Báo cáo khoa học: Identification of two cysteine residues involved in the binding of UDP-GalNAc to UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1) ppt

Identification of two cysteine residues involved in the binding
of UDP-GalNAc to UDP-GalNAc:polypeptide
N
-acetylgalactosaminyltransferase 1 (GalNAc-T1)
Mari Tenno
1
, Shinya Toba
1
, Fere
´
nc J Ke
´
zdy
3
,A
˚
ke P. Elhammer
3
and Akira Kurosaka
1,2
1
Department of Biotechnology Faculty of Engineering, and
2
Institute for Comprehensive Research, Kyoto Sangyo University,
Kamigamo-motoyama, Kyoto, Japan;
3
Pharmacia Corporation, Kalamazoo, Michigan, USA
Biosynthesis of mucin-type O-glycans is initiated by a family
of UDP-GalNAc:polypeptide N-acetylgalactosaminyl-
transferases, which contain several conserved cysteine resi-
dues among the isozymes. We found that a cysteine-specific

reagent, p-chloromercuriphenylsulfonic acid (PCMPS),
irreversibly inhibited one of the isozymes (GalNAc-T1).
Presence of either UDP-GalNAc or UDP during PCMPS
treatment protected GalNAc-T1 from inactivation, to the
same extent. This suggests that GalNAc-T1 contains free
cysteine residues interacting with the UDP moiety of the
sugar donor. For the functional analysis of the cysteine
residues, several conserved cysteine residues in GalNAc-T1
were mutated individually to alanine. All of the mutations
except one resulted in complete inactivation or a drastic
decrease in the activity, of the enzyme. We identified only
Cys212 and Cys214, among the conserved cysteine residues
in GalNAc-T1, as free cysteine residues, by cysteine-specific
labeling of GalNAc-T1. To investigate the role of these two
cysteine residues, we generated cysteine to serine mutants
(C212S and C214S). The serine mutants were more active
than the corresponding alanine mutants (C212A and
C214A). Kinetic analysis demonstrated that the affinity of
the serine-mutants for UDP-GalNAc was decreased, as
compared to the wild type enzyme. The affinity for the
acceptor apomucin, on the other hand, was essentially
unaffected. The functional importance of the introduced
serine residues was further demonstrated by the inhibition of
all serine mutant enzymes with diisopropyl fluorophosphate.
In addition, the serine mutants were more resistant to
modification by PCMPS. Our results indicate that Cys212
and Cys214 are sites of PCMPS modification, and that these
cysteine residues are involved in the interaction with the
UDP moiety of UDP-GalNAc.
Keywords: cysteine; GalNAc-transferase; mucin; O-glyco-

sylation; UDP-GalNAc.
Mucin-type O-glycosylation is an important post-transla-
tional modification that is widely distributed on many
secretory and membrane glycoproteins [1,2]. The initial step
of this glycosylation is catalyzed by the UDP-GalNAc:poly-
peptide N-acetylgalactosaminyltransferases (GalNAc-trans-
ferases; EC 2.4.1.41). These enzymes transfer GalNAc from
UDP-GalNAc to serine or threonine residues of proteins [3].
Recent progress in molecular cloning has revealed that the
GalNAc-transferases constitute a large gene family, with 10
distinct isozymes identified to date [4–14], and that they are
type II membrane proteins with a short N-terminal
cytoplasmic tail, a hydrophobic transmembrane anchor, a
luminal stem region, and a large luminal putative catalytic
domain (Fig. 1). The luminal putative catalytic domain
contains two distinct subdomains; a central catalytic
domain and a C-terminal lectin-like domain. The central
catalytic domain can be further subdivided into two regions.
The N-terminal half is represented by a glycosyltransferase 1
(GT1) motif that is conserved among a wide range of
glycosyltransferases [15]. The extreme C-terminal end of the
GT1 motif contains a so-called DXH motif, which corres-
ponds to the DXD sequence common to many glyco-
syltransferases [16]. The C-terminal half of the catalytic
domain contains a so-called Gal/GalNAc-T motif, a
sequence segment where significant homology can be seen
between b1,4-galactosyltransferases and GalNAc-transfer-
ases [15,17]. A C-terminal lectin-like domain, called the
(QXW)
3

repeats, occurs exclusively in the GalNAc-trans-
ferases [18,19].
Although recent reports show that the GalNAc-transfer-
ases all have common structural features and the conserved
motifs described above, the exact role of each domain in
catalysis remains largely unknown. Moreover, these
enzymes are also characterized by the presence of highly
conserved cysteine residues, several of which are positioned
in and around the conserved motifs (Fig. 1). In order to
obtain more detailed information on the structure–function
relationship of the GalNAc-transferases, we investigated
the possible role(s) of the conserved cysteine residues
in GalNAc-T1. We used site-directed mutagenesis, in
Correspondence to A. Kurosaka, Department of Biotechnology,
Faculty of Engineering, Kyoto Sangyo University,
Kamigamo-motoyama, Kita-ku, Kyoto 603-8555, Japan.
Fax: + 81 75 705 1914, Tel.: +81 75 705 1894,
E-mail:
Abbreviations: ABD-F, 4-(aminosulfonyl)-7-fluoro-2, 1, 3-benzoxa-
diazole; DFP, diisopropyl fluorophosphate; GalNAc, N-acetylgalac-
tosamine; GalNAc-transferase, UDP-GalNAc:polypeptide
N-acetylgalactosaminyltransferase; GT1, glycosyltransferase 1; NEM,
N-ethylmaleimide; PCMPS, p-chloromercuriphenylsulfonic acid;
UBD, UDP-binding domain; TFA, trifluoroacetic acid.
Enzymes: UDP-GalNAc:polypeptide N-acetylgalactosaminyl-
transferases (GalNAc-transferases; EC 2.4.1.41).
(Received 16 May 2002, revised 9 July 2002, accepted 18 July 2002)
Eur. J. Biochem. 269, 4308–4316 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03123.x
combination with identification of free cysteine residues
(defined as cysteine residues not involved in the formation of

a disulfide bond), by cysteine-specific labeling, to study the
mechanistic involvement of the conserved cysteine residues
in the function of GalNAc-T1. Our results demonstrate that
Cys212 and Cys214, which are located at the C terminus of
the DXH motif, are free cysteine residues that interact with
the nucleotide moiety of UDP-GalNAc, possibly through
hydrogen bonding.
EXPERIMENTAL PROCEDURES
Preparation of soluble bovine GalNAc-T1
A soluble form of bovine GalNAc-T1 was expressed in
High Five cells using the baculovirus expression system. The
molecule was purified to homogeneity by apomucin-
Sepharose chromatography as described previously [20].
Construction of soluble rat recombinant GalNAc-T1
and expression in COS7 cells
Rat GalNAc-T1 cDNA was obtained as outlined by Hagen
et al. [21]. For the construction of soluble GalNAc-T1, rat
GalNAc-T1 full-length cDNA was subcloned into pcDNA4
to create the vector, prT1. prT1 was linearized with BamHI,
andthendigestedwithBal31 nuclease. The Bal31 digest was
blunt-ended and digested with NotI. The resulting digest
was ligated into the EcoRV and NotIsitesofpcDNA4,
obtaining pDN42 that encodes GalNAc-T1 with 42
N-terminal amino acid residues, including a cytoplasmic
tail and a transmembrane domain, deleted. A NheI-SmaI
fragment of the plasmid pGIR201protA (a gift from H.
Kitagawa, Kobe Pharmaceutical University) [22,23],
containing a cDNA encoding the insulin signal sequence
and the Protein A-IgG binding domain, was inserted into
NheI-HindIII digested pcDNA3.1, producing the vector,

pInsProA. pDN42 was digested with BamHI and NotI, and
was subcloned into pInsProA, generating the plasmid
pInsProADN42 containing truncated rat GalNAc-T1 fused
with IgG-binding domain of Protein A (P-DN42).
Site-directed mutagenesis
Site-directed mutagenesis was performed on pInsProADN42
using the LA PCR
TM
in vitro mutagenesis kit, using the
primers listed. Nucleotides shown in italic are nucleotides
mutated to convert conserved cysteine residues into either
alanine or serine residues. C106a, 5¢-TAGAGGGGGCTA
AAACAAAA-3¢; c212a, 5¢-ACTGTGCACTCGGCGTG
AGC-3¢; c214a, 5¢-ACTGTGGCCTCGCAGTGAGC-3¢;
c235a, 5¢-TAGGAGCCACCACTGTCCTC-3¢; c330a,
5¢-CAGAGTCCCTCCAGCCTGCC-3¢; c339a, 5¢-GGAG
GCCGTCACTATTTCCA-3¢; c408a, 5¢-GAAAGGCTTG
GCCTGTAGTT-3¢;c212s,5¢-GCTCACAGCGAGTGCA
CAGT-3¢; c214s, 5¢-GCTCACTGCGAGAGCACAGT-3¢;
c212s/c214s, 5¢-GCTCACAGCGAGAGCACAGT-3¢.
Expression of P-DN42 and mutant P-DN42 in COS7 cells
Expression construct (pInsProADN42 or mutant pInsPro-
ADN42) was transfected into COS7 cells using
FuGENE
TM
6 Transfection Reagent. Three days after the
transfection, the culture medium was collected and the
Fig. 1. Schematic representation of the domain structure and the position of the cysteine residues in the cloned GalNAc-transferases. Arrows indicate
cysteine residues and the numbers indicate residues mutated in this study. The amino acid residue numbering is based on the GalNAc-T1 sequence.
Highlyconservedcysteineresiduesarerepresentedbydottedlines.b,r,hT1,bovine,rat,andhumanGalNAc-T1;hT2,humanGalNAc-T2;hT3,

human GalNAc-T3; hT4, human GalNAc-T4; rT5, rat GalNAc-T5; hT6, human GalNAc-T6; hT7, human GalNAc-T7; hT8, human GalNAc-T8;
hT9, human GalNAc-T9; rppGaNTase-T9, rat ppGaNTase-T9.
Ó FEBS 2002 Cys residues in GalNAc-T1 interact with UDP-GalNAc (Eur. J. Biochem. 269) 4309
secreted enzyme was purified on IgG-Sepharose. For
analysis by SDS/PAGE, the resins adsorbed with the
secreted enzyme were boiled in SDS/PAGE loading buffer.
The resulting supernatant was loaded directly on the gel.
For Western blotting, the proteins on the membrane were
visualized by incubating the blot with an affinity purified,
alkaline phosphatase-conjugated, rabbit antibody to mouse
IgG, followed by staining with nitrobluetetrazolium and
5-bromo-4-chloro-3-indolylphosphate. The protein bands
on the immunoblots was quantified by densitometry
scanning and the intensity of each band was determined
using the NIH Image software. The enzymatic activity of
the P-DN42 and mutant P-DN42 gene products was
determined as described below. The activity levels were
corrected for enzyme protein concentration in the medium.
Assay for GalNAc-transferase activity (PD-10 assay)
The enzyme activity was determined in a reaction mixture
composed of 50 m
M
imidazole buffer (pH 7.2), 10 m
M
MnCl
2
, 0.1% Triton X-100, 6 nmol UDP-
3
H-GalNAc
(approximately 10 000 d.p.m.), 150 lg apomucin [24],

and an appropriate amount of enzyme. The mixture was
incubated for 30 min at 37 °C, and the reaction was stopped
by adding 0.25
M
EDTA. The reaction mixture was then
separated on a PD-10 column. The void fraction containing
3
H-labeled apomucin was recovered and the radioactivity
was determined.
Modification of GalNAc-T1 with PCMPS and DFP
Purified soluble bovine GalNAc-T1 or a recombinant
mutant rat GalNAc-T1 was treated with p-chloromercuri-
phenylsulfonic acid (PCMPS) in 40 m
M
imidazole buffer
(pH 7.2) for 90 min at room temperature, or with diiso-
propyl fluorophosphate (DFP) in 40 m
M
imidazole buffer
(pH7.2)for30minat37°C. Following treatment, the
reaction mixture was dialyzed against 25 m
M
imidazole
buffer (pH 7.2) containing 300 m
M
NaCl, 10% glycerol,
and 0.1% taurodeoxycholate. The enzymatic activity of the
samples was determined using the PD-10 assay (see above).
To study the influence of UDP-GalNAc or UDP on the
effect of PCMPS, the treatment was carried out in 0.1 m

M
PCMPS in the presence of UDP-GalNAc or UDP.
Identification of free cysteine residues
Labeling of bovine GalNAc-T1 with ABD-F and fraction-
ation of the labeled peptides were carried out as described
[25,26]. Briefly, GalNAc-T1 was first labeled with ABD-F,
followed by reduction with tributylphosphine and S-carbo-
xymethylation with iodoacetic acid. The alkylated protein
was digested with endoproteinase Lys-C. The digest was
then fractionated by HPLC on a C
18
HPLC column. The
fluorescent peptides were purified by rechromatography on
aC
8
column and sequenced with an automated Edman
sequencer.
Kinetic analysis
K
m
for UDP-GalNAc was obtained by varying the
concentration of UDP-GalNAc from 1.5 to 43.5 l
M
in
the presence of 1.88 mgÆmL
)1
apomucin. To determine the
K
m
for apomucin, GalNAc-transferase activity was assayed

in the presence of 7.5 l
M
UDP-GalNAc and
0.625–8.75 mgÆmL
)1
apomucin. Calculation of kinetic
parameters was done from double reciprocal plots (1/v vs.
1/[S]), using standard procedures.
RESULTS
Involvement of the free cysteine residues of GalNAc-T1
in catalysis
To investigate the functional role of the cysteine residues
(Fig. 1), we first modified GalNAc-T1 with a cysteine-
specific reagent, PCMPS. We then examined the influence
of the modification on the GalNAc-transferase activity. A
purified bovine GalNAc-T1, expressed as a secreted protein
in High Five cells, was used for this experiment [20]. As
shown in Fig. 2, PCMPS caused a marked, concentration
dependent decrease in enzyme activity, with a K
i
of
0.03 m
M
. This suggests that free cysteine residues, possibly
located at the catalytic site of GalNAc-T1, might be
involved in the catalytic function of the enzyme.
To investigate whether the cysteine residues modified by
PCMPS are involved in the binding of UDP-GalNAc,
we treated recombinant GalNAc-T1 with PCMPS in the
presence of either UDP-GalNAc or UDP. To increase the

sensitivity in this experiment, the cysteine modification
was performed with the minimal PCMPS concentration
(0.1 m
M
) required for complete inhibition of GalNAc-T1
(Fig. 2). Fig. 3 shows that GalNAc-T1 retained enzymatic
activity in the presence of either UDP or UDP-GalNAc.
This suggests that the sulfhydryl groups of free cysteine
residues modified by PCMPS may interact with UDP-
GalNAc, or at least be located in the UDP-GalNAc binding
cleft. Furthermore, the data suggest that the cysteine
residues predominantly interact with a UDP moiety of
UDP-GalNAc, as UDP and UDP-GalNAc were equally
effective at protecting the enzyme from inactivation.
Fig. 2. Inhibition of GalNAc-T1 with PCMPS. Purified bovine
GalNAc-T1 was incubated with increasing concentrations of PCMPS
for 90 min at room temperature. Following incubation, the treated
enzyme was dialyzed to remove excess PCMPS, and assayed for
activity as described in Experimental procedures.
4310 M. Tenno et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Mutagenesis of the cysteine residues in and around
the GT1 and Gal/GalNAc-T motifs
To investigate which cysteine residues are involved in the
catalytic function of GalNAc-T1, site-directed mutagenesis
was carried out on the conserved cysteine residues in the
catalytic domain. A rat GalNAc-T1 cDNA, cloned by PCR
as outlined by Hagen et al. [21], was used for this
experiment. Rat GalNAc-T1 is 98% identical to the bovine
ortholog and all of the cysteine residues are conserved
between the two enzymes (Fig. 1). For ease of purification

and detection, 42 N-terminal amino acid residues containing
the cytoplasmic tail and the transmembrane region were
deleted from the rat isozyme, and an insulin signal sequence
and a Protein A-IgG binding domain were fused to the
resulting N terminus of the sequence. The recombinant
truncated GalNAc-T1 was then expressed in COS7 cells and
the secreted fusion protein was purified from the culture
medium on IgG-Sepharose. The purified recombinant,
truncated rat GalNAc-T1, designated P-DN42, retained full
enzymatic activity and had kinetic properties almost identi-
cal to those of soluble bovine GalNAc-T1 [27]. Hence, it was
used for the following site-directed mutagenesis studies. The
amount of fusion protein secreted into the medium was
quantified by Western blotting in combination with densi-
tometric scanning of the bands on the blotting membrane.
The enzymatic activities were correlated with the concen-
tration of recombinant proteins in the media. This was done
to evaluate the effects of the mutations on both the specific
activity and the absolute levels of the secreted mutant
enzymes. In a first experiment, we mutated Cys106, Cys212,
Cys214, and Cys235 in P-DN42, individually, to alanine.
These residues are located in (Cys212 and Cys214), and
around (Cys106 and Cys235) the GT1 motif. Mutation of
C106A, C212A, and C214A, resulted in a considerable
decrease in secretion of the mutant proteins (Fig. 4),
indicating that the replacement of cysteine with alanine
significantly affected enzyme stability and/or efficiency of
secretion. Apomucin was used as the acceptor when
comparing the activity of the secreted mutants. As shown
in Fig. 4, C106A was completely inactive. The relative

activities of C212A and C214A were drastically decreased,
to 6% and 17% of that of P-DN42, respectively. These
results indicate that the conserved Cys106, Cys212, and
Cys214 residues are essential for efficient enzyme function.
In contrast, the C235A mutant retained almost full activity,
as well as a high level of secretion into the culture medium.
Hence, Cys235 appears not to be required for GalNAc-T1
activity or secretion.
The Gal/GalNAc-T motif contains one conserved
cysteine residue, Cys330. In addition, there are two
conserved cysteine residues (Cys339 and Cys408) at the
C-terminal side of this motif. Each of these cysteine residues
was also mutated to alanine. Secretion of the mutants,
especially C339A, decreased significantly. Moreover, there
was a complete loss of activity in all three mutant enzymes
(Fig. 4). These results demonstrate that the cysteine residues
in positions 330, 339 and 408 are important for both
secretion and function of GalNAc-T1.
Identification of free cysteine residues in GalNAc-T1
The inactivation observed for several of the GalNAc-T1
mutants may result either from conformational changes
caused by the disruption of disulfide bridges or from
mutational effects of cysteine residues involved in enzyme
function. However, the results from modification of
GalNAc-T1 with PCMPS (Fig. 2) strongly suggest the
presence of essential, free cysteine residues. To identify
the free cysteine residues in the native enzyme, we
labeled soluble bovine GalNAc-T1 with a cysteine specific
Fig. 3. Protection of GalNAc-T1 from PCMPS inactivation. GalNAc-
T1wastreatedwith0.1m

M
PCMPS in the presence of increasing
concentrations of UDP-GalNAc (d)andUDP(s). Following incu-
bation, the enzyme activity was determined as described in Fig. 2.
Fig. 4. Enzyme activity of GalNAc-T1 with mutated cysteine residues.
Each mutant was expressed in COS7 cells and the secreted recom-
binant protein was recovered from the culture medium. The amount of
the secreted protein was determined by Western blotting followed by
the densitometric scanning (lower panel). The enzymatic activity
secreted in the medium was corrected for the amount of mutant
proteins in the medium and expressed as activity relative to that of the
wild-type, P-DN42. Solid bars represent percent enzyme activity
relative to that of P-DN42 (hatched bars).
Ó FEBS 2002 Cys residues in GalNAc-T1 interact with UDP-GalNAc (Eur. J. Biochem. 269) 4311
fluorescent reagent, 4-(aminosulfonyl)-7-fluoro-2, 1, 3-ben-
zoxadiazole (ABD-F), in the absence of reducing agent
[25,26]. ABD-F is nonfluorescent until it reacts with thiols.
Therefore, free cysteine residues can be identified as carrying
a fluorescent label following ABD-F treatment of a protein.
Purified bovine GalNAc-T1 was first labeled with ABD-F,
followed by reduction and S-carboxymethylation. The
labeled, reduced enzyme was then cleaved with endopro-
teinase Lys-C, and the resulting peptide fragments were
fractionated by HPLC on a C
18
column. As shown in Fig. 5,
a number of peptide peaks absorbing at 220 nm, were
detected but only a few were fluorescent. All of the major
fluorescent peaks (indicated by arrows) were collected and
re-fractionated by HPLC using a C

8
column. This revealed
that peaks 1, 2, and 3 were artifacts as all of them separated
into several small peaks on the C
8
column and none of these
(secondary peaks) contained any polypeptide sequence
detectable by sequence analysis (data not shown). By
contrast, peak 4 produced a single peak on the C
8
column.
Edman analysis showed that this peak contained an ABD-F
labeled peptide. The peptide contained the N-terminal
sequence G202QVITFL
DAHC212EC214TV. The sequence
includes the GalNAc-T1 DXH motif (underlined above), a
region believed to be involved in coordination of a divalent
cation or the binding of UDP-GalNAc [16,28,29]. The two
cysteine residues in the sequence, Cys212 and Cys214, were
both labeled by ABD-F (Fig. 6), as demonstrated by the
presence of a peak corresponding to fluorescent cysteine and
the complete absence of a peak corresponding to carbo-
xymethylated cysteine, in the sequence analysis of the
peptide. Consequently, both Cys212 and Cys214 can be
considered free cysteine residues that probably are exposed
on the surface of the UDP-binding pocket. The other
essential cysteine residues, which were identified by muta-
tional analysis but not labeled by ABD-F, may form
intramolecular disulfide bonds required for proper folding
of the enzyme. Cys235, on the other hand, does not seem to

be involved in formation of a disulfide bond, as mutation at
this site did not affect the activity of the enzyme (Fig. 4).
Why this residue was not labeled with ABD-F is not clear.
Fig. 5. HPLC fractionation of endoproteinase
Lys-C digest of ABD-F labeled GalNAc-T1.
Soluble bovine GalNAc-T1 was labeled with
ABD-F, reduced with tributylphosphine,
alkylatedwithiodoaceticacid,andthen
digested with endoproteinase Lys-C. The
digest was loaded onto a C
18
column
(4.6 · 250 mm) equilibrated with 0.1%
trifluoroacetic acid. Elution was carried out
with a linear gradient from 0 to 50%
acetonitrile in 0.1% trifluoroacetic acid, at
flow rate of 1 mLÆmin
)1
. The chromatograms
were monitored by (A) relative absorbance at
220 nm and (B) by relative fluorescence
(excitation at 385 nm, emission at 520 nm).
The peaks labeled 1–4 were pooled and
re-fractionated by C
8
column chromatogra-
phy for subsequent amino acid sequence
analysis.
Fig. 6. Amino acid sequence analysis of the ABD-F labeled peptide. The
amino acid sequence of the fluorescent peptide (peak 4 in Fig. 5) was

determined with an automated Edman sequencer. The solid and the
open arrows indicate the elution position of ABD-F labeled cysteine,
and S-carboxymethylated cysteine, respectively. HPLC profiles of (A)
cycle11(Cys212)and(B)13(Cys214)areshown.
4312 M. Tenno et al. (Eur. J. Biochem. 269) Ó FEBS 2002
As the fluorescent labeling was performed without dena-
turing reagents, it is possible that only free cysteine residues
exposed to the solvent were labeled by the ABD-F.
Expression and kinetic studies of cysteine-to-serine
mutant GalNAc-T1 enzymes
The findings that the activities of the C212A and C214A
mutants were drastically decreased and that UDP or UDP-
GalNAc prevented PCMPS inactivation of GalNAc-T1
suggest that electrostatic interactions through the polar
sulfhydryl groups of these cysteine residues may be
involved in the interaction(s) between the nucleotide moiety
of UDP-GalNAc and GalNAc-T1. To examine this
hypothesis, we generated two single point mutants,
C212S and C214S, and one double point mutant, C212S/
C214S. In these mutants, the cysteine residues (212 and
214) were replaced by serine residues, thereby generating
proteins with hydroxyl, instead of sulfhydryl, side chains at
positions 212 and 214. This should allow retained hydro-
gen bonding capacity at these positions and at least
theoretically, if this capacity is an essential function of these
residues, result in functional enzyme. The results shown in
Fig. 7 suggest that this is indeed the case. C212S and
C214S retained approximately 60% and 80% of parent
enzyme activity, respectively. This is significantly higher
than the activity of the corresponding alanine mutants (6%

and 17%, respectively) (Fig. 4). The activity of the double
mutant, C212S/C214S, was lower but still amounted to
40% of the parent enzyme activity.
In a more in-depth evaluation of the function of the three
serine mutants, the kinetic properties of the mutant enzymes
were compared to those of the parent enzyme, P-DN42. As
showninTable1,theK
m
values of all three mutants, for
apomucin, were essentially the same as that of P-DN42,
indicating that Cys212 and Cys214 are not involved in
the recognition of the acceptor. By contrast, the affinity of
the mutant enzymes for UDP-GalNAc was affected quite
significantly. When serine was substituted for Cys214, the
increase in K
m
was only slight ( 1.2-fold that of P-DN42).
On the other hand, C212S showed a 3.4-fold increase in K
m
.
Furthermore, the effect of mutating the two cysteine
residues appear to be cooperative as the double mutant,
C212S/C214S, shows an even higher increase in K
m
(5-fold).
These results demonstrate that while Cys212 may be a
major site of interaction with UDP-GalNAc, Cys214 is also
involved in binding.
PCMPS and DFP modification of the serine-mutants
of GalNAc-T1

We also examined the sensitivity of the three serine mutants
to PCMPS inactivation (shown in Fig. 8). C214S, which
contains free Cys212, was inactivated by PCMPS with a K
i
of 0.03 m
M
almost identical to that of native GalNAc-T1.
On the other hand, C212S was more resistant to the
treatment. The K
i
of  0.65 m
M
, may be due to a lower
reactivity of Cys214 as compared to Cys212. Moreover, no
inhibition was observed for C212S/C214S, even in the
presence of a large excess of PCMPS (1 m
M
), that resulted
in the complete inactivation of P-DN42. These results show
that both Cys212 and Cys214 are modified by PCMPS, and
that, consistent with the kinetic data, Cys212 is the most
Fig. 7. Enzymatic activity of cysteine-to-serine mutant GalNAc-T1
enzymes. Enzyme activity measurements and Western blotting of
mutant proteins were carried out as described in Fig. 4.
Table 1. Comparison of donor and acceptor K
m
values (apparent) for the
parent and mutant enzymes. Values shown are means of three separate
determinations.
UDP-GalNAc Apomucin

K
m
(l
M
) -fold K
m
(mgÆmL
)1
) -fold
P-DN42 5.1 ± 0.8 1.0 4.7 ± 0.1 1.0
C212S 17.4 ± 3.3 3.4 4.6 ± 0.9 1.0
C214S 6.0 ± 1.1 1.2 4.0 ± 0.3 0.9
C212S/C214S 25.0 ± 3.7 5.0 4.0 ± 0.4 0.9
Fig. 8. Inhibition of cysteine-to-serine mutant GalNAc-T1 enzymes with
PCMPS. Mutant enzymes expressed in COS7 cells were purified on
IgG-Sepharose from the conditioned medium. Proteins adsorbed by
the resins were treated with increasing concentrations of PCMPS.
Following treatment the resins were washed with buffer and the
enzymatic activity of the mutant proteins were determined as described
in Experimental procedures. d, C212S; s, C214S; m, C212S/C214S.
Ó FEBS 2002 Cys residues in GalNAc-T1 interact with UDP-GalNAc (Eur. J. Biochem. 269) 4313
important site for the interaction with UDP-GalNAc.
Complete loss of PCMPS inactivation in the double mutant
suggeststhattherearenoPCMPSreactivesitesinnative
GalNAc-T1, other than Cys212 and Cys214, and that
ABD-F and PCMPS modify the same cysteine residues in
GalNAc-T1.
We also investigated the function of the serine residues
introduced at positions 212 and 214 by modifying the
mutant proteins with DFP, a reagent specific for active

serine residues. Native bovine GalNAc-T1 is totally insen-
sitive to DFP treatment, and thus appears not to contain
any serine residues important for enzyme function. By
contrast, all three serine mutants were inactivated by DFP
to some extent (Fig. 9). The inhibition was more efficient for
C212S than C214S, again demonstrating that position 212 is
the more important site for substrate interaction. The
double mutant, C212S/C214S, was most susceptible to
DFP, confirming the cooperative involvement of the two
sites observed in the kinetic analysis (Table 1).
Taken together, the results from the kinetic, mutational
and chemical modification studies presented in this report
strongly suggests that the sulfhydryl groups at Cys212 and
Cys214, but primarily at Cys212, are involved in substrate
binding, possibly as hydrogen bond partners with UDP.
DISCUSSION
The primary aim of this study is to evaluate the functional
role(s) of the conserved cysteine residues found in the
GalNAc-transferase family. Using site-directed mutagene-
sis, in combination with identification of free cysteine
residues by cysteine-specific labeling, we demonstrated that
Cys212 and Cys214, but predominantly Cys212, is involved
in the binding of the nucleotide portion of UDP-GalNAc,
most probably through hydrogen bonding. This is consis-
tent with our previous inhibition study on GalNAc-T1
using various nucleotides and nucleotide sugars [30],
showing that the enzyme primarily recognizes the UDP
portion of the sugar donor. Recent crystallographic studies
on glycosyltransferases indicate that several interactions are
involved in binding of the UDP portion of the sugar donors

to the enzymes [31–37]. In GalNAc-T1, hydrogen bonding
with Cys212 and Cys214 appears to be predominant
interactions with UDP. Other interactions may contribute
in a more modest fashion, as replacement of Cys212 or
Cys214 with alanine resulted in a substantial decrease, but
not in a complete loss, of the activity (Fig. 4). We also found
that the C212S mutant enzyme works relatively efficiently,
with a 3.4-fold difference in the K
m
value for UDP-GalNAc,
as compared to P-DN42, while the activity of the corres-
ponding alanine mutant, C212A, was too low for deter-
mination of kinetic parameters. This relatively modest
difference in efficiency between the wild-type enzyme and
the C212S mutant could be a matter of the size of the
hydrogen bond forming group: a hydroxyl group (–OH) is
smaller than a sulfhydryl group (–SH). If hydrogen bonding
is involved in the interaction with the nucleotide sugar,
substituting -OH for -SH may increase bond length slightly,
thereby making it less efficient and decreasing the affinity.
Although we show that the interaction of Cys212 and
Cys214 with the sugar donor is important for the GalNAc-
T1 activity, it should be noted that some GalNAc-transfer-
ases do not have cysteine residues at the corresponding
positions. As shown in Fig. 1, GalNAc-T1, -T2, -T3, -T4,
and -T6 all contain both cysteine residues, but other
GalNAc-transferases contain different amino acids at these
positions, such as DSH
VEC (GalNAc-T5), DAHCEV
(GalNAc-T7), DAH

IEV (GalNAc-T8), DAHVEF
(GalNAc-T9), and DSHCE
A (ppGaNTase-T9). Of these
isozymes with variation at the two cysteine sites, GalNAc-
T5, -T7 and ppGaNTase-T9 are catalytically active. This
indicates that the two cysteine residues C-terminal to the
DXH motif may not be crucial for the basic catalytic
function of the GalNAc-transferases, but rather are
important in defining the catalytic properties of specific
isozymes. In fact, the interaction of UDP-GalNAc with
GalNAc-T5, which has a valine residue at the Cys212 site, is
less efficient than with GalNAc-T1. GalNAc-T5 has a
significantly lower affinity for UDP-GalNAc (K
m
¼ 55 l
M
)
[9], than P-DN42 and its serine-mutants (Table 1). The low
affinity of UDP-GalNAc for GalNAc-T5 may, at least in
part, be ascribed to the substitution with valine at the
Cys212 site. The two isozymes GalNAc-T8 and GalNAc-T9
lack cysteine residues at both position 212 and 214.
Consequently they may also have a low affinity for
UDP-GalNAc and consistent with this, no enzymatic
activity has so far been reported for these molecules. It is
possible that the activity of these isozymes cannot be
measured under the standard assay conditions used for
other GalNAc-transferases. Similarly, the importance of
four histidine residues for GalNAc-T1 activity has been
demonstrated by Wragg et al. [17], using site-directed

mutagenesis. Of these residues, His211 and His341 are
conserved in all isozymes cloned to date. Some GalNAc-
transferases, however, do not contain the other two histidine
residues, His125 and His341. His125 and His341 are found
in six and eight isozymes out of 10, respectively. Aspartic
acid at the DXH motif in GalNAc-T1 is reported to be
essential for GalNAc-T1 activity, because the mutation at
this site results in inactivation of the enzyme [15]. Contrary
to this observation, GalNAc-T4 does not contain the DXH
Fig. 9. Inactivation of cysteine-to-serine mutant GalNAc-T1 enzymes
with DFP. DFP treatment of mutant proteins was performed as
described in Fig. 8. No change in pH was observed during the reaction.
This indicates that the inactivation of the mutants was not due to
acidification of the incubation but to modification of the active serine
residues. d, C212S; s, C214S; m, C212S/C214S.
4314 M. Tenno et al. (Eur. J. Biochem. 269) Ó FEBS 2002
sequence, but contains YXH instead [7]. All these findings
indicate that essential amino acid residues for some
isozymes are not necessarily conserved in other isozymes.
The variations in the primary sequence found in different
members of the GalNAc-transferase family could provide
each isozyme with distinct kinetic properties, thereby
enabling the specific reaction catalyzed by these enzymes
in vivo. Sequence variation is also found in the
fucosyltransferases. a1,3/4-Fucosyltransferases III, V, and
VI are inhibited by the cysteine specific reagent, N-ethyl-
maleimide (NEM). The presence of a free cysteine residue
has been reported for the NEM sensitive enzymes, whereas
those that are insensitive to NEM contain a serine (a1,3/4-
fucosyltransferase IV) or a threonine (a1,3/4-fucosyltrans-

ferase VII) residue at the corresponding site. Importantly,
the NEM-sensitive cysteine residue is reported to be located
in or near the binding site for GDP-Fuc [38–41], in analogy
to what was found for GalNAc-T1.
The known glycosyltransferases have been classified into
52 different families, based both on sequence similarity and
substrate/product stereochemistry (inverting or retaining),
at the carbohydrate-active enzymes server on world
wide web, URL: />db.html [42,43]. The crystal structures of several glyco-
syltransferases belonging to different groups have been
determined recently [31–37]. Although these proteins have
no sequence identity or related functional features, the
crystallographic studies show that some of them share a
domain structure, called a UBD (UDP-binding domain)
[44], also known as a SGC (SpsA N-acetylglucosaminyl-
transferase I core) domain [28,32]. The UBD is predicted to
consist of alternating a-helices and b-sheets, constituting an
a-b-a sandwich [31,44]. The UBD of glycosyltransferases
also contains a DXD motif. In all crystallized enzymes, the
DXD motifs are located at positions closely related to one
anotherandareexpectedtobeindirectcontactwiththe
sugar donor or interact with UDP-sugars through binding
with a divalent ion [28,29]. The two cysteine residues at
positions 212 and 214 in GalNAc-T1, identified in this study
as being involved in sugar donor binding, are in the GT1
motif. A DXH motif that precedes Cys212 and Cys214 is
located at the C-terminal end of the GT1 motif. The
hydrophobic cluster analysis of several glycosyltransferases
demonstrates that the DXH motif corresponds to the DXD
motif found in most other glycosyltransferases [29]. More-

over, it has been reported that the GT1 motif forms a
five-stranded parallel b-sheet flanked by four a-helices and
that the amino acids essential for enzymatic activity as well
as the DXH motif are located near the C-terminal ends of
the putative b-strands, lining the face of the predicted active
site cleft [15]. These proposed structural features of the GT1
motif in GalNAc-T1 are similar to the UBD, raising the
possibility that catalytic mechanisms similar to those
described above are conserved in the GalNAc-transferases
as well. It therefore appears likely that Cys212 and Cys214
at the C-terminal end of the DXH motif in GalNAc-T1 are
located at the active site of the enzyme, and interact with
UDP-GalNAc through hydrogen bonding.
The results presented in this report offer new insights into
the catalytic mechanism of GalNAc-T1. Identification of
amino acid residues essential for activity will help us to
understand the functions of the different domains in
GalNAc-transferases and will allow the development of
strategies for engineering new GalNAc-transferases with
altered or modified donor and/or acceptor specificities.
Together with information on the three-dimensional struc-
ture of the GalNAc-transferases, this will allow an under-
standing of the catalytic mechanism(s) of these enzymes that
in turn can be used for development of isozyme-specific
inhibitors and thereby for investigations of the functional
roles of mucin carbohydrates.
ACKNOWLEDGEMENTS
This work was supported in part by the Research Foundation for
Pharmaceutical Science, Sasakawa Scientific Research Grant, and the
Foundation for Bio-venture Research Center from the Ministry of

Education, Culture, Sports, Science, and Technology, Japan.
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