From the Department of Molecular Biology and
Molecular and Experimental Medicine, Scripps Research Institute, San
Diego, California 92037 and the ¶ Department of Molecular
Genetics, University of California, San Diego, California 92121
Received for publication, November 21, 2000, and in revised form, January 22, 2001
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ABSTRACT |
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The sialyltransferase gene family is comprised of
16 cloned enzymes. All members contain two conserved protein domains,
termed the S- and L-sialylmotifs, that participate in substrate
binding. Of only six invariant amino acids, two are cysteines, with one found in each sialylmotif. Although the recombinant soluble form of
ST6Gal I has six cysteines, quantitative analysis indicated the
presence of only one disulfide linkage, and thiol reducing agents
dithiothreitol and The sialyltransferase gene family represents a group of enzymes
that transfers sialic acid from its nucleotide-sugar donor, CMP-NeuAc,
to carbohydrate groups of various glycoproteins and glycolipids. So
far, 16 enzymes have been cloned (1-3), each of which exhibits unique
specificity for its acceptor substrates and forms one of four sialic
acid linkages, Neu5Ac Although comparison of any two sialyltransferases reveals ~30% amino
acid identity, there are only six amino acids that are invariant in all
16 sialyltransferases. Two of the invariant amino acids are cysteines,
with one found in each sialylmotif (Fig. 1). Our previous analysis by
site-directed mutagenesis of a secreted form of ST6Gal I showed that
alanine substitution of either of the invariant cysteines,
Cys181 or Cys332, produced immunoprecipitated
protein but no detectable enzyme activity (12-14). The results raised
the possibility that these two cysteines may be involved in the
formation of a conserved intrachain disulfide bond.
-mercaptoethanol inactivated the enzyme. Analysis of site-directed mutants showed that alanine or serine mutants
of invariant Cys181 or Cys332 exhibit no
detectable activity, either by direct assay or by staining of the
transfected cells with Sambucus nigra agglutinin, which
recognizes the product NeuAc
2,6Gal
1,4GlcNAc on glycoproteins. In
contrast, alanine mutations of charged residues adjacent to either
cysteine showed little or no effect on enzyme activity. Immunofluorescence microscopy showed that although the wild type sialyltransferase is properly localized in the Golgi apparatus, the
inactive cysteine mutants are retained in the endoplasmic reticulum.
The results suggest that the invariant cysteine residues in the L- and
S-sialylmotifs participate in the formation of an intradisulfide
linkage that is essential for proper conformation and activity of
ST6Gal I.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2,6Gal, Neu5Ac
2,3Gal, Neu5Ac
2,6GalNAc, or
Neu5Ac
2,8Neu5Ac (4-6). The sialyltransferases are localized in the
Golgi apparatus and are type II membrane proteins with a short
cytoplasmic domain, an N-terminal signal anchor, and a large lumenal
catalytic domain, characteristic of all glycosyltransferases localized
to the secretory pathway (7, 8). They contain two conserved homologous
regions, termed L (long)- and S (short)-sialylmotifs, which are present
in the catalytic domain (9-11). Mutational analysis has suggested that
the sialylmotifs are involved in binding the donor and acceptor
substrates (12-14).
View larger version (39K):
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Fig. 1.
Amino acid alignment of the L- and
S-sialylmotifs of all the known cloned sialyltransferases gene
family. Sialyltransferase sequences are grouped according to the
linkage formed by these enzymes (for nomenclature, see Ref. 1).
Sequences were aligned by the Clustal Method using the McVector 6.5 (Oxford Molecular) software program. The sequences are from the rat
ST6Gal I (22), the porcine ST3Gal I (54), the rat ST3Gal II (55), the
rat ST3Gal III (56), the human ST3Gal IV (57), the human ST3Gal V (58),
the human ST3Gal VI (3), the chick ST6GalNAc I (11), the chick
ST6GalNAc II (59), the rat ST6GalNAc III (60), the mouse ST6GalNAc IV
(2), the mouse ST8Sia I (61), the rat ST8Sia II (9), the mouse ST8Sia
III (62), the hamster ST8Sia IV (63), and the mouse ST8Sia V (64). The
consensus sequence consists of invariant and highly conserved amino
acids. The invariant cysteine residue in each of the sialylmotifs is
shown in bold type.
In this report, we provide evidence that the soluble recombinant ST6Gal
I contains a single disulfide bond. Moreover, deletion of either of the
conserved cysteine residues results in inactive recombinant enzyme,
which fails to exit from the endoplasmic reticulum and be transported
to the Golgi apparatus (15), presumably because it is improperly
folded. The combined results, suggest that the invariant cysteine
residues in the L- and S-sialylmotifs of ST6Gal I form an intrachain
disulfide bond that is essential for maintaining an active conformation
of the enzyme.
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EXPERIMENTAL PROCEDURES |
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Construction of Expression Vector--
For expression of wild
type ST6Gal I and its mutants (Fig. 2),
the mammalian expression vector pcDNA3 (Invitrogen, San Diego, CA)
was used after modifications as follows; plasmid DNA for the vector
pcDNA3 was digested with KpnI and EcoRV and
then ligated using T4 DNA ligase in the presence of T7 DNA polymerase
(unmodified with 3'-5' exonuclease activity) using synthesis buffer
(0.5 mM each of deoxynucleoside triphosphate, 1 mM ATP, 10 mM Tris, pH 7.4, 5 mM
MgCl2, 1.5 mM dithiothreitol). This treatment
deleted the unique restriction sites BamHI and
EcoRI, besides KpnI and EcoRV, from
the modified pcDNA3, hereafter denoted pcDNA3mod. A
dog proinsulin signal sequence was then subcloned into this modified
vector as follows. The plasmid DNA for pGIR201Nhe3' containing proinsulin signal sequence (a gift from K. Drickamer; see Ref. 16) was
digested with NheI, and the smaller fragment for the insulin
signal sequence was gel purified and then subcloned into XbaI-digested calf intestine phosphatase-treated
pcDNA3mod, following the usual procedure (17). Prior to
this treatment, the plasmid DNAs were propagated through a dam negative
strain of Escherichia coli GM2163, the competent cells of
which were made according to Hanahan's procedure (18). The clone
containing the proinsulin signal sequence in the 5'-3' orientation was
selected by sequencing, hereafter termed pcDNAins. Two
oligonucleotides were designed to incorporate into this vector
pcDNAins, sites for nickel binding and enterokinase
cleavage. The oligos (sense,
5'-CTAGCACATCATCATCATCATCATGATGATGATGATAAAGATTCTAGAGAATTCGAG-3', and
antisense, 5'-GATCCTCGAATTCTCTAGAATCTTTATCATCATCATC
ATGATGATGATGATGATGTG-3') were annealed at 37 °C and ligated with the
XbaI- and BamHI-digested pcDNAins, using standard molecular biological
techniques (17). The resultant vector, termed
pcDNAins-His, was confirmed by sequencing.
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For subcloning the soluble form of ST6Gal I into pcDNAins-His vector, two restriction sites, XbaI and BamHI, were introduced into its cDNA by polymerase chain reaction (PCR)1 as follows: a forward primer, 5'-GCTCTAGAATTCCAATCCTCAGTTACCACAG-3' (nucleotides 214-236) with an internal XbaI site (underlined) and a reverse primer, 5'-CCAGGAGAGGATCCATAAAATGAC-3' (nucleotides 1270-1247), with an internal BamHI site were used for amplification using as template the cDNA for the rST6Gal I (for nomenclature, see Ref. 1) previously subcloned in pBluescript (19). The conditions for PCR were 94 °C for 30 s, 56 °C for 1 min, and 73 °C for 2 min for 20 cycles. Gel analysis showed the generation of a single band of 1.05 kilobases. This band was purified using Geneclean II (Bio 101, San Diego, CA), digested with XbaI and BamHI, and then subcloned into similarly digested mammalian expression vector pcDNAins-His, described above. The sequence of the resulting expression vector, termed His-spST, was confirmed by dideoxy double-stranded sequencing (20) of the entire subcloned fragment, including the restriction sites used.
For construction of the full-length form of the wild type ST6Gal I, the cDNA was released from a previously made construct in pBluescript (19) by digestion with BamHI and XhoI and then subcloned into similarly digested pcDNA3. Sequencing was performed to check for the desired 5'-3' orientation of this construct, termed pcDNA:ST6Gal I.
Construction of Mutants-- Single point mutations were introduced in the above His-spST by a two-step PCR, following the procedure outlined previously (21). The cDNA for rST6Gal I (22), previously subcloned in pBluescript (19), was used as a template for introduction of these mutations. The mutagenic antisense oligonucleotides (substituted nucleotides underlined) used for construction of the corresponding cDNAs were 5'-CCTGGTCAGACAGCGTCATC-3' (nucleotides 1003-984) for C332S, and 5'-TATCTACCTGGGCACACAGCGTCAT-3' (nucleotides 1009-985) for D333A. For C181A, C332A, and R180A, the mutagenic antisense oligonucleotides used were described previously (12-14). In the first step of PCR, 25-50 pmol of each of a forward primer 5'-GCTCTAGAATTCCAATCCTCAGTTACCACAG-3' (sense, nucleotides 214-236) and the mutant antisense oligonucleotide for the desired mutation were used to generate a megaprimer using Pfu DNA polymerase (Stratagene, San Diego, CA). The conditions used were 94 °C for 30 s, 56 °C for 1 min, and 73 °C for 2 min for 20 cycles. Gel analysis showed the generation of a single band for each mutation. This double-stranded DNA fragment was purified using Geneclean II and used as a forward primer in the second step of PCR. After an initial five cycles of linear amplification with the megaprimer, the reverse primer, 5'-CCAGGAGAGGATCCATAAAATGAC-3' (nucleotides 1270-1247), was added into the reaction tube, and the reaction was continued at 94 °C for 1 min, 68 °C for 1 min and 73 °C for 3 min for 20 cycles. Products were analyzed by agarose gel electrophoresis, which showed the generation of a major single band of expected size (1.05 kilobases). This band was purified by agarose gel electrophoresis, followed by Geneclean II to separate it from the megaprimer. For the cDNAs of R180A and C181A, the gel purified product was digested with two unique restriction enzymes DraIII (at nucleotide 477) and BstBI (at nucleotide 824), and the smaller 350-base pair fragment was purified using the MERmaid kit (Bio101, San Diego, CA). The fragment for each mutation was then subcloned into a similarly digested and purified larger fragment of His-spST. Similarly, for C332A, C332S, and D333A, the gel purified product was digested with BstBI and BspEI (at nucleotide 1188), and the 330-base pair fragment was purified and subcloned. The mutation was confirmed by dideoxy double-stranded sequencing of the entire fragment that was subcloned, including the restriction sites used.
To construct the full-length form of the mutants C181A and C332A, the cDNAs were digested with BspEI (at nucleotide 1188) and PflMI (at nucleotide 477), and the smaller 711-base pair fragment for each mutant was gel purified and then subcloned into a similarly digested larger fragment of pcDNA:ST6Gal I, using standard molecular biological techniques.
Expression and Selection of Stable CHO-K1 Cell Line Transfected
with His-spST--
Transfection of CHO-K1 cells was performed using
the calcium phosphate method as described previously (23). Cells were
plated at a density of 1-2 × 106 cells/100-mm dish
and cultured for 20-24 h before transfection with 2.0 µg of plasmid
DNA/100-mm dish. During transfection, serum free DMEM/Ham's F-12
medium (Life Technologies, Inc.) was used. Stable transfectants were
selected 48-60 h post-transfection with 1 mg/ml of G418 sulfate (Life
Technologies, Inc.) and 0.1 mg/ml RCA120 (Sigma) in the
medium for at least 2 weeks. Stable clones were isolated and then
further selected by ELISA, using anti- rat Gal1,4GlcNAc
2,6-sialyltransferase antibody, characterized previously (15).
For ELISA, stable clones (~ 104 cells/well) resistant to
both G418 and RCA120 were individually plated in 24-well
plates containing DMEM/Ham's F-12 medium (500 µl) with 5% fetal
calf serum. After overnight growth, 100 µl of the medium from each
stable clone were added to the wells of a 96-well microtiter plate and
incubated at 4 °C overnight. The media were removed, and the plates
were blocked with PBS containing 1% bovine serum albumin
at room temperature. After washing the plates with PBS
three times, 50 µl of anti-rat Gal
1,4GlcNAc
2,6-sialyltransferase antibody (1:500 in PBS
containing 1% bovine serum albumin) were added and incubated at room
temp for 2 h. The plates were then washed thrice with PBS
, and 50 µl of horseradish peroxidase-conjugated
anti-human IgG (Sigma; 1:1000 in PBS
containing 1%
bovine serum albumin) were added, and the plate was incubated at room
temperature for 1 h. After washing the plates, the color was
developed with 50 µl/well 3,3'5,5'-tetramethylbenzidine peroxidase
substrate (Pierce). Color was quenched with 1 M phosphoric acid (50 µl/well), and readings were taken at 450 nm using a
microtiter ELISA reader (Labsystems, Titertex Multiscan MCC/340).
Treatment by Thiol-specific Reagents-- ST6Gal I (0.5-0.7 µM) purified as described under "Results" was incubated in 0.1 M phosphate buffer (pH 7.5) containing varying concentration of each thiol specific reagent. After incubation for 20-30 min at room temperature, the reaction was stopped by dilution with the assay mixture, and the remaining activity was assayed immediately, as described previously (12). Control experiments were conducted under the same conditions but without thiol reagents.
Quantitation of Disulfides and Free Sulfhydryl Groups-- Purified ST6Gal I, obtained from stable CHO-K1 cell line expressing the protein, was used (0.18-0.2 mg) for this assay. Quantitative estimation of the disulfide bonds were done using 5,5'-dithiobis(2-nitrobenzoic) acid, following the procedure described by Cavallini et al. (24). The reaction was carried out in a solution of monopotassium phosphate containing 1.6 mM EDTA, 16.6 mM HCl, 24 mmol urea at pH 7.4, in the absence or presence of sodium borohydride. The reaction was monitored at 412 nm until a constant absorbance reading was achieved.
Transient Expression of the Wild Type and Mutant Sialyltransferase-- For expression of the wild type His-spST and its mutants, COS-1 cells (1-2 × 106 cells/100-mm dish) were transfected with ~2.0 µg of plasmid DNA using LipofectAMINETM (Life Technologies, Inc.) as described previously (12, 14). Expression of the proteins was allowed to continue for 40-60 h post-transfection before harvesting the cells. For the analysis of the soluble expressed proteins, the culture medium was collected and used for the enzyme assay as described previously (12, 14). These transfected cells were also used for metabolic labeling. The transfection experiments and subsequent analysis were repeated at least three times for each mutant using plasmid DNAs from different preparations.
For the analysis of the membrane bound proteins, the transfected COS-1
cells were harvested 40-60 h post-transfection. For sialyltransferase
assays, cells were washed with PBS (Irvine Scientific,
Irvine, CA) and lysed as previously described (19).
Pulse-Chase Labeling of Transfected COS-1 Cells and Analysis of
the Transiently Expressed Proteins--
Metabolic labeling of cells
and immunoprecipitation of expressed proteins were performed
essentially following the procedure of Colley et al. (25)
with modification as follows. 36-48 h post-transfection, the
transfected COS-1 cells were washed thrice with methionine-free DMEM
(Life Technologies, Inc.) and then incubated in the same medium for
1-2 h in the 5% CO2 incubator at 37 °C. Medium was
removed, and fresh medium containing 100 µCi/ml Trans 35S-label (PerkinElmer Life Sciences) was added to each
dish. After 3 h of incubation in the 5% CO2 incubator
at 37 °C, the medium was removed, and the cells were washed at least
thrice with DMEM containing methionine. Fresh DMEM (5 ml) containing
methionine and 10% fetal bovine serum was added, and the cells were
incubated for 16-18 h (chase period) at 37 °C in a humidified 5%
CO2 incubator. The radiolabeled media were then collected
and used for detection of ST6Gal I protein by immunoprecipitation, as
described previously (14) using affinity purified rabbit anti-rat
ST6Gal I. In some experiments, the radiolabeled proteins were also
analyzed by binding with Ni-NTA agarose (Qiagen). Sialyltransferase
fusion protein present in the radiolabeled medium was pelleted with
Ni-NTA agarose by centrifugation and detected as follows. The medium
(100 -500 µl) from the transfected cells was mixed with equal amounts
of 50 mM MES (pH 6.0) containing 0.1% Triton CF-54, 25%
glycerol, 0.15 M NaCl, 2 mM
-mercaptoethanol, and 100 mM imidazole. Ni-NTA agarose
(20 µl) in a slurry was added, incubated by rotation at room
temperature for 1-2 h, and centrifuged. The pellet was used for
SDS-polyacrylamide gel electrophoresis after washing twice with the
above buffer. Proteins were eluted from the pellet by boiling for 5 min
in 40 µl of 1× Laemmli gel sample buffer (26) containing 10%
-mercaptoethanol and 25 mM EDTA. Ni-NTA agarose bound
fusion proteins were electrophoresed on 10% SDS-polyacrylamide gels,
according to the method of Laemmli (26). Radiolabeled proteins were
visualized by fluorography using 2,5-diphenyloxazole/dimethyl sulfoxide
(27), and the gels were exposed to Kodak XAR-5 film at
80 °C.
Western Blot--
This was carried out using unlabeled medium
from transfected COS-1 cells as described previously (12) with the
following modifications. The sialyltransferase protein samples were
separated from the transfected medium using Ni-NTA agarose, as
described above. Samples were subjected to SDS-polyacrylamide gel
electrophoresis and then transferred to a nitrocellulose membrane,
following the usual technique (28). The membrane blot was developed by
adsorption of the rabbit anti-rat Gal1,4GlcNAc
2,6-sialyltransferase antibody (1: 500), followed by horseradish
peroxidase-conjugated goat anti-rabbit secondary antibody (1:1000;
Amersham Pharmacia Biotech). The protein bands were finally visualized
by ECL, as suggested by the supplier of the reagents (Amersham
Pharmacia Biotech).
Immunofluorescence Microscopy--
This was performed as
described by Colley et al. (25) with the following
modifications. Approximately 0.5 × 105 COS-1 cells
were plated on poly-D-lysine coated circular (18 mm)
microscope cover glasses in 500 µl of DMEM with 10% fetal bovine
serum and allowed to reach 60-70% confluence by overnight incubation.
Cells were transfected using LipofectAMINETM, as described
by the supplier (Life Technologies, Inc.), with 1-2 µg of plasmid
DNA. After 36-60 h of transfection, cells were washed twice with
PBS and fixed in freshly prepared 2% paraformaldehyde
for 35-50 min at room temperature. For detection of expressed wild
type ST6Gal I and its mutants, the cells were washed twice with 0.1 M glycine and then permeabilized with 0.1% Triton X-100
for 10 min. Fixed cells were incubated for 45 min in blocking buffer at
room temperature. Blocking buffer was removed, and 150 µl of a 1:100
dilution of affinity purified rabbit anti-rat Gal
1,4GlcNAc
2,6-sialyltransferase antibody in blocking buffer were added to each
well, and incubation was continued for 45 min at room temperature.
Cells were washed four times for 5 min with 500 µl of
PBS
at room temperature, a 1:100 dilution of
FITC-conjugated goat anti-rabbit IgG (Sigma) in blocking buffer was
added, and the incubation was allowed to continue for 45 min. For
co-localization experiments, the cells were washed four times with
PBS
(without calcium and magnesium salts), and then a 1:
200 dilution of tetramethylrhodamine isothiocyanate (TRITC)-conjugated
lectin from Sambucus nigra (SNA; EY Laboratories, Inc., San
Mateo, CA) in blocking buffer (0.5% goat serum in PBS) containing 5%
bovine serum albumin, 1 mM
Ca2+/Mg2+, and 0.1 M lactose was
added to stain the cells for detection of NeuAc
2,6Gal
1,4GlcNAc
containing glycoproteins. Cells were again washed four times for 5 min
each with PBS
and then mounted on microscope slides using
~20 µl of Vectashield mounting media (Vector Laboratories Inc.,
Burlingame, CA). Cells were visualized on a Nikon Microphot-FXA
microscope and pictures taken with Ektachrome (P1600) film.
Other Methods--
The protein determination using the
bicinchonic acid protein assay reagent kit (Pierce) and
sialyltransferase assay, and the kinetic analysis were done essentially
as described previously (12-14).
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RESULTS |
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Selection of Stable CHO-K1 Cell Line Expressing Wild Type ST6Gal I
and Purification of the Protein--
The cDNA for the wild type
ST6Gal I sialyltransferase, His-spST, was transfected into CHO-K1,
which lacks the endogenous ST6Gal I enzyme (19). In addition to G418,
we have also used Ricinus communis agglutinin,
RCA120, for selection of the transformants expressing the
enzyme. It was noted that while using only RCA120, the
frequency of obtaining untransfected CHO-K1 resistant cells was
0.001%. This lectin is known to bind terminal galactose on the
oligosaccharide bound to the glycoprotein. Because CHO-K1 cells have
endogenous sialyltransferases other than ST6Gal I that can sialylate
the terminal Gal, some untransfected cells also become resistant to
RCA120, although the frequency is very low. Using both G418
and RCA120, not a single resistant clone was obtained using
106 untransfected cells. With G418 selection only,
~10-30% of the cells were resistant. In contrast, about 0.1% cells
became resistant to both G418 and RCA120 when transfected
cells were used, presumably because of increased expression of ST6Gal
I. At least 30 resistant clones were selected for further screening by
ELISA. The isolated resistant clones were grown individually in 48-well
plates containing DMEM/Ham's F-12 medium supplemented with G418,
RCA120, and fetal calf serum. After about 48-36 h of
growth, 100 µl of the medium from individual wells were used for
ELISA using rabbit anti-rat ST6Gal I antibody to select the clone
expressing the highest level of sialyltransferase, which was finally
subcloned by limit dilution. ST6Gal I protein was purified from the
culture medium of this clone using CDP-hexanolamine Sepharose as
described previously (29). The purity of the enzyme obtained was >90%
as judged by SDS-polyacrylamide gel electrophoresis (not shown).
Effect of Thiol Modifying Agents on ST6Gal I Enzyme
Activity--
The purified sialyltransferase was subjected to the
thiol reducing reagents dithiothreitol and -mercaptoethanol, as
described under "Experimental Procedures." Both reagents completely
abolished enzyme activity in a dose-dependent manner with
50% inhibition achieved at 0.35 mM dithiothreitol and 1.0 mM of
-mercaptoethanol (Table
I). This inhibition was irreversible
because no activity was regained following removal of these reducing
agents by dialysis. The same results were obtained for the enzyme
purified from rat liver (not shown). The results suggest that a
disulfide linkage is essential for the enzyme activity. In contrast,
the free sulfhydryl reagents, 5,5'-dithiobis(2-nitrobenzoic) acid (5 mM), N-ethylmaleimide (25 mM), and
iodoacetamide (25 mM), had no effect on enzyme activity. Under the same reaction conditions, 0.5 mM
14C-labeled N-ethylmaleimide covalently reacted
with the enzyme as detected by SDS-PAGE (not shown).
5,5'-Dithiobis(2-nitrobenzoic) acid also covalently reacted with the
enzyme as detected spectrophotometrically (Table
II). The results demonstrate the presence
of free sulfhydryls and suggest that they are not essential for enzyme
activity.
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Quantitative Estimation of the Number of Disulfide
Linkages--
The number of disulfide linkage(s) was estimated using
the purified secreted form of ST6Gal I. The deduced amino acid sequence showed the presence of 6 cysteine residues in the secreted form of the
enzyme. Using 13,600 M1 Cm
1 as
the extinction coefficient (30) of the 2-nitro-5-thiobenzoic acid
modified enzyme, the number of cysteine residues obtained was 5.5 before reduction and 7.0 after reduction (Table II). The difference of
1.5 cysteines between nonreduced and reduced ST6Gal I is consistent
with a single disulfide bond. Alternatively, the difference could be
accounted for by multimerization of the enzyme by interchain disulfide
bonds. Bovine serum albumin, which was used as control, showed the
presence of 17 disulfide linkages in accord with the experimental
value, obtained by x-ray crystallography and NMR (31, 32).
The Soluble Form of ST6Gal I Is a Monomer--
It was shown
earlier that the intracellular membrane bound ST6Gal I exists as both
monomer and dimer (33). While the monomer was active, the dimer was
found to be inactive, although it could act as a galactose binding
lectin (33). To establish whether the secreted form of this enzyme also
exists in both monomer and dimer form, His-spST was expressed in COS-1
cells. As described under "Experimental Procedures," the
35S-radiolabeled ST6Gal I was analyzed by nonreducing and
reducing SDS-PAGE followed either by purification from the medium using Ni-NTA resin (using the His tag sequence) or by immunoprecipitation with polyclonal antibody to the enzyme. As a control, medium of COS-1
cells transfected with vector alone was used for comparison. As shown
in Fig. 3 (A and
B), both before and after reduction with 10%
-mercaptoethanol, the Ni-NTA resin bound enzyme showed a single band
with a molecular mass of ~39 kDa, as predicted for the
monomer. The other band was for nonspecific binding of a protein, plausibly serum albumin, which was also present in the medium of cells
transfected with vector only. Similar results were observed for the
immunoprecipitated enzyme (Fig. 3, C and D).
Thus, it does not appear that the soluble recombinant ST6Gal I forms
dimer or higher multimers via interchain disulfide bonds. Indeed, a slight shift in mobility between the reduced and nonreduced enzyme (Fig. 3, A and B) provides additional evidence
for the presence of an intradisulfide linkage.
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Analysis of Site-directed Mutants of ST6Gal I--
Analysis of
alanine (or serine) mutants of the two invariant cysteines
(Cys181 and Cys332) and their adjacent charged
amino acids described previously (12, 14) was extended to further probe
the role of these residues. In the present study, each mutant cDNA
was placed in the expression vector pcDNAins-His as
described under "Experimental Procedures." To assess the effect of
the mutations on the enzymatic activity, the cDNAs for the wild
type ST6Gal I and the mutant proteins R180A, C181A, C332A, C332S, and
D333A were transiently expressed in COS-1 cells. SDS-PAGE of
immunoprecipitated radiolabeled proteins showed that all mutant
sialyltransferases were expressed and exhibited similar molecular mass
compared with that of the wild type sialyltransferase. Fig.
4A is shown here as a
representation of mutants. We observed that the level of expression of
cysteine mutants was low compared with that of the wild type. It is
possible that these mutant proteins were degraded rapidly after
expression. Medium containing the sialyltransferase was also used to
assess the enzymatic properties of the mutant sialyltransferases.
Endogenous levels of sialyltransferase activity in COS-1 cells
"mock" transfected with the expression vector,
pcDNAins-His, were typically less than 2% that of the expressed wild type sialyltransferase. The sialyltransferase assay using similar amount of protein (as judged by SDS-PAGE) showed that the
cysteine mutants C181A, C332A, and C332S were enzymatically inactive.
In contrast, mutants of the adjacent charged residues, R180A and D333A,
were active, exhibiting 53 and 45% of the activity, respectively,
compared with that of the wild type enzyme (Fig. 4B). As
previously reported for the alanine mutant of Arg180 (13),
kinetic analysis of the alanine mutants of Arg180 and
Asp333 revealed no significant change in
Km values for either the donor substrate CMP-NeuAc
or the acceptor substrate asialo 1-acid glycoprotein
(Table III). The fact that the mutation
of these adjacent charged residues has little effect on the kinetics of
the sialyltransferase suggests that the invariant cysteine residues may
not be directly involved in substrate binding.
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Evaluation of Sialyltransferase Activity and Subcellular
Localization in Transfected COS-1 Cells--
One explanation for the
secreted C181A and C332A sialyltransferase mutants being inactive is
that they were unstable. To further evaluate the activity and
subcellular localization of the mutants, pcDNA3 constructs of the
full-length enzymes containing the N-terminal signal anchor were
prepared for transfection into COS-1 cells. Following transfection,
activity was examined indirectly by detecting the formation of the
NeuAc2,6Gal
1,4GlcNAc product of the enzyme using TRITC-conjugated
SNA, which binds this structure on glycoproteins and glycolipids (34,
35). The sialyltransferase protein was detected by immunofluorescence,
using FITC-conjugated secondary antibody. As shown in Fig.
5, the vector transfected COS-1 cells did
not stain with either SNA or sialyltransferase antibody, consistent with the fact that COS-1 cells lack ST6Gal I enzyme activity. In
contrast, the wild type ST6Gal I transfected cells were stained for
both the sialyltransferase product (red TRITC stained) and protein
(green FITC stained), as previously demonstrated by Colley et
al. (36). Expression of the protein in transfected cells was
variable, ranging from low to very high. Fig. 5 shows transfected COS-1
cells with two different expression levels of ST6Gal I. As reported
earlier (36), for cells expressing moderate levels of the wild type
ST6Gal I protein, staining was localized primarily to a perinuclear
crescent on one side of the nucleus, consistent with the localization
to the Golgi apparatus. For cells expressing high levels of the wild
type protein, FITC staining of the Golgi region was intense but was
also found diffusely localized throughout the cytoplasm in reticular
structures characteristic of endoplasmic reticulum. All cells
expressing the wild type ST6Gal I also stained positive with the
TRITC-conjugated SNA, indicating the formation of the
Neu5Ac
2,6Gal
1,4GlcNAc product by the enzyme (Fig. 5). The
TRITC-conjugated SNA staining of the intracellular organelles is due to
the permeabilization procedure used for staining the cells.
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In contrast to the wild type enzyme that was primarily localized to the
Golgi apparatus, the cysteine mutants, C181A and C232A, were localized
diffusely throughout the cytoplasm, with more intense staining of the
membrane surrounding the nuclear area, characteristic of localization
to the endoplasmic reticulum (Fig. 5). This staining pattern was seen
for all levels of expression. Despite the fact that the level of
expression of the mutant proteins appeared to be the same as that of
the wild type enzyme, as judged by the FITC staining intensity,
TRITC-conjugated SNA gave very weak staining, if any, which was often
indistinguishable from background staining, of the cells transfected
with the cysteine mutants. Thus, the cysteine mutants appear to produce
an inactive sialyltransferase.
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DISCUSSION |
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In this report we have investigated the role of the conserved cysteine residues of the sialyltransferase ST6Gal I, Cys181 and Cys332. Sialyltransferase mutants replacing either invariant cysteine with Ala or Ser results in the production of sialyltransferase polypeptide when expressed in COS-1 cells either as a soluble recombinant form of the enzyme (residues 71-403) or as the full-length membrane bound form (Fig. 3). However, none of these cysteine mutants exhibit activity. In contrast to the Golgi localization of the wild type sialyltransferase, the full-length mutants accumulate primarily in the endoplasmic reticulum, suggesting that they are retained as improperly folded proteins (see reviews in Refs. 37 and 38). These results do not distinguish between the possibility that the sialyltransferase is initially produced as a properly folded active enzyme, which is then rapidly denatured, and the possibility that the enzyme never reaches an active conformation. However, in either case it appears that the two conserved cysteines, Cys181 and Cys332, are critical for production and maintenance of an active conformation of the sialyltransferase.
In total, the full-length wild type sialyltransferase contains seven
cysteine residues, and the recombinant secreted enzyme studied here
contains only six of these cysteines, because of deletion of
Cys24, which is present in the transmembrane domain.
Quantitation of free cysteines using 2-nitro-5-thiobenzoic acid (Table
II) showed a difference of 1.5 mol/mol before and after reduction,
consistent with the presence of a single disulfide bond. Although
reduction of the enzyme using either dithiothreitol or
-mercaptoethanol abolishes enzyme activity, agents that react with
free cysteines had no effect on activity. These results, combined with
the fact that site-directed mutagenesis of either of the two conserved cysteines, Cys181 and Cys332, produces an
inactive enzyme, provides strong evidence that these conserved
cysteines are involved in the formation of the disulfide bond, which is
essential for enzyme activity. Because each of the two sialylmotifs
carry one of these two cysteines, the disulfide bond would physically
link them in close proximity in the active conformation of the enzyme
as proposed previously (13, 14). Site-directed mutagenesis of conserved
amino acid residues in the two sialylmotifs suggested that the
S-sialylmotif is involved in binding the acceptor substrate and that
they are both involved in binding the donor substrate CMP-NeuAc (14).
The existence of a disulfide bond joining the two sialylmotifs provides
further credence for how the two motifs would participate in binding
the substrates of ST6Gal I. Although only ST6Gal I was studied in this
report, we propose that a disulfide bond between the corresponding invariant cysteines would similarly join the sialylmotifs in each of
the 16 sialyltransferases cloned to date.
Although there is little sequence homology between sialyltransferases
and other families of glycosyltransferases, they all have a common
topology with a short N-terminal cytoplasmic domain, a transmembrane
signal anchor sequence, and a lumenal stem region and catalytic domain
(8, 25, 39). Within each homologous glycosyltransferase family there
are regions of high conservation (8, 13, 39), and conserved cysteines
have been reported for 1,4-galactosyltransferases (8),
1,3-galactosyltransferases (8), fucosyltransferases (40-43),
N-acetyl-galactosaminyl-transferases (44-46), and
N-acetyl-glucosaminyltransferases (47, 48). However, examination of the pattern of conserved cysteines in these families does not reveal an obvious pattern of disulfide bonds as a conserved structural motif across all glycosyltransferases.
The cysteines of the
N-acetylglucosamine-1,4-galactosyltransferase (or
4GalT1) have been studied most extensively of any glycosyltransferase to date. Early chemical modification studies with
the bovine enzyme (49, 50) suggest that two of the five cysteines are
involved in a disulfide linkage (residues 134 and 247). Similar
conclusions were reached in studies involving site-directed mutagenesis
of cysteines of the bovine and human galactosyltransferase, followed by
expression in E. coli (51, 52). The recent crystal structure
of the N-acetylglucosamine-
1,4-galactosyltransferase (53)
revealed two pairs of cysteine residues, residues 134 176 and residues
247 and 266, were close enough (~5 Å) to permit disulfide bond
formation. The apparent discrepancy between the crystal structure data
and the earlier studies is not entirely clear. However, the site-directed mutagenesis studies are not necessarily inconsistent with
the crystal structure data. Expression of wild type
galactosyltransferase in E. coli typically produces
partially active enzyme requiring purification of active fractions
and/or denaturation and renaturation steps prior to analysis (51, 52).
Thus, deletion of a cysteine that participates in a disulfide bond need
not abolish activity providing that at least part of the enzyme can
assume an active conformation in its absence. Moreover, there are now
seven members reported for the
4GT family (8). Of the five cysteines
in the human and bovine
4GalT1, only one, Cys129
(corresponding to the bovine Cys134), is found in human
4GalT7. Thus, for one member of the
4GalT family, neither of the
disulfide bonds is required for proper folding and maintenance of activity.
Analysis of glycosyltransferase sequences has revealed the possibility
of protein motifs that cross glycosyltransferase families (39).
Although it may ultimately be shown that these enzymes as a class will
have similar peptide folds, it is premature to speculate that this is
the case, based on the sequence information and limited
structure-function analysis currently available. As additional
glycosyltransferase structures become available, it will be of interest
to determine whether common structural motifs are associated with their
common function.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Kurt Drickamer for GIR201Nhe3' and Dr. Drickamer, Dr. James Rini, and Dr. Louis Gastinel for helpful discussions. We also express gratitude to the laboratory of Dr. Ajit Varki for help in the immunofluorescence experiments.
![]() |
FOOTNOTES |
---|
* This work was supported in part by United States Public Health Service Grant GM27904 (to J. C. P.).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.
§ To whom correspondence should be addressed: Dept. of Molecular Biology, MEM-L71, 10550 N. Torrey Pines Rd., San Diego, CA 92037. Tel.: 858-784-9634; Fax: 858-784-9690; E-mail: jpaulson@scripps.edu.
Present address: Unidade de Oncologia Experimental- Ludwig
Institute for Cancer Research, Universidade Federal de Sao Paulo, SP Brazil.
Published, JBC Papers in Press, January 29, 2001, DOI 10.1074/jbc.M010542200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PCR, polymerase chain reaction;
SNA, S. nigra agglutinin;
PAGE, polyacrylamide gel electrophoresis;
ELISA, enzyme-linked immunosorbant
assay;
PBS, Dulbecco's phosphate-buffered saline
solution;
DMEM, Dulbecco's modified Eagle's medium;
MES, 2-(N-morpholino)ethanesulfonic acid;
Ni-NTA, nickel-nitrilotriacetic acid;
RCA120, R.
communis agglutinin;
CHO, Chinese hamster ovary;
FITC, fluorescein
isothiocyanate;
TRITC, tetramethylrhodamine
isothiocyanate.
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