From CNRS UPR 9024, 31 Chemin Joseph Aiguier,
F-13402 Marseille Cedex 20, France, § Centre de
Recherche des Macromolécules Végétales-CNRS, Domaine
Universitaire, F-38041 Grenoble Cedex 9, France, ¶ INSERM U260, 27 Boulevard J. Moulin, F-13335 Marseille Cedex 5, France,
Institut de Chimie Moleculaire et Organique UMR 8614, Université de Paris-Sud Batiment 420, F-91405 Orsay
Cedex, France, and the * Institute of Physiology, University of Zurich,
Winterthurerstrasse 190, CH-8057 Zürich, Switzerland
Received for publication, January 30, 2001
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ABSTRACT |
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Human Glycosyltransferases belong to a family of hundreds of enzymes
that catalyze the transfer of a sugar from a sugar nucleotide donor to glycoproteins and glycolipids in eukaryotic cells and to
polysaccharides in bacteria. Many of these enzymes are in charge of
assembling cell-surface glycoconjugates, for which the extent of
sialylation has been increasingly recognized as the molecular determinant of a wide array of biological recognition processes as
different as viral and bacterial adhesion, immune response, and
neuronal outgrowth (1, 2). These events equally involve all types of
glycoconjugates and different glycosidic linkages, but the key
specificity of such complex molecular recognition is still unclear to
us. It thus appears of primary importance to elucidate how glycan
expression at the cell surface is controlled by glycosyltransferases.
Especially within a transferase family sharing similar catalytic
properties, the determinants governing specificity for their substrates
and their underlying respective acceptor(s) need to be identified to
understand the structural information that may be encoded by these
enzymes through such a remarkable diversity.
Sialyltransferases constitute a family of at least 15 distinct
glycosyltransferases that catalyze the transfer of sialic acid from
CMP-NeuAc to glycoproteins or glycolipids during biosynthesis in
animals and man (3, 4). A number of them have been cloned across the
animal kingdom, including bacteria, suggesting a broad function
throughout evolution (1). In eukaryotes, they share with the other
glycosyltransferases a typical type II architecture with a short
cytoplasmic N-terminal tail, a transmembrane fragment followed by a
stem of variable length, and a C-terminal catalytic domain facing the
lumen of the Golgi apparatus (5). Unlike other glycosyltransferase
families, they display significant sequence homology in their catalytic
domain, viz. three conserved peptide sequences designated as
sialyl motifs L and S (6) and VS (7). Mutagenesis of the L motif,
consisting of ~50 amino acids present in the middle of the catalytic
domain, showed that it is largely involved in donor recognition (8),
whereas the S motif, located closer to the C terminus, contributes to
the binding of both donor and acceptor substrates (9).
Sialyltransferases are generally named and classified according to the
nature of the monosaccharide substrate and the type of linkage formed
(10). Within a subfamily catalyzing the same glycosidic bond,
sialyltransferase members may exhibit very low amino acid sequence
identity as well as major differences in acceptor substrate
recognition, i.e. in their requirement for simple or more
complex acceptor structures (11). A typical example demonstrating such
keen specificity in substrate recognition resides in the
ST6GalNAc1 group. ST6GalNAcI,
likely to be involved in the synthesis of the sialosyl-Tn
antigen, exhibits the broadest specificity on (NeuAc Other examples showing distinct acceptor preferences can be found
among sialyltransferase families. Among the six enzymatically distinct
human Very recently, three x-ray crystallographic structures of eukaryotic
glycosyltransferases have been reported (19-21) showing common
structural features, which are as follows: (i) the occurrence of a
structurally very similar catalytic domain consisting of a three-layer
Materials--
Asialofetuin and human protein acceptors,
neuraminidase, CMP-NeuAc, p-nitrophenyl phosphate, and
cacodylic acid were purchased from Sigma. Synthetic acceptor substrate
(LacNAc-O-spacer-biotin) was purchased from
Syntesome. Biotinylated Sambucus nigra lectin was from EY
Laboratories. Streptavidin-alkaline phosphatase conjugates were
purchased from Jackson ImmunoResearch Laboratories, Inc. Restriction
enzymes, purified rat liver Cloning of Human ST6GalI and Expression of Soluble and Membrane
Variants--
The cDNA encoding ST6GalI was isolated from an HL-60
cDNA library and further cloned in the FLAG-cytomegalovirus
expression vector containing the preprotrypsinogen signal peptide
upstream from the FLAG sequence. Sequencing of the insert showed the
same sequence as the one published earlier (28). The full-length sequence was also deleted of the same N-terminal portion for the soluble form to be constructed in stably transfected CHO-K1 cells.
Various truncated forms of human ST6GalI lacking the
transmembrane fragment were cloned into the pFLAG expression vector. The
The cell culture media were collected over a 48-72-h period and
further concentrated >10-fold by Centriprep centrifugation. Each batch
was assayed for activity on asialofetuin to calibrate enzyme production
and stored in small aliquots of similar activity at
Human soluble ST6GalI was produced in Pichia pastoris after
truncation of the N terminus and the transmembrane fragment as described (29). This deletion was therefore very similar to the
FLAG-ST6GalI construct. The activity present in the supernatant was 0.8 milliunits/mg of yeast protein.
Transfection--
Transfection of cells was carried out using
FLAG-cytomegalovirus plasmids containing the different ST6GalI
constructs and the LipofectAMINE (Life Sciences) procedure as
recommended by the manufacturer. Semiconfluent cells were routinely
transfected with ~5 µg of plasmid DNA. Stably transfected CHO-K1
cells were selected using Geneticin at a concentration of 500 µg/ml
over a time period of at least 4 weeks. Transient expression was
routinely performed in COS cells using a lower selection pressure of
the antibiotics.
Solid-phase Assay for ST6GalI Acceptor Specificity--
The
preference of ST6GalI for glycoprotein acceptors was assayed according
to a solid-phase procedure described earlier by us (30) using the
sialic acid-specific lectin S. nigra (SNA) as reported by
Mattox et al. (31). Briefly, coating of microtiter plates
was carried out using increasing amounts of protein acceptors in 100 µl of 50 mM PBS (pH 7.5) overnight at 4 °C. After
saturating wells with 2% bovine serum albumin and washings, coated
glycoproteins were treated with neuraminidase from Clostridium
perfringens overnight at room temperature (30). The extent of
sialic acid removal was assessed on each plate using fetuin (1 µg)
for comparison with a standard preparation of asialofetuin taken as a
reference. Washings were performed with PBS containing 0.1% bovine
serum albumin and 0.05% Tween 20. Incubation with the various forms of
ST6GalI was carried out in 50 mM cacodylate buffer (pH 6.5) containing 32 µM CMP-NeuAc and 2 mM
MnCl2 in a final volume of 100 µl for 1-2 h at 35 °C.
Cell supernatants containing soluble forms of ST6GalI were concentrated
>10-fold using Centriprep columns. Immunopurification of
sialyltransferase variants was performed by incubating the concentrated
supernatant overnight with anti-FLAG antibodies coupled to agarose
beads and further released by 25 µg of FLAG peptide at 4 °C prior
to use. Intracellular full-length membrane ST6GalI forms were extracted
with 0.5% Triton X-100 in 50 mM PBS for 45 min at 0 °C,
and a 10,000 × g supernatant was immediately tested
for activity.
Sialic acid linked in the Liquid-phase Assays of Secreted Soluble ST6GalI
Variants--
Standard reactions were conducted at 37 °C for 15-60
min in a final volume of 50 µl in the presence of 50 µM
CMP-NeuAc, 3.5 µM CMP-[14C]NeuAc (110,000 cpm), 1 mM LacNAc-O-spacer-biotin or
asialofetuin (2 mg/ml) as acceptor, and 2 mM
MnCl2 in 50 mM cacodylate buffer (pH 6.5). The
reaction was initiated by addition of the cell supernatant. In assays
with the LacNAc derivative, the reaction was stopped by addition of 3 ml of cold water, and the mixture was applied to a Waters Sep-Pak
C18 reverse-phase chromatography cartridge. After
washing with 15 ml of water, the radiolabeled product was eluted with 3 ml of methanol and counted with 3 volumes of liquid counting
scintillant (Ultima-FloM, Packard Instrument Co.). In assays
with asialofetuin, the reaction was stopped by addition of 0.5 ml of
cold water and applied to NanosepTM 10 kDa
centrifugal cartridges (Pall Filtron Corp.) to remove unreacted
CMP-[14C]NeuAc by diafiltration. Radioactivity associated
with the protein acceptor was assessed by adding 3 volumes of aqueous
counting scintillant (Amersham Pharmacia Biotech). The apparent
Km value for CMP-NeuAc was obtained using 8-250
µM CMP-NeuAc with 8 µM
CMP-[14C]NeuAc and 1 mM LacNAc acceptor. The
Km value for the LacNAc acceptor was determined
using 0.1-4 mM LacNAc-O-spacer-biotin with 100 µM CMP-NeuAc and 5.25 µM
CMP-[14C]NeuAc. The Km value for the
glycoprotein acceptor was determined using 0.025-0.25 mM
asialofetuin in the presence of 100 µM CMP-NeuAc and 5.25 µM CMP-[14C]NeuAc. In all assays,
consumption of substrates was kept below 15-20% to ensure accurate
initial rate measurements, and each test was done in duplicate.
Fluorescence-assisted Carbohydrate Electrophoresis--
To
analyze sialic acid transfer to N-glycans of different
structure, carbohydrate chains from asialofetuin and native or
recombinant glycoprotein hormones were released by
N-glycanase and further labeled with a 2-aminobenzoic acid
chromophore as recommended by Bio-Rad. Labeled glycans were incubated
with or without soluble ST6 forms from CHO cells or yeast.
The reaction mixture was then precipitated with ethanol overnight at
Fluorescence Microscopy--
Double labeling with FITC-SNA and
anti-FLAG monoclonal antibody M2 was performed as follows. FITC-SNA
labeling was assayed essentially as reported (32). Briefly, cells were
fixed for 5 min with 0.5% paraformaldehyde in calcium- and
magnesium-containing PBS and incubated with 50 µg/ml lectin for 60 min on ice. For intracellular distribution of FLAG-tagged enzymes,
FITC-SNA-stained cells were fixed with 3.5% paraformaldehyde in PBS,
permeabilized with 0.5% Triton X-100 in PBS containing 1% fetal calf
serum, and incubated with 10 µg/ml anti-FLAG monoclonal antibody M2
for 1 h. They were then rinsed three times and incubated with
rhodamine isothiocyanate-conjugated goat anti-mouse IgG antibody for 1 additional h, and staining was visualized by confocal microscopy on a
Leica instrument (Uniblitz Sutter Instrument Co.). Confocal images were processed with Metamorph Imaging System Version 3.5. Volumes were originally raytraced as 24-bit TrueColor images and transferred to Adobe Photoshop as 24-bit RGB TIFF files.
Protein Sequence Analysis--
Protein sequences were retrieved
from the GenBankTM/EBI Data Bank and analyzed using BLAST
programs (33) and ClustalW (34). Secondary structure predictions were
performed using programs available on the NPS and JPred servers.
Differential Activity of Full-length and Soluble Forms of
ST6GalI--
The specificity of the rat enzyme has been extensively
characterized (5-9); and since sequence analysis of rat liver and human ST6GalI showed 88% identity, it is assumed that the specificity of both enzymes is very similar. Of particular interest is the earlier
observation by us that recognition of the human enzyme for LacNAc
residues includes the branch downstream of the
First, we investigated the influence of the branching pattern on enzyme
specificity based on the earlier results that fetuin, human
transferrin, and human
We thus first compared the specificity for these acceptors of native
and soluble forms of ST6GalI produced in mammalian cells or yeast. When
the desialylated glycoproteins were incubated with membrane ST6GalI in
the presence of CMP-NeuAc and the reaction products were probed by
lectin binding (Fig. 1), efficient
transfer was recorded in all cases, with affinity values similar to
those reported previously, with the membrane enzyme having a
slightly better recognition of asialofetuin compared with other
desialylated glycoproteins (Fig. 1A). These results with the
rat transferase are similar to those previously reported (37), and this
assay has been repeated here for comparative purposes. Interestingly, the soluble forms of similar length produced in CHO cells (Fig. 1B) or yeast (Fig. 1C) equally recognized these
acceptors, independently of the expression system. These findings show
that the soluble enzymes did not differ in binding asialoglycoprotein
acceptors and therefore suggest that differential acceptor recognition
by the full-length transferase has been altered by truncation of the
membrane anchor.
Transfer Efficiency of Soluble FLAG-ST6GalI for the
Glycoprotein Hormone Family--
To understand whether or not the
enzyme may recognize a determinant present in the polypeptide backbone
that could influence branch specificity, we investigated a family of
structurally related proteins sharing bi- or triantennary glycans
differing in terminal glycosylation. Glycoprotein hormones are
heterodimers sharing a common
When incubated in the presence of soluble FLAG-ST6GalI, both urinary
hCG (Fig. 2A) and human
pituitary follicle-stimulating hormone (Fig. 2B) behaved as
efficient acceptors in their native state, indicating that their
glycans display sites available for receiving additional sialic acid.
As anticipated, desialylated forms were found to be better acceptors,
demonstrating that their branching pattern has no influence in the
transfer reaction and that the soluble enzyme does not discriminate
LacNAc groups originally sialylated in the
Pituitary hTSH was anticipated to be a very poor acceptor because of
its high content of sulfated sugar (Fig.
3A). However, when recombinant
hTSH was tested as an acceptor, the native preparation could readily be
over-sialylated to the same extent as the hormone treated with
neuraminidase (Fig. 3B). In this regard, this glycoprotein hormone reacted similarly to asialofetuin and
asialo-follicle-stimulating hormone, indicating that the recombinant
product could be extensively sialylated in vitro. Similar
data were obtained with the ST6GalI Sialic Acid Transfer to Free N-Glycans and Derivatized
Disaccharides--
To further investigate to what extent soluble forms
of ST6GalI are able to sialylate N-linked glycans, we
analyzed if these enzymatic forms could achieve the completion of
complex oligosaccharides. Glycans released from glycoprotein acceptors
by endoglycosidase treatment were incubated with the soluble forms
produced in CHO cells or yeast and further derivatized for separation
on gels according to their sialic acid content. Fig.
4 presents evidence that the glycans
originally present in asialofetuin (lane 2) could be
sialylated to a similar extent as the most negatively charged originally present in the fetuin preparation (lane 5).
Urinary hCG glycans (lane 4) were similarly
completed (lane 3) by the enzyme, further confirming
that the protein acceptor is totally dispensable for sialic acid
transfer. Whether these glycans were of low sialic acid content
in their native form or desialylated by neuraminidase treatment did not
affect enzyme efficiency. Therefore, it appeared again that the soluble
transferase fully recognized the entire panel of glycan structures
whatever the expression system used, yeast or CHO cells (lanes
7 and 8).
To further elucidate the contribution of the glycan moiety to the
specificity of soluble ST6GalI, we studied the capacity of soluble
enzymes to transfer sialic acid to
Gal Effect of Deleting the Stem Sequence of ST6GalI on Acceptor
Preference--
Various protein sequence analysis methods were used to
tentatively determine the boundary between the stem and the catalytic domain to identify the minimal size of the catalytic domain of the
transferase. Comparison of known ST6GalI peptide sequences from
different species showed the highest similarity in a region starting at
about residue 100 down to the C-terminal end. In addition, predictive
methods based on hydrophobic cluster analysis indicated that this
region is composed of alternating, regular secondary structure
elements, whereas the preceding segment (amino acids 50-100) appeared
to be less organized and composed mostly of coiled regions. We
therefore decided to progressively delete the N terminus up to residue
100 to assess to what extent this segment is involved in enzyme
activity and to elucidate whether or not this portion may interact with
the catalytic domain and affect acceptor preference. It was expected
that selective acceptor recognition would be abolished, whereas
catalytic properties would be maintained. To compare the activity of
the truncated ST6GalI variants, the activities of all enzymatic forms
were matched using asialofetuin under conditions in which sugar
transfer was displayed as suboptimal lectin binding and increased as a
function of time. Under these conditions, sialic acid transfer was
quantitatively similar for all dose-response curves and independent of
enzyme concentration in the medium. Since multiple
Five additional deletions in the stem regions were carried out
and tested for activity; but for clarity, only those starting from
amino acids
To obtain a better understanding of the structural basis of increased
transfer efficiency, we determined the kinetic parameters of the
shortest and longest soluble truncated forms in liquid-phase assays
using a radioactive donor and similar enzyme concentrations. Kinetic
analyses were performed using both a synthetic acceptor (Gal
Altogether, these data demonstrate that releasing ST6GalI as a soluble
enzyme significantly altered the conformation of the transferase to
broaden its acceptor specificity and to accelerate the transfer
reaction. It is therefore proposed that in the full-length enzyme, the
stem region interacts with the catalytic domain to expose a specific
recognition site for glycoprotein acceptors. Removing this modulatory
region probably released steric constraints and induced a
conformational change that opened up the active site and thus
facilitated sugar transfer.
To elucidate whether or not membrane anchorage can indeed contribute to
such an interaction, a chimeric protein was constructed by fusing the
shortest and most active truncated variant to an unrelated N-terminal
moiety of another transferase. The catalytic domain of ST6GalI was thus
extended with the N-terminal portion of the core 2
To tentatively compare the acceptor recognition of the full-length,
chimeric, and secreted forms of ST6GalI, detergent extracts of
different CHO clones were assayed in solid-phase assays for their
respective activity for asialofetuin (Fig.
9A) or asialotransferrin (Fig.
9B). We observed that human full-length ST6GalI had lower activity for transferrin like the rat enzyme presented in Fig. 1A. The chimeric enzyme in which the N-terminal anchor and
the stem region were substituted generated a transfer activity equally efficient for both fetuin and transferrin acceptors. The half-maximal transfer rate was found to be similar to that for the corresponding truncated variant, again reproducing increased efficiency compared with
that of the full-length enzyme. These findings further support the
conclusion that the membrane anchor does not participate by itself in
acceptor specificity, but may rather constrain the stem region through
lipid packing and somehow sustain its interaction with the catalytic
domain of the transferase.
This study was aimed at exploring the acceptor preference of the
human ST6GalI sialyltransferase and infers that recognition of the
glycan, followed by sugar transfer, is governed by a short peptide
sequence present in the juxtamembrane portion of the protein. Among
glycosyltransferases, the Until recently, very little information was available concerning the
acceptor preference of glycosyltransferases, but ongoing cloning of
many new enzymes and characterization of their catalytic activity
unraveled a very complex redundancy. It appears that each class of
transferases can be divided in subfamilies displaying several
activities capable of constructing the same sugar linkage on distinct
glycoconjugates and that exhibit high but not necessarily exclusive
preference for an acceptor. To date, a key step is therefore to
understand the structural features that allow each glycosyltransferase within a family to discriminate among their glycoprotein or glycolipid acceptors. Consequently, it is also an intriguing question to understand how a cell equipped with a wide array of such enzymes can
control the many combinatorial possibilities of glycosylation of its
cell-surface glycoconjugates and/or secretory glycoproteins. Since
full-length and soluble transferases transfected in mammalian cell
lines were found to be differentially active at glycosylating cell
proteins (26), it has been suggested that membrane anchorage and
intracellular localization are also important for enzyme activity. As
an example, transfecting the full-length rat Using site-directed mutagenesis, several laboratories have elucidated
the location of substrate-binding sites of sialyl- and fucosyltransferases over the last few years. Structure-function relationships of the rat liver enzyme ST6GalI have been well advanced, especially the role of the two sialyl motifs in catalysis (6, 8, 9). In
this regard, it was shown that LacNAc was less effective than
glycoprotein acceptors, partly because enzyme specificity extends over
glycan branching (35, 36, 41). Although the S sialyl motif is involved
in the recognition of both the donor and the lactosaminyl substrates
(9), it is anticipated that regions located elsewhere in the protein
should also contribute to the display of glycan specificity through
more distal interactions. In this regard, recent work on homologous
Regarding ST6GalI, our data also favor the participation of the
stem region in the selective recognition of glycan acceptors. Removal
of this region proved to alter interactions with the catalytic domain
that are essential for restricting access to acceptor subsite and for
accommodating well defined glycans. This in turn affects the catalytic
domain probably through further conformational change to readily
proceed with sugar transfer, as if the enzyme has been turned on to a
more open functional state. Since constructing a chimeric molecule
containing the stem and anchor portions of an unrelated transferase
(core 2 While this work was in progress, the crystal structures of three
eukaryotic glycosyltransferases were elucidated (19-21). These proteins were crystallized in the presence of UDP, thereby revealing the portion of the protein interacting with the nucleotide moiety and
the involvement of the DXD motif previously identified in many glycosyltransferases (22). Despite the lack of sequence identity,
these enzymes adopt a similar topology ( Whether or not the stem region plays a physiological role in regulating
glycosyltransferase action is still unknown. Since enzyme
diversification occurs during evolution (2), it may very well be that
modulatory sites could have been added to restrain catalytic properties
of transferases to well defined classes of glycoconjugates and to
create acceptor preference. Such a fine mechanism may allow
site-specific sialylation like that found in the glycoprotein hormone
family, for which alternate sulfate signaling and sialylation control
hormonal activity and pulsatility (38). Also, the
1,4-galactoside
2,6-sialyltransferase I (ST6GalI) recognition of glycoprotein
acceptors has been investigated using various soluble forms of the
enzyme deleted to a variable extent in the N-terminal half of the
polypeptide. Full-length and truncated forms of the enzyme have been
investigated with respect to their specificity for a variety of
desialylated glycoproteins of known complex glycans as well as related
proteins with different carbohydrate chains. Differences in transfer
efficiency have been observed between membrane and soluble enzymatic
forms, indicating that deletion of the transmembrane fragment induces
loss of acceptor preference. No difference in substrate recognition
could be observed when soluble enzymes of similar peptide sequence were
produced in yeast or mammalian cells, confirming that removal of the
membrane anchor and heterologous expression do not alter enzyme folding and activity. When tested on free oligosaccharides, soluble ST6GalI displayed full ability to sialylate free N-glycans as well
as various N-acetyllactosaminyl substrates. Progressive
truncation of the N terminus demonstrated that the catalytic domain can
proceed with sialic acid transfer with increased efficiency until 80 amino acids are deleted. Fusion of the ST6GalI catalytic domain to the N-terminal half of an unrelated transferase (core 2
1,6-N-acetylglucosaminyltransferase) further showed that
a chimeric form of broad acceptor specificity and high activity could
also be engineered in vivo. These findings therefore
delineate a peptide region of ~50 amino acids within the ST6GalI stem
region that governs both the preference for glycoprotein acceptors and
catalytic activity, thereby suggesting that it may exert a steric
control on the catalytic domain.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2,3)0-1(Gal
1,3)0-1GalNAc-O-Ser/Thr,
whereas ST6GalNAcIII was shown to be specific for
NeuAc
2,3Gal
1,3GalNAc
/
-R without discriminating between
- and
-linked GalNAc (11, 12). As a result, this enzyme can
synthesize the GD1a antigen (12). ST6GalNAcII presents an
intermediate specificity and is active on (NeuAc
2,3)
0-1Gal
1,3GalNAc-O-Ser/Thr (13), and isoforms I and II are considered to be responsible for mucin synthesis. The
recently cloned ST6GalNAcIV displays a similar substrate specificity to
that of ST6GalNAcIII, but prefers O-glycans to glycolipids (14). Another candidate for GD1a synthase activity has been identified in mouse and is referred to as ST6GalNAcV (15).
Comparison of peptide sequences reveals interesting features since
ST6GalNAcIII appears to be virtually devoid of a stem region, whereas
ST6GalNAcI exhibits the longest variable region. It thus
appears that in this group, the broader the acceptor specificity, the
longer the stem region.
2,3-sialyltransferases cloned to date, galactoside
2,3 sialyltransferase ST3GalI and ST3GalII display similar catalytic activity for Gal
1,3GalNAc substrates common to O-linked
glycans and glycolipids. ST3GalI prefers glycoproteins to glycolipids, whereas ST3GalII prefers glycolipids (16). In addition, ST3GalIII was
found to utilize Gal
1,3GlcNAc more efficiently than Gal
1,4GlcNAc in vitro (17). Other studies also demonstrated that mouse
2,8 sialyltransferase II requires the presence of internal
fucose on N-glycans as well as the polypeptide region to
sialylate variants of the neural adhesion molecule at special IgG-like
domains (18). It is thus believed that these enzymes should display
specific structural features to transfer sugars to appropriate
acceptors and to exhibit such exquisite specificity.
/
/
-fold and (ii) the presence of a large pocket on one face
capable of accommodating both the donor and acceptor substrates,
surrounded by conserved motifs identified for a given glycosyltransferase family (22). It appears that the recognition of
donor substrate is mediated by residues that are found in the most
conserved regions, but very little experimental background has so far
delineated those residues that specify acceptor recognition. For
several
2,3-fucosyltransferases, a region located in a hypervariable segment at the N-terminal end of the catalytic domain was shown to
account for differences in acceptor recognition (23-25). It has also
been demonstrated that soluble forms of some transferases are less
efficient in vivo than their membrane-bound counterparts in
glycosylating endogenous acceptors (26), whereas no difference was
observed for other classes of enzyme (27). These findings suggest that
acceptor recognition may reside in variable regions, and as a result,
we investigated whether or not it could be located within the stem
region. We therefore compared the activity of membrane and
soluble forms of human ST6GalI (EC 2.4.99.1) for glycoprotein acceptors
of known glycan structure and show here that deleting the stem region
of this transferase results in loss of acceptor preference together
with increased transfer efficiency. We thus tentatively conclude that,
in vivo, the juxtamembrane region of ST6GalI restricts
enzyme specificity and activity.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2,6-sialyltransferase, and
Taq polymerase (high fidelity) were from Roche Molecular
Biochemicals. FLAG-cytomegalovirus plasmid was from Eastman Kodak Co.
pcDNA3 was from Invitrogen. fluorescent-assisted carbohydrate
electrophoresis gels were from Bio-Rad. All culture reagents
were from Life Technologies, Inc. Oligonucleotide primers were from and
plasmid sequencing was carried out by Eurogentec.
1-28 form of ST6GalI was stably transfected in CHO-K1 cells for
comparison with the membrane ST6GalI form, and a chimeric form was
constructed with the N-terminal (amino acids 1-52) membrane portion of
the rat core 2
1,6-GlcNAc-transferase and
1-70 of human ST6GalI
and also tagged at the N terminus. The six soluble forms of
ST6GalI deleted to a variable extent (
1-28,
1-35,
1-48,
1-60,
1-80, and
1-100) were transiently expressed in COS
cells as summarized in Scheme 1.
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Scheme 1.
Scheme of various human
ST6GalI constructs. The N-terminal peptide portion of human
ST6GalI was progressively truncated up to residue, 100, which was
predicted to correspond to the boundary between the stem and the
catalytic domain (indicated by shading). The nomenclature
used indicates the number of amino acids deleted. The chimeric
construct C2GnT-ST6GalI 1-70 (where C2GnT is core 2
1,6
N-acetylglucosaminyltransferase) was obtained by joining the
N-terminal peptide segment of human core 2
6-N-acetylglucosaminyltransferase (amino acids 1-52) to
the truncated ST6GalI form (
1-70). The checkered boxes
indicate the transmembrane domain (TMD).
20 °C. When
necessary, the amount of the tagged protein was further estimated by
competitive immunoassays using anti-FLAG antibodies. The stable cell
line was found to secrete ~10 ng of FLAG-ST6GalI/106 cells/h. The specific activity of
this enzymatic form was estimated to be 7.5 units/mg of protein.
2,6-position was measured by amplifying
SNA binding to the reaction product using a streptavidin-alkaline phosphatase conjugate as previously described (31). All assays were
performed in quadruplicates, and the data are expressed as the means of
these values. Nonspecific binding was assessed using non-transfected
cell media or extracts and was generally found to give an absorbance of
0.2-0.3. This value was deduced from the assays in all graphs. Since
different forms of the ST6GalI enzyme were assayed on several acceptors
containing glycans exhibiting variable content of available galactose
residues, the activity of each transferase form had to be calibrated.
For the commercial enzyme, 1 unit of activity is the amount of enzyme
catalyzing the transfer of 1 µmol of NeuAc to asialofetuin/min at
37 °C. In the first step, we standardized this activity to a
standard calibrator preparation of this protein under the above
conditions to determine the comparative activity of soluble forms of
the rat liver enzyme. Under these conditions, solid-phase assays were designed to contain at least 0.7 milliunits of recombinant soluble forms when tested on asialofetuin, reaching an absorbance of 0.8 ± S.D. When various deletion variants were compared, the activity of
each enzyme was adapted to this absorbance per 1 µg of acceptor/60 min, a period of time of linear velocity over which sialic acid is
transferred as a function of time.
20 °C. The 15,000 × g supernatants were
evaporated and further dissolved in water. The labeled glycans were
then separated by gel electrophoresis and detected at 540 nm using a
Bio-Rad GlycoImager. Size calibration was carried out using a glucose
ladder and an N-glycan library provided by the manufacturer.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-linked mannose (35),
confirming the earlier observation that, like the rat enzyme, ST6GalI
can discriminate glycan acceptors on the basis of their branching
pattern (36). Preliminary testing showed that the purified liver
preparation was more stable than the native form of the human enzyme
extracted from transfected cells using a detergent. We therefore used
the rat liver transferase to compare the acceptor preference of the
same soluble forms of human ST6GalI expressed in two different systems
for various glycoproteins of known glycan structure.
1-acid glycoprotein are efficient acceptors for the native enzyme (37). These glycoprotein acceptors all
contain complex-type N-glycans with terminal LacNAc groups, but differ in branching pattern and sialylated bonds. Human transferrin contains biantennary glycans sialylated in the
2,6-position; fetuin
contains 2,2,4-triantennary glycans with both
2,3- and
2,6-linked
sialic acids; and
1-acid glycoprotein contains
2,2,4,6-tetraantennary N-glycans with both
2,3- and
2,6-sialic acid. The presence of
2,6-linkages was confirmed by
SNA binding to native preparations, and loss of lectin binding
confirmed the removal of sialic acid by the neuraminidase treatment
(data not shown).
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Fig. 1.
Comparative activities of membrane-bound and
soluble forms of ST6GalI. Neuraminidase-treated fetuin ( ),
transferrin (
), and
1-acid glycoprotein (
) were
assayed using a purified preparation of rat liver membrane ST6GalI
(A) or soluble FLAG-ST6GalI produced in CHO-K1 cells
(B) or in yeast (C).
-subunit and differing in their
-subunit (38, 39). Placental hCG contains biantennary glycans;
urinary hCG contains mostly triantennary oligosaccharides with
2,3-sialic acid as terminal sugars; and follicle-stimulating hormone
is known to display triantennary glycans terminating in both
2,3-
and
2,6-sialic acids. Pituitary hTSH contains biantennary
glycans largely capped with sulfated GalNAc, but also terminating in
2,3-linked sialic acid. When produced in CHO cells, recombinant hTSH
was found to exhibit triantennary glycans with low
2,3-sialic acid
content (38, 40).
2,3-position. It was
concluded that like the plasma proteins tested above, these
glycoprotein acceptors do not appear to contain any structural features
governing sialic acid transfer in the
2,6-position to specific
lactosaminyl groups. These data are in total agreement with previous
structural work on hCG remodeling using soluble bovine ST6GalI
(41).
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Fig. 2.
In vitro sialylation of
gonadotropins. A, a highly purified preparation of
urinary hCG ( ) was treated with (
) or without (
) neuraminidase
before incubation with soluble FLAG-ST6GalI (
and
)
produced in CHO-K1 cells as described under "Experimental
Procedures." B, a highly purified preparation of native
human pituitary follicle-stimulating hormone (hFSH) (
)
was treated with (
) or without (
) neuraminidase before incubation
with soluble FLAG-ST6GalI produced in CHO-K1 cells as described under
"Experimental Procedures."
1-28 form produced in
yeast (data not shown), confirming again that heterologous expression
per se does not affect the catalytic properties of the
transferase. Rather, it is likely that the transferase deleted of its
membrane portion is no longer able to recognize features present in the
various acceptors necessary to specify sialic acid transfer.
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Fig. 3.
In vitro sialylation of hTSH.
Highly purified preparations of pituitary hTSH (A, ) or
recombinant hTSH (B,
) were treated with (
) or without
(
) neuraminidase before incubation with soluble FLAG-ST6GalI
produced in CHO-K1 cells as described under "Experimental
Procedures."
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Fig. 4.
Gel (fluorescent-assisted carbohydrate
electrophoresis) electrophoresis analysis of N-glycans
resialylated using soluble forms of ST6GalI produced in CHO cells or
yeast. Endoglycosidase-released glycans from asialofetuin or
native urinary hCG, incubated or not with soluble forms of ST6GalI
expressed in CHO-K1 cells or yeast, were separated according to their
negative charge and molecular size after derivatization with a
fluorescent chromophore. Lane 1, ladder of glucose-labeled
polymer; lane 2, fetuin asialoglycans; lane 3,
native hCG glycans sialylated with FLAG-ST6GalI; lane 4,
native hCG glycans; lane 5, native fetuin glycans;
lane 6, purified biantennary N-glycans containing
0, 1, or 2 residues of sialic acid/chain; lane 7, fetuin
asialoglycans sialylated with FLAG-ST6GalI; lane 8, fetuin
asialoglycans sialylated with the soluble yeast transferase.
1,4GlcNAc
-O(CH2)6-NH-dansyl and
Gal
1,3GlcNAc
-O(CH2)6-NH-dansyl
derivatives. When the reaction was carried out in solution,
dansylated substrates were allowed to follow the reaction as a function
of time by fluorescence-assisted carbohydrate electrophoresis since
only the sialylated trisaccharide products migrated on the gel because
of the negative charge. Fig. 5 shows that
both types of dansylated substrates can be sialylated over a period of
2-24 h. The benzyl-Gal
1,4GlcNAc and octyl-Gal
1,3GlcNAc glycosides were also tested by solid-phase assays to determine if
coating the acceptors on a solid phase can affect the kinetics of
sialic acid transfer. Soluble FLAG-ST6GalI
1-28 could
sialylate both disaccharides as a function of time over a 3-h period as detected by lectin binding (data not shown). Again, no difference in
substrate recognition could be observed for the soluble forms whether
they had been expressed in CHO cells (Fig.
6A) or yeast (Fig.
6B). Analysis of the dansylated products by mass
spectrometry after methylation indicated that both compounds were
sialylated on the galactose residue through an
2,6-linkage, but some
substitution could be detected on the GlcNAc
residue.2 These data thus
confirmed that the soluble catalytic domain of ST6GalI is able to
recognize a substrate as small as a disaccharide. They are in good
agreement with earlier observations on the full-length transferase
showing that, in vitro, the 6-OH of galactose and the amide
group of the GlcNAc residue are the only groups essential for efficient
transfer of sialic acid (42).
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Fig. 5.
Time dependence of sialic acid
transfer to dansyl disaccharide derivatives.
FLAG-ST6GalI was incubated with saturating amounts
of dansylated Gal 1,4GlcNAc (lanes 1-4) or
Gal
1,3GlcNAc (lanes 6-9) over a time period of 2 h
(lanes 1 and 6), 4 h (lanes 2 and
7), 8 h (lanes 3 and 8), or
24 h (lanes 4 and 9). The products were
analyzed by gel electrophoresis. Lane 10 represents the
glucose-labeled ladder.
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Fig. 6.
Solid-phase assays measuring sialic acid
transfer to LacNAc glycosides using soluble ST6GalI.
Benzyl-Gal 1,4GlcNAc (A) and benzyl-Gal
1,3GlcNAc
(B) coated on plates were incubated with FLAG-ST6GalI in CHO
cells (
) or yeast (
) for 2 h as described under
"Experimental Procedures."
-Gal residues are
present in different glycan structures for each glycoprotein acceptor,
no classical kinetic parameters can be accurately measured in this
approach, but only relative activity.
28 to
60 are shown in Fig.
7. Four variants with deletions over the
first 80 amino acids were found to be enzymatically active, whereas
truncation of ~100 amino acids abolished activity. Of interest, the
transfer reaction was found to be increased as the deletion augmented
from
1-28 to
1-60 (Fig. 7, A-C). For all three
glycoproteins, optimal transfer was reached at lower acceptor
concentration for the shortest enzymatic variant. Additional truncation
between amino acids
60 and
80 did not further modify this property,
but a more extensive deletion of 100 amino acids led to inactivation
(data not shown). Half-maximal activity of the five active variants
over the three acceptors indicated that transfer efficiency was
augmented by a factor of 3-5 when the stem region was shortened (Table
I). However, since these acceptors differ
in LacNAc content, no conclusion could be drawn as to whether the removal of the N-terminal region affected acceptor recognition and/or the catalytic properties of the transferase.
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Fig. 7.
Comparative activities of truncated forms of
ST6GalI for various glycoprotein acceptors. Fetuin (A),
transferrin (B), and 1-acid glycoprotein
(C) were treated with neuraminidase prior to incubation for
90 min with various truncated forms of ST6GalI expressed in COS cells:
1-27(
),
1-35 (
),
1-48 (
),
1-60 (
). Matched
activities of each variant were used in these assays with respect to
asialofetuin as acceptor as described under "Experimental
Procedures."
Comparative efficiencies of membrane and deleted forms of human ST6GalI
for glycoprotein acceptors
1,4-GlcNAc-O-spacer-biotin) and a glycoprotein
acceptor (asialofetuin). As shown in Table
II, there appeared to be no
difference in affinity between both enzymatic forms for the common
donor and the two acceptors. They both exhibited Km
values of ~18 µM for CMP-NeuAc, 1 mM for
LacNAc, and 150 µM for asialofetuin, in the same order of
magnitude as those reported in the literature for the rat enzyme (42).
Thus, removing ~60 amino acids upstream from the catalytic domain did
not alter the recognition parameters, but rather modified the velocity
of sialic acid transfer. Catalytic efficiency was found to be augmented
by a factor of 3 for CMP-NeuAc, by a factor of 36 for LacNAc, and by a
factor of 10 for asialofetuin. To ensure that truncation did not modify
biosynthesis and secretion of the variants during transfection, Western
blot analysis of the intracellular content was carried out using the
same amount of total proteins and showed no difference in the various
truncated forms of ST6GalI. Similarly, cell supernatants showed no
significant variation in the secretory rate of the variants in the
medium (data not shown). Gel electrophoresis carried out under mild
reducing conditions did not display FLAG-tagged proteins of high
molecular weight, ruling out that enzyme activity can be related to
association with unknown proteins or enzyme oligomerization. In
addition, confocal microscopy using FITC-SNA binding further showed
that each transfected cell exhibited a higher content of sialylated glycoconjugates at the cell surface for the
1-80 variant compared with the
1-28 variant,3
further indicating that both forms were also differentially active on
endogenous acceptors.
Kinetic parameters for FLAG-ST6GalI1-28 and FLAG-ST6GalI
1-80
enzymatic forms
6-GlcNAc-transferase tagged with the FLAG epitope (Scheme
1), and the chimeric form was stably
transfected in CHO cells. The activity was then compared for these
full-length membrane enzymes both in vivo and in
vitro. Fig. 8 (A and
B, panels 2) shows the subcellular
immunolocalization of these transferases using anti-FLAG antibodies.
Both enzymes were localized to the Golgi apparatus very similarly. The
respective activities of the full-length forms were then compared
in vivo and in vitro. As shown in Fig. 8
(A and B, panels 1), both ST6GalI and
the chimeric transferase proved to be able to sialylate endogenous
cell-surface glycoconjugates to a large extent. However, full-length
ST6GalI led to SNA binding distributed in the Golgi and periplasmic
compartment, whereas the cell surface exhibited spotted labeling (Fig.
8A, panel 1). In striking contrast, the cell
surface of cells expressing the chimera was intensely
highlighted by the fluorescent lectin (Fig. 8B,
panel 1). It was concluded that the chimeric form was less
restrictive and more efficient in sialylating cell-surface acceptors
than the wild-type enzyme.
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Fig. 8.
Immunolocalization of full-length and
chimeric forms of ST6GalI in stably transfected CHO-K1 cells.
Cells expressing either full-length ST6GalI (A) or the
C2GnT-ST6GalI 1-70 chimeric form (B) were labeled with
fluorescent lectin and anti-tag antibodies to be visualized by confocal
microscopy. FITC-SNA labeling is shown in panels 1,
anti-FLAG/rhodamine isothiocyanate labeling in panels 2, and
dual labeling in panels 3.
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Fig. 9.
Comparative activities of full-length ST6GalI
and truncated and chimeric forms. Fetuin (A) and
transferrin (B) were treated with neuraminidase prior to
incubation for 90 min with detergent extracts of full-length ST6GalI
( ), soluble FLAG-ST6GalI (
), and chimeric C2GnT-ST6GalI
1-70
(
) expressed in CHO-K1 cells as described under "Experimental
Procedures."
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2,6-sialyltransferase has been extensively
characterized because this enzyme terminates most of the
N-glycans in human serum proteins, thereby controlling both
their duration in blood and metabolic clearance (1). Like most of the
glycosyltransferases, the rat liver transferase is a type II membrane
Golgi protein of a well characterized domain structure. It has been
widely assumed that the stem region protrudes into the lumen because
proteolytic degradation has been shown very early on to release an
active soluble form missing 62 amino acids (36). Within a homologous
glycosyltransferase group, the length and primary structure of
catalytic domains are relatively well conserved, and variability in the
molecular size of these enzymes is often attributable to differences in
the length of the stem region. This polypeptide portion tethers the
catalytic domain to the membrane anchor and generally displays highly
variable amino acid sequence. It is thought to consist of a flexible
stretch of little secondary organization. The longest stem reported so far (416 amino acids) was observed in
N-acetylgalactosaminyltransferase, a region rich in
hydroxyamino acids containing many potential glycosylation sites (43).
Conversely, ST6GalNAcIII was found to have a very short stem region, if
any (12). Earlier on, it was proposed that the stem region may
contribute to Golgi localization and/or enzyme oligomerization (44). It
has also been very recently shown that the ST6GalI stem region is
directly involved in Golgi retention and contains at least two possible
cleavage sites responsible for enzyme secretion (45). However, because
this portion has been found to be dispensable for enzyme activity
in vitro, it has not been considered to be functionally
related to the catalytic properties of the transferase, but rather to
facilitate its access to the glycan acceptor by acting as a spacer arm.
2,6-sialyltransferase in mammalian cells was shown to result in limited addition of
2,6-linked sialic acid, indicating a weak competition with
endogenous enzymes (46). Indeed, the mode of action of
glycosyltransferases in vivo is far from being fully
identified, but it is widely admitted that the remarkable diversity of
the cell glycosylation machinery should contribute to the synthesis of
glycan structures with exquisite specificity. This study has deciphered
the minimal catalytic domain of ST6GalI and showed that it is active
in vivo in both membrane and soluble forms. Preliminary work
has also revealed that all truncated variants of ST6GalI (
1-28 to
1-80) are fully active on cell-surface acceptors in vivo
and clustered in the Golgi apparatus together with endogenous
sialylated acceptors.3 These findings further confirm that
discrimination of cell-surface glycoconjugates heavily relies on
intracellular compartmentalization of the transferase. While this work
was in progress, a second Golgi retention signal was reported for
ST6GalI (45), which is in total agreement with our observation that, in
the soluble enzymes, Golgi localization and activity are still
interdependent for the newly synthesized glycoproteins trafficking
in the trans-Golgi network to acquire their final glycan structure.
3-fucosyltransferases demonstrated that residues important for
discriminating related substrates are located within a hypervariable
segment of the catalytic domain close to the stem region (23-25).
Using subdomain swapping with human
1,3-fucosyltransferases
III and VI, a hypervariable region of 11 amino acids (positions
103-153) at the N terminus of the catalytic domain has been found to
contain structural information for Gal
1,3GlcNAc-R versus
Gal
1,4GlcNAc specificity (23). When the stem region was deleted in
human fucosyltransferases V (amino acids 76-374) and III (amino acids
62-361), which then exhibited 93% identity, two regions in the
N-terminal half of the catalytic domain were also identified to induce
an
1,3- to
1,4-switch, representing a difference in 8 and 12 amino acids between the two protein sequences, respectively (24). These
studies therefore delineate the existence of discrete subsites within
this family of enzymes sharing similar
1,3/4-activity that are
responsible for the various aspects of enzyme specificity. Recently,
site-directed mutagenesis demonstrated that, in bovine
fucosyltransferase b-encoded
1,3-transferase, a single amino
acid substitution at position 115 is sufficient to switch the substrate
specificity, whereas a mutation at position 116 regulates the
transferase efficiency of bovine
1,3-fucosyltransferase and human
1,3/4-fucosyltransferase III (25). This work lends further support
to the possibility that, in fucosyltransferases, the stem region may
participate in transferase activity by creating a highly specific
acceptor site in the spatial vicinity of the catalytic domain. Such a
modulatory region allows several transferases involved in Lewis antigen
synthesis to discriminate structurally related disaccharide substrates
while catalyzing a similar fucose transfer through highly homologous catalytic domains.
6-GlcNAc-transferase) showed that this latter transmembrane
fragment does not reverse this effect, we also concluded that the role
of the stem region may be specific for the transferase.
/
/
-fold), with the key
amino acids involved in UDP binding apparently located at equivalent
positions. Since only the catalytic domains of these glycosyltransferases have been crystallized, there is still no indication of a possible role of the stem region in acceptor
recognition. In addition, whether or not sialyltransferases can adopt a
similar topology is still difficult to assess for at least two reasons: (i) they use a different sugar donor (CMP); and (ii) they do not contain the DXD, motif but instead contain other
specifically conserved motifs (6-7, 22). Nevertheless, since
acceptor-binding subsites may involve regions upstream of the catalytic
domain for at least
3-fucosyltransferase and, as reported here, for ST6GalI, there are definitely structural features to be searched in the
variable region that control the transfer reaction. Even though
the stem region is predicted to be highly flexible, it often contains
cysteine residues as well as several N- and/or O-glycosylation sites, which can contribute to a local
conformation stabilized by membrane anchorage. Since truncated variants
exhibited unchanged affinity for the donor and acceptor substrates, but displayed increased velocity parameters, it was concluded that they
lost peptide elements crucial for the active site that possibly maintain the catalytic domain in a closed state. Alternatively, removal
of this modulatory region may induce local changes that facilitate
positioning of critical amino acids within the catalytic site to
proceed with the transfer reaction. Further work based on
crystallization of the full-length transferase in the presence of its
acceptor will thus be required to delineate both the location of the
acceptor-binding site and the catalytic mechanism. Accordingly, variation in stem length and sequence within a glycosyltransferase subfamily may reflect variation in acceptor preference among
transferases sharing similar properties like glycan specificity,
recognition of the underlying glycosylation site, and/or the nature of
the glycoconjugate acceptor.
2,8-sialyltransferase subfamily was found to exhibit exquisite
glycosylation very specific for neural cell adhesion molecules during
development (47) since polysialylation by
2,8 sialyltransferase II
was demonstrated to require core fucosylation of neural-cell adhesion
molecule variants, showing the highest preference of all
transferases known to date, i.e. for both the glycan and
protein acceptors (18). Furthermore, mice produce four isoforms of
1,3-galactosyltransferase that differ in the length of this region,
indicating that polymorphism of a transferase polypeptide may
physiologically occur, possibly introducing new regulatory features
(48). Such a length polymorphism has also been described to occur in
ST3GalIII (49). Moreover, it has been found that two naturally
occurring
2,6-sialyltransferases differing by a single amino acid in
their catalytic domain can differ in their enzymatic properties (50).
If these findings prove to be applicable to all classes of
glycosyltransferases, such functional complexity may very well have
important implications during biosynthesis of cell-surface
glycoconjugates in coordinating the action of multiple enzymes in a
tissue-specific manner and in displacing the physiological balance in
favor of those enzymes overexpressed under pathological conditions.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. J. C. Michalski for mass spectrometric analysis and to C. Dubois, C. Brossette, B. Brioude, and C. Lamagna for helpful collaboration. Thanks are also due to Dr. D. Lombardo for constant interest and to Dr. R. Verger for fruitful discussion of enzymatic catalysis.
![]() |
FOOTNOTES |
---|
* This work was carried out as part of the French GTrec Network and was supported by Action Concertée Chimie et Sciences de la Vie Grant 951411 from Ministère de l'Education Nationale, de la Recherche, et de la Technologie, the Physique et Chimie du Vivant Program of CNRS, and Grant 5000-57797 from the Swiss National Science Foundation (to E. G. B.).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. Tel.:
33-4-911-645-37; Fax: 33-4-917-428-15; E-mail:
ronin@irlnb.cnrs-mrs.fr.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M100860200
2 J. C. Michalski and C. Ronin, unpublished data.
3 J.-C. Guillemot, C. Lamagna, and C. Ronin, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ST6GalNAc, 1,3N-acetylgalactosamine
2,6-sialyltransferase;
ST6Gal,
1,4-galactoside
2,6-sialyltransferase;
LacNAc, N-acetyllatosamine;
CHO, Chinese hamster ovary;
SNA, S.
nigra agglutinin;
PBS, phosphate-buffered saline;
FITC, fluorescein isothiocyanate;
hCG, human chorionic gonadotropin;
hTSH, human thyroid-stimulating hormone;
dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.
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