(Received for publication, May 10, 1995; and in revised form, June 6, 1995)
From the
We now report that
Glycosyltransferases catalyze the transfer of sugar from a sugar
nucleotide donor to an acceptor glycone or aglycone(1) . One of
the best studied glycosyltransferases is lactose synthetase (EC
2.4.1.22), which is composed of a galactosyltransferase that transfers
Gal from UDP-Gal to Glc in the presence of the regulatory protein
Glycosyltransferases have a preferred sugar nucleotide donor, and
for In recent years we and others have
described oligosaccharides in glycoproteins containing the terminal
sequence GalNAc While assaying extracts of vertebrate cell lines for the
presence of a glycosyltransferase that could transfer GalNAc from
UDP-GalNAc to GlcNAc, we identified an activity apparently specific for
UDP-GalNAc. In testing whether this activity might in fact be the known
Figure 1:
Dependence of N-acetylgalactosaminyltransferase activity of
This striking change in sugar
nucleotide specificity is best shown by data in Fig. 2, where
the activity of
Figure 2:
Switch in sugar nucleotide specificity of
We also determined the affinity of the disaccharide toward the
immobilized plant lectin W. floribunda agglutinin, a
lectin previously shown to bind with high affinity to oligosaccharides
containing terminal GalNAc in Taken together, these results demonstrate that the product
is the disaccharide GalNAc
Previous studies have shown that Until now,
the major effect that Many studies have been
conducted on the kinetic mechanisms of The location of regions in The lack of transfer of GalNAc to Glc suggests
that the conformation of the complex E Some glycosyltransferases exhibit a lower K In preliminary experiments we found that Several glycoproteins from bovine milk,
including bovine Terminal GalNAc
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-lactalbumin (
-LA) has a novel
effect on bovine milk UDP-Gal:GlcNAc-
1,4-galactosyltransferase
(
1,4-GT) and induces the enzyme to efficiently utilize UDP-GalNAc
as a donor. In the presence of
-LA the enzyme transfers GalNAc to
free GlcNAc to produce GalNAc
1-4GlcNAc at a rate 55% of that
compared to the rate when UDP-Gal is the donor in the absence of
-LA. The stimulation by
-LA is dependent on the
concentrations of
-LA, acceptor, and sugar nucleotide.
Interestingly,
1,4-GT is unable to transfer GalNAc to Glc with or
without
-LA.
-LA also stimulates the transfer of GalNAc from
UDP-GalNAc to various chitin oligomers, although the degree of
stimulation decreases as the acceptor size increases. Thus, bovine milk
1,4-GT has an inherent ability to utilize two different sugar
nucleotides and the sugar nucleotide preference is regulatable by
-LA.
-lactalbumin
(
-LA)(
)(2, 3, 4) . The
galactosyltransferase component of lactose synthetase is the enzyme,
UDP-Gal:
-D-GlcNAc
-1,4-galactosyltransferase
(
1,4-GT) (EC 2.4.1.38). In non-lactating tissue,
1,4-GT
catalyzes the incorporation of galactose in
1,4-linkage to GlcNAc
residues at the nonreducing termini of certain glycoproteins and
glycolipids(3, 5) . The enzyme can also efficiently
transfer galactose to the monosaccharide GlcNAc to form the
disaccharide N-acetyllactosamine (Gal
1-4GlcNAc). It
has been proposed that in addition to these biosynthetic functions,
1,4-GT may also participate in cell adhesion (6) .
1,4-GT this has been shown to be UDP-Gal. Several studies have
indicated, however, that
1,4-GT can slowly and inefficiently
transfer glucose, arabinose, and N-acetylgalactosamine from
their UDP derivatives to N-acetylglucosamine in
vitro. For example,
1,4-GT uses UDP-Glc and UDP-GalNAc at
only 0.3 and 0.19%, respectively, of the rate at which it utilizes
UDP-Gal (7, 8) .
1,4GlcNAc
1-R (9, 10, 11) . The biological functions of the
sequence GalNAc
1-4GlcNAc-R in oligosaccharides are unclear,
but it may be important for interactions between glycoproteins and
specific receptors(12, 13) . The enzymes responsible
for synthesizing the terminal
1-4GalNAc structures have been
found in several sources(14, 15, 16) , but
there are many questions about the diversity of these enzymes and
uncertainties as to whether the known
1,4-GT might be responsible
for some of the observed N-acetylgalactosaminyltransferase
activities.
1,4-GT or related to it, we examined the effect of
-LA and
conducted corresponding control experiments with bovine milk
1,4-GT. During these studies we made the unexpected observation
that
1,4-GT was tremendously stimulated to use UDP-GalNAc as a
donor and GlcNAc as the acceptor in the presence of
-LA. We now
describe our studies of this unusual sugar nucleotide specificity of
the
1,4-GT in response to the regulatory protein
-LA.
Materials
UDP-Gal, UDP-GalNAc, UDP-Glc,
NAD, MnCl
, p-nitrophenyl sugar
derivatives, UDP-Gal:GlcNAc
1,4-GT from bovine milk (6.0 units/mg
protein), nucleoside-5`-diphosphate kinase, uridine-5`-diphosphate
glucose pyrophosphorylase, uridine-5`-diphosphate glucose
dehydrogenase, ATP,
-LA, lysozyme, and jack bean
-N-acetylhexosaminidase were obtained from Sigma. Dowex
AG-1 X8 was purchased from Bio-Rad.
UDP-[4,5-
H]Gal (50 Ci/mmol) and
UDP-[1-
H]GalNAc (6.3 Ci/mmol) were purchased from
DuPont NEN. Wisteria floribunda agglutinin-agarose (2 mg/ml)
was purchased from Vector and concanavalin A-Sepharose was obtained
from Pharmacia Biotech Inc.
Assay of
A radioactive assay for
1,4-GT
1,4-GT was conducted as described(17) . The reaction
mixture normally contained the following materials in a total volume of
50 µl: bovine milk
1,4-GT diluted to 2-10
milliunits/µl in phosphate-buffered saline containing 1.0 mg/ml
bovine serum albumin, 20 mM MnCl
, 5 mM ATP, 2.5-20 mMN-acetylglucosamine as the
acceptor molecule, 10-20 µM UDP-[
H]Gal (40,000-60,000 cpm/nmol) or
similar amounts of UDP-[
H]GalNAc,
-LA at
different concentrations, and 10 mM sodium cacodylate buffer,
pH 7.5. The reaction mixtures were incubated at 37 °C for 1-2
h and stopped by adding 1.0 ml of water and stored on ice. The product
was isolated by passage of the reaction mixture over a 1.0-ml column of
Dowex AG 1-X8 (chloride form) equilibrated with 5% borate in water (17) and washed with water to neutrality before each use. Each
column was eluted 5 times with 2.0 ml of Milli-Q water and mixed with
2.0 ml of scintillation fluid and the amount of radioactivity was
determined by liquid scintillation counting. Control incubations were
done without an acceptor and the radioactivity eluting from the above
column was subtracted as background. All assays were done in duplicate
and basic statistical analysis was applied to the data. Acceptor p-nitrophenyl sugar derivatives were used at concentrations of
10 mM and products were isolated as described above on a
column of Dowex AG 1-X8.
DEAE-Sephacel Chromatography and Spectrophotometric Assay
for
The 1,4-GT
1,4-GT was suspended at 2
milliunits/µl in 100 mM cacodylate buffer, pH 7.5,
containing 1.0 mg/ml bovine serum albumin. A DEAE-Sephacel column (1
10 cm) was equilibrated with 100 mM cacodylate buffer,
pH 7.5. A total of 400 milliunits of enzyme was applied and the column
was washed with 50 ml of equilibration buffer. The bound enzyme was
eluted with a linear gradient of 0-300 mM NaCl gradient
in equilibration buffer. Fractions (1 ml) were collected and assayed
for Gal and GalNAc transferase activity by a spectrophotometric assay (18) . This assay measures the production of UDP through a
coupled enzyme reaction leading to formation of NADH. The
spectrophotometric assay consisted of the following components: 0.5 ml
of 20 mM MnCl
, 0.28 ml of 100 mM cacodylate buffer, pH 7.5, 0.01 ml of nucleoside-5`-diphosphate
kinase (1000 units/ml), 0.01 ml of UDP-glucose pyrophosphorylase (25
units/ml), 0.01 ml of UDP-glucose dehydrogenase (2 units/ml), 0.067 ml
of NAD (10 mg/ml), 0.05 ml of ATP (10 mg/ml), 0.02 ml of 5 mM UDP-Gal (or UDP-GalNAc), 0.02 ml of 1 MN-acetylglucosamine, and 200 µl of each fraction pool.
After mixing, the tubes were incubated for 2 h at 37 °C and the
absorbance at 340 nm was recorded in a Beckman spectrophotometer DU
640. For GalNAc transferase activity, UDP-GalNAc was added instead of
UDP-Gal and
-LA (8 mg/ml) was included. Control assays were
performed by conducting reactions in the absence of acceptor
monosaccharide.
Lectin Affinity Column Chromatography
W.
floribunda agglutinin-agarose (2 mg/ml) was placed in a column
constructed from two 1-ml plastic disposable pipettes linked together
(0.5 30 cm), and equilibrated in phosphate-buffered
saline/NaN
. The disaccharide product
GalNAc
1-4GlcNAc was applied to the column and bound
materials were eluted with 5 mM GalNAc in phosphate-buffered
saline/NaN
. Fractions (1 ml) were collected and
radioactivity was determined by liquid scintillation counting.
Paper Chromatography and Exoglycosidase
Treatment
Descending paper chromatography for separation of
oligosaccharides was performed in solvent n-butanol:pyridine:water (6:4:3) for 72 h on
borate-impregnated paper or ethyl acetate:pyridine:glacial acetic
acid:water (5:5:1:3) for 24 h(19, 20) . The
distribution of radioactivity on the paper chromatogram was determined
by cutting the paper strips into 1-cm sections and measuring the
radioactivity associated with each section in a liquid scintillation
counter (Beckman). Treatment of oligosaccharides with jack bean
-N-acetylhexosaminidase was performed with 20 milliunits
of enzyme in 0.1 M sodium acetate, pH 5.6, for 20 h at 37
°C. The reaction mixtures were spotted on Whatman paper and
analyzed again by descending paper chromatography.
Preparation of Chitin Oligomers and Amine-adsorption
HPLC
Chitin oligomers were purified as described previously (21) and further purified by amine-adsorption HPLC on an AX5
column (4.0 mm 30 cm). The AX5 column was washed with water at
a flow rate of 1.0 ml/min and equilibrated with 70% acetonitrile. The
mixture of chitin oligomers (500 µg) was injected and 0.5 ml of
fractions were collected using a linear gradient of 70 to 40%
acetonitrile for 20 min. Each fraction was subsequently reanalyzed for
purity by thin layer chromatography. By this procedure we purified N,N`-diacetylchitobiose, N,N`,N"-triacetylchitotriose, and N,N`,N",N‴-tetraacetylchitotetraose. The
oligosaccharides were lyophilized and utilized as acceptors. The
disaccharide product GalNAc
1-4GlcNAc was also analyzed using
the same amine-adsorption HPLC system.
GalNAc Transfer by
Bovine milk 1,4-GT Is Stimulated by
-LA
1,4-GT transfers GalNAc to GlcNAc very
poorly in vitro, but high amounts of the enzyme have been used
to synthesize glycoconjugates terminating with
GalNAc
1-4GlcNAc-R(8, 12) . We found,
however, that the rate of reaction with UDP-GalNAc as a donor and free
GlcNAc as an acceptor was greatly stimulated by
-LA (Fig. 1A). Low concentrations of
-LA had little
effect, but higher concentrations above 5 mg/ml were stimulatory.
Following this result, we used 8 mg/ml
-LA in routine assays. This
stimulation of GalNAc transferase activity was dependent on the
concentration of GlcNAc (Fig. 1B) and UDP-GalNAc (Fig. 1C). The K
values
of
1,4-GT for GlcNAc and UDP-GalNAc were 21.6 mM and 52
µM, respectively, in the absence of
-LA. In the
presence of
-LA the K
values of
1,4-GT for GlcNAc and UDP-GalNAc were 15.5 mM and 45
µM, respectively.
-LA increased the apparent V
at least 30-fold for UDP-GalNAc from
approximately 0.8 to 23 pmol/h.
1,4-GT on
-LA. The reactivity of
1,4-GT was measured as a function of
-LA (A), GlcNAc (B), and UDP-GalNAc (C). All enzyme assays were performed as described under
``Experimental Procedures.'' In B and C the
-LA concentration was 8 mg/ml and in A and C the
GlcNAc concentration was 20 mM.
1,4-GT for UDP-Gal and UDP-GalNAc are compared
directly in the presence or absence of
-LA using GlcNAc as the
monosaccharide acceptor. As expected, in the presence of
-LA,
1,4-GT is inhibited in transferring Gal from UDP-Gal to GlcNAc.
However, the activity of
1,4-GT obtained with UDP-GalNAc as a
donor was highly stimulated in the presence of
-LA and was
approximately 55% of that obtained with UDP-Gal as a donor in the
absence of
-LA.
1,4-GT by
-LA. Enzyme assays were performed as described
under ``Experimental Procedures'' using GlcNAc (20
mM) as an acceptor, either UDP-[
H]Gal or
UDP-[
H]GalNAc as sugar nucleotide donors (70
cpm/pmol), and
1,4-GT (4 milliunits) in the absence and presence
of
-LA (2 mg/ml).
Analysis of the Disaccharide Product
The product
of the 1,4-GT reaction using radioactive
UDP-[
H]GalNAc as a donor and GlcNAc as acceptor
in the presence of
-LA was anticipated to be the disaccharide
GalNAc
1-4GlcNAc. The product migrated as a single peak and
moved slightly slower than N-acetyllactosamine during
descending paper chromatography on borate-impregnated paper in the
6:4:3 solvent system but co-migrated with N-acetyllactosamine
during descending paper chromatography on regular paper in the 5:5:1:3
solvent system (data not shown). These results indicate that the
product is a disaccharide. To further confirm this, the disaccharide
product was treated with jack bean
-hexosaminidase and then
re-analyzed by descending paper chromatography on borate paper. All
radioactivity was recovered as the monosaccharide N-acetylgalactosamine following the exoglycosidase treatment.
Upon analysis by amine-adsorption HPLC the disaccharide product eluted
in the region where standard disaccharides elute (data not shown).
1,4-linkage to GlcNAc (22, 23) . All the disaccharide bound tightly to the
lectin column and was eluted with 5 mM GalNAc (data not
shown).
1-4GlcNAc. This conclusion is
consistent with the results of others(8) , in which commercial
1,4-GT was used to transfer GalNAc to GlcNAc-R, where R is a
hydrophobic aglycone, albeit at a low rate given that
-LA was
absent. In that study they also reported that the product formed with
UDP-GalNAc as a donor and GlcNAc-R as an acceptor was
GalNAc
1-4GlcNAc-R.
DEAE-Sephacel Chromatography of
To
confirm that the single enzyme 1,4-GT
1,4-GT was able to efficiently
transfer both GalNAc and Gal from UDP-GalNAc and UDP-Gal, respectively,
we determined whether the Gal and GalNAc transferase activities
co-eluted upon ion exchange column chromatography.
1,4-GT (400
milliunits total; 6 units/mg) was suspended in 100 mM cacodylate buffer, pH 7.5, containing 1 mg/ml bovine serum albumin
and was applied to a column of DEAE-Sephacel and eluted with a linear
gradient of 0-300 mM NaCl. For convenience the
activities of the enzyme were measured by a spectrophotometric
assay(18) . In this assay the UDP produced by the enzyme from
either UDP-Gal or UDP-GalNAc utilization is coupled to the production
of NADH which is measured at an absorbance of 340 nm. Both the Gal and
GalNAc transferase activities co-eluted with 70-100 mM NaCl during the gradient elution (data not shown). This elution
behavior is consistent with previous reports(24) . UDP-Gal
utilization was measured in the absence of
-LA, whereas UDP-GalNAc
utilization was measured in the presence of
-LA. No significant
activity was observed when UDP-GalNAc was used in the absence of
-LA. These results demonstrate that both the Gal and GalNAc
transferase activities reside in
1,4-GT.
Specific Effect of
-LA
-LA and c-type
lysozymes have 40% homology in amino acid sequence and are very similar
in three-dimensional structure(25, 26) . Lysozyme
degrades bacterial cell wall and does not bind to
1,4-GT(27) . To test the specific effect of
-LA for
enhancing GalNAc transfer, we added lysozyme to the enzyme reaction.
GalNAc transferase activity toward GlcNAc was specifically stimulated
by
-LA (11,534 cpm of product) but not by lysozyme (not
significant cpm of product).
Lack of Transfer of GalNAc to Glc by
We further tested for acceptor specificity of
1,4-GT
1,4-GT in the presence of
-LA. As expected,
-LA caused
the
1,4-GT to efficiently utilize Glc as an acceptor with UDP-Gal
as the donor (Table 1). However, there was no significant
transfer of GalNAc to Glc in the presence of
-LA (Table 1).
Transfer of GalNAc to Oligosaccharides
We tested
whether 1,4-GT could transfer GalNAc from UDP-GalNAc to oligomers
of GlcNAc and whether
-LA was also stimulatory for this reaction.
-LA stimulated the GalNAc transferase activity of the enzyme
toward oligomers of GlcNAc up to N,N`,N",N‴-tetraacetylchitotetraose (GlcNAc)
(Table 2). The stimulatory effect declined, however, as the
size of the oligosaccharide increased. Surprisingly, the enzyme
transferred GalNAc to p-nitrophenyl-
-D-GlcNAc
(GlcNAc
-O-pNP) and to p-nitrophenyl-
-D-chitobiose
(GlcNAc-GlcNAc-
-O-pNP) without added
-LA,
although
-LA stimulated transfer somewhat with these acceptors (Table 2). As controls we tested the acceptors p-nitrophenyl-
-D-xylose and p-nitrophenyl-
-D-GalNAc and neither was
efficiently utilized by
1,4-GT with or without
-LA (data not
shown).
1,4-GT exhibits a
marked preference for UDP-Gal as the sugar nucleotide donor and poorly
utilizes alternative donors like UDP derivatives of glucose, N-acetylgalactosamine, deoxyglucose, and arabinose. The rates
of reactions of
1,4-GT with these other donors are only
0.2-4% as efficient as with UDP-Gal(8, 28) .
Despite this poor reactivity with UDP-GalNAc, the
1,4-GT has been
used to enzymatically synthesize interesting glycoconjugates containing
the terminal sequence
GalNAc
1-4GlcNAc-R(8, 12) .
-LA has been considered to have on
1,4-GT is to cause a switch in monosaccharide acceptor specificity
from GlcNAc to Glc. The K
of
1,4-GT
for Glc is reduced about a 1000-fold in the presence of
-LA from 2 M down to 2 mM(29, 30) . As already
known,
-LA inhibits bovine
1,4-GT from efficiently utilizing
UDP-Gal to transfer Gal to GlcNAc. Our study shows, however, that
-LA induces the
1,4-GT to switch to the alternative donor
UDP-GalNAc when GlcNAc is the acceptor.
1,4-GT and the effects of
-LA on enzyme activity and specificity. It has been proposed that
1,4-GT operates by a partially ordered and sequential reaction
mechanism(31, 32) , in which the enzyme binds first to
Mn
and UDP-Gal to form the E
Mn
UDP-Gal complex, and this
complex then interacts with either a monosaccharide acceptor GlcNAc or
-LA and N-acetyllactosamine synthesis is inhibited or
stimulated depending on the relative concentrations of these latter two
components. However, many aspects of the interaction of
1,4-GT
with
-LA and the specific mechanism of action of
1,4-GT are
poorly understood.
1,4-GT involved
in binding to sugar nucleotide donor and
-LA have been studied by
differential labeling and chemical
modification(33, 34, 35) . Recent studies
using site-directed mutagenesis(36) , revealed that
-LA
has two aromatic clusters and that cluster I is involved in binding to
1,4-GT. A model was proposed whereby
-LA binding to
1,4-GT creates a ``monosaccharide bridge'' for the
binding of glucose. Precisely how our finding on the switch to
UDP-GalNAc as a donor in the presence of
-LA fits in with these
observations is unknown. Interestingly, we found no evidence that
1,4-GT can transfer GalNAc to Glc in either the presence or
absence of
-LA.
Mn
UDP-GalNAc may be different
from the conformation of the complex E
Mn
UDP-Gal in that
-LA
cannot expose the binding pocket for glucose in the former complex.
Instead, the enzyme complex E
Mn
UDP-GalNAc must still be
capable of interacting with
-LA to promote transfer of GalNAc to
GlcNAc. This implies that the GlcNAc-binding site is preserved in the
complex E
Mn
UDP-GalNAc
-LA.
Since
1,4-GT has a single binding site for UDP, we presume that
UDP-Gal and UDP-GalNAc occupy the same catalytic site. However, the
effect of UDP-GalNAc on the enzyme complex with
-LA must be
different from the effect of UDP-Gal, since
-LA blocks transfer of
Gal from UDP-Gal to GlcNAc. It is also possible that
-LA could
stimulate the GalNAc transfer activity of the enzyme by altering the
rate of product release when GalNAc terminating products are
synthesized. For example, it was previously observed that covalent
cross-linking of
-LA with
1,4-GT reduced the maximum velocity
by a factor of 100 for lactose synthesis compared to the
non-cross-linked enzyme for N-acetyllactosamine synthetase
activity(37) . Many further experiments are needed to elucidate
the precise mechanism by which
-LA causes the sugar nucleotide
switch of
1,4-GT.
for acceptors containing a hydrophobic
aglycone than for the free oligosaccharide
acceptor(38, 39) . This may relate to our observation
that p-nitrophenyl-
-D-GlcNAc was more efficient
than free GlcNAc as an acceptor for the GalNAc transferase activity of
1,4-GT even without added
-LA (Table 2). As mentioned
above, it has been suggested that aromatic amino acids in
-LA are
important for both
-LA interaction with
1,4-GT and induction
of the substrate glucose binding pocket, and these two sites are in
close proximity(36) . Our results suggest the possibility that
the aromatic aglycone moiety of the acceptor p-nitrophenyl-
-D-GlcNAc might substitute for the
aromatic cluster of
-LA and thereby stimulate
1,4-GT to
transfer GalNAc to GlcNAc in the absence of
-LA. This phenomenon
needs further study, since the nature of the aglycone may be important
and aromatic aglycone derivatives may be more active in this regard
than non-aromatic aglycones. For example, it has been observed (8) that
1,4-GT could transfer GalNAc from UDP-GalNAc to
the 8-methoxycarbonyloctyl hydrophobic aglycone derivative of GlcNAc,
but the rate was 0.19% of that obtained when UDP-Gal was the donor.
1,4-GT could transfer
GalNAc from UDP-GalNAc to biantennary acceptor glycopeptides and the
reaction was stimulated by
-LA, but the degree of stimulation was
not as great as observed with the chitin-like acceptors. This may
result from the large size of the acceptors that interfere with
-LA binding to the
1,4-GT, as previously observed for other
acceptors when UDP-Gal is the
donor(32, 40, 41) . We are currently
examining in more detail the interesting effect of
-LA on the
ability of
1,4-GT to transfer GalNAc to glycoprotein acceptors. In
addition, we are studying whether this effect of
-LA extends to
glycosphingolipid acceptors.
-LA, bovine lactotransferrin, and bovine milk
epithelial glycoprotein IV, contain oligosaccharides with the terminal
sequence GalNAc
1-4GlcNAc-R (42, 43, 44, 45) . Terminal
1,4-linked GalNAc is a common residue in Asn-linked
oligosaccharides in many membrane glycoproteins from bovine milk fat
globule (45) and these glycoproteins may acquire more GalNAc
during lactation(46) . Our results suggest the possibility that
under certain conditions within the lactating mammary gland, where the
concentration of
-LA is high, the
1,4-GT might be able to
generate glycoproteins containing terminal GalNAc structures.
1-4GlcNAc-R has been found in many other
non-mammary gland-derived
glycoproteins(47, 48, 49, 50, 51, 52, 53, 54) and in
glycoproteins derived from the parasites Schistosoma mansoni and Dirofilaria immitis(11, 55) . A
1,4-N-acetylgalactosaminyltransferase distinct from
1,4-GT has been shown to be important in the synthesis of terminal
1,4-GalNAc structures in pituitary glycoprotein
hormones(14) , but other N-acetylgalactosaminyltransferase activities have also been
reported(15, 16) . The degree to which the effects we
have observed on
1,4-GT are related to the synthesis of terminal
1,4-GalNAc structures is unknown.
-LA is highly expressed in
the lactating mammary gland where its expression is associated with
lactose production, but the UDP-GalNAc utilization induced by
-LA
is unrelated to lactose production, since Glc is not an acceptor when
UDP-GalNAc is the donor. Whether there are other modifier proteins
expressed by non-mammary gland-derived cells, capable of mimicking
-LA and altering the sugar nucleotide specificity of
1,4-GT,
remains to be determined. In addition, we do not know whether the
effect we have observed is specific only for
1,4-GT or whether
some other glycosyltransferases may be induced to use alternative sugar
nucleotide donors in the presence of
-LA or some modifier proteins
or factors.
-LA,
-lactalbumin;
1,4-GT, UDP-Gal:
-D-GlcNAc
-1,4-galactosyltransferase; GlcNAc, N-acetylglucosamine;
GalNAc, N-acetylgalactosamine; Gal, galactose; HPLC, high
performance liquid chromatography.
We thank Dr. A. Kwame Nyame for critically reading the
manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.