From the
A mammalian expression vector was designed to express a secreted
soluble form of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (polypeptide GalNAc
transferase) with a metal binding site (HHWHHH) at the NH
The initial step in mucin type O-linked glycosylation
is catalyzed by the enzyme UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (EC 2.4.1.41) (polypeptide
GalNAc transferase)
Previous characterization of the enzyme has demonstrated
that the monomeric soluble enzyme requires the divalent cation
manganese for its activity(3, 4, 5) . The donor
specificity is restricted to UDP-GalNAc (3, 5); in contrast, substrate
specificity studies indicate that the enzyme binding site is large (8
or 9 residues) and can accommodate a wide range of amino acids that
flank the glycosylation site(6, 7) . However, the
kinetics of the enzyme in terms of the mechanism and the order in which
the two substrates bind have not yet been defined.
Available
evidence suggests that more than one polypeptide GalNAc transferase may
exist and that these may exhibit overlapping
specificities(5, 8, 9, 10) . Therefore,
in the present study, we have purified to apparent homogeneity a
recombinant form of this enzyme. We have measured kinetic parameters of
this enzyme; these are consistent with the polypeptide GalNAc
transferase binding to its substrates in a random sequential order
prior to catalysis. We further demonstrate that the presence of a
hydroxyamino acid is not essential for the interaction of the
polypeptide GalNAc transferase with peptide substrates.
The
harvested media were clarified of cellular debris by low speed
centrifugation and diluted to SBB conditions (150 ml). The pool was
applied to columns of NiCl
Data were plotted graphically to show the
initial velocity and fitted to Equation 1, corresponding to the initial
velocity of a two-substrate
reaction(14) .
On-line formulae not verified for accuracy
where V is the maximum velocity, K is the
Michaelis constant for the varied substrate, and A and B are the concentrations of A and B.
Data were plotted
graphically to show inhibition patterns and fitted to Equations
2-4 corresponding to competitive, noncompetitive, and
uncompetitive inhibition,
respectively(14) .
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
where I is the concentration of inhibitor I, K is the Michaelis constant, and K
We have transiently expressed a secreted recombinant form of
a polypeptide GalNAc transferase indistinguishable from the native
enzyme with respect to kinetic parameters (K
Our data suggest that
the polypeptide GalNAc transferase reaction proceeds via a random
ordered sequential mechanism (Fig. S1). This mechanism is
consistent with the observation that bovine colostrum polypeptide
GalNAc transferase can be purified on a 5-Hg-UDP-GalNAc affinity matrix
in the absence of protein substrate (9). Further, the estimated kinetic
constants K
In this study, the K
The data
from the competitive inhibition between EPO-T and EPO-G provided
insight into the mechanism of site selection of the enzyme. The
similarity of the K
All assays
were carried out as described under ``Experimental
Procedures.''
We thank Dr. Eric Phizicky for thoughtful comments
about this work, Brian VanWuyckhuyse for the amino acid analysis and
NH
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
terminus. The recombinant enzyme was purified to homogeneity from
COS-7 cell media by sequential chromatography on columns of
NiCl
-chelating Sepharose, Affi-Gel blue, and Sephacryl
S-100. Kinetic parameters of recombinant and native polypeptide GalNAc
transferase were comparable for the donor UDP-GalNAc and for the
peptide acceptors AcTPPP, EPO-T (PPDAATAAPLR), and HVF (PHMAQVTVGPGL).
Initial velocity and product inhibition studies were carried out with
purified recombinant polypeptide GalNAc transferase and the substrates
UDP-GalNAc and peptide EPO-T. Initial velocity data was consistent with
a sequential type mechanism in which binding of both substrates
precedes product release. Product inhibition analysis using UDP showed
competitive inhibition against UDP-GalNAc and a noncompetitive
inhibition against peptide EPO-T. The dead end peptide analogue EPO-G
(PPDAAGAAPLR) was a noncompetitive inhibitor of UDP-GalNAc and a
competitive inhibitor of peptide EPO-T. Collectively, the results
suggest that the most probable kinetic mechanism for the enzyme is one
in which both substrates must bind in a random order prior to
catalysis. Interestingly, the K
for EPO-T
is similar to the K
for EPO-G, suggesting
that peptide interaction with the polypeptide GalNAc transferase does
not require a hydroxyamino acid.
(
)and results in the covalent
attachment of GalNAc to the hydroxyamino acids threonine and serine in
an
-anomeric linkage. The two-substrate/two-product reaction
utilizes the nucleotide sugar UDP-GalNAc and a polypeptide acceptor and
produces free UDP and glycosylated protein in a yet undefined
mechanism(1) . The polypeptide GalNAc transferase is the first
enzyme in a series of glycosyltransferases leading to the stepwise
assembly of protein-linked complex carbohydrate structures (2). As
such, it represents a potentially important early control point in O-glycosylation, and it is therefore important to define its
mechanism.
Plasmid Construction
An insulin secretion signal
sequence (gift of Dr. K. Drickamer) and the coding region of the bovine
placental polypeptide GalNAc transferase were introduced into the
mammalian expression vector pSVL (Pharmacia Biotech Inc.) as described
in Hagen et al.(9) . Synthetic oligonucleotides were
used to engineer a metal binding site with the amino acid sequence
HHWHHH (modified from Ref. 11) immediately downstream from the insulin
processing site (Fig. 1A). The secreted recombinant
enzyme encoded by plasmid pSVL-inGNT1 is thus designed with an in
vivo cleavable processing site that produces the
NH-terminal amino acid sequence FVHMHHWHHH preceding the
native soluble form of the polypeptide GalNAc transferase.
Figure 1:
Sequence of
the NH-terminal region of the recombinant polypeptide
GalNAc transferase. A, nucleotide and amino acid sequence of
the first 156 nucleotides of the recombinant polypeptide GalNAc
transferase. The first 78 nucleotides encode the insulin secretion
signal from pGIR-199 (19). Changes introduced in the bovine cDNA during
cloning are underlined. The remainder of the cDNA (not shown)
is identical to that reported in Ref. 9. The metal binding site is boxed. The arrow points to the insulin signal peptide
processing site. B, NH
-terminal amino acid
sequence of the secreted recombinant polypeptide GalNAc transferase.
The first 14 amino acids were determined experimentally, while the
remainder were inferred from the conceptually translated sequence. C, NH
-terminal sequence of the secreted form of
the naturally secreted bovine colostrum GalNAc transferase enzyme
(9).
Expression of the Recombinant Polypeptide GalNAc
Transferase
Plasmid pSVL-inGNT1 was used to transfect COS-7
cells. COS-7 cells were grown to 80-90% confluence (approximately
1 10
cells) at 37 °C in Dulbecco's
modified Eagle's medium (Life Technologies, Inc.) with 10% fetal
calf serum, 6% CO
in 10 100-mm culture dishes. Cells were
transfected with 8 µg DNA using a liposome-mediated transfection
protocol (8 µl of LipofectAMINE Reagent, Life Technologies, Inc.).
At 72 h post-transfection, the media were harvested and replaced with
conditioned Dulbecco's modified Eagle's medium. At 96 h
post-transfection, the media were harvested.
Purification of Recombinant Polypeptide GalNAc
Transferase from COS-7 Cell Supernatants
Sterile buffers used in
the enzyme purification were SBB (30 mM MOPS, 10 mM MgCl, 100 mM NaCl, 20 mM imidazole,
pH 6.5), wash buffer A (50 mM MES, 100 mM NaCl, 10
mM MgCl
, pH 5.7), wash buffer B (100 mM
MES, 100 mM NaCl, 10 mM MgCl
, pH 4.7),
binding buffer (30 mM MOPS, 10 mM MgCl
,
100 mM NaCl, pH 7.1), buffer D (25 mM MES, 5
mM MnCl
, 0.001% Triton X-100 (hydrogenated form),
pH 6.5), and buffer E, 25 mM MES, 80 mM NaCl, 10%
glycerol, 0.05% Triton X-100 (hydrogenated form), pH 6.5.
-chelating Sepharose (Pharmacia)
equilibrated with SBB and fractionated as described in the legend to Fig. 3.
Figure 3:
Purification of the recombinant
polypeptide GalNAc transferase. All fractions were monitored for
absorbance at 280 nm (hatchedline) and assayed for
transferase activity (closedcircles). A,
harvested media were loaded on a column of NiCl-chelating
Sepharose (1.5
20 cm) equilibrated with SBB. The column was
developed sequentially with a 32-ml linear gradient of wash buffer A to
B (arrow 1), 34 ml of wash buffer B (arrow 2), and 70
ml of binding buffer, 50 mM imidazole (arrow 3).
Bound transferase was eluted with 35 ml of binding buffer, 250 mM imidazole (arrow 4). Enzyme-containing fractions were
pooled and diluted 1:3 in buffer D. B, the enzyme-containing
material was loaded onto a column of Affi-Gel blue (0.7
10 cm)
equilibrated with buffer D. The column was developed sequentially with
10 ml of buffer D, 100 mM NaCl (arrow 1) and 15 ml of
buffer D, 100 mM NaCl, 20% glycerol (arrow 2). Bound
transferase was eluted from the column with a 16-ml linear salt
gradient of 0.1-1 M NaCl in buffer D and 20% glycerol (arrow 3). The enzyme-containing fractions were pooled and
dialyzed against buffer E. C, the dialyzed material was
concentrated 20-fold using a Centricon-30 (Amicon) and loaded onto a
Sephacryl S-100 gel filtration column (1
120 cm) equilibrated
in buffer E. The active fractions were pooled, concentrated 41-fold
using a Centricon-30, supplemented with sodium azide to 0.02%, and
stored at -70 °C.
Protein Determination and Amino Acid
Analysis
Prior to analysis, aliquots from each purification step
were buffer-exchanged for water using Micron 10 ultrafiltration devices
(Amicon). The proteins were then hydrolyzed under standard conditions
(vapor phase HCl, 106 °C, 20 h), and the hydrolyzed material was
analyzed on a Hewlett-Packard amino acid analyzer with Amino Quant II
software.
SDS-PAGE of Protein
Aliquots of the pools from the
recombinant transferase purification were subjected to 10%/3% Tricine
SDS-polyacrylamide gel electrophoresis(12) . To detect proteins,
samples were dried down and resuspended in 100 mM Hepes, 6 M urea, 20 mM NaCNBH, and a 10-fold
excess of [
C]formaldehyde to lysine in
protein(13) . After incubation at 37 °C for 24 h, the
samples were ethanol-precipitated (80%) and resuspended in Tricine
SDS-PAGE gel loading buffer (4% SDS, 12% glycerol (w/v), 50 mM Tris, pH 6.8, 2% mercaptoethanol (v/v), 0.01% Serva Blue G).
Electrophoresis of 10% gels was performed at 15-20 mA and was
followed by soaking of the gel in En
Hance (DuPont NEN). The
gel was dried down, and radiolabeled protein was visualized by
autoradiography on Kodak XAR film.
Amino Acid Sequence Data
To obtain
NH-terminal amino acid sequence data, 8 pmol of purified
recombinant transferase from step 3 was exchanged into water using a
Centricon-10 (Amicon) ultrafiltration device. The sample was applied to
a Polybrene-coated trifluoroacetic acid-activated precycled glass fiber
filter and sequenced on an Applied Biosystems 473A protein sequencer.
Determination of Polypeptide GalNAc Transferase Activity
and Kinetic Parameters
The recombinant polypeptide GalNAc
transferase was assayed for synthesis of GalNAc-substituted peptide in
25-50-µl reactions containing 40 mM sodium
cacodylate, 0.1% Triton X-100, 4 mM -mercaptoethanol,
10-15 mM MnCl
, pH 6.5. The acceptor peptides
EPO-T (PPDAATAAPLR), HVF (PHMAQVTVGPGL), and AcTPPP have been described
previously (8) and used in a concentration range of 0.02-5
mM. The UDP-[
C]GalNAc (DuPont NEN) and
UDP-GalNAc (Sigma) concentration range was 1-1600
µM. The UDP (Sigma) stock solution concentration was
determined spectrophotometrically (extinction coefficient (260 nm)
= 10 optical density units/mM). The purified
recombinant transferase was diluted 1:20 in 50 mM cacodylate,
100 mM NaCl, 50% glycerol, pH 6.5 added to the reactions. The
reactions were incubated at 37 °C for a period of time such that
less than 10% of the substrate UDP-[
C]GalNAc was
consumed. At this point, the incubation was terminated by addition of
10 µl of 150 mM EDTA and passed through anion exchange
spin columns (Dowex AG 1-X8, Bio-Rad), and the counts were determined
by liquid scintillation spectroscopy. The kinetic experiments were
performed at least twice.
and K
are the slope and
intercept constants, respectively. Starting estimates of the parameters
were obtained from linear regression, and fits were made using the NLIN
procedure of SAS with the Gauss-Newton method(15) .
Expression of the Recombinant Polypeptide GalNAc
Transferase
The plasmid construct pSVL-inGNT1 contains an
insulin secretion signal preceding a metal binding site and polypeptide
GalNAc transferase coding sequence as defined in Fig. 1A. In vivo processing of the signal
peptide in COS-7 cells directs secretion of the soluble form of the
recombinant transferase with an NH-terminal metal anchor (Fig. 1B) into the culture medium. COS-7 cells
transiently transfected with pSVL-inGNT1 expressed and secreted
sufficient GalNAc transferase, such that 1 µl of culture medium
incorporated 2,000-10,000 cpm/h of
UDP-[
C]GalNAc into peptide substrates under
standard assay conditions. Splitting cells 18 h post transfection
resulted in a reduction in the concentration of expressed enzyme
activity relative to unsplit cells. Growth at 100% confluence for 5
days was not detrimental to this transient expression system (Fig. 2). In an experiment to determine the duration of gene
expression, maximal expression of the recombinant gene was achieved at
48 h post-transfection, whereas de novo recombinant enzyme
synthesis was minimal by 96 h (data not shown), indicating that the
plateau for enzyme activity observed in Fig. 2was due to a
decrease in gene expression and not to instability or degradation of
the recombinant enzyme. Indeed, the soluble recombinant enzyme was
stable at 37 °C for at least 3 days, since a 37 °C incubation
of the clarified culture medium did not result in loss of recombinant
enzyme activity (data not shown).
Figure 2:
Transient
expression of a soluble recombinant polypeptide GalNAc transferase from
COS-7 cells. Enzyme activity duplicate measurements were made at 18,
40, 63, 117, and 168 h post transfection. Opensymbols indicate cells grown to 100% confluence and not split. Closedsymbols indicate cells split 1:6 18 h post-transfection.
Media were clarified, and 1 µl was assayed for activity under
standard conditions using acceptor peptide
AcTPPP.
Recombinant Polypeptide GalNAc Transferase
Purification
A summary of the purification of this enzyme from
approximately 10 COS-7 cells is given in .
Metal chelate chromatography (step 1) was used to selectively remove
the recombinant form of the transferase from the media supernatants by
virtue of the affinity tag (HHWHHH) at the NH
terminus of
the enzyme (Fig. 3A). Chromatography on Affi-Gel blue
(step 2) eliminated high molecular weight contaminants (Fig. 4, lane3) and increased the specific activity of the
material 7-fold (Fig. 3B). Gel filtration on Sephacryl
S-100 (step 3) (Fig. 3C) yielded a recombinant
transferase fraction that migrated as a single band on Tricine SDS-PAGE (Fig. 4, lane4). The apparent molecular weight
of the recombinant GalNAc transferase was 68,000 Da, a value consistent
with that of the native bovine colostrum GalNAc transferase(9) .
The NH
-terminal sequence of the recombinant polypeptide
GalNAc transferase was determined by NH
-terminal protein
sequencing (Fig. 1B). The presence of the processed
insulin secretion signal and the metal binding sequence indicate that
the protein was intact.
Figure 4:
10%/3% Tricine SDS-PAGE analysis of
reductively C-methylated aliquots of pooled fractions. Lane 1, COS-7 cell supernatant; lane 2, pooled
material from metal chelate chromatography; lane 3, pooled
material from Affi-Gel blue; lane 4, pooled material from gel
filtration; lane 5, bovine serum albumin control as size
marker; lane 6,
C-labeled molecular weight
markers.
Kinetic Properties of the Recombinant Polypeptide GalNAc
Transferase
To determine whether the addition of a metal binding
domain and secretion by COS-7 cells into tissue culture medium had any
effect on the properties of the enzyme, kinetic parameters K and V
of the
purified recombinant GalNAc transferase were compared with those of the
purified native enzyme (). The K
and V
for UDP-GalNAc were obtained
with peptide AcTPPP at saturating conditions with both enzymes; the
recombinant GalNAc transferase displayed an acceptor specificity
similar to that of the native GalNAc transferase with regard to the K
values for the nucleotide sugar and for
peptides AcTPPP and HVF. Similarly, the V
values
for both the nucleotide sugar and peptide AcTPPP were the same. These
results indicated that the kinetic properties of the recombinant
polypeptide GalNAc transferase were not affected by the presence of the
metal binding domain at its NH
terminus.
Initial Velocity Pattern
Two-substrate enzyme
reactions can proceed by one of three mechanisms: (a) ping-pong, (b)
compulsory ordered sequential, or (c) random ordered
sequential(17) . To identify the mechanism preferentially used
by the polypeptide GalNAc transferase, experiments were performed in
which the concentrations of peptide EPO-T and UDP-GalNAc were varied.
Six concentrations of UDP-GalNAc ranging from 1 to 80 µM and six concentrations of peptide EPO-T ranging from 0.07 to 2
mM (0.2-8 K for UDP-GalNAc
and 0.2-5 K
for EPO-T) were used.
Concentrations of peptide beyond 2 mM were avoided because
problems of peptide solubility arise and because the velocity of the
reaction begins to decrease at values slightly greater than 2
mM. The data were graphed in a double-reciprocal plot (Fig. 5) and produced a family of intersecting lines typical of a
sequential mechanism. A plot of 1/vversus 1/[EPO-T] also produced a family of intersecting lines
(data not shown). Data obtained were fit to the initial velocity
equation (Equation 1), which can be modified to specifically describe
each of the two-substrate enzyme reaction mechanisms outlined above.
The ping-pong model is a special case of Equation 1 in which K
= 0(14) . When the K
is 0, the slope expression of the
double-reciprocal form of Equation 1 becomes insensitive to changes in
the concentration of the second substrate in the reaction. On a
double-reciprocal plot, this produces the typical parallel line pattern
of the ping-pong mechanism. The estimate of K
(I) was found to be more than 6.7 standard
errors away from 0 (95% confidence interval (0.0111, 0.0207)) and is
thus inconsistent with the ping-pong mechanism. The equilibrium-ordered
mechanism is also a special case of Equation 1 in which K
= 0(14) . However, the
estimate of K
(I) was found
to be more than 6.1 standard errors away from 0 (95% confidence
interval (0.0067, 0.0133)), and this is inconsistent with the
equilibrium-ordered sequential model.
Figure 5:
Double-reciprocal plot of initial velocity versus UDP-GalNAc concentration at fixed variable EPO-T
concentrations. Peptide EPO-T concentration range was 0.2-5 K (0.07-2 mM), and UDP-GalNAc concentration range was
0.2-8 K (1-80 µM). The assays were
performed as described under ``Experimental
Procedures.''
Product/Dead End Inhibition
To further distinguish
between an ordered sequential and random mechanism, we first examined
the inhibitory effect of UDP. UDP is a reaction product known to
inhibit the reaction(3) . Fig. 6A shows that UDP
was a competitive inhibitor of UDP-GalNAc. The slope effect, combined
with the absence of an intercept effect, typical of competitive
inhibition, was further emphasized by the replot (Fig. 6A, inset). Fig. 6B shows
the effect of UDP with peptide EPO-T as the varied substrate. The
replot (Fig. 6B, inset) shows both a slope and
intercept effect consistent with UDP being a noncompetitive inhibitor
of the peptide.
Figure 6:
Product inhibition by UDP with variable
UDP-GalNAc concentrations (A) and with variable EPO-T
concentrations (B). Substrates and the inhibitor UDP were
added simultaneously to the enzyme, and assays were performed as
described under ``Experimental Procedures.'' In A,
the peptide EPO-T concentration was 0.4 mM. UDP concentrations
were 0 (), 0.056 (
), 0.142 (
), 0.28 (
), 0.7
(⊞), and 2 mM (
). In B, the UDP-GalNAc
concentration was 20 µM. UDP concentrations were 0
(
), 0.037 (
), 0.23 (
), 0.46 (
), 1.15
(⊞), and 2 mM (
). Insets show secondary
replots of slopes and intercepts versus UDP
concentration.
To unambiguously distinguish between a random
ordered and ordered sequential mechanism, a dead end peptide analogue
in which the threonine residue of EPO-T was replaced with a glycine
residue (EPO-G: PPDAAGAAPLR) was used in inhibitor studies. If
UDP-GalNAc participates in an ordered sequential mechanism, a pattern
of uncompetitive inhibition would be seen when the concentrations of
UDP-GalNAc and EPO-T were varied and held constant, respectively. This
would graphically translate as a family of parallel lines on a
double-reciprocal plot. However, Fig. 7A shows that a
series of parallel lines is not generated. The replot (Fig. 7A, inset) indicates that there was a
slope effect as well as a slight intercept effect providing evidence
against an ordered sequential mechanism. The statistical fit of the
data was consistent with EPO-G being a noncompetitive inhibitor of
UDP-GalNAc (I). At a fixed subsaturating concentration of
UDP-GalNAc, only changes in slope consistent with competitive
inhibition were observed with varying concentrations of EPO-G (Fig. 7B and inset). The absence of
uncompetitive inhibition between the dead end analogue EPO-G and either
of the substrates ruled out an ordered mechanism in which there is
compulsory binding of one substrate before the other to form a ternary
complex. Instead, the patterns of inhibition of the dead inhibitor
EPO-G were consistent with a random sequential mechanism.
Figure 7:
Dead end substrate inhibition by peptide
EPO-G with variable UDP-GalNAc concentrations (A) and variable
peptide EPO-T concentrations (B). Substrates and the peptide
inhibitor EPO-G were added simultaneously to the enzyme, and assays
were performed as described under ``Experimental
Procedures.'' In A, the peptide EPO-T concentration was
0.4 mM. EPO-G concentrations were 0 (), 0.18 (
),
0.4 (
), 1 (
), 2 (⊞), and 4 mM (
). In B, the UDP-GalNAc concentration was 20 µM. EPO-G
concentrations were 0 (
), 0.15 (
), 0.4 (
), 1
(
), 1.5 (⊞), 2.5 (
), and 4 mM (
). Insets show secondary replots of slopes and intercepts versus peptide EPO-G concentration.
, V
). Since
several lines of evidence have suggested that the polypeptide GalNAc
transferase may be expressed in multiple forms with overlapping
specificities, the characterization of a homogeneous form of the enzyme
represents an initial step in determining if alternate forms of this
enzyme exist. An insulin secretion signal was used to target the
recombinant enzyme for secretion(19) , thereby allowing us to
distinguish the cloned activity from the endogenous activity of COS-7
cells. The metal binding affinity tag engineered in the amino terminus
of the recombinant enzyme provided a facile method to remove over 98%
of contaminant proteins from cell culture media by metal chelate
chromatography. Approximately 5 µg of homogeneous recombinant
polypeptide GalNAc transferase was prepared from the culture
supernatants of 10
COS-7 cells.
and K
for UDP-GalNAc (10 µM, 16 µM) and
EPO-T (0.408 mM, 0.648 mM) were similar to one
another (I); the similarity of the K
and K
of a substrate is an
indication that their binding to the enzyme occurs independently of one
another(17) .
Figure S1:
Scheme
1.
To our knowledge, only one other nucleotide
sugar:protein transferase has been described in terms of reaction
mechanism. UDP-D-xylose:core protein
-D-xylosyltransferase catalyzes the first step in
chondroitin sulfate proteoglycan O-glycosylation. The reaction
in which xylose, donated by the nucleotide sugar UDP-xylose, is added
to serine has been determined to proceed via an ordered sequential
mechanism(20) . It remains unclear why two enzymes that catalyze
similar committed steps of O-glycosylation do so using
different mechanisms.
of UDP was 380 µM, as determined by slope and
intercept effects (I). This value is one order of
magnitude greater than the K
of a
polypeptide GalNAc transferase purified from an ascites hepatoma and
published previously (26 µm; Ref. 3). The discrepancy might be
because the two enzymes represent different proteins. The effect of UDP
on the activity of the polypeptide GalNAc transferase described here
was not as potent as for the polypeptide GlcNAc transferase catalyzing
the addition of GlcNAc residues to hydroxyamino acids; more than 50% of
the activity of this cytosolic enzyme could be abolished by as little
as 0.5 µM UDP(21) . Although both nucleotide
sugar:protein transferases glycosylate polypeptides via the transfer of
a saccharide donated by a UDP-based nucleotide-sugar, the data here
suggested that UDP may not be an important means of regulation of
polypeptide GalNAc transferase activity. However, similar to the lack
of effect of free GlcNAc on the activity of the polypeptide GlcNAc
transferase(21) , the presence of various concentrations of free
GalNAc had no significant effect on the activity of the polypeptide
GalNAc transferase (data not shown), suggesting a requirement by the
enzyme for GalNAc to be attached to a uridine diphosphate.
value for EPO-T
(0.408 mM) and the K
value for
the dead end inhibitor EPO-G (0.770 mM) indicates that the
enzyme binds to both peptides with similar affinity. Substrate
selection may be a two-part process in which the enzyme can bind to a
substrate that meets the flanking sequence requirements, followed by
binding to the target itself, i.e. the hydroxyamino acid. We
speculate that the competition observed in vitro between EPO-T
and EPO-G can occur in vivo in that both hydroxyamino
acid-bearing peptides and peptides devoid of hydroxyamino acids can
bind to the enzyme but can also exchange with one another, thus
creating a potential point of regulation. We are currently conducting
studies, in vivo, to test this hypothesis.
Table: 0p4in
Due to
the small amount of material available for analysis, the total protein
was estimated by averaging the results of amino acid analysis (12.6
µg), NH
-terminal sequencing initial yield values (2.3
µg), and scanning densitometry (1.03 µg) of Fig. 4.
Table: Estimated kinetic constants for the
recombinant polypeptide GalNAc transferase substrates
Table: Estimated kinetic constants for the
recombinant polypeptide GalNAc transferase
-terminal sequencing, Dr. Richard Raubertas for help with
the statistical analysis, and Pat Noonan for preparing the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.