel was
stained with silver staining.
Figure 3:
Effect of G activator protein
and G
protein on the hydrolysis of G
by
clostridial sialidase. A, each tube contained 40 µM G
, 10 units of clostridial sialidase, and 2.5
µM of the indicated activator protein in a final volume of
100 µl as described under ``Experimental Procedures.''
The incubations were carried out at 37 °C for 16 h. M2,
G
; E, clostridial sialidase; P2,
G
activator protein; P2A, G
protein. B, the incubation conditions were identical to
that of A except using [
H]G
at three different concentrations as indicated. The mixtures were
incubated at 37 °C for 3 h. The black bars represent the
analyses performed in the absence of an activator protein, the white bars, in the presence of 2.5 µM G
protein, and the hatched bars, in the presence of 2.5
µM G
activator
protein.
Figure 4:
Hydrolysis of GalNAc-G by
clostridial sialidase in the presence of G
protein,
G
activator protein, or saposin B. A, each tube
contained 20 µM GalNAc-G
, 10 units of
clostridial sialidase, and the activator protein as indicated in a
final volume of 100 µl as described under ``Experimental
Procedures.'' The incubations were carried out at 37 °C for 16
h. ND1a, GalNAc-G
; D1a,
G
; E, clostridial sialidase; P2,
G
activator protein, 2.5 µM; P2A,
G
protein, 2.5 µM; P1, saposin B,
20 µM in lane 7 and 10 µM in lane 8. B, the incubation conditions were identical to A except each incubation mixture contained 20 µM of
each activator protein.
The above results strongly
suggest that G protein is also able to recognize the
NeuAc residue in the G
epitope and its mode of action in
stimulating the liberation of NeuAc from G
by clostridial
sialidase is identical to that of G
activator protein. In
view of the facts that the amino acid sequence of G
protein (except VST) is identical to the sequence of G
activator protein from 1 to 109 and that both proteins have the
same stimulatory activity for the hydrolyses of the NeuAc from G
and GalNAc-G
, it is logical to assign the NeuAc
recognition domain of G
activator protein to be within
amino acids 1-109.
Figure 5:
Effect of G activator protein
and G
protein on the hydrolysis of G
by
-hexosaminidase A. A, TLC analysis showing the conversion
of G
to G
. The incubations were carried out
at 37 °C for 3 h. M2, G
; E,
-hexosaminidase A; P2, G
activator protein; P2A, G
protein. The numbers in parentheses (0.5 and 2.5) showed concentration
(µM) of activator proteins used. The detailed incubation
conditions are described under ``Experimental Procedures.'' B, quantitative analysis of the liberation of GalNAc from
G
by
-hexosaminidase A in the presence of activator
proteins. The incubation mixtures were essentially the same as A except [
H]G
at three indicated
concentrations were used. The black bars represent the
experiments performed in the absence of an activator protein, the white bars, in the presence of 2.5 µM G
protein, and the hatched bars, in the presence of 2.5
µM G
activator
protein.
As reported previously, G mRNA was found in both human placenta and fibroblasts, although
in a much lower abundance than the mRNA of G
activator
protein(12) . This suggests that the existence of an
alternative splicing for G
activator RNA may be
physiologically important. Nature may use the alternative splicing of
G
activator RNA to direct the catabolism of
G
. The production of G
activator mRNA or
G
mRNA should lead to the production of G
activator protein or G
protein, respectively. In
the presence of G
activator protein, the catabolism of
G
may preferentially go through the cleavage of GalNAc
residue, which is a well established pathway for the catabolism of
G
. This pathway explains the biochemical bases of
Tay-Sachs diseases caused by the deficiency or the defect of
-hexosaminidase A, or G
activator protein. However,
in the presence of G
protein the catabolism of G
might shift to a possible alternate pathway,
G
G
, since G
protein can
only stimulate the hydrolysis of NeuAc from G
by
clostridial sialidase, but not the hydrolysis of GalNAc. This would
mean that the control of the production of G
activator
mRNA and G
mRNA could be the branching point to direct
the G
hydrolysis to G
G
or
G
G
pathways. The possible in vivo pathway for the conversion of G
to G
has
been proposed by Riboni et al.(31) in studies on the
Neuro2a cell line. The authors suggested that the G
pathway was carried out by a specific sialidase which could
convert G
to G
. It has also been suggested by
Fingerhut et al.(32) that the degradation of
gangliosides by lysosomal sialidase also required an activator protein.
The question of the physiological role of G
protein will
remain unanswered until the native G
protein is isolated.
By SDS-PAGE and Western blotting analysis, we have observed the
presence of a protein band corresponding to 13 kDa which reacted with
the antibody against G
activator protein in the partially
purified placental G
activator protein preparation. At the
present time, the isolation of G
protein is hampered by
the lack of an assay method which can distinguish G
protein from G
activator protein.