(Received for publication, April 4, 1995; and in revised form, January 24, 1996)
From the
The 2,6-sialyltransferase is a terminal glycosyltransferase
localized in the trans Golgi and trans Golgi network. Here we show that
30% of the total rat liver Golgi
2,6-sialyltransferase forms a
disulfide-bonded 100-kDa species that can be converted to the 50-kDa
monomer form of the enzyme upon reduction. Limited proteolysis of both
enzyme forms demonstrates that the 100-kDa species is a
disulfide-bonded homodimer of the
2,6-sialyltransferase. The
2,6-sialyltransferase disulfidebonded dimer is found in bovine
liver Golgi membranes and in Golgi membranes prepared and solubilized
in the presence of 100 mM iodoacetamide, suggesting that it is
not unique to rat liver or formed aberrantly upon membrane lysis. The
dimer form of the enzyme possesses no significant catalytic activity
and has a much lower affinity for CDP-hexanolamine-agarose compared
with the monomer form. In contrast, both the
2,6-sialyltransferase
monomer and the disulfide-bonded dimer bind strongly to galactose and
galactose-terminated substrates. These results suggest that the
2,6-sialyltransferase disulfide-bonded dimer lacks catalytic
activity due to a weak affinity for its sugar nucleotide donor,
CMP-NeuAc, and that this catalytically inactive form of the enzyme may
act as a galactose-specific lectin in the Golgi.
The oligosaccharide structures found on proteins have been shown to be important for their correct folding, stability, and biological function (reviewed in (1) and (2) ). Specific oligosaccharide structures are also found to mediate cell-cell interactions during development and disease (reviewed in (3, 4, 5) ). The activities of the terminal Golgi glycosyltransferases, including the N-acetylglucosaminyltransferases, galactosyltransferases, fucosyltransferases, and sialyltransferases, are responsible for the large variation in glycoprotein oligosaccharide structures. The levels of galactosyltransferase and the sialyltransferases are controlled primarily by transcriptional mechanisms(6, 7, 8, 9) . Because these glycosyltransferases require specific sugar nucleotide donors and acceptor substrates, another way of controlling their activity is through compartmentation in the Golgi cisternae. The Golgi glycosyltransferases of the rat hepatocyte are localized across the Golgi stacks in the order in which they act to add sugar residues to the oligosaccharide chains of the nascent protein(10) . This arrangement ensures that each enzyme has access to the correct substrates and sugar nucleotide donors. In other cell types, including cancer cells and immortalized cell lines, this strict compartmentation breaks down and many enzymes are found in overlapping cisternae (11, 12, 13) . How this organization influences the type of oligosaccharide structures observed on the cell surface is not known.
The signals and mechanisms that mediate the
localization of proteins throughout the secretory pathway have been
studied intensely over the past several years (reviewed in (14) and (15) ). Unlike the localization signals of
soluble and membrane-associated endoplasmic reticulum (ER) ()proteins that are composed of specific linear amino acid
sequences, the localization signals of Golgi glycosyltransferases are
much more complex(16) . Although some studies have concluded
that the primary Golgi localization signals are found in the
transmembrane regions of these
enzymes(17, 18, 19) , other studies have
suggested that sequences in the cytoplasmic tails, transmembrane
regions, and luminal regions are required for efficient Golgi
localization(20, 21, 22, 23, 24) .
Several different mechanisms of Golgi protein localization have been suggested(25, 26, 27) . A receptor-mediated retention of the glycosyltransferases has been ruled out by the observation that the overexpression of these proteins does not saturate a potential retention receptor leading to their expression on the cell surface (20) . Bretscher and Munro (26) suggested the possibility that the relatively short transmembrane regions of Golgi proteins might prevent their partitioning into cholesterol-rich transport vesicles destined for the plasma membrane. Although early observations favored this model (22) , more recent experiments (21) and the observation that the transmembrane regions of Golgi and plasma membrane proteins overlap in length suggest that this cannot be the sole mechanism for Golgi retention.
Because several Golgi proteins require sequences in their cytoplasmic tails, transmembrane regions, and luminal domains for their Golgi localization(20, 21, 22, 23, 24) , it seems likely that their localization signals are conformation-dependent. From this standpoint, one mechanism that seems attractive is oligomerization. In this model, the environment of a specific cisterna would induce an oligomerization of one protein or a set of colocalized Golgi proteins. Work by Nilsson et al.(28) has suggested that glycosyltransferases that overlap in localization may be able to form specific complexes leading to their retention. Other investigators studying early Golgi proteins, such as the M protein of the infectious bronchitis virus(29) , have presented results that suggest that the formation of insoluble oligomers correlates with the Golgi retention of these proteins.
Little is known about the oligomerization status of the
-galactoside
2,6-sialyltransferase (
2,6-ST), a terminal
glycosyltransferase localized in the trans Golgi and trans Golgi
network. Radiation inactivation experiments performed by Fleischer et al.(30) suggested that the active form of the
2,6-ST in Golgi membranes is a dimer, although these experiments
cannot rule out the formation of larger oligomers. Here we report that
approximately one third of the total
2,6-ST in rat and bovine
liver Golgi membranes is found as an inactive, disulfide-bonded dimer.
The disulfide-bonded dimer form of the
2,6-ST exhibits a low
affinity for the sugar nucleotide donor, CMP-NeuAc. As a result, this
form has little to no sialyltransferase activity. Interestingly, both
the
2,6-ST monomer and disulfide-bonded dimer have similar
affinities for galactose and galactose-terminated substrates. These
results suggest that the
2,6-ST disulfide-bonded dimer may act as
a galactose-specific lectin in the Golgi.
Figure 1:
A portion of the Golgi
2,6-ST forms a higher molecular mass disulfide-bonded form. Golgi,
rough ER (rER), and smooth ER (sER) membranes were
purified from rat liver according to the method of Fleischer and
Kervina(31) . Preparations were performed in the presence of
100 mM iodoacetamide to prevent aberrant disulfide bond
formation. Membrane lysates were electrophoresed on SDS-polyacrylamide
gels under reducing and nonreducing conditions. The gels were then
transferred to nitrocellulose filters, and an affinity purified
anti-
2,6-ST antibody was used to detect the
2,6-ST by
immunoblotting. All reduced samples contained 10% BME and were heated
at 100 °C for 5 min (+BME). Sucrose (0.25 M)
and glycerol (20%) refer to the storage conditions for each membrane
sample.
The reduced form of the 2,6-ST appeared to be
very sensitive to proteolytic degradation, especially if the sample was
stored in sucrose containing buffers (Fig. 1, +BME, sucrose). This was particularly noticeable
in the reduced rough ER and smooth ER membrane fractions. To ensure
that the 100-kDa band contains the 50-kDa
2,6-ST monomer and is
not an antigenically related protein that is being preferentially
degraded upon heating under reducing conditions, we separated the 50-
and 100-kDa bands on a SDS-polyacrylamide gel, excised these bands, and
extracted both proteins from the gel slices by incubating for 16 h at
37 °C in Laemlli sample buffer containing BME (Fig. 2).
Immunoblot analysis of the extracted proteins demonstrates that the
100-kDa band collapses to the 50-kDa band after heating the sample to
100 °C in the presence of BME (Fig. 2, Excised Monomer and Excised Dimer). In addition, simply heating the
detergent-solubilized Golgi membranes to 100 °C for 5 min (Fig. 2, GM, +100 °C, -BME) or treating the detergent solubilized Golgi
membranes with 10% BME without heating (Fig. 2, GM, -100 °C, +BME) do not deplete the
amount of the 100-kDa form. These results suggest that the 100-kDa
immunoreactive band observed in Golgi membranes contains the 50-kDa
2,6-ST monomer.
Figure 2:
The
100-kDa immunoreactive band contains the 2,6-ST monomer. Isolated
rat liver Golgi membranes (GM) were solubilized and were
prepared for SDS-polyacrylamide gel electrophoresis with (+BME) or without (-BME) 10% BME and with (+100 °C) or without (-100 °C)
heating to 100 °C for 5 min. In addition, the 50-kDa form of the
2,6-ST (Excised Monomer) and the 100-kDa form of the
2,6-ST (Excised Dimer) were excised from the
SDS-polyacrylamide gel and eluted with sample buffer containing 10% BME
for 16 h at 37 °C, and recovered proteins were prepared for
electrophoresis as described previously. Following electrophoresis,
2,6-ST proteins were detected by immunoblotting as described under
``Experimental Procedures.''
To determine whether the 100-kDa form of the
2,6-ST is a dimer of two identical
2,6-ST monomers, we
performed limited proteolysis of both enzyme forms (Fig. 3).
First, rat liver Golgi membrane 50- (M) and 100-kDa (D) immunoreactive bands were excised from gels and subjected
to proteolysis with 10-200 µg/ml V8 protease, as described
previously(34) . Immunoblots of the resulting peptide maps were
identical, confirming that the 100-kDa immunoreactive band contains the
2,6-ST monomer (Fig. 3A). Next, FTO2B rat hepatoma
cells were metabolically labeled for 6 h with
S-Express
protein labeling mix, and the
2,6-ST was immunoprecipitated from
cell lysates. Immunoprecipitates were electrophoresed on a
SDS-polyacrylamide gel under nonreducing conditions, and radiolabeled
bands corresponding to the
2,6-ST 50- (M) and 100-kDa (D) forms were excised. Following a 10-µg/ml V8 protease
digestion of both the radiolabeled monomer and unreduced 100-kDa
disulfide-bonded bands, we found that the 100-kDa disulfide-bonded form
of the enzyme was more completely digested than was the monomer form
and looked very similar to the immunoblots of the rat liver Golgi
2,6-ST digested with 200 µg/ml V8 protease (in Fig. 3,
compare B, left side (D) with A, 200 µg/ml protease). In contrast, digestion of the
radiolabeled
2,6-ST monomer form with 10 µg/ml V8 protease led
to a peptide pattern similar to that observed for the rat liver Golgi
enzyme digested at lower protease levels (in Fig. 3, compare B, left side (M) with A, 10 and 50 µg/ml protease). Because the 100-kDa form of
the
2,6-ST was very difficult to recover by immunoprecipitation,
we believe that this result simply reflects differing amounts of
protein added to the proteolytic digests of the 50- and 100-kDa
proteins. Using higher amounts of V8 protease to digest the more
abundant 50-kDa monomer form, we found that the 50-kDa band was
digested into a pattern similar to that of the 100-kDa form of the
enzyme (in Fig. 3, compare the left side of B (D) with the right side of B, identical
bands are indicated by asterisks). No obvious unique peptide
bands were observed in the digestion pattern of the
2,6-ST 100-kDa
form, suggesting that this form is comprised of only the
2,6-ST.
These results strongly suggest that the 100-kDa form is a homodimer of
two
2,6-ST 50-kDa monomers.
Figure 3:
Limited V8 protease digestion of
2,6-ST monomer and disulfide-bonded forms suggests that the
100-kDa form of the enzyme is a disulfide-bonded homodimer. A,
rat liver Golgi
2,6-ST 50- (M) and 100-kDa (D)
forms were excised from SDS gels and subjected to digestion with
10-200 µg/ml V8 protease for 90 min at 37
°C(34) . Digestion patterns were visualized by
immunoblotting. B, left side, FTO2B rat hepatoma
cells were metabolically labeled for 6 h with 100 µCi/ml of
S-Express protein labeling mix, the
2,6-ST monomer (M) and 100-kDa disulfide-bonded form (D) were
immunoprecipitated, and these bands excised from a nonreducing
SDS-polyacrylamide gel. Proteins were eluted from the gel slices as
described (34) and incubated with 10 µg/ml V8 protease for
90 min at 37 °C. Proteolysis was stopped by bringing the samples to
final concentrations of SDS and BME of 2 and 10%, respectively, and
heating for 5 min at 100 °C. Proteolytic peptides were separated on
12.5% polyacrylamide gels and visualized by fluorography(32) .
Molecular masses of bands with asterisks (from the largest
peptide) were 32, 30, 29, and 24.5 kDa. Right side,
immunoprecipitated radiolabeled
2,6-ST monomer was subjected to
proteolysis with 10-200 mg/ml V8 protease, and peptides were
analyzed by polyacrylamide gel electrophoresis and fluorography as
described above. The asterisks indicate peptides common to both the
monomer and disulfide-bonded forms of the enzyme. Molecular masses of bands with asterisks (from the largest peptide) were 32.5, 30,
29, and 24 kDa. Molecular mass standards indicated by hash marks in B are identical to those in A.
To ensure that the presence of the
2,6-ST disulfide-bonded dimer is not a rat liver-specific
phenomenon, we purified bovine liver Golgi membranes and subjected
these to immunoblot analysis under reducing and nonreducing conditions.
The immunoblot analysis shows that the bovine liver
2,6-ST is also
found as a high molecular mass form that is sensitive to BME reduction (Fig. 4). We also were able to detect the dimer form of the
2,6-ST in Cos-1 cells expressing the exogenous enzyme and in FTO2B
and H-4-II-E rat hepatoma cells ( Fig. 3and data not shown).
Notably, the 100-kDa immunoreactive protein was only observed in Cos-1
cells transfected with
2,6-ST cDNA, demonstrating that the
presence of the 100-kDa protein depends on the expression of the 50-kDa
2,6-ST monomer (data not shown). These data suggest that a
significant amount of the
2,6-ST exists as a 100-kDa
disulfide-bonded dimer in the Golgi complex in the liver of different
species and in different cell types.
Figure 4:
The disulfide-bonded form of the
2,6-ST is also found in bovine liver Golgi membranes. Bovine liver
Golgi membranes were isolated and detergent solubilized as described
under ``Experimental Procedures.'' Prior to
SDS-polyacrylamide gel electrophoresis, the bovine Golgi membrane
lysates were prepared with (+BME) or without (-BME)
10% BME and with (+100 °C) or without (-100
°C) heating for 5 min at 100 °C. The
2,6-ST was
detected by immunoblotting.
Figure 5:
Glycerol gradient analysis of the native
forms of the 2,6-ST. To determine whether the monomer and dimer
forms of the
2,6-ST associate into higher molecular mass
oligomers, we performed glycerol gradient sedimentation analysis. Rat
liver Golgi membranes were solubilized in 1.0% Triton X-100 and 100
mM iodoacetamide on ice for 30 min and then loaded on a
12-35% continuous glycerol gradient. Gradient fractions were
collected and electrophoresed on SDS-polyacrylamide gels under
nonreducing conditions. The
2,6-ST was detected by immunoblot
analysis. BSA (67 kDa) and lactate dehydrogenase (140 kDa) were used as
molecular mass markers and detected on SDS-polyacrylamide gels by
silver staining.
Figure 6:
The 2,6-ST dimer is not as
catalytically active as the monomer form. Sialyltransferase assays were
performed on partially separated
2,6-ST monomer and dimer forms to
determine whether both forms possessed catalytic activity. Rat liver
Golgi membranes were solubilized in 1.0% Triton X-100 and loaded on a
12%-35% glycerol gradient. Gradient fractions were collected and
analyzed by immunoblotting and for sialyltransferase activity.
Asialofetuin was used as the acceptor substrate in this assay. Top
panel, the separation of
2,6-ST monomer and dimer forms
assessed by densitometric scanning of a film exposed to an immunoblot
of gradient fractions developed using ECL reagents. Bottom
panel, sialyltransferase activity found in these gradient
fractions using asialofetuin as an acceptor
substrate.
Figure 7:
The 2,6-ST dimer binds
CDP-hexanolamine-agarose much more weakly than the monomer form. To
determine whether the
2,6-ST dimer form has altered affinity for
its CMP-NeuAc donor, we applied solubilized rat liver Golgi membranes
to a 1-ml CDP-hexanolamine-agarose column. The column was washed using
buffer E (Wash Through Fractions 1-5) and buffer H (Wash Through Fractions 6-24) and eluted with buffer H
plus 5 mM CDP (5 mM CDP Elution
Fractions)(35, 36) . Fractions were electrophoresed on
a SDS-polyacrylamide gel, and the
2,6-ST was detected by
immunoblotting.
Figure 8:
Both the 2,6-ST monomer and dimer
forms bind strongly to galactose-Sepharose and asialofetuin-Sepharose.
To determine whether the
2,6-ST monomer and dimer forms have the
same affinity for galactose-terminated substrates, we investigated
their binding to galactose-Sepharose (galactose-Seph) and
asialofetuin-Sepharose 4B (asialofetuin-Seph). Detergent
solubilized rat liver Golgi membranes were incubated with 1 ml of each
type of affinity beads for 2 h at 4 °C. Then the flow through was
collected, and the affinity beads were washed extensively in buffer E
(flow through and wash through, W). Both sets of affinity
beads were rotated with 500 mM galactose in buffer H for 30
min at 4 °C (elution fraction, E). The wash through (W), elution (E), and 500 µl of the affinity
beads (B) were electrophoresed on a nonreducing
SDS-polyacrylamide gel, and the
2,6-ST in these fractions was
detected by immunoblotting.
Figure 9:
The 2,6-ST monomer and dimer forms do
not differ significantly in the extent of their N-linked
glycosylation. To determine whether the N-linked
oligosaccharides of the monomer and dimer forms of the
2,6-ST were
processed differently, we analyzed these oligosaccharide structures
using Endo H, peptide N-glycosidase F, and V. cholerae neuraminidase. Rat liver Golgi membranes were solubilized in 0.1%
Triton X-100 and resuspended in buffers appropriate for the enzyme
digestions. Endo H (10 milliunits), peptide N-glycosidase F (2
units), or V. cholerae neuraminidase (10 milliunits) were
added to one sample of each pair, and the treated and untreated samples
were incubated for either 16 h (Endo H and peptide N-glycosidase F) or 2 h (V. cholerae neuraminidase)
at 37 °C. Following digestion, samples were electrophoresed on
SDS-polyacrylamide gels under nonreducing conditions, and the
2,6-ST was detected by immunoblotting.
Studies of the signals required for 2,6-ST Golgi
localization have led to the identification of sequences in the
cytosolic, transmembrane, and luminal regions that play a role in this
process (17, 18, 19, 20, 21, 22, 23, 24) .
The lack of a saturable Golgi retention receptor coupled with the
ambiguous retention ``signal'' suggests that the inherent
characteristics of the ST may lead to its specific retention in the
trans Golgi and trans Golgi network. Although several hypotheses
concerning the mechanism of Golgi retention have been proposed (25, 26, 27) , we have chosen to focus on one
of these that suggests that the specific environment of the late Golgi
causes oligomerization of the
2,6-ST and that this leads to its
retention. Our studies of
2,6-ST oligomerization led to the
identification of a 100-kDa form of the enzyme that comprises
30%
of the total Golgi
2,6-ST. Reduction of the 100-kDa form results
in the appearance of the 50-kDa
2,6-ST monomer and suggests that
this larger form is a disulfide-bonded dimer of the enzyme ( Fig. 1and Fig. 2). Limited proteolysis of isolated
monomer and dimer forms confirmed that the 100-kDa form is a homodimer
of two 50-kDa
2,6-ST monomers (Fig. 3). The dimer form of
the enzyme is observed in rat and bovine liver, in H-4-II-E and FTO2B
rat hepatoma cells, and in Cos-1 cells transfected with
2,6-ST
cDNA (Fig. 1, Fig. 3, and Fig. 4and data not
shown), suggesting that dimerization is not a characteristic of a
particular species of cell type. Analysis of the catalytic activity of
both
2,6-ST forms demonstrated that the disulfide-bonded dimer has
little to no sialyltransferase activity using asialofetuin,
asialotransferrin, or asialo-
1-acid glycoprotein as substrates ( Fig. 6and data not shown). Substrate and donor binding assays
suggest that the disulfide-bonded dimer's lack of catalytic
activity probably reflects its relatively low affinity for its sugar
nucleotide donor, CMP-NeuAc (Fig. 7). However, both the monomer
and the disulfide-bonded dimer bind very strongly to
galactose-Sepharose and asialofetuin-Sepharose, suggesting that the
disulfide-bonded dimer's primary activity in the Golgi may be as
a galactose-binding lectin (Fig. 8).
What differs between the
2,6-ST molecules that remain as monomer and those that form a
disulfide-bonded dimer is not clear. Analysis of both monomer and dimer
forms with Endo H, peptide N-glycosidase F, and V.
cholerae neuraminidase suggest that the pattern of glycosylation
is generally the same in the two forms (Fig. 9). One distinct
possibility is that the sub-Golgi localization of these two forms
differs. The rat liver
2,6-ST was previously localized in the
trans cisternae of the Golgi and the trans Golgi network by
immunoelectron microscopy(42) . It is possible that only 30% of
the total rat liver
2,6-ST reaches the trans Golgi network and in
this compartment forms a disulfide-bonded molecule. Unfortunately, at
this time we have no antibodies that distinguish the monomer and dimer
forms of the enzyme to test this hypothesis.
Our results suggest
that the disulfide-bonded dimer's lack of catalytic activity
reflects its low affinity for its sugar nucleotide donor, CMP-NeuAc.
Consistent with this observation, preliminary experiments suggest that
the disulfide bond involved in dimer formation occurs between two
catalytic domains of 2,6-ST monomers. (
)All the
sialyltransferases cloned to date contain a consensus sequence in the
catalytic domain called the ``sialyl motif''(43) .
Datta and Paulson have demonstrated that this region is involved in
CMP-NeuAc binding(44) . Within the sialyl motif is a Cys
residue (Cys
) that is conserved in all
sialyltransferases(45) . Alteration of this residue and other
Cys residues in the
2,6-ST catalytic domain lead to inactivation
of the enzyme(44) . (
)It is possible that this Cys
residue in the sialyl motif does participate in the disulfide bond
formed between two catalytic domains and leads to inactivation of the
enzyme. Future studies will focus on identifying the Cys residues
involved in the
2,6-ST dimer's disulfide bond.
Fleischer et al.(30) used radiation target inactivation
analysis to show that the active form of the Golgi 2,6-ST is a
dimer. Because this technique relies on the inactivation of catalytic
activity, we presume that the dimer identified is a noncovalent dimer
of
2,6-ST monomers and that the inactive, disulfide-bonded dimer
we have identified was not detected in this analysis. Taken together,
our results and those of Fleischer et al.(30) suggest
an in vivo situation where the
2,6-ST exists as both
covalently and noncovalently associated dimers in the Golgi membrane.
Our inability to detect a noncovalently associated dimer form by
glycerol gradient or sucrose gradient sedimentation may be simply due
to a very weak association of these dimers and again points to the
possibility that larger oligomers are also formed but are unstable in
the nonionic detergents used during the sedimentation analyses.
Several other Golgi localized proteins have been identified as
dimers both biochemically and by radiation target inactivation. Moremen et al.(46) found that the active form of the Golgi
-mannosidase II is a disulfide-bonded homodimer. The disulfide
bonds of the enzyme are formed by Cys residues in the luminal domain
because proteolytic removal of the amino-terminal cytosolic tail and
transmembrane region results in a disulfide-bonded, soluble form of the
enzyme. A similar disulfide-bonded form has been observed for the
G
/G
1,4N-acetylgalactosaminyltransferase expressed in
CHO cells. (
)Interestingly, purified Golgi
1,4-galactosyltransferase has been observed to exist as both a
monomer and a disulfide-bonded dimer(47) , and radiation target
inactivation also demonstrated that the active form of this
glycosyltransferase is a dimer(30) . These observations are
remarkably similar to those made by our laboratory and Fleischer et
al.(30) for the
2,6-ST and suggest that this may be
a common situation for glycosyltransferases localized in the late Golgi
cisternae.
The presence of two forms of the Golgi 2,6-ST in
vivo leads us to consider how they are related and what function
the enzyme disulfide-bonded dimer might play. One possibility is that
the
2,6-ST dimer sialylates other substrates or makes other types
of anomeric linkages. However, because this form does not bind
CMP-NeuAc with high affinity, this is not likely. A second possibility
is that this disulfide bond formation is a mechanism of down-regulation
that is controlled by the availability of donor or substrate molecules.
If the availability of CMP-NeuAc in the Golgi cisternae is limiting,
the
2,6-ST monomer form (noncovalent dimer) having a higher
affinity for CMP-NeuAc will perform the transferase function. In this
situation, the disulfide-bonded dimer form of the
2,6-ST, having a
much lower affinity for CMP-NeuAc, remains essentially inactive but
will still be able to bind galactose-terminated substrates. This leads
to a third possibility that is that the disulfide-bonded
2,6-ST
dimer acts as a galactose-specific lectin in the Golgi. This form of
the enzyme might act to retain unsialylated molecules and pass them off
to the active, noncovalently associated
2,6-ST dimers for
sialylation. In this way, the disulfide-bonded dimer would be acting
essentially as a chaperone molecule by preventing the exit of
asialoglycoproteins from the late Golgi. Other lectin-like proteins are
thought to play important roles in the secretory
pathway(47, 48, 49) . For example, the
chaperone protein calnexin is also a lectin that recognizes the
Glc
Man
GlcNAc
carbohydrate structure
on unfolded or misfolded proteins and prevents their exit from the
ER(49, 50) .
We predict that the cellular
conditions may control the formation of the 2,6-ST
disulfide-bonded dimer, its down-regulation, and potential conversion
into a galactose-specific lectin. A somewhat similar situation is
observed in the control of the dimerization and activity of the
heme-regulated eIF-2
kinase by levels of heme in reticulocytes
(reviewed in (51) ). In heme deficiency, the heme-regulated
eIF-2
kinase is an active noncovalent dimer whose activity will
ultimately lead to the inhibition of protein synthesis initiation.
However, when levels of heme are high, the noncovalent dimers of
heme-regulated eIF-2
kinase become covalently associated via
disulfide bonds leading to an inactivation of the kinase and allowing
the initiation of protein synthesis. In this way protein synthesis will
only commence when the reticulocyte has adequate levels of heme.
Analogously, low levels of CMP-NeuAc may lead to the formation of
inactive disulfide-bonded dimers of the
2,6-ST. However, to
compensate for a decreased sugar nucleotide donor pool, this covalent
dimerization may also convert the enzyme into a galactose-specific
lectin can that control the exit of undersialylated glycoproteins from
the Golgi.
The significant amount of the 2,6-ST
disulfide-bonded dimer form in the Golgi and its relative absence from
the ER suggest that it may be formed in the Golgi. While studying the
biosynthesis of the
2,6-ST in dexamethasone-treated H-4-II-E
cells, Bosshart and Berger (52) identified a high molecular
mass form of the enzyme that appeared after 10 min of pulse labeling
and 1-2 h of chase. Analysis of the oligosaccharide structures on
the
2,6-ST protein suggested that this high molecular mass form
was made in the Golgi, and they suggested that it might be an enzyme
dimer. Formation of the disulfide-bonded
2,6-ST dimer in the Golgi
supports the above hypothesis that the
2,6-ST dimer may be formed
in response to donor or substrate levels in the Golgi. One possibility
is that Cys residues are intimately involved in the
2,6-ST
catalytic mechanism. If some noncovalently associated dimers are not
supplied with adequate sugar nucleotide donor or substrate, the
reactive sulfhydryl groups in the catalytic domain of one molecule may
interact with other reactive sulfhydryl groups in the catalytic domain
of another molecule. Only those noncovalently associated dimers that
are continuously supplied with donor and/or substrate resist disulfide
bond formation and remain catalytically active. In this way enzyme
activity and putative galactose-specific chaperone activities could
respond to cellular conditions and availability of substrate and/or
donor molecules. Further experiments must be performed to study the
location and mechanism of
2,6-ST disulfide-bonded dimer formation
and determine the role the dimer form of this enzyme plays in the
process of N-linked glycosylation.