Galactosyltransferase Acceptor Specificity of the Lactose Synthetase A Protein

Abstract

The galactosyl acceptor specificity of the highly purified A protein (galactosyltransferase) isolated from bovine milk was examined in the absence and presence of α-lactalbumin. α-Lactalbumin inhibits the transfer of galactose to N-acetylglucosamine but does not appreciably inhibit the transfer to oligomers of N-acetylglucosamine and other β-1,4 linked glycosides such as cellobiose, cellobiulose (glucosyl-β-1,4-fructose), β-d-methylglucose, glucosyl-β-1,4-mannose, indoxyl-β-d-glucose, and ovalbumin. α-Glycosides are poor substrates in the presence of α-lactalbumin but are not substrates in its absence. The biosynthesis of lactose and the formulation of the Gal-β-1,4-GlcNAc linkage in the carbohydrate side chain of glycoproteins are compatible and are carried out by the same galactosyltransferase.

Full text

THE JOURNAL 08 B~OLOCWAL CHEMISTRY
Vol.245, No.19,Issue of October 10, pp.5057-5061, 1970
Printed in U.S.A.
Galactosyltransferase Acceptor Specificity of
the Lactose Synthetase A Protein*
(Received for publication, March 25, 1970)
F. L.
SCHANBACHER~ AND
Ii. E.
EBNER$
From the Department of Biochemistry, Agricultural Experiment Xtation, Oklahoma &ate University, Xtillwater,
Oklahoma
740’74
SUMMARY
The galactosyl acceptor specificity of the highly purified A
protein (galactosyltransferase) isolated from bovine milk
was examined in the absence and presence of cY-lactalbumin.
cr-Lactalbumin inhibits the transfer of galactose to N-acetyl-
glucosamine but does not appreciably inhibit the transfer to
oligomers of N-acetylglucosamine and other p-1,4 linked
glycosides such as cellobiose, cellobiulose (glucosyl-p-1,4-
fructose), fl-D-methylghtcose, ghtcosyl-p-1,4-maMOSe, in-
doxyl+-D-glucose, and ovalbumin. oc-Glycosides are poor
substrates in the presence of oc-lactalbumin but are not sub-
strates in its absence. The biosynthesis of lactose and the
formulation of the Gal-/?-1,4-GlcNAc linkage in the carbohy-
drate side chain of glycoproteins are compatible and are
carried out by the same galactosyltransferase.
Lactose synthetase (UDP-galactose:n-glucose l-galactosyl-
transferase; EC 2.4.1.22) catalyzes the biosynthesis of lactose
(Equation 1). The enzyme occurs in a soluble form in bovine
UDP-galactose + glucose + lactose + UDP (1)
milk (2,3) and was resolved by Brodbeck and Ebner (4) into two
protein fractions, designated as A and B, which were both re-
quired for significant enzymatic activity. The B protein was
identified as oc-lactalbumin, the common milk whey protein (5,
6). Furthermore, it has been shown that there is a high degree
of homology in the amino acid sequence (7) and tertiary struc-
ture (8) of bovine ar-lactalbumin and hen’s egg-white lysozyme,
although lysozyme has no effect on the lactose synthetase re-
action. Brew, Vanaman, and Hill (9) showed that the A pro-
tein in the absence of a-la&albumin catalyzes the following
reaction (Equation 2) which is appreciably inhibited by oc-lactal-
bumin.
* This work was supported in part by Grant AM 10764 from the
National Institutes of Health, by Grant GB 7975 from the Na-
tional Science Foundation, and by Grant P-420 from the American
Cancer Society. A preliminary report has been published (1).
$ This work was submitted in partial fuE.llment for the Ph.D.
degree to the Graduate College, Oklahoma State University.
8 Career Development Awardee lK04 GM 42396 of the Na-
tional Institutes of Health.
UDP-galactose + N-acetylglucosamine -+
N-acetyllactosamine + UDP
(2)
The A protein occurs in many tissues and appears to act as a
galactosyltransferase to terminal N-acetylglucosaminyl residues
in the carbohydrate portion of glycoproteins (10). Hill
et al.
(10) have examined briefly the acceptor substrate specificity of
a partially purified bovine ,4 protein and concluded that the A
protein had limited specificity to a variety of galactosyl acceptors,
although it did transfer galactose to orosomucoid.
Recent studies in this laboratory have shown that a highly
purified A protein isolated from bovine milk can catalyze the
formation of lactose in the absence of ol-lactalbumin and that
there is a reciprocal relationship between ol-lactalbumin and the
concentration of glucose used in the assay (11). The observation
that the A protein acts as a galactosyltransferase to glycopro-
teins (10) and that ar-lactalbumin inhibits the formation of N-
acetyllactosamine (Reaction 2) suggested that it may be an
inhibitor of the transfer of galactose to glycoproteins. If this
were the case, then it would appear that lactose biosynthesis,
which requires or-lactalbumin for significant rates, and the trans-
fer of galactose to the carbohydrate portion of glycoproteins
would not be compatible in the mammary gland. Accordingly,
a detailed study of the substrate specificity of various galactosyl
acceptors and the effect of or-lactalbumin on these acceptors
was undertaken. These studies support the view that the A pro-
tein of lactose synthetase is a general galactosyltransferase and
that it transfers galactose to a variety of P-1,4-glycosides and
that ar-lactalbumin does not appreciably inhibit this reaction.
Also, certain cr-1,4-glycosides become poor substrates only in
the presence of oc-lactalbumin. Thus, in the mammary gland
the biosynthesis of lactose and the transfer of galactose to the
carbohydrate side chains of certain glycoproteins are compatible.
EXPERIMENTAL PROCEDURE
Materials-The A protein (11) and or-lactalbumin (6) were
prepared as previously described.’
Glucosamine, melibiose, gentiobiose, cellobiose, @-methyl-n-
glucoside, N-acetylmannosamine, N-acetylmuramic acid, and
indoxyl-P-n-glucoside were purchased from Sigma. Maltose
was obtained from Fisher. Mannose, n-fucose, cr-methyl-n-
glucoside, 2-deoxy-n-glucose, and ovalbumin (twice crystallized)
were purchased from Mann. Cellobiulose (glucosyl - ,8 - 1,4-
1 The authors thank Dr. R. Mawal and Dr. B. Colvin for prepa-
ration of the A protein.
5057
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6058 Galactosyltransferase Specijkity
Vol. 245, No. 19
12 24 36
pg A-PROTEIN
0 01 02 03
IhM N-ACETYLGLUCOSAMINE I/ug a- LACTALEUMIN pg a LACTALBUMIN/ml
FIG.
1. Linearity between the A protein of lactose synthetase and the formation of UDP whenN-acetylglucosamine was the substrate.
Lyophilized A protein was the source material (11) and the assay contained 20 mM N-acetylglucosamine and 0.1
M
KC1 (text).
FIG.
2. Lineweaver-Burk plot of the galactosyltransferase activity of the A protein of lactose synthetase when N-acetylglucosamine
was the galactosyl acceptor. Eighteen units of the A protein were used in the standard assay (text). O-O, no or-lactalbumin;
O-0, 10 pg of oc-lactalbumin per ml;
A-A,
25 pg of ol-lactalbumin per ml; and O-0, 50 pg of oc-lactalbumin per ml.
FIG.
3. Tertiary plot of the reciprocal of the intercept (V is the observed intercept a,nd V, is the intercept when the concentration of
a-la&albumin is zero) against the reciprocal of the Lu-lactalbumin concentration. The data. were obtained from the secondary plot of
the intercept versus a-lactalbumin concentration (Fig. 2).
FIG. 4. Percentage of inhibition of N-acetylglucosamine, ovalbumin, and (G~cNAc)~ as galactosyl acceptors of the A protein of
lactose synthetase by a-lactalbumin. Eighteen units of A protein were used in each assa.y. *m, AT-acetylglucosamine, 20 mM,
1OOyh equals 14 units; O-O, ovalbumin, 0.4 mM, 1007, equals 4.9 units; O---O, (GlcNAc)s, 2.5 mM, 1007, equals 15 units.
fructose) and glucosyl-p-1,4-mnnnose were obtained chromato-
graphically pure from Dr. J. M. Leatherwood of the Department
of Animal Science, North Carolina State University, Raleigh,
North Carolina. GlcNAc-ManAcZ was a gift from Dr. Nathan
Sharon, Weisman Institute, Rehovoth, Israel. The GlcNAc
oligomers, gifts from Dr. Patrick Guire of this department, were
prepared by molecular sieve chromatography. All other chem-
icals were of reagent grade quality. Maltose, gentiobiose, (Y-
methylglucoside, and P-methylglucoside were chromatographed
on Silica Gel Plates (MN-Polygram Silica Gel N-HR from
Brinkmann Instruments, Inc., Westbury, New York) using pro-
panol-HZO, 7: 1, v/v, as the solvent and aniline phthalate as the
spray. There was no evidence for glucose in these compounds.
The technique could readily detect less than
17*
contaminant.
The presence of glucose in a potential substrate could lead to
erroneous results when the reaction was performed in the pres-
ence of a-lactalbumin. If a potential substrate at 250 mM
contained lyO glucose, the rate due to this amount of glucose
in the presence of or-lactalbumin would be 5yc of the rate
when glucose was the saturating substrate.
Enzymatic activity of the A protein (LacNhc synthetase)
was assayed spectrophotometrically in a revised procedure by
measuring UDP formation enzymatically (12). In addition,
the assay cont,ained 0.1
M
KC1 which made the assay more
reproducible because pyruvate kinase in the coupling system
requires K+. Normally, 18 units of lyophilized A protein (11)
were added to each assay which had a final volume of 1.0 ml.
The various galactosyl acceptors were substituted for N-acetyl-
glucosamine in the assay. Enzymatic rates are initial velocities
and are normally expressed as millimicromoles of UDP formed
per min. Concentrations of oc-lactalbumin up to 1 mg per ml
did not inhibit the enzymatic coupling system used for the anal-
ysis of UDP. The
K,
values and
V,n,,
are apparent constants
2 The abbreviation used is: LacNAc, N-acetyllactosamine.
and were determined under the conditions described. A unit of
enzyme is the amount required to form 1 mpmole of UDP per
min under the conditions described.
RESULTS
The linearity of the rate of the LacNAc synthetase assay
(Reaction 2) as a function of A protein concentration is pre-
sented in Fig. 1, and the assay is linear to at least 16 mpmoles of
UDP per min per ml or 36 pg of bovine A protein.
The inhibition of the LacNAc synthetase activity by oc-lactal-
bumin was investigated and preliminary experiments showed
that the activity was inhibited 82% by 100 pg of oc-lactalbumin
per ml at both 10 and 20 mM N-acetylglucosamine. The effect
of varying fixed levels of a-lactalbumin on the LacNAc syn-
thetase reaction is shown in a Lineweaver-Burk plot in Fig. 2.
The apparent
K,
values of N-acetylglucosamine at 0, 10, 25,
and 50 pg of cr-lactalbumin per ml were 12.5, 2.2, 1.2, and 1.0
InM,
respectively. Thus, cY-lactalbumin lowers both the ap-
parent
K,
and
V,,l,,
of the reaction (Fig. 2). The secondary
plots of the slope and the intercept versus ar-la&albumin from
Fig. 2 are nonlinear, but the tertiary plot of the reciprocal of
the slope and intercept versus l/oc-lactalbumin is linear (Fig. 3),
indicating that the inhibition by cY-lactalbumin is hyperbolic.
or-Lactalbumin is an efficient inhibitor of the LacNAc syn-
thetase reaction and approximately 15 pg per ml will inhibit the
reaction 50% (Fig. 4). These results suggest that the transfer
of a galactosyl group to GlcNAc could be controlled by cr-lactal-
bumin. However, additional experiments with polymers of
GlcNAc, all p-1,4 linked, indicated that this suggestion was in-
correct. With (GlcNAc)? the
K, was
the same in the presence
or absence of cr-lactalbumin (25 pg per ml) while the Ti,11,, was
slightly decreased from 132 to 121 mpmoles of UDP formed
per min (Table I). Similar results were obtained with tri- and
tetra-GlcNAc. The effect of a-lactalbumin when tri-GlcNAc
was the galactosyl acceptor is presented in Fig. 4 and shows that
1 mg of oc-lactalbumin per ml inhibited the reaction 327& -1
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Issue of October 10, 1970
F. L. Xchanbacher and K. E. Ebner 5059
TABLE
I TABLE II
Apparent K,,, and V,,,
values
of galactosyl acceptors in presence and
absence of &actalbumin Galactosyl acceptor activity of A protein of lactose synthetase in
presence and absence of a-lactalbumin
Each assay contained 18 units of the A protein of lactose syn-
thetase (see text).
Acceptor
GlcNAc.
(GlcNAc) 2.
(GlcNAc)n
(GlcNAc)c
GlcNAc-NAM. _.
Cellobiose. .
Maltose.
Glucose............
Ovnlbumin..
-
a-Lactal- + a-Lactal
bumin bumin
?lzM
8.3
0.62
1.0
1.2
1.6
833
14li
1.66
-
_-
t
1
1.7
0.62
1.13
1.4
2.2
833
5000
5
1.66
VlS.X
- a-Lac- + a-La
albumin talbumin
n,moles UDP/min
26.9
131.1
17.6
23.7
20.2
124.2
0
26.9
8.3
120.3
19.7
23.7
20.2
124.2
129.0
19.4
20.2
4mount of
a-lactal-
bumin
present
aghl
25
25
25
25
100
100
100
100
100
number of other oligosaccharides were tested as galactosyl
acceptors and the apparent K, values and V,,,,, values were
determined in the absence and presence of ar-lactalbumin (Table
I). The reciprocal plots for the GlcNAc oligomers, glucose,
and ovalbumin were linear, whereas the plots for GlcNAc-N-
acetylmuramic acid and cellobiose were curvilinear at less than
0.25 mM. The apparent K, for ovalbumin was unaffected by
100 pg per ml of cr-lactalbumin; however, the maximum velocity
was slightly reduced. In a separate experiment, 600 pg of
cu-lactalbumin per ml inhibited the reaction 48% when the oval-
bumin concentration was 0.4
InM
(Fig. 4).
Maltose (Table I) was not a substrate in the absence but was
a poor substrate in the presence of cr-lactalbumin. These results
prompted the investigation of other potential galactosyl acceptors
differing in structure and in the anomeric bond configuration.
These results, as well as the effect of cr-lactalbumin on these
reactions, are presented in Table II. Mannose, L-fucose, meli-
biose, and UDP-N-acetylglucosamine were not acceptors at
the concentrations tested. 2-Deoxyglucose, maltose, and
ar-methylglucose were acceptors only in the presence of cw-lactal-
bumin. A variety of p-1,4-linked glucose derivatives were
galactosyl acceptors and the rates were not appreciably affected
by cr-lactalbumin, and indeed ovalbumin was a good galactosyl
acceptor. Ovalbumin has a single carbohydrate chain which
terminates in a ,&linked GlcNAc residue (13) and based on the
above studies with model compounds it is not surprising to find
that it was a good galactosyl acceptor and relatively insensitive
to inhibition by ol-lactalbumin.
The observation that glucosyl (and derivatives) residues,
nonterminal and linked as a /3-1,4-glucoside, were relatively
insensitive to inhibition by a-la&albumin suggested that the
transfer of galactose to carbohydrate side chains of glycoproteins
and to glucose may occur concurrently. An experiment was
designed to determine whether galactosyl transfer from UDP-
galactose to glucose and ovalbumin in the presence of oc-lactal-
bumin could occur concomitantly. The concentration of sub-
strates used was less than their K, values which would be nearer
intracellular concentrations. Each standard assay contained 18
units of the A protein and 100 pg of a-lactalbumin per ml. When
the galactosyl acceptor was glucose (2.0 mrvr), the rate was 1.24
mpmoles per min; with ovalbumin (0.16 mM) the rate was 1.06
mpmoles per min; and with both glucose (2.0 mM) and ovalbumin
(0.16 mM) the rate was 2.30 mpmoles per min. Under these
GlcNAc .
Glucose.................
Glucose.................
2-Deoxy-o-glucose .
Mannose
L-Fucose
Glucosamine
N-Acetylmannosamine.
N-Acetylmuramic acid..
ol-Methylglucose .
&Methylglucose
p-Indoxylglucose
Maltose.
Cellobiose
Cellobiulose
Glucosylmannose
Gentiobiose
Melibiose...............
UDP-GlcNAc
a-1
P-1
B-1
(Y-1,4
P-l,4
P-124
o-1,4
P-l,6
P-l,6
a!-1
Acceptor
:oncentra-
tion
.w
0.02
0.02
0.25
0.25
0.25
0.25
0.25
0.25
0.119
0.25
0.25
0.02
0.25
0.25
0.10
0.10
0.25
0.25
0.0004
Activity in
pg per ml 100 pg per
f la;ldd- ml of a-lpc-
talbumm
18.7 3.15
0 17.4
0 18.0
0 1.72
0 0
0 0
0.13 0.55
0.19 0.66
8.26 6.85
0 1.12
1.54 1.62
4.34 3.24
0 4.23
2.17 2.17
0.76 0.72
0.23 0.26
1.04 6.7
0 0
0 0
-
conditions the rates are additive suggesting that in the mam-
mary gland the same enzyme is able to carry out concomitantly
the synthesis of lactose in the presence of cu-lactalbumin and
the transfer of galactose t.o the carbohydrate side chains of
glycoproteins.
DISCUSSION
The studies on the galactosyl acceptor specificity of the
galactosyltransferase of lactose synthetase provide further
insight into the mechanism of the lactose synthetase reaction.
In addition to catalyzing the synthesis of lactose in the presence
of ac-lactalbumin, the galactosyltransferase of the lactose syn-
thetase reaction appears to have its major role in the biosyn-
thesis of certain carbohydrate side chains of glycoproteins.
oc-Lactalbumin is a good inhibitor of the transfer of galactose
to GlcNAc and the inhibition appears to be hyperbolic. How-
ever, /3-1,4-glycosides of GlcNAc (di-, tri-, and tetra-GlcNAc,
GlcNAc-N-acetylmuramic acid, and ovalbumin) were not ap-
preciably inhibited by oc-lactalbumin at concentrations of 100
pg per ml (Table I). However, the concentrations of cr-lactal-
bumin required to inhibit the transfer of galactose by 50% to
tri-GlcNAc was 1500 pg per ml; to ovalbumin, 700 pg per ml,
whereas to GlcNAc it was 15 pg per ml (Fig. 4). The intra-
cellular concentration of cu-lactalbumin in bovine mammary
tissue is at least 35 pg per g of tissue (6) and the concentration
of GlcNAc in tissues is very low (14) if it is present at all. Hence,
the physiological function of the galactosyltransferase is most
likely to transfer galactose to the nonreducing end of a heterosac-
charide of a glycoprotein terminating in GlcNAc, particularly
because the linkage Gal-GlcNAc (/l-1,4) is the most prevalent
one in the carbohydrate side chains of glycoproteins (15).
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Galactosyltransferase Specificity
Vol. 245, No. 19
Derivatives of GlcNAc (/3-1,4 linked) are the best galactosyl
acceptors in the galactosyltransferase reaction. The a-l,4
derivatives are not substrates but did exhibit very poor activity
in the presence of a-lactalbumin.
A variety of P-linked glucosides were tested as acceptors in
the presence and absence of or-lactalbumin. All of these were
acceptors and P-indoxylglucose was the best but none was as
good as GlcNAc or its oligomers. In general, these compounds
were not affected appreciably by cr-lactalbumin. Glucose is
a very poor substrate but in the presence of cr-lactalbumin it
becomes a good substrate because oc-lactalbumin drastically
lowers the K, of glucose (11). Other galactosyl acceptors (2-
deoxy-n-glucose, glucosamine, N-acetylmannosamine, a-l,4
glucosides, and gentiobiose) exhibit higher rates in the presence
of cr-la&albumin.
p-1,4-Glycosides of GlcNAc are the best galactosyl acceptors.
2-Deoxy-n-glucose is not an acceptor in the absence of Lu-lactal-
bumin, whereas glucosamine and N-acetylmannosamine are
poor acceptors. The acetamido group as in GlcNAc is highly
essential for good acceptor activity. N-Acetylmuramic acid
is a fairly good acceptor which suggests that the bulky 3’-O-
lactyl group reduces the specificity for the GlcNAc conformation.
However, in the presence of cr-la&albumin the specificity for
the GlcNAc conformation in position 2 is greatly reduced. Thus,
cY-lactalbumin modifies the galactosyltransferase so that glucose
and some of its derivatives become better substrates.
The galactosyltransferase of lactose synthetase appears to
have an absolute specificity for the 4-hydroxy in the glucose
conformation of the terminal nonreducing residue because fucose
and melibiose were not acceptors.
The observation that ovalbumin is a good acceptor is predict-
able from the specificity studies because the oligomers of GlcNAc
glycosides act in a similar manner. Also, a carbohydrate oli-
gomer (minus sialic acid) obtained from an ovarian cyst (16)
was an excellent substrate. These studies clearly implicate
that the galactosyltransferase is involved in the synthesis of
Gal+l,4-GlcNAc residues in the carbohydrate side chains of
glycoproteins.
In the mammary gland, the same galactosyltransferase ap-
pears to be involved in the biosynthesis of lactose and glycopro-
teins. The present study shows that both of these syntheses
may occur concurrently because both glucose and ovalbumin are
equally good acceptors in the presence of or-lactalbumin at a
concentration which allows sufficient synthesis of lactose and
no appreciable inhibition of the transfer of galactose to oval-
bumin. It is possible that there may be two independent sites
on the galactosyltransferase, but this appears to be rather un-
likely because both activities are lost in a parallel manner by
heat inactivation (9) and the conditions for measuring activities
are similar (12).
Several galactosyltransferases have been described and par-
tially purified from several sources and these have properties in
common with the galactosyltransferase isolated from bovine
milk. The galactosyltransferase isolated from goat colostrum
by McGuire et al. (17) was active with a variety of P-linked
GlcNAc acceptors including GlcNAc, P-methyl-GlcNAc,
(G~cNAc)~, and (GlcNAc)z, although glucose was reported not
to be an acceptor. This enzyme transferred galactose to a
variety of glycoprotein acceptors having a /3-N-acetylglucos-
aminyl end group including ovalbumin and orosmucoid pre-
viously treated with sialidase and P-galactosidase. Iyer and
Carlson (16) also showed transfer of galactose to GlcNAc and
to an ovarian cyst glycoprotein previously treated with sialidase.
The galactosyltransferase from bovine milk also transfers
galactose to this protein and to the carbohydrate side chain
derived from this protein. Zinderman et al. (18) showed that
particles prepared from rabbit gastric mucosa would transfer
galactose to GlcNAc, (G~cNAc)~, and P-methyl-GlcNAc. Spiro
and Spiro (14) partially purified a galactosyltransferase from
calf thyroids and reported a K, for GlcNAc of 1.9 X lo-* M
and a K, for a sialic acid and galactose-free peptide from thyro-
globulin as 3.3 x lop3 M which is similar to the results obtained
with the bovine milk galactosyltransferase (Table II) with a
variety of substrates. However, Spiro and Spiro (14) suggest
the existence of two separate enzymes (one transferring galactose
to GlcNAc and the other transferring to a glycopeptide acceptor)
based on differing ratios of activity during purification. They
do suggest that if there was one enzyme the N-acetylglucosamine
present in glycopeptides is probably the natural substrate. The
present study with the bovine milk galactosyltransferase shows
that this enzyme catalyzes both reactions and that the N-acetyl-
glucosaminyl-P-1,4 glycosides are the best substrates for the
enzyme because GlcNAc is probably not a physiologically sig-
nificant substrate.
Babad and Hassid (3) showed that GlcNAc, cellobiose, and
glucose were galactosyl acceptors with the mammary enzyme
from milk but their preparation probably contained unspecified
amounts of cY-lactalbumin because it was not separated into the
two proteins. Brew et al. (9) and Hill et al. (10) reported
that a
partially purified galactosyltransferase from bovine milk would
transfer galactose to GlcNAc and this reaction was inhibited
by a-lactalbumin. In addition, they showed galactosyl transfer
to orosomucoid previously treated with sialidase and P-galac-
tosidase but studies with other acceptors were inconclusive.
These studies suggest that the principal function of the galac-
tosyltransferase isolated from bovine milk is to transfer galactose
to a heterosaccharide of a glycoprotein terminating in p-1,4-
linked GlcNAc. The mammary gland is unique in that it has
the ability to synthesize or-lactdlbumin which allows glucose to
become an effective galactosyl acceptor but the presence of
cr-lactalbumin does not appreciably inhibit the transfer of
galactose to heterosaccharides of glycoproteins.
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