From the
Glycogenin is the autocatalytic, self-glucosylating protein that
initiates glycogen synthesis in muscle and other tissues. We have
sequenced the cDNA for rabbit muscle glycogenin and expressed and
purified the protein in high yield as well as two mutant proteins in
which Phe or Thr replaces Tyr-194, the site of glucosylation. While the
wild-type protein can self-glucosylate, the mutants cannot, but all
three utilize alternative acceptors by intermolecular glucose transfer
for which the mutants have altered specificity. Tyr-194 is therefore
not essential for the catalytic activity of glycogenin. All three
proteins also hydrolyze UDP-glucose to glucose at rates comparable with
the rate of self-glucosylation. The hydrolysis is competitive with
glucose transfer to p-nitrophenyl
Glycogenin is a protein first obtained from rabbit muscle
glycogen, to which it is covalently bound in one molecular proportion
via the novel glucose-tyrosine bond, where its presence in this form
suggested that it was the long sought primer for glycogen synthesis
(for reviews, see Refs. 1 and 2). Glycogen-free proteins with similar
properties were detected in muscle and other tissues. When purified to
homogeneity they were found to be autocatalytic, undergoing
self-glucosylation in the presence of µM UDP-glucose and
Mn
We are
interested in the functionality of glycogenin, such questions as the
nature of its active site, where and how it binds UDP-glucose, where
ATP, a powerful inhibitor(3) , binds, how the first glucose
residue is attached to tyrosine(4, 5) , and whether the
phosphorylation of serine (6) and tyrosine (7) residues
plays a role in the regulation of self-glucosylation. Molecular
cloning, mutagenesis, and the expression of recombinant proteins are
the preferred methodologies with which to pursue such questions.
Accordingly, we have cloned the cDNAs for glycogenin from human and
rabbit muscle, compared the deduced amino acid sequences, and expressed
these proteins. We report here only on the rabbit muscle enzyme, which
we have purified to homogeneity and tested for functionality. The
similar cloning, expression, and purification of the rabbit protein and
two mutant proteins have already been reported by Roach and
co-workers(8, 9) . Asking whether it is necessary for
Tyr-194 to be present for glycogenin to function as a transglucosylase,
we have expressed the same recombinant proteins as Roach and
co-workers, in which Phe and Thr replace Tyr-194. Contrary to their
report that these two mutants are enzymically inactive because Tyr-194
is ``essential for activity''(9) , we have already
reported elsewhere that both mutants display intermolecular
transglucosylase activities(4) . That of the Phe-194 mutant is
comparable in its catalytic efficiency with that of wild-type
glycogenin. Here we report a much more detailed study, with different
substrates, of the ability of wild-type and mutant glycogenins to carry
out intermolecular transglucosylation. What this has revealed is that
the self-glucosylation, previously claimed to be intramolecular, could
be intermolecular and that the transglucosylase activity of glycogenin
functions irrespective of whether Tyr-194 is fully glucosylated or is
replaced by Phe or Thr. A new catalytic activity of glycogenin that we
report here is the hydrolysis of UDP-glucose at a rate significant in
comparison with that at which glycogenin uses UDP-glucose in
autocatalysis, while the homogeneous recombinant enzymes break down
spontaneously to 32-kDa species. We also note a difference between
native and recombinant glycogenin in respect to inhibition by ATP.
The commercial sources of biochemical reagents and kits for
molecular cloning and expression are mentioned at their point of use.
Otherwise the supplier was Sigma. Oligonucleotides were synthesized by
the Biotechnology Facility at the University of Florida, Gainesville,
and by Dr. R. Werner of our department. 1
When the E. coli cultures containing
expressed proteins (see below) were tested for the presence of
glycogenin, 1-ml portions of the cultures were centrifuged, and the
pellets were resuspended in 500 µl of lysis buffer (see below),
sonicated, and centrifuged at 12,000 rpm for 10 min. Portions (65
µl) were used for self-glucosylation reactions (10) in
100-µl digests where the
[
Given the strong expression of the induced wild-type rabbit muscle
protein, we decided to examine this protein and mutants derived from
its cDNA where Phe and Thr replaced Tyr-194. All three proteins were
purified to homogeneity by a two-step column procedure that we have
described elsewhere(4) . A comparison with the data reported by
Cao et al.(9) for their rabbit protein expression
system reveals the superiority of ours in terms of level of expression.
So much glycogenin was expressed in our system that only a 2.5-fold
purification was necessary to obtain a homogeneous product in a
two-step process ( Fig. 1and Ref. 4) in a yield of 49%.
Previously (9) a five-step process was necessary to achieve an
8.4-fold purification to homogeneity in a 14.4% yield. (Compare Fig. 1A, lane1, in Ref. 9 with our Fig. 1A, lane3). Fig. 1D depicts the Coomassie Blue stains of the purified proteins after
SDS-PAGE. There is a clear difference in M
We measured the initial rate of self-glucosylation by
wild-type glycogenin under the standard conditions (10) over a
range of enzyme concentration from 0.01 to 1 µM. The rate
was uniform during the 5-min incubation. The results are shown in Fig. 2.
Rabbit muscle glycogenin was expressed and detected in
lysates of the E. coli host, where induced and uninduced
cultures were compared (Fig. 1, ). Both lysates
contained immunologically positive, active glycogenin of the correct M
This variability reflects not so much the amount of glycogenin
protein, but instead reflects the level to which the protein is already
glucosylated and how much more glucose is to be added to bring the
maltosaccharide chains to an average of eight glucose units. Roach and
co-workers (8, 9) had noted for the recombinant rabbit
protein that glucosylation had already occurred in the expression
system. We agree, noting the higher M
In order to explore the
mechanism of self-glucosylation of glycogenin, we constructed mutants
in which Phe or Thr replaced Tyr-194 and tested them with UDP-glucose.
No self-glucosylation occurred, but the mutants could still be capable
of transglucosylation. This proved to be the case. The p-nitrophenyl
The ability of the mutant proteins to
transglucosylate but not self-glucosylate should be of assistance in
exploring the catalytic mechanism of transglucosylation. Hitherto only
the wild-type enzyme has been available, acting both to donate and to
accept transferred glucose. Now, in the mutant proteins, we have an
enzyme that is no longer a substrate. Correspondingly, the availability
of low molecular weight saccharide acceptors shows that the aglycone
does not have to be a protein but can be a very small molecule. Thus,
glucose has no detectable acceptor activity(11) , while p-nitrophenyl
The discovery that the Phe-194 and Thr-194 mutants
are capable of intermolecular transglucosylation made us realize that
this property, which is shared by the wild-type enzyme, is independent
of self-glucosylation. The question arises whether there is an
endogenous receptor substrate, other than glycogenin itself, for such
intermolecular transglucosylation. It is clear that maltose is such a
substrate(9, 11) . Second is the question whether the
activity is significant. It is certainly capable of being more rapid
than self-glucosylation. The rate of transfer of glucose to DBM at 2
mM UDP-glucose is 0.92 µmol of glucose transferred/min/mg
of protein, some 200 times greater than the maximal rate of
self-glucosylation shown in Fig. 2. Whether self-glucosylation
also occurs by intermolecular transfer has not previously been decided,
despite claims to this effect. Both Pitcher et al.(18) and Cao et al.(9) reported
first-order kinetics for self-glucosylation and concluded that it is
intramolecular. However, the Pitcher et al. laboratory had
reported that glycogenin is a dimer(19, 20) , while Cao et al.(9) did not comment on the size of their
recombinant enzyme. When we found that homogeneous muscle glycogenin
underwent self-glucosylation, we also noted that it was oligomeric (10) and did not attempt to draw a conclusion as to intra- or
inter-molecular transfer. When the intermolecular glucosylation of the p-nitrophenyl saccharides was discovered, we pointed out the
implication that this had for the self-glucosylation reaction,
suggesting that glycogenin, in its autocatalysis, ``may act by
intermolecular glycosylation between aggregated protein molecules and
that the aggregation may be purposeful''(11) .
Using
wild-type recombinant enzyme we reexamined the self-glucosylation
reaction kinetics, paying particular attention to the reaction rates at
the lower end of the range of glycogenin concentration that Cao et
al.(9) employed (Fig. 2). The reaction only assumes
first-order kinetics above 0.5 µM enzyme concentration.
Below this, the rate falls away, suggestive of the dissociation of an
interactive complex. Therefore, previous conclusions, based on
kinetics, that the reaction is intramolecular (9, 20) are not justified on that evidence alone, and
the question remains open.
With respect to the mass of the
glycogenin monomer, we noted that unless peptidase inhibitors were
added to the E. coli lysates during purification and
subsequent storage of the purified enzyme, the wild-type and mutant
proteins each broke down to active species with M
Finally, we report two additional properties of
glycogenin, the first that it hydrolyzes UDP-glucose. The second is the
only difference between native and recombinant glycogenin so far noted.
Homogeneous wild-type glycogenin and the two mutants hydrolyze
UDP-glucose (I). The Thr-94 mutant is the most active. The
rate of hydrolysis is comparable with the rate of self-glucosylation.
Thus, in a 20-min period during which the wild-type enzyme would have
utilized 2.0 mol of UDP-glucose in self-glucosylation(4) , the
enzyme would have hydrolyzed 0.6 mol of UDP-glucose. Therefore, the
hydrolytic capacity of glycogenin has to be considered of potential
significance, and in light of the fact that the concentrations of
UDP-glucose employed for its assay are in the low micromolar range,
investigators who have previously engaged in prolonged incubation of
glycogenin with micromolar UDP-glucose in order to learn how much
glucose the enzyme would add to itself have not known that the enzyme
was simultaneously hydrolyzing the substrate. Therefore, reported
extents or rates of glucosylation may have been in error because the
substrate had disappeared.
The hydrolysis of UDP-glucose was also seen in experiments
in which the Phe-194 mutant protein was allowed to transglucosylate p-nitrophenyl
Lomako et al.(3) reported that ATP is a powerful
inhibitor of native rabbit muscle glycogenin such that at physiological
ATP concentration (5 mM) there was almost complete inhibition.
Cao et al.(9) however, found that in 10 mM ATP, recombinant muscle glycogenin is inhibited only 45%, while
UTP is much more inhibitory. We find similar levels of inhibition of
recombinant glycogenin by ATP and UTP. A reason for the difference
between the two forms of glycogenin, native and recombinant, could be
protein phosphorylation. Since it was not exposed to mammalian protein
kinases, we would not expect the recombinant protein expressed in E. coli to be phosphorylated; but native glycogenin from
rabbit muscle glycogen contained 0.8 mol.proportions of phosphate, and
additional phosphate could be introduced at Ser-43 by protein kinase
and [
We thank Erik Lagzdins and John Rodriguez for carrying
out the initial studies with DBM, Michele Montejo and Michael Muench
for the molecular sieving of recombinant glycogenin, and Dr. Lennart
Roden and colleagues for providing information about DBM prior to
publication(12) .
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-maltoside.
Self-glucosylation, glucosylation of other acceptors, and hydrolysis
all appear to be catalyzed by the same active center. In the absence of
peptidase inhibitors, the homogenous recombinant proteins of M
37,000 break down to equally active species
having M
32,000. The kinetics of
self-glucosylation catalyzed by the wild-type enzyme suggest that the
reaction could be intermolecular rather than, as previously reported,
intramolecular. The wild-type recombinant enzyme and native muscle
glycogenin, which is phosphorylated, are inhibited quite differently by
ATP at physiological concentration.
, creating a maltosaccharide chain of eight glucose
residues that functioned as a primer for glycogen synthesis.
SSC
(
)was used in more concentrated or less concentrated
forms such as 2
SSC, 0.5
SSC. Glycogenin
self-glucosylating activity was measured by incorporating glucose from
UDP-[
C]glucose (DuPont) into the protein and
precipitating it with trichloroacetic acid(10) . For Western
blotting we used an antibody to native rabbit muscle
glycogenin(10) . Transglucosylase activity was measured with p-nitrophenyl saccharides (Ref. 11, and see below) or with n-dodecyl
-maltoside (DBM) as acceptor substrate (4, 12) and UDP-[
C]glucose as
donor. Treatment of glycogenin with insoluble
-amylase prior to
UDP-[
C]glucose () was carried out
with cell lysates in 100 mM acetate buffer, pH 7.0, 10 mM CaCl
and 2 units of insoluble enzyme for 1 h at room
temperature with shaking. The enzyme was removed by centrifugation
through SpinX filter units (Costar), and the filtrate was used for
self-glucosylation in comparison with a corresponding volume of
untreated lysate.
C incorporated was measured
by trichloroacetic acid precipitation and counting () or by
SDS-PAGE and radioautography (Fig. 1). The
C
counts/min in represent the total
[
C]glucose that can be incorporated, not the
relative rates. Western blotting (10) was carried out on
10-µl aliquots of the lysates.
Figure 1:
Properties of recombinant glycogenins
as shown by SDS-PAGE, radioautography, and immunoblotting. A,
Coomassie Blue-stained gel (M markers on the left) of E. coli lysates in which human muscle
glycogenin (induced (lane 1) or uninduced (lane 2))
rabbit muscle glycogenin (induced (lane3)
or uninduced (lane4)) had been expressed. B, radioautograph of the gel in A. C,
Western blot of the same lysates after SDS-PAGE. PanelD contains the Coomassie Blue-stained purified wild type (lane1), Phe-194 (lane2), and Thr-194
mutants (lane3) studied
here.
Cloning of Rabbit Muscle Glycogenin cDNA and Expression of the
Protein
The cloning procedures were performed independently of
and prior to the appearance of the publications by Viskupic et al.(8) and Cao et al.(9) . A 728-base pair
probe was used to screen a rabbit muscle ZAP II cDNA library
(Stratagene). The probe was obtained by PCR utilizing two
oligonucleotide primers derived from the primary sequence of rabbit
muscle glycogenin (2) and the library itself as the DNA
template. The sense primer (5`-AACGACGCCTA(C/T)GC(C/G)AA(G/A)GG-3`)
originated from residues 11-17, while the antisense primer
(5`-GTGGTGAAGATGTC(C/A)CACC-3`) was derived from amino acids
248-254. The probe, with its sequence confirmed by dideoxy
sequencing (13) using Sequenase version 2.0 (U.S. Biochemical
Corp.), was labeled by the PCR incorporation of digoxigenin 11-dUTP
(Boehringer Mannheim)(14) . Digoxigenin is an intermediary in
chemiluminescent signal emission, detected by the procedures specified
by the manufacturer. Approximately 10
phage particles from
the cDNA library were screened by standard techniques(15) .
After three rounds of plaque purification, putative positive clones
were excised in vivo from
ZAP II to yield recombinant
pBluescript phagemid. The in vivo excision protocol provided
by the manufacturer (Stratagene) was followed. The clones were
confirmed by dideoxy sequencing of both strands(13) . The
subcloning of the full-length cDNA into the prokaryotic expression
vector was done essentially as by Viskupic et al.(8) ,
except for the use of a different expression vector: pET-11d (Novagen).
The resulting expression construct was designated pET-R. The expression
of the wild-type and mutagenized forms of glycogenin was done as by
Studier et al.(16) . Small samples of the culture
expressing the wild-type protein were subjected to self-glucosylation
assays, SDS-PAGE, and Western blotting (see above) (Fig. 1, ). The purification of the three forms of glycogenin has
been described elsewhere(4) .
Substitution of Tyr-194 by Phe or Thr
The
``megaprimer'' method of PCR-mediated site-directed
mutagenesis was employed(17) . The sequences of the primers for
the Phe and Thr substitutions of Tyr-194 in glycogenin were,
respectively, 5`-GCATTTCTATATTCTCCTACCTCCCAGC-3` and
5`GCATTTCTATAACCTCCTACCTCCCAGC3`, spanning residues 190-199. The
oligonucleotide primers employed in the subcloning of the wild-type
cDNA into pET-11d (see above) were used along with the mutagenic
primers and a preparation of pET-R plasmid DNA in the two-step PCR
procedure to generate the mutant molecules. These were subcloned into
pET-11d as above to yield the pET-RF (Phe-194 mutant) and pET-RT
(Thr-194 mutant) expression vectors. Sequence confirmation was done by
the dideoxynucleotide method(13) .
Glucosylation of p-Nitrophenyl
In order to measure the
glucosyltransferase activities of the wild type and its mutagenized
forms, p-nitrophenyl -Saccharides by
Wild-type and Mutant Forms of Glycogenin
-glucoside,
-maltoside,
-maltotrioside,
-maltotetraoside, and
-maltohexaoside
were used as glucose acceptor substrates(11) . The digests (100
µl each) contained 1 µM homogeneous recombinant
protein (Fig. 1D), 10 mM UDP-glucose, 10 mMp-nitrophenyl
-saccharide, 5 mM MnCl
, and 50 mM Tris-HCl buffer, pH 7.4.
After incubation for 18 h at room temperature, 5 µl or 10 µl of
each digest was loaded onto a C18 column and fractionated by reverse
phase high performance liquid chromatography using an ascending
gradient of acetonitrile (10-20%; 1 ml/min for 20 min). The
increased concentration of UDP-glucose and the time of incubation were
designed to ensure that a sufficient mass of product was formed for
accurate measurement. The wild-type enzyme was completely stable during
the 18-h incubation. The formation of p-nitrophenyl saccharide
species containing n + 1 glucose residues with respect to
the acceptor substrate from which they originated, was monitored and
quantitated by absorbance at 320 nm (). The results in were calculated from the relative areas under the curves
for each peak, determined by the integrator.
C]Glucose arising from hydrolysis of the
[
C]UDP-glucose substrate (see below) was
measured concomitantly with intermolecular transfer by the Phe-194
mutant enzyme of [
C]glucose to p-nitrophenyl
-maltoside at concentrations of the latter
ranging from 0.1 µM to 10 mM. The reaction
mixtures (100 µl) were as above, except that the enzyme
concentration was 0.2 µM and the concentration of
UDP[
C]glucose was 2 µM. Incubation
was for 2 h at room temperature, after which excess UDP-glucose was
removed by applying the digest to a column of mixed bed resin (Bio-Rad
AG 501-X8(D), 0.8 ml) and eluting with 1 ml of water. The effluent (200
µl) was fractionated by reverse phase high performance liquid
chromatography as above, p-nitrophenyl
-maltoside and
-maltotrioside being mixed in to permit their appearance to be
detected by absorbance at 320 mn. [
C]Glucose was
collected in the fractions immediately postinjection. The
C-labeled p-nitrophenyl
-maltotrioside, formed from
the maltoside, was also collected (see ).
Hydrolysis of UDP-Glucose by Recombinant
Proteins
Each of the three recombinant proteins (20 pmol, wild
type, Phe-194, and Thr-194 mutants) was incubated in a 100-µl
digest containing 2 µM UDP-[C]glucose, 50 mM Tris-HCl, pH
7.4, in the presence and absence of 5 mM Mn
.
After 2 h at room temperature, the digests were applied to mixed bed
resin columns (0.5 ml, Bio-Rex RG 501-X8 resin, Bio-Rad) and eluted
with 2 ml of water, collecting 0.5-ml fractions in which
[
C]glucose released by hydrolysis was counted.
In the case of wild-type protein, an additional column was used to
remove self-[
C]glucosylated protein from free
[
C]glucose. The mixed-bed resin column was
connected to a Sep-Pak C
cartridge (Millipore) prewetted
with acetonitrile. Elution was done with 5 ml of water in 0.5-ml
fractions in which the [
C]glucose was counted.
Control digests omitting enzyme were processed in the same way to
ensure that in the absence of enzyme no
[
C]glucose was released. The results are shown
in I.
Cloning and Sequencing the cDNAs for Rabbit Muscle
Glycogenin
A full-length clone of the cDNA for rabbit muscle
glycogenin was obtained and sequenced by standard procedures. The clone
contained 1809 base pairs encoding a protein of 332 residues. The
sequences were identical both with respect to the nucleotide coding and
the amino acid sequence with those already reported by Viskupic et
al.(8) . We simultaneously cloned and expressed the human
muscle protein. The results, which will be reported elsewhere, showed
the lengths of both proteins to be the same, with 34 differences
between the amino acid sequences.
Expression and Purification of Recombinant
Proteins
We expressed both the human and rabbit proteins and
proceeded with study of the latter because of a much greater level of
expression. The wild-type expression vectors were introduced into an Escherichia coli BL 21/DE 3 host. Protein synthesis was
induced with isopropyl -thiogalactopyranoside. Glycogenin was
detected in lysates of cultures by means of
Mn
-dependent self-glucosylation with a
UDP-[
C]glucose donor. After SDS-PAGE, prominent
bands at M
37,000 could be seen on the stained
gel. Fig. 1is a composite picture of the properties of lysates
of cultures of recombinant glycogenins. In Fig. 1A,
stained for protein, the high level of expression of induced rabbit
protein (lane3, M
37,000) can
be seen. These lysates had been preincubated with
UDP-[
C]glucose, and Fig. 1B is a
radioautograph of Fig. 1A. Radioglucosylated protein was
seen in all cases. A qualitative comparison of relative amounts of
glycogenin in each lysate is afforded by Fig. 1C, a
Western blot using antibody to glycogenin from rabbit muscle
glycogen(10) . Compared with the induced lysates (Fig. 1C, lanes 1 and 3), the amounts
of glycogenin in the uninduced lysate (Fig. 1C, lanes 2 and 4) were much smaller, although active
glycogenin was certainly present (Fig. 1B, lanes 2 and 4). By contrast, there was a much greater relative
degree of radiolabeling of the uninduced versus induced
proteins (compare Fig. 1B and Fig. 1C).
This is shown quantitatively in , where the relative
amounts of [
C]glucose incorporated by rabbit
protein lysates are compared. The lysates were also treated with
insolubilized
-amylase before incubation with
UDP-[
C]glucose, and the results are compared.
between
the wild-type protein and the mutants, which can be related to the
presence (wild type) or lack (Phe-194 and Thr-194 mutants) of glucose
carbohydrate. In this respect, the wild-type enzyme was able to
glucosylate itself and, when homogeneous (Fig. 1), different
preparations added glucose equivalent to 0.8-2 mol.proportions,
suggesting that 6-7 mol.proportions of glucose were already
present. The mutant enzymes did not appear to self-glucosylate. During
storage of the lysate or during concentration of the homogeneous
proteins, each was converted into a product having M
32,000, but no change in activity occurred. When a mixture of
peptidase inhibitors such as is used to prevent breakdown of muscle
glycogenin during purification (10) was included at all stages
of purification, and during subsequent storage, no breakdown occurred.
We report here that the same behavior has been noted in wild-type
carbohydrate-free glycogenin expressed in a different strain of E.
coli(5) , but we did not observe breakdown with purified
native rabbit muscle glycogenin(10) . The recombinant enzymes,
stored with peptidase inhibitors, were stable for up to 3 months when
refrigerated.
Figure 2:
Dependence of glycogenin
self-glucosylation on protein concentration. The initial rate of
glucose incorporation was measured at 5 min under standard conditions
(10) with 20 µM UDP-[C]glucose in
the presence of bovine serum albumin at 1 mg/ml over the range
0.01-1 µM enzyme. The rates with 0.01-0.3
µM enzyme were also measured at 0.1 mg/ml bovine serum
albumin. The results for 0.01-0.3 µM enzyme are the
averages of four determinations, and the results for 0.5 and 1
µM enzyme are the averages of
duplicates.
Transglucosylation by Mutant Proteins
Although the
two mutants could not self-glucosylate, there was the possibility that
they could use an alternative glucose acceptor. p-Nitrophenyl
-glucoside and
-maltosaccharides act as alternative glucose
acceptors for the muscle enzyme, competing with self-glucosylation
(11), and proved to be acceptors not only for the recombinant wild-type
enzyme but also for the Phe-194 and Thr-194 mutants ().
The mutant proteins were even superior to the wild-type enzyme in
respect to their relative abilities to glucosylate p-nitrophenyl
-glucoside, but not so for the maltoside,
which is the preferred substrate for the wild-type enzyme ().
UDP-Glucose Hydrolysis
All three recombinant
proteins hydrolyzed UDP-glucose in an Mn-dependent
manner (I). The Thr-194 mutant protein was most active.
When the Phe-194 mutant protein was allowed simultaneously to
glucosylate p-nitrophenyl
-maltoside, the hydrolytic
release of glucose was inversely related to the maltoside concentration ().
Inhibition by Nucleoside Triphosphates
In
agreement with Cao et al.(9) , the wild-type glycogenin
was powerfully inhibited by UTP (98.5% in 10 mM UTP) and less
so by ATP (53% in 10 mM ATP). Cao et al.(9) reported 95% inhibition by UTP and 46% by ATP at the
same inhibitor concentrations. These results for ATP stand in marked
contrast to those for the inhibition exerted by ATP on native rabbit
muscle glycogenin(3) , where the glycogenin is virtually
inactive in 5 mM ATP.
as shown by Mn
-dependent
[
C]glucosylation and Western blotting (Fig. 1, B and C). The induced lysate contained
much more glycogenin protein than the corresponding uninduced lysate (Fig. 1C). That in the induced lysate was such that it
could easily be seen after SDS-PAGE and protein staining (Fig. 1A, lane3), amounting to almost
half the total soluble protein. Therefore, we used this system to
obtain purified enzyme and to construct and express the Phe-194 and
Thr-194 mutants. The relative amounts of
[
C]glucosylation seen in the induced lysates of
the wild-type enzyme in three experiments varied 4-fold ().
, presumably
due to carbohydrate, of the wild type versus the Phe-194 and
Thr-194 mutants (Fig. 1D) and the fact that prior
-amylolysis of the induced rabbit protein resulted in its ability
to incorporate much more [
C]glucose (). Therefore,
-amylase-degradable maltosaccharide was
already present in the expressed protein. An unexpected feature of the
comparison between lysates of the wild-type enzyme was the relative
degree of [
C]glucosylation as between the
induced and uninduced proteins. In different sets of lysates the
uninduced lysate incorporated 4-10 times as much
[
C]glucose as did the corresponding induced
lysate (). This occurred even though there was less
glycogenin present in the uninduced lysate (Fig. 1C).
The induced glycogenin was therefore already glucosylated to a greater
degree than the uninduced protein, meaning that in vitro the
latter would self-glucosylate with UDP[
C]glucose
to a greater extent. This comparison suggested that the uninduced
glycogenin was largely unglucosylated, and this is borne out by
comparing the M
values of the proteins (Fig. 1C). An unexpected feature of the ability of the
two types of protein to self-glucosylate was seen when they were first
treated with
-amylase to shorten the preexisting maltosaccharide
chains. The induced glycogenin now incorporated 5-7 times more
C than before
-amylolysis. The uninduced glycogenin,
however, almost lost its capacity to glucosylate (). This
phenomenon requires further examination.
-maltosaccharides and the
-glucoside
are efficient competitive acceptors of glucose transferred from
UDP-glucose by the rabbit muscle enzyme(11) . The mutants also
used these acceptors (). In other words, the removal of
Tyr-194 only prevents the glucosylation of glycogenin. The catalytic
activity is retained in the mutants, but their acceptor substrate
preferences are changed. The mutants use the glucoside as the preferred
substrate, whereas wild-type glycogenin prefers the maltoside. What the
mutants share in common is the absence of a carbohydrate chain.
Therefore, the mutant proteins are capable of intermolecular
transglucosylation, with altered specificity but comparable catalytic
activity. The ability to transglucosylate does not reside to any
important extent in Tyr-194. These findings contradict the claim by Cao et al.(9) , who also expressed the Phe-194 and Thr-194
mutants and, finding them unable to self-glucosylate, or to glucosylate
a 30-residue synthetic peptide surrounding Tyr-194, stated that
``Tyr-194 is essential for function [and] essential for
activity.''
-glucoside is a powerful acceptor ().
about 32,000.
(
)This occurred even after
the M
37,000 proteins had been purified to
apparent homogeneity. The breakdown occurred without change of
activity. This phenomenon was not noted by Cao et
al.(9) .
(
)In contrast to our
findings, others have stated, but without providing evidence, that
``we have excluded the possibility that glycogenin itself has any
form of glycosidase activity''(9, 24) . We
disagree. No mention was made in these reports of the substrates
tested.
-maltoside over a range of concentrations
of the acceptor from 0.1 µM to 10 mM. As the
acceptor concentration increased and more p-nitrophenyl
-maltotrioside was formed, the amount of glucose, formed by
hydrolysis, decreased (). When the native enzyme is
incubated with
-maltoside, there is also an inverse relation
between the degree of self-glucosylation and
-maltoside
concentration(11) . Therefore, we may conclude that
self-glucosylation, which may be intermolecular (Fig. 2),
intermolecular transglucosylation to simple saccharides, and
UDP-glucose hydrolysis are each catalyzed by the same active center.
P]ATP(6) .
Table: [C]Glucosylation
of proteins in lysates of E. coli expressing rabbit-muscle glycogenin
Table: Intermolecular transglucosylation of
p-nitrophenyl saccharides by wild-type and recombinant glycogenins
Table: Hydrolysis of UDP-glucose by
recombinant proteins
Table: Simultaneous
hydrolysis of UDP-glucose and glucose transfer to p-nitrophenyl
-maltoside by Phe-194 mutant glycogenin
-maltoside; PAGE,
polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.
32,000 (21, 22), but it should be noted that the kidney protein
failed to give a positive immunoblot with two antibodies to muscle
glycogenin, one of which gave a positive blot with the M
32,000 breakdown product of muscle glycogenin (23) and, we now
report, with the M
32,000 breakdown product of
wild-type recombinant glycogenin as described here or when
carbohydrate-free (5).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.