Generation of a mutant mucor hiemalis endoglycosidase that acts on core-fucosylated N-Glycans

Article abstract of DOI:10.1074/jbc.M116.737395

Endo-?-N-acetylglucosaminidase M (Endo-M), an endoglycosidase from the fungus Mucor hiemalis, is a useful tool for chemoenzymatic synthesis of glycoconjugates, including glycoprotein-based therapeutics having a precisely defined glycoform, by virtue of its transglycosylation activity. Although Endo-M has been known to act on various N-glycans, it does not act on core-fucosylated N-glycans, which exist widely in mammalian glycoproteins, thus limiting its application. Therefore, we performed site-directed mutagenesis on Endo-M to isolate mutant enzymes that are able to act on mammalian-type core-?1,6-fucosylated glycans. Among the Endo-M mutant enzymes generated, those in which the tryptophan at position 251 was substituted with alanine or asparagine showed altered substrate specificities. Such mutant enzymes exhibited increased hydrolysis of a synthetic ?1,6-fucosylated trimannosyl core structure, whereas their activity on the afucosylated form decreased. In addition, among the Trp-251 mutants, the W251N mutant was most efficient in hydrolyzing the core-fucosylated substrate. W251N mutants could act on the immunoglobulin G-derived core-fucosylated glycopeptides and human lactoferrin glycoproteins. This mutant was also capable of transferring the sialyl glycan from an activated substrate intermediate (sialyl glycooxazoline) onto an ?1,6-fucosyl-N-acetylglucosaminyl biotin. Furthermore, the W251N mutant gained a glycosynthase-like activity when a N175Q substitution was introduced and it caused accumulation of the transglycosylation products. These findings not only give insights into the substrate recognition mechanism of glycoside hydrolase family 85 enzymes but also widen their scope of application in preparing homogeneous glycoforms of core-fucosylated glycoproteins for the production of potent glycoprotein-based therapeutics.

Full text of DOI:10.1074/jbc.M116.737395

JBC Papers in Press. Published on September 14, 2016 as Manuscript M116.737395
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.737395
Endo-M Variant Acting on Core-fucosylated N-Glycans
Generation of a Mutant Mucor hiemalis Endoglycosidase that Acts on Core-fucosylated N-Glycans*
Toshihiko Katoh1, Takane Katayama1,2, Yusuke Tomabechi3, Yoshihide Nishikawa4, Jyunichi
Kumada4, Yuji Matsuzaki4, and Kenji Yamamoto3
From the 1Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan; 2Host-Microbe
Interaction Research Lab., Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, Japan;
3Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi,
Ishikawa 921-8836, Japan; 4Tokyo Chemical Industry Co., Ltd., 6-15-9 Toshima, Kita-ku, Tokyo
114-0003, Japan.
*Running title: Endo-M Variant Acting on Core-fucosylated N-Glycans
To whom correspondence should be addressed: Toshihiko Katoh, Graduate School of Biostudies, Kyoto
University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan. Telephone: +81-75-753-9430;
FAX: +81-75-753-9242; E-mail: tkatoh@lif.kyoto-u.ac.jp
Keywords:
Endo-M,
Core
fucose,
N-linked
glycosylation,
Transglycosylation,
Endo-β-N-acetylglucosaminidase, Glycoside hydrolase
ABSTRACT
mammalian-type core-α1,6-fucosylated glycans.
Among the Endo-M mutant enzymes generated,
those in which the tryptophan at position 251
was substituted with alanine or asparagine
showed altered substrate specificities. Such
mutant enzymes exhibited increased hydrolysis
of a synthetic α1,6-fucosylated trimannosyl
core structure, whereas their activity on the
afucosylated form decreased. In addition,
among the W251 mutants, the W251N mutant
was most efficient in hydrolyzing the
core-fucosylated substrate. W251N mutants
could act on the immunoglobulin G-derived
core-fucosylated glycopeptides and human
Endo-M, an endoglycosidase from the
fungus, Mucor hiemalis, is a useful tool for
chemoenzymatic synthesis of glycoconjugates,
including glycoprotein-based therapeutics
having a precisely defined glycoform, by virtue
of its transglycosylation activity. Although
Endo-M has been known to act on various
N-glycans, it does not act on core-fucosylated
N-glycans, which exist widely in mammalian
glycoproteins, thus limiting its application.
Therefore,
we
performed
site-directed
mutagenesis on Endo-M to isolate mutant
enzymes that are able to act on
1
Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Endo-M Variant Acting on Core-fucosylated N-Glycans
compromise the host defense system, contributing
to the virulence of pathogenic bacteria (7–9).
There are two main classes of these
endoglycosidases based on their amino acid
sequences, within the carbohydrate-active
lactoferrin glycoproteins. This mutant was also
capable of transferring the sialyl glycan from
an activated substrate intermediate (sialyl
glyco-oxazoline)
onto
an
α1,6-fucosyl-N-acetylglucosaminyl
biotin.
enzymes
(CAZy)
database
Furthermore, the W251N mutant gained a
glycosynthase-like activity when a N175Q
substitution was introduced and it caused
(http://www.cazy.org
)—
Glycoside Hydrolase
(GH) families, GH18 and GH85. GH18
endoglycosidases, including the enzymes used
frequently in glycoprotein analysis, such as
Endo-H from Streptomyces plicatus (10), Endo F1,
-F2, and -F3 from Elizabethkingia meningoseptica
(11), EndoS from Streptococcus pyogenes (8), and
Endo-T from Tricoderma reesei (12), normally
show only hydrolytic activity, whereas GH85
enzymes, including Endo-M from Mucor hiemalis,
Endo-A from Arthrobacter protophormiae (13),
Endo-D from Streptococcus pneumoniae (14),
Endo-Om from Ogataea minuta (15), and
Endo-CC1, -CC2 from Coprinopsis cinerea (16),
show transglycosylation activity in vitro in the
presence of suitable acceptor substrates as well as
hydrolytic activity.
accumulation
of
the
transglycosylation
products. These findings not only give insights
into the substrate recognition mechanism of
glycoside hydrolase family 85 enzymes but also
widen their scope of application in preparing
homogeneous glycoforms of core-fucosylated
glycoproteins, for the production of potent
glycoprotein-based therapeutics.
INTRODUCTION
Endo-β-N-acetylglucosaminidases
(ENGases, EC 3.2.1.96) are enzymes that
hydrolyze β-1,4 glycosidic linkages within the
N,N’-diacetylchitobiose moiety in the N-glycan
core structure and liberate a large part of the
glycan,
thus
leaving
the
innermost
Based on mutation analyses of GH85
N-acetylglucosamine residue (often attached to the
core fucose) on the aglycon. These enzymes are
distributed in a wide range of organisms—from
bacteria living in various ecological niches to
higher eukaryotes, including humans. In
eukaryotes, the endoglycosidase activity of this
enzyme is thought to be involved in the
processing of free oligosaccharides in the cytosol
(1–5), and in bacterial species, they may play
roles in acquisition of carbohydrates as energy
sources from the environment (6, 7), or
enzymes (17–19) and X-ray crystallographic
studies of Endo-D (20) and Endo-A (21, 22), it has
been postulated that the reaction mechanism of
GH85 involves substrate-assisted catalysis. The
reaction comprises two steps with the retention of
anomeric configuration. In the first step, an
enzymatic carboxylic acid (Glu 177 in Endo-M,
Fig. 1D) acts as a general acid, with the
concomitant nucleophilic attack by the carbonyl
oxygen of the 2-acetamide group in
N-acetylglucosamine (substrate), to form a sugar
2

Endo-M Variant Acting on Core-fucosylated N-Glycans
in minimum degradation of the reaction products
(33, 35, 36). Nonetheless, Endo-M and its mutants
show limited activity toward core-fucosylated
glycans.
oxazoline intermediate. In the second step, the
resulting intermediate is broken down by a
process that is almost the reverse of the first step,
accompanied either by hydrolysis (in the presence
of water) or transglycosylation (in the presence of
an appropriate acceptor molecule instead of water)
(23). Abbott et al. proposed a catalytic mechanism
involving an unusual proton shuttle in which the
imidic acid tautomer of another catalytically
important residue, asparagine (Asn 175 in
Endo-M), may be involved in the orientation of
the 2-acetamido group of the substrate and the
Asn residue also acts as a general base to facilitate
the formation of an oxazoline intermediate (20).
Endo-M serves as a particularly useful
Core fucosylation is a modification often
found in natural and recombinant glycoproteins
that affects N-glycan conformation and regulates
their
biological
activity
(37).
Notably,
Immunoglobulin (Ig) G1 with the fucose-deficient
N-glycan of Asn 297 in its Fc region shows
increased binding affinity for the Fcγ receptor III
on the effector cells, resulting in highly enhanced
antibody-dependent cellular cytotoxicity (ADCC)
(38, 39). Endoglycosidase-catalyzed modification
of
core-fucosylated
glycans
has
been
demonstrated using glycosynthase mutants of
Endo-D (40) and some GH18 enzymes.
Transglycosylation onto α1,6-fucosyl GlcNAc
moieties by endoglycosidase was first reported in
Endo-F2 and Endo-F3 (41). Recently Endo-F3
was converted to a glycosynthase and the mutant
enzyme was capable of transferring bi- and
tri-antennary complex type N-glycans using sugar
oxazoline donor substrates to synthesize
core-fucosylated complex glycopeptides (42).
EndoS and its glycosynthase which specifically
and efficiently acts on the IgG-Fc domain of
N-glycans have been used for chemoenzymatic
synthesis of IgGs with structurally defined
glycoforms for functional studies (43–45). More
recently, Endo-S2 was shown to have a more
flexible substrate specificity and high efficiency in
transferring complex, hybrid, and high-mannose
type N-glycans onto core-fucosylated or
non-fucosylated IgG molecules (46).
GH85 enzyme because it exhibits significant
transglycosylation activity towards complex-type
oligosaccharides, including sialo-oligosaccharides
(24–29). Glycoforms of glycoproteins play
important roles in regulating their biological
activities. Especially, sialosides generally affect
the half-lives of glycoproteins in the body fluid
and they often modulate immune responses
(30–32). Thus, regulation of glycoforms in
glycoprotein-based therapeutics (biomedicines),
including antibodies and cytokines, have drawn
attention in the pharmaceutical industry. This
enzyme has been explored extensively to expand
its utility, by protein engineering using
site-directed mutagenesis, resulted in the isolation
of efficient glycosynthase-like mutant enzymes,
such as N175Q (17, 33, 34). These mutant
enzymes exhibit both effective synthetic rates
using sugar oxazoline as the glycosyl donor
substrate, and lack of hydrolytic activity resulting
3

Endo-M Variant Acting on Core-fucosylated N-Glycans
to
the
trimannosylated
3GlcNAc2Fuc1).
Comparison of the surface structures of the
core
structure
In this study, we generated Endo-M mutant
enzymes that are able to act on core-fucosylated
substrates. These mutant enzymes including
W251N exhibited an altered substrate preference
(Man
catalytic sites of Endo-A and Endo-D revealed the
differences in the distribution of amino acid
residues. In Endo-A, a couple of tryptophan
residues (Trp 216 and Trp 244) are distributed
around a presumable subsite corresponding to the
innermost GlcNAc residue of the N-glycan
substrate, whereas in Endo-D, an asparagine (Asn
413) and a histidine (His 384) residue are present
instead. Interestingly, these tryptophan residues of
Endo-A are conserved in Endo-M (Trp 228 and
Trp 251) (Fig 1D). We also focused on a proline
residue at position 128 and a tyrosine residue at
position 131 of Endo-A, since they stretch over
the glycan substrate bound to the catalytic site.
These residues might determine substrate
specificity; however, Pro 128 and Tyr 131 are not
conserved in the amino acid sequence of Endo-M
(Gly 125 and Gln 128) or in that of Endo-D (Trp
292 and Ser 295) (Fig. 1D). To examine the roles
of these residues in determining the substrate
preference of Endo-M, six Endo-M mutant
enzymes—G125W, Q128A, Q128S, W228H,
W251A, and W251N—were generated in an E.
coli expression system and used to investigate
their substrate preferences.
for
core-fucosylated
glycan
substrates,
demonstrating their potential to act on more
diverse glycoproteins. To the best of our
knowledge, this is the first report that the substrate
specificity of an endoglycosidase was altered by
site-directed mutagenesis. These findings may
provide new insights into the mechanism of the
interaction between enzymes and glycan
substrates, giving
endoglycosidase
engineering.
a
basis for further
improvement by protein
RESULTS
Structural comparison of GH85 enzymes
To determine the amino acid residues
critical for substrate preference of Endo-M with
respect to α1,6-linked core-fucosylated glycans,
we first carried out a comparison of the GH85
enzymes. In light of the differences in substrate
specificities and the availability of tertiary
structures, we compared Endo-A (22) (Protein
Data Bank [PDB] ID: 3FHQ) and Endo-D
(20)(PDB ID: 2W92) (Fig. 1A-C). Since the
structural data for Endo-M is not available, we
assumed Endo-A to be ‘virtual Endo-M’,
considering that neither of them cleave
core-fucosylated glycans. On the other hand,
Endo-D has been reported to act on
core-fucosylated glycans when they are trimmed
Evaluation of hydrolytic activities of
Endo-M mutants on biotinylated substrates
The six mutants were assayed for
hydrolytic activity using biotinylated trimannosyl
core structure, with and without an α1,6-linked
4

Endo-M Variant Acting on Core-fucosylated N-Glycans
exhibited 350-fold increase in specific
core fucose residue (Man3GlcNAc2Fuc1-biotin and
Man3GlcNAc2-biotin, respectively) as the
substrate (Fig. 2A). TLC analysis demonstrated
that the wild-type enzyme and all the mutant
enzymes (1 μg) could hydrolyze 10 μg of
Man3GlcNAc2-biotin within 20 minutes (Fig. 2B).
However, they could hardly hydrolyze
Man3GlcNAc2Fuc1-biotin, except the two W251
mutants, W251A and W251N, which could
hydrolyze most of the core fucosylated substrate.
a
hydrolytic activity with the core-fucosylated
substrate, compared to the wild-type enzyme,
indicating that Trp 251 of Endo-M is important for
determining specificity for the glycan core
structure. Since W251 mutants showed increased
activity with Man3GlcNAc2Fuc1-biotin, we next
examined the effect of replacement with an amino
acid other than alanine and asparagine. All
Endo-M W251 variants, except W251C (the
expression of which impaired E. coli growth),
were generated and their relative hydrolysis ratios
The
products
were
confirmed
by
MALDI-TOF/MS; the observed masses for
Man3GlcNAc1 and Fucα1-6GlcNAc-biotin are m/z
730.6 [M + Na]+ (calculated; m/z 730.2) and m/z
673.6 [M + Na]+ (calculated; m/z 673.3),
respectively (Fig. 2C). The reaction products were
then quantified by HPLC analysis and the specific
activities of the mutants with each substrate were
obtained. As shown in Table 1, all the mutants
exhibited lower specific hydrolytic activity with
Man3GlcNAc2-biotin, than the wild-type enzyme
(6.49 μmol/min/mg). In particular, G125W and
the two W251 mutants showed about 40-fold
reduction. In contrast, the wild-type enzyme
exhibited marginal specific hydrolytic activity
with Man3GlcNAc2Fuc1-biotin (less than 0.2% of
the specific activity observed with the
afucosylated substrate). G125W, Q128A, Q128S,
and W228H showed undetectable or marginal
activities similar to the wild-type enzyme.
However, consistent with the result of TLC
analysis, W251A and W251N showed greater
for
Man3GlcNAc2-biotin
and
Man3GlcNAc2Fuc1-biotin, were compared (Fig. 3).
We found that W251N showed the highest activity
with the core-fucosylated substrate, among W251
variants. Interestingly, the wild-type and W251N
mutant enzymes showed a sharp contrast in their
substrate preference, i.e., they prefer either
afucosylated or core-fucosylated glycans. The
W251F variant showed substrate preference rather
like the wild-type enzyme, implying the
involvement of aromatic side chains in substrate
recognition; however, W251Y showed reduced
activity with the afucosylated substrate.
Furthermore, W251H retained approximately 20%
activity with the afucosylated substrate and
acquired
hydrolytic
activity
with
the
core-fucosylated substrate.
Alteration of substrate specificity of the
W251N mutant
substrate
preference
for
core-fucosylated
trimannosyl glycans (0.13 and 1.3 μmol/min/mg,
To investigate the possibility of its
respectively) than the wild-type enzyme. W251N
enzymatic application, the Endo-M W251N
5

Endo-M Variant Acting on Core-fucosylated N-Glycans
variant was further characterized. The optimal pH
for hydrolysis (pH 6.0) was similar for the
wild-type enzyme with Man3GlcNAc2-biotin and
W251N with Man3GlcNAc2Fuc1-biotin. The
kinetic parameters of the wild-type enzyme and
W251N variant with both substrates are shown in
Table 2. There is little difference in the Km values
for Man3GlcNAc2-biotin between the two
enzymes. However, the Km value of W251N for
the core-fucosylated substrate is more than
90-fold higher than that for the afucosylated one.
The kcat values of the wild-type and W251N
enzymes for their respective preferred substrate
are not very different.
activities of W251N with biotinylated,
afucosylated glycans and pyridylaminated ones, is
probably due to the open ring of the GlcNAc
residue at the reducing termini. Taken together,
the substrate specificity shifted from afucosylated
glycans to core-fucosylated glycans, owing to a
single amino acid substitution, W251N, with
minimum impact on the recognition of the
structures at the non-reducing end.
Hydrolytic activity of W251N variant on
glycopeptides and glycoproteins
We next examined whether the W251N mutant is
able to release N-glycans having a core fucose
from glycopeptide and glycoprotein substrates.
Rituximab is an anti-CD20 IgG1 mAb, which is
produced by a Chinese Hamster Ovary cell line. It
is known that 95% of the N-glycans attached to
Asn 297 of the Fc region of rituximab is core
fucosylated (47); we confirmed this by MS
analysis of PNGase F-released N-glycans (Fig. 4A
and STable S1). We prepared a mixture of
N-glycopeptides/peptides from rituximab by
tryptic digestion, for assaying the activity of the
W251N mutant enzyme. The rituximab peptide
mixture was incubated with Endo-M wild-type
enzyme, the W251N variant, and the IgG-specific
endoglycosidase EndoS (8). The released glycans
were collected, derivatized by permethylation, and
analyzed by MALDI-TOF/MS (Fig. 4B-D). In the
reaction with the wild-type enzyme (Fig. 4B), the
released N-glycans could be scarcely detected,
owing to the resistance of core fucosylated
glycans to Endo-M wild-type enzyme, whereas
6
Next, to determine whether the W251N
substitution
affects
the
specificity
for
non-reducing terminal structures of N-glycan
substrates, we examined the relative activities
with various pyridylaminated (PA-) glycan
substrates (Table 3). The wild-type enzyme acted
well on the afucosylated substrates, but not on the
core-fucosylated glycans; its specific activities
with the substrates were in the following
decreasing order: Man3, Man5, asialo-, sialo-, and
agalacto-biantennary
complex-type
glycans.
However, the W251N variant, which could act on
afucosylated PA-Man3 substrates unlike on
afucosylated biotinylated substrates, showed
approximately 30% of the wild-type enzyme’s
activity with afucosylated PA-Man3, thus showing
a pattern similar to that of the wild-type enzyme;
the order of preference for core-fucosylated
substrates was similar between the wild-type
enzyme and the W251N variant (M3F > asialo- >
agalacto-). The difference between the enzymatic

Endo-M Variant Acting on Core-fucosylated N-Glycans
confirmed that both enzymes could release
various N-glycans, including Man5 and complex
biantennary structures, from hLF (Fig. 5C). Note
that the signal intensities of glycans from W251N
enzyme treatment were greater than that of
glycans from the wild-type enzyme treatment,
probably due to the increased availability of core
fucosylated N-glycans; this is consistent with the
result of the SDS-PAGE analysis.
they could be detected in the reaction with the
W251N variant (Fig. 4C). As indicated in the
figures, the MALDI-TOF/TOF analysis revealed
that the deduced structures correspond to
endoglycosidase-released structures with a single
GlcNAc residue at the reducing ends of N-glycan.
The glycan signal pattern was almost identical to
that from the reaction mixture with EndoS (Fig
4D), indicating that W251N can act on most of the
glycovariants of the IgG glycopeptide.
Evaluation of the transglycosylation
activity of W251N variant and the generation of
glycosynthase-like enzyme N175Q/W251N
Human lactoferrin (hLF) is a glycoprotein
that has mostly core-fucosylated glycans at the
two N-glycosylation sites (48, 49)—this was
confirmed by MS analysis (Fig. 5A, and STable
S2). We used hLF to characterize further the
hydrolytic activity of W251N. Human LF was
treated with EndoS, wild-type Endo-M, and
Endo-M W251N variant and then separated by
SDS-PAGE and stained by Coomassie Brilliant
Blue (Fig. 5B). In the untreated control, a major
part of the hLF was observed slightly above a
80-kDa protein marker. EndoS treatment removed
only a single sugar chain, as revealed by the
emergence of a lower band. This result was
unchanged by treatment with increasing amounts
of EndoS—up to 1,000 U (approximately 1.5 μg)
(data not shown). Treatment with wild-type
Endo-M (2 μg) showed a pattern similar to that of
EndoS treatment. On the other hand, upon
treatment with Endo-M W251N variant, a new
band emerged further below, indicating the
presence of a completely deglycosylated form of
hLF. Subsequent MALDI-TOF/MS analyses of
permethylated glycans that were released by
treatment with wild-type Endo-M and W251N
One of the advantages of using Endo-M is
that its transglycosylation activity enables the
synthesis of novel sialoglycoconjugates. Hence,
we next examined the ability of the W251N
mutant enzyme to facilitate transglycosylation.
Incubation
of
W251N
with
Fucα1-6GlcNAc-biotin as the acceptor and
sialylglyco-oxazoline (SG-oxazoline) as the donor
resulted in the detection of a new product peak in
HPLC analysis. Fig. 6A shows a representative
HPLC profile of the transglycosylation reaction
products produced by the N175Q/W251N double
mutant, which will be described below. The
molecular mass of the product was m/z 2719 [M +
3Na]+
, according to MALDI-TOF/MS analysis.
Moreover, in the MALDI-TOF/TOF-MS analysis
of the product peak, the signature ions for
sialylated glycans were detected, indicating that
the
sialylglycoconjugate
(Neu5Ac
product
was
a
newly
generated
2Gal2GlcNAc2Man3GlcNAc2Fuc1-biotin)
7

Endo-M Variant Acting on Core-fucosylated N-Glycans
(Fig. 6B). The product was hydrolyzed by Endo
F3 which can hydrolyze core fucosylated
biantennary complex-type N-glycans, but not by
expected, the double mutant showed the
characteristics of both enzymes: similar
(maximum) yield with both fucosylated and
afucosylated acceptor substrates, but no apparent
yield decline during the prolonged incubation time
because of the loss of its hydrolytic activity (Fig.
6C and D), suggesting that both effects of N175Q
and W251N on the reaction are independent of
each other. Furthermore, to increase the product
yield, we tested the effect of adding
wild-type Endo-M, thus confirming that
a
N,N’-chitobiose core was formed in the
transglycosylation reaction by W251N (data not
shown). The yield of the transglycosylation
product with 5 mM Fucα1-6GlcNAc-biotin and 5
mM SG-oxazoline (1:1 ratio) reached up to about
5% after a 2-h incubation; however, this was
followed by a decline after a prolonged reaction
time, presumably due to the degradation of the
transglycosylation product by the inherent
hydrolytic activity of the W251N enzyme (Fig.
6C). On the other hand, N175Q, a Endo-M
glycosynthase-like mutant, scarcely synthesized
the transglycosylation product with the
fucosylated acceptor (Fig. 6C). In contrast, with
the GlcNAc-biotin as the acceptor substrate,
N175Q generated more than 95% yield in a 1-h
reaction with a marginal reduction of the product
over the incubation time (Fig. 6D), suggesting that
Trp 251 is critical for the recognition of
Fucα1-6GlcNAc-biotin as an acceptor. Moreover,
the relatively low yield of W251N with
GlcNAc-biotin (less than 10%) indicates that the
W251N substitution reduces the preference for
GlcNAc as the acceptor substrate. This was
supported by the difference in their Km values—
3.5 ± 0.8 mM for N175Q with 4-nitrophenyl
(pNP-) GlcNAc (33) and 226 ± 29.2 mM for
W251N with GlcNAc-biotin. As mentioned above,
the N175Q mutant retains its transglycosylation
activity but lacks hydrolytic activity. We then
generated a double mutant, N175Q/W251N. As
dimethylsulfoxide
(DMSO)
to
the
transglycosylation reaction. Adding 5% or 10%
DMSO in the reaction solution enhanced the
transglycosylation yield at 2 h (approximately
20% increase in 10% DMSO compared to the
reaction without DMSO), likely due to increased
substrate solubility. The effect of donor substrate
concentration was also examined using the
N175Q/W251N double mutant (Fig. 6E). The
yields were almost proportionally increased by
more than 22% when 20 mM SG-oxazoline was
added, in which the donor: acceptor ratio was 4:1.
This result indicated that the Endo-M
N175Q/W251N double mutant may be practically
useful for synthesizing various core-fucosylated
glycoconjugates.
DISCUSSION
The lack of substrate specificity of
wild-type Endo-M for core-fucosylated glycans
limits its scope of application for glycoproteins
that are core-fucosylated. In the present study, we
found that Trp 251 of Endo-M is an important
residue that determines its substrate preference for
8

Endo-M Variant Acting on Core-fucosylated N-Glycans
has an alanine residue at this position, and plant
Endo-Os from rice Oryza sativa, which has a
threonine residue at this position (53). The
tryptophan residues often interact with residues of
the glycan substrates and stabilize the binding of
the substrate in the subsite. Therefore, we
speculate that Trp 251 of wild-type Endo-M may
be interacting with the innermost GlcNAc residue
of the glycan, possibly due to the proximity
between these residues. Fucosylation of the
innermost GlcNAc residue might inhibit its
interaction. In addition, future elucidation of the
three-dimensional structure of the W251N mutant
enzyme will give insights into the role of the
asparagine residue in its interaction with
core-fucosylated glycan residues and reveal the
factors that define its substrate preference.
core-fucosylated glycan substrates. Among W251
mutants, the W251N variant showed the highest
hydrolytic activity with core-fucosylated glycans
and exhibited transglycosylation activity in a
reaction mixture with α1,6-fucosyl GlcNAc-biotin
as the acceptor. The yield of the transglycosylation
product with the fucosylated acceptor substrate
reached up to 22% relative to the control reaction.
Our findings will facilitate the efficient production
of homogeneous glycoforms of various
glycoconjugates
including
glycopeptides/glycoproteins.
The replacement of a single amino acid
residue from tryptophan to asparagine at position
251 of Endo-M changed the substrate preference
of this enzyme from afucosylated glycans to
core-fucosylated glycans. As shown in Table 4,
the asparagine residue at this position is conserved
among several GH85 enzymes, including bacterial
Endo-D, Endo-CE from nematode (50), and some
mammalian ENGases (2). On the other hand, the
tryptophan residue is conserved among enzymes
such as bacterial Endo-A, Endo-BH from Bacillus
halodurans C-125 (51), EndoBB from
Bifidobacterium longum DJO10A (BLD_0197)
(52), fungal Endo-M, Endo-CC1 (16), and
Endo-Om from methylotrophic yeast Ogatae
minuta (15). Although there appears to be no clear
correlation between the Trp/Asn residue and
substrate specificity of these enzymes, it is likely
that the enzymes with a tryptophan residue at that
position do not act on core-fucosylated glycans,
whereas the enzymes having an asparagine at that
position can act on both types of substrates. There
are exceptions, such as fungal Endo-CC2, which
Wang and colleagues have extensively
explored the usefulness of endoglycosidases in a
convergent chemoenzymatic approach for the
N-glycosylation of innately core-fucosylated
glycoproteins, including the IgG molecule. The
Endo-D glycosynthase N322Q mutant is capable
of transferring a trimannosyl sugar donor onto an
α1,6-fucosyl GlcNAc on IgG (40). The EndoS
glycosynthase D233Q mutant has been used
successfully for the conversion of N-glycans of
the Fc region in IgG (43). Endoglycosidase
Endo-F3 from Elizabethkingia meningosepticum
and its glycosynthase variant have been shown to
exhibit transglycosylation activity to synthesize
core-fucosylated sialoglycopeptides (41, 42).
Recently, Endo-S2 was reported to possess a more
relaxed substrate specificity for donor and
acceptor substrates with a higher efficiency than
9

Endo-M Variant Acting on Core-fucosylated N-Glycans
GlcNAc-biotin),
EndoS (46). Efforts to develop an enzyme that
acts on various N-glycan donor species and
various glycoconjugates including glycoproteins
are successfully expanding the applicability of this
chemoenzymatic approach.
α1,6-fucosyl-N-acetylglucosaminyl biotin (F1021:
Fucα1-6GlcNAc-biotin), trimannosyl chitobiosyl
biotin (Man
3GlcNAc2-biotin, M3-biotin), and
core-α1,6-fucosylated trimannosyl chitobiosyl
biotin (Man3GlcNAc2Fuc1-biotin, M3F-biotin)
were supplied by Tokyo Chemical Industry Co.,
Ltd. (Supplemental Scheme S1 and Fig. S1).
Pyridylaminated (PA-) oligosaccharide substrates
were purchased from TaKaRa Bio Inc. (Otsu,
Japan) or Masuda Chemical Industries Co., Ltd.
(Takamatsu, Japan). Rituximab (Rituxan®), an
anti-CD20 monoclonal antibody, was obtained
from Zenyaku Kogyo Co., Ltd. (Japan).
(Neu5Ac1Gal1GlcNAc1)2Man3GlcNAc-oxazoline
(SG-oxazoline) was prepared, as described
previously (36). Human lactoferrin and Endo F3
were purchased from Sigma-Aldrich (St. Louis,
MO, USA). PNGase F was purchased from Roche
Diagnostics GmbH (Mannheim, Germany).
EndoS was purchased from New England
BioLabs (Ipswich, MA, USA).
By site-directed mutagenesis, we isolated an
Endo-M mutant enzyme exhibiting altered
substrate specificity compared to wild-type
Endo-M. To the best of our knowledge, this is the
first report that endoglycosidase substrate
specificity was altered by site-directed
mutagenesis. This finding will lead to a novel
approach for developing new enzymes with
broader substrate specificity, rather than isolating
novel enzymes from nature. The Endo-M W251N
variant could act on core fucosylated
glycopeptides derived from IgGs and the native
form of human lactoferrin. Although intact IgG
molecules may not be as good substrates for
W251N as that for EndoS, glycopeptides and
some globular glycoproteins like hLF, may be
good substrates. This finding can open up new
avenues and provide novel options for enzymatic
tools in the field of glycobiology. It is one option
available for efficient use in convergent
chemoenzymatic synthesis of glycoprotein-based
therapeutics that have specific glycoforms for
enhanced potency.
Site-directed Mutagenesis
Site-directed mutagenesis to generate
Endo-M mutant enzymes was performed by PCR
using KOD-Plus DNA polymerase (Toyobo,
Japan), according to the procedure prescribed in
the QuikChange site-directed mutagenesis kit
(Agilent Technologies, Santa Clara, CA, USA). A
pET23b-Endo-M plasmid containing the cDNA
fragment of wild-type Endo-M fused with a
hexahistidine tag at the C-terminus, was used as
the template DNA (17). The following primers
EXPERIMENTAL PROCEDURES
Reagents
N-Acetylglucosaminyl biotin (G0297:
10

Endo-M Variant Acting on Core-fucosylated N-Glycans
and their complementary strands were used: 5’-
GTGTTTGGTACTTTTTTAGTAGAATGGAATA
by centrifugation. The clear supernatant obtained
was then loaded onto a 1-mL Ni2+-charged
His-Trap chelating column (GE healthcare, Little
Chalfont, UK) pre-equilibrated with the binding
buffer (20 mM sodium phosphate, pH 7.5,
containing 0.5 M sodium chloride and 10 mM
imidazole). Unbound proteins were washed out
with the binding buffer. Endo-M mutants were
eluted with the elution buffer (20 mM sodium
phosphate, pH 7.5, containing 0.5 M sodium
chloride and 100 mM imidazole). The eluates were
desalted and buffer-changed to 20 mM sodium
phosphate, pH 7.2, using Amicon Ultra filter
device (30K, Millipore). Endo-M mutants were
further purified by Mono Q anion exchange
column chromatography. The enzyme solutions
were loaded onto a Mono Q 5/50 GL column (GE
healthcare) pre-equilibrated with 20 mM sodium
phosphate, pH 7.2. The elution was carried out
using a linear gradient of 20 mM sodium
phosphate, pH 7.2, containing 1 M sodium
chloride (concentrated up to 50%), in 20 column
volumes. The eluates were analyzed by
SDS-PAGE/Coomassie Brilliant Blue staining and
the fractions containing purified Endo-M mutant
enzymes were combined and concentrated using
Amicon Ultra filter devices (30K). The protein
concentrations were quantified using the
bicinchoninic acid (BCA) protein assay kit
(Thermo Scientific) and used in the following
enzyme assay.
ACCAAATGCATG-3’ (for
GTAGAAGGAAATAACGCAATGCATGAAAT
GGAAGCCTTGC-3’ (for Q128A); 5’-
GTAGAAGGAAATAACTCAATGCATGAAATG
GAAGCCTTGC-3’ (for Q128S); 5’-
G125W);
5’-
GACAAATGAAGGAGAAATCCACCACCAGA
ACCAGCTCACATGG-3’ (for W228H); 5’-
CGGATGGTATTTTTTTGAATTATGCGTGGAA
AAAAGAATACCCTG-3’ (for W251A); and 5’-
CGGATGGTATTTTTTTGAATTATAACTGGAA
AAAAGAATACCCTG-3’ (for W251N). For
saturation mutagenesis of the Trp 251 residue, a
forward
primer,
5’-CGGATGGTATTTTTTTGAATTATNNSTGG
AAAAAAGAATACCCTG-3’, and
primer,
a
reverse
5’-
CAGGGTATTCTTTTTTCCASNNATAATTCAA
AAAAATACCATCCG-3’, were used. Mutations
were confirmed by DNA sequencing.
Protein Expression in Escherichia coli and
Purification of Recombinant Endo-M
E. coli strain BL21 (DE3) was transformed
with the plasmid containing mutated Endo-M
genes, and the transformants were cultured in
Luria-Bertani medium containing 100 mg/liter
ampicillin, at 19°C for 38 h to express Endo-M
mutants at a basal level. The cells were harvested
and lysed in BugBuster Master Mix (Merck
Millipore, Darmstadt, Germany) supplemented
with 1 mM phenylmethylsulfonyl fluoride, and the
cell debris and insoluble materials were removed
Enzyme Assays
Assays for the hydrolytic activity of the
11

Endo-M Variant Acting on Core-fucosylated N-Glycans
Endo-M enzymes on biotinylated oligosaccharides
were performed by incubating 3 mM M3-biotin or
M3F-biotin (as the substrate) with 20-200 ng of
the enzyme in 30 mM sodium phosphate buffer
(pH 6.0), at 30°C. The reaction was stopped by
heating at 95°C for 5 min. The reaction mixtures
were analyzed by thin-layer chromatography or
high performance liquid chromatography, as
described in the below sections. To examine the
relative activity of the mutant enzyme with
PA-oligosaccharides as the substrate, 4 mM
PA-oligosaccharides were incubated with either
400 ng wild-type or 1 μg W251N mutant enzyme
in 20 mM sodium phosphate buffer, pH 6.0, at
30°C for 20 min. The reactions were stopped by
heating at 95°C for 5 min. The reaction mixtures
were analyzed by high performance liquid
chromatography.
visualize the saccharides by color development,
the plate was sprayed with orcinol-sulfuric acid
reagent and heated in a toaster.
High
Performance
Liquid
Chromatography (HPLC) Analysis
Analytical
reversed-phase
high
performance liquid chromatography (RP-HPLC)
was performed on a Waters e2695 separation
module with a Cosmosil 5C18-AR-II column (4.6
× 150 mm; Nacalai Tesque, Japan) at 40°C. The
biotinylated oligosaccharides were eluted using an
isocratic solvent system—90% solvent A (0.1%
trifluoroacetic acid)/10% solvent
B
(0.1%
trifluoroacetic acid in acetonitrile)—at a flow rate
of 0.5 mL/min, along with UV monitoring at 214
nm using a Waters 2998 Photodiode Array
Detector. For PA-oligosaccharides, RP-HPLC was
carried out using a Cosmosil 5C18-AR-II column
(4.6 × 150 mm) at 40°C and the elution was done
using an isocratic solvent system—96% solvent A
(100 mM acetic acid-triethylamine, pH 4.0)/4%
solvent B (100 mM acetic acid-triethylamine, pH
4.0, containing 0.5% n-butanol) at a flow rate of
1.0 mL/min, accompanied by monitoring the
fluorescence of the pyridylamino group
(excitation, 320 nm; emission, 400 nm) using a
Waters 2475 Multi-wavelength Fluorescence
Detector.
Assay for the transglycosylation activity of
the Endo-M mutant enzyme was performed at
30°C by incubating 5–20 mM SG-oxazoline (as
the glycan donor substrate) with
5
mM
Fucα1-6GlcNAc-biotin or GlcNAc-biotin (as the
acceptor substrate) and 0.66 μg Endo-M mutant
enzyme, in 25 mM sodium phosphate buffer, pH
6.5. The reaction was stopped by heating at 95°C
for 5 min.
Thin-layer
Analysis
Chromatography
(TLC)
TLC was performed using silica gel
60-coated aluminum plates (Merck). The samples
were spotted on the plates and developed in a
solvent (n-butanol:acetic acid:water::3:2:2). To
Preparation
of
Tryptic
Glycopeptide/peptide Mixture of Rituximab and
Human Lactoferrin
12

Endo-M Variant Acting on Core-fucosylated N-Glycans
Rituximab (800 μg) or human lactoferrin
(200 μg) was incubated in 40 mM ammonium
bicarbonate at 55°C for 45 min in the presence of
10 mM dithiothreitol. After it cooled down to
room temperature, iodoacetamide was added to a
final concentration of 30 mM and the tube was
incubated at room temperature for 1 h in the dark.
The alkylated glycoprotein was then digested
using 5 μg of Sequencing Grade Modified Trypsin
(Promega) at 37°C. The tryptic peptides were
dried, reconstituted in 5% acetic acid, and applied
out, according to the standard method (54),
followed by Matrix-assisted laser desorption
ionization time-of-flight (MALDI-TOF)/mass
spectrometry (MS) and MALDI-TOF/TOF-MS
analyses, as described below.
Enzymatic
Lactoferrin
Treatment
of
Human
Ten micrograms of human lactoferrin
(hLF) was treated overnight with EndoS
(100-1,000 U), wild-type Endo-M (2 μg), or the
W251N variant (2 μg). Aliquots of reaction
mixtures were resolved by SDS-PAGE on 7.5%
acrylamide gel and visualized by Coomassie
Brilliant Blue staining. The released N-glycans
were purified, as described above, and analyzed
by MALDI-TOF/MS.
onto
a
C18 cartridge column (Waters)
pre-equilibrated with 5% acetic acid, followed by
sequential elution with 20% 2-propanol/5% acetic
acid and 40% 2-propanol/5% acetic acid. The
eluates were evaporated to dryness in a Speed-Vac
concentrator.
Release of N-Glycans from Glycopeptides
Matrix-assisted
Laser
Desorption
by Peptide : N-glycanase F or Endoglycosidases
Ionization Time-of-flight (MALDI-TOF) Mass
Spectrometry Analysis
For N-glycans of rituximab, the
glycopeptide/peptide mixture equivalent to 200 μg
rituximab was treated overnight with 1 U of
PNGase F, 4 μg of wild-type Endo-M, 4 μg of
W251N mutant Endo-M, and EndoS (100 U). For,
N-glycans of lactoferrin, the glycopeptide mixture
from 200 μg of lactoferrin was treated with 1 U of
PNGase F. These reaction mixtures were
The permethylated glycan samples were
reconstituted in 2,5-dihydroxybenzoic acid
(DHBA) matrix solution (10 mg/mL DHBA in
50% methanol) and then analyzed by
MALDI-TOF/MS or MALDI-TOF/TOF-MS
using UltrafleXtreme (Bruker Daltonics, Billerica,
MA, USA) in the positive ion mode.
evaporated
in
Speed-Vac
concentrator,
reconstituted in 5% acetic acid, and applied onto a
C18 cartridge column (Waters). The released
N-glycans were collected in the flow-through
fractions and lyophilized. Permethylation of the
endoglycosidase-released N-glycans was carried
13

Endo-M Variant Acting on Core-fucosylated N-Glycans
Acknowledgements: We thank Dr. Yoshinobu Kimura, Okayama University, for valuable discussions.
This work was partly funded by the Ministry of Economy, Trade and Industry of Japan for the ”Project
focused on developing key technology of discovering and manufacturing drug for next-generation
treatment and diagnoses” and partly by a Grant-in-Aid for Scientific Research from the MEXT
KAKENHI Grant Number 25292063. A part of this work was conducted in Japan Advanced Institute
of Science and Technology (JAIST), supported by the Nanotechnology Platform Program (Molecule and
Material Synthesis) of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT),
Japan. Employment of T. Katayama was supported by the Institute for Fermentation, Osaka.
Conflict of interest: The following authors report that they have no disclosures relevant to this
publication: T. Katayama and Y. T. The following authors disclose actual or potential conflicts of a patent
application filed by Tokyo Chemical Industry Co., Ltd.: T. Katoh, and K. Y. The three co-authors, Y. N., J.
K., and Y. M., are employees of Tokyo Chemical Industry Co., Ltd.
Author contributions: T. Katoh, T. Katayama, and K. Y. designed the study and wrote the manuscript. T.
Katoh, T. Katayama, Y. T., Y. N., J. K., and Y. M. performed the experiments and discussed the data. All
authors reviewed the results and approved the final version of the manuscript.
14

Endo-M Variant Acting on Core-fucosylated N-Glycans
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sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor
hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline. Biochim. Biophys.
Acta. 1800, 1203–1209
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André, S., Kozár, T., Schuberth, R., Unverzagt, C., Kojima, S., and Gabius, H.-J. (2007)
Substitutions in the N-glycan core as regulators of biorecognition: the case of core-fucose and
bisecting GlcNAc moieties. Biochemistry. 46, 6984–6995
Shields, R. L., Lai, J., Keck, R., O’Connell, L. Y., Hong, K., Meng, Y. G., Weikert, S. H. a, Presta,
L. G., Gloria Meng, Y., Weikert, S. H. a, and Presta, L. G. (2002) Lack of fucose on human IgG1
N-linked oligosaccharide improves binding to human FcγRIII and antibody-dependent cellular
toxicity. J. Biol. Chem. 277, 26733–26740
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Shinkawa, T., Nakamura, K., Yamane, N., Shoji-Hosaka, E., Kanda, Y., Sakurada, M., Uchida, K.,
Anazawa, H., Satoh, M., Yamasaki, M., Hanai, N., and Shitara, K. (2003) The absence of fucose
but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type
oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J.
Biol. Chem. 278, 3466–3473
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43.
Fan, S. Q., Huang, W., and Wang, L. X. (2012) Remarkable transglycosylation activity of
glycosynthase mutants of endo-D, an endo-β-N-acetylglucosaminidase from Streptococcus
pneumoniae. J. Biol. Chem. 287, 11272–11281
Huang, W., Li, J., and Wang, L. X. (2011) Unusual Transglycosylation Activity of
Flavobacterium meningosepticum Endoglycosidases Enables Convergent Chemoenzymatic
Synthesis of Core Fucosylated Complex N-Glycopeptides. ChemBioChem. 12, 932–941
Giddens, J. P., Lomino, J. V., Amin, M. N., and Wang, L.-X. (2016) Endo-F3 glycosynthase
mutants enable chemoenzymatic synthesis of core fucosylated tri-antennary complex-type
glycopeptides and glycoproteins. J. Biol. Chem. 291, 9356-9370.
Huang, W., Giddens, J., Fan, S. Q., Toonstra, C., and Wang, L. X. (2012) Chemoenzymatic
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glycoengineering of intact IgG antibodies for gain of functions. J. Am. Chem. Soc. 134,
12308–12318
44.
45.
Parsons, T. B., Struwe, W. B., Gault, J., Yamamoto, K., Taylor, T. A., Raj, R., Wals, K.,
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Glycosylation of a Therapeutic Antibody. Angew. Chemie. Int. Ed. 55, 2361-2367
Kurogochi, M., Mori, M., Osumi, K., Tojino, M., Sugawara, S. I., Takashima, S., Hirose, Y.,
Tsukimura, W., Mizuno, M., Amano, J., Matsuda, A., Tomita, M., Takayanagi, A., Shoda, S. I.,
and Shirai, T. (2015) Glycoengineered monoclonal antibodies with homogeneous glycan (M3, G0,
G2, and A2) using a chemoenzymatic approach have different affinities for FcγRIIIa and variable
antibody-dependent cellular cytotoxicity activities. PLoS One. 10, 1–24
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Li, T., Tong, X., Yang, Q., Giddens, J. P., and Wang, L.-X. (2016) Glycosynthase Mutants of
Endoglycosidase S2 Show Potent Transglycosylation Activity and Remarkably Relaxed Substrate
Specificity for Antibody Glycosylation Remodeling. J. Biol. Chem. 291, 16508–16518
Shang, T. Q., Saati, A., Toler, K. N., Mo, J., Li, H., Matlosz, T., Lin, X., Schenk, J., Ng, C. K.,
Duffy, T., Porter, T. J., and Rouse, J. C. (2014) Development and application of a robust N-glycan
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glycoproteins. J. Pharm. Sci. 103, 1967–1978
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van Berkel, P. H., van Veen, H. A., Geerts, M. E., de Boer, H. A., and Nuijens, J. H. (1996)
Heterogeneity in utilization of N-glycosylation sites Asn624 and Asn138 in human lactoferrin: a
study with glycosylation-site mutants. Biochem. J. 319, 117–122
Yu, T., Guo, C., Wang, J., Hao, P., Sui, S., Chen, X., Zhang, R., Wang, P., Yu, G., Zhang, L., Dai,
Y., and Li, N. (2011) Comprehensive characterization of the site-specific N-glycosylation of
wild-type and recombinant human lactoferrin expressed in the milk of transgenic cloned cattle.
Glycobiology. 21, 206–224
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Kato, T., Fujita, K., Takeuchi, M., Kobayashi, K., Natsuka, S., Ikura, K., Kumagai, H., and
Yamamoto, K. (2002) Identification of an endo-β-N-acetylglucosaminidase gene in
Caenorhabditis elegans and its expression in Escherichia coli. Glycobiology. 12, 581–587
Fujita, K., Takami, H., Yamamoto, K., and Takegawa, K. (2004) Characterization of
Endo-β-N-acetylglucosaminidase from Alkaliphilic Bacillus halodurans C-125. 68, 1059–1066
Garrido, D., Nwosu, C., Ruiz-Moyano, S., Aldredge, D., German, J. B., Lebrilla, C. B., and Mills,
D. A. (2012) Endo-β-N-acetylglucosaminidases from Infant Gut-associated Bifidobacteria Release
Complex N-glycans from Human Milk Glycoproteins. Mol. Cell Proteomics. 11, 775–785
Nakamura, K., Inoue, M., Maeda, M., Nakano, R., Hosoi, K., Fujiyama, K., and Kimura, Y.
(2009) Molecular cloning and gene expression analysis of tomato endo-β-N-acetylglucosaminidase,
an endoglycosidase involved in the production of high-mannose type free N-glycans during tomato
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Endo-M Variant Acting on Core-fucosylated N-Glycans
fruit ripening. Biosci. Biotech. Bioch. 73, 461–464
54.
Anumula, K. R., and Taylor, P. B. (1992) A comprehensive procedure for preparation of partially
methylated alditol acetates from glycoprotein carbohydrates. Anal. Biochem. 203, 101–108
FOOTNOTES
The following abbreviations have been used in this report: ENGase, endo-β-N-acetylglucosaminidase;
Fuc, L-fucose; Gal, galactose; GlcNAc, N-acetylglucosamine; Man, mannose; MS, mass spectrometry;
Neu5Ac, N-acetylneuraminic acid.
FIGURE LEGENDS
FIGURE 1. Comparison of the protein structures of GH85 enzymes A. The protein surface structure
around the catalytic core of Endo-A complexed with a trimannosyl thiazoline (Protein Data Bank
accession number: 3FHQ). B. The protein surface structure around the catalytic core of Endo-D
complexed with a thiazoline (Protein Data Bank accession number: 2W92). C. Superimposition of the
Endo-A and Endo-D structures (stereoview). The four amino acids of interest are indicated. D. Alignment
of the amino acid sequences around the proposed catalytic core of Endo-M, Endo-A, and Endo-D.
Residues Gly 125, Gln 128, Trp 228, and Trp 251 of Endo-M are highlighted in light gray. Residues Asn
175 and Glu 177, the putative catalytic residues, are highlighted in pink.
FIGURE 2. Evaluation of the hydrolytic activity of Endo-M mutants using core fucosylated
synthetic substrate A. Schematic of the reaction of trimannosyl biotinylated synthetic substrates with
and without core fucose, catalyzed by Endo-M. Abbreviations used are as follows: M, D-mannose; GN,
D-GlcNAc; F, L-fucose. B. TLC analysis of the hydrolysis of each substrate by Endo-M variants. C.
MALDI-TOF/MS analysis of the reaction mixtures of the W251N mutant enzyme with a core fucosylated
synthetic substrate, Man3GlcNAc2Fuc1-biotin. Upper panel, before adding the enzyme; lower panel, after
incubation with the enzyme. Cartoons of the estimated structures represent the following: blue square,
D-GlcNAc; green circle, D-mannose; red triangle, L-fucose.
FIGURE 3. Relative hydrolytic activities of Endo-M Trp 251 mutants on synthetic substrates
Endo-M W251 mutants were obtained by saturation mutagenesis, except the W251C mutant. The relative
20

Endo-M Variant Acting on Core-fucosylated N-Glycans
hydrolysis rates for both synthetic substrates (Man3GlcNAc2-biotin, open bars; Man3GlcNAc2Fuc1-biotin,
filled bars) were measured and the highest values were taken as 100%. Data were obtained using a single
assay.
FIGURE 4. MALDI-TOF/MS analyses of the glycans from rituximab glycopeptides, released by
treatment with various enzymes Glycopeptides of rituximab were prepared and treated with PNGase F
(A), wild-type Endo-M (B), W251N variant (C), and EndoS (positive control) (D). The released
oligosaccharides were collected, permethylated, and analyzed by MALDI-TOF/MS, as described in the
Experimental Procedures section. Cartoons of the estimated structures represent the following: purple
diamond, Neu5Ac; yellow circle, D-galactose; blue square, D-GlcNAc; green circle, D-mannose; red
triangle, L-fucose.
FIGURE 5. Hydrolysis of the N-glycans of human lactoferrin by the W251N variant A.
MALDI-TOF/MS spectrum of permethylated N-glycans released from the tryptic glycopeptides of human
lactoferrin by PNGase F. B. Human lactoferrin (hLF, 10 μg) was treated either with EndoS (100 U),
wild-type Endo-M (2 μg), or Endo-M W251N mutant (2 μg), and then the aliquots were separated by
SDS-PAGE and stained with Coomassie Brilliant Blue. The singly glycosylated hLF (the upper
deglycosylated (DG)-hLF band) was observed upon treatment with EndoS and wild-type Endo-M, as
indicated. However, the non-glycosylated hLF (the lower DG-hLF band) as well as singly glycosylated
hLF were detected upon treatment with the Endo-M W251N variant. C. MALDI-TOF/MS spectra of
permethylated glycans released from hLF samples by wild-type Endo-M (left panel) and the W251N
variant (right panel). The estimated glycan structures based on the m/z values of precursor ion mass and
MS/MS fragment (data not shown) are depicted in the figures. Cartoons of the estimated structures
represent the following: purple diamond, Neu5Ac; yellow circle, D-galactose; blue square, D-GlcNAc;
green circle, D-mannose; red triangle, L-fucose. Asterisks indicate the hexose polymer peaks.
FIGURE 6. Transglycosylation by the W251N mutant enzyme A. A reversed phase-HPLC profile of
the transglycosylation reaction of N175Q/W251N mutant enzyme with Fucα1-6GlcNAc-biotin as the
acceptor substrate and SG-oxazoline as the donor substrate after
a 1-h incubation. B.
MALDI-TOF/TOF-MS spectra of the transglycosylation products. A precursor MS peak at m/z 2719,
Neu5Ac2Hex5HexNAc4dHex1-biotin, [M + 3Na]+, was subjected to MALDI-TOF/TOF-MS analysis. The
fragment ion peaks in the MS/MS spectrum of m/z 2719 corresponds to the signature ions for sialylated
glycans: loss of a terminal Neu5Ac plus a sodium ion (m/z 2407), loss of two pairs of terminal Neu5Ac
plus a sodium ion (m/z 2093); loss of a Neu5Ac plus a sodium ion and a Neu5Ac-Hex-HexNAc moiety
plus a sodium ion (m/z 1727); a sodium adduct of dHex-HexNAc-biotin (m/z 695); and a sodium adduct
21

Endo-M Variant Acting on Core-fucosylated N-Glycans
of Neu5Ac (m/z 335). Cartoons of the structures represent the following; diamond, Neu5Ac; filled circle,
galactose; filled square, GlcNAc; open circle, mannose; triangle, L-fucose. C and D. Yields of the
transglycosylation product in the reaction of Endo-M mutants with either Fucα1-6GlcNAc-biotin (C) or
GlcNAc-biotin (D) as the acceptor substrate and SG-oxazoline as the donor substrate. Data represents the
average of three independent reactions with mean ± SD. E. Transglycosylation product yields from the
reaction of the Endo-M N175Q/W251N double mutant with increased amounts of SG-oxazoline donor
substrate in optimized reaction conditions.
22

Endo-M Variant Acting on Core-fucosylated N-Glycans
Table 1. Comparison of the hydrolysis activity of wild-type and mutant enzymes
Man3GlcNAc2-biotin
Man3GlcNAc2Fuc1-biotin
Endo-M
variants
Specific hydrolysis activitya Relative activityb
Specific hydrolysis activitya Relative activityb
μmol min-1mg-1
%
100
2.4
13
μmol min-1mg-1
<0.01
ndc
%
<0.2
-
Wild type
G125W
Q128A
Q128S
6.49
0.15
0.82
1.3
<0.01
<0.01
<0.01
0.13
<0.2
<0.2
<0.2
2.0
20
W228H
W251A
W251N
2.9
45
0.18
0.16
2.7
2.5
1.3
20
aThe hydrolysis activity was determined by a single assay using 2 μg/mL enzyme with 3 mM Man3GlcNAc2-biotin,
or 20 μg/mL enzyme with 3 mM core fucosylated Man3GlcNAc2-biotin.
bThe relative activity of each enzyme was calculated with respect to the activity of wild type enzyme with
Man3GlcNAc2-biotin as a substrate, which was set to 100.
cnd, not detected.
23

Endo-M Variant Acting on Core-fucosylated N-Glycans
Table 2. Kinetic parameters of the hydrolysis of biotinylated substrates catalyzed by wild-type and
W251N Endo-M
Man3GlcNAc2-biotin
Man3GlcNAc2Fuc1-biotin
Endo-M
kcat
Km
kcat/Km
mM-1s-1
46
kcat
s-1
Km
mM
kcat/Km
s-1
mM
mM-1s-1
WT
17.5 ± 2.5a
0.39 ± 0.11
0.21 ± 0.05
ndb
nd
nd
W251N
0.46 ± 0.04
2.2
27.7 ± 0.40
19.6 ± 0.53
1.4
aThe values were shown with standard errors (n = 3).
bnd, not determined.
24

Endo-M Variant Acting on Core-fucosylated N-Glycans
Table 3. Relative hydrolytic activity of wild-type Endo-M and W251N mutant on
PA-oligosaccharides
PA-oligosaccharide
Relative activity (%)a
Wild-type
W251N
100
40
PA-trimannosyl core (M3)
PA-oligomannose (M5)
100
33
PA-agalactosylbiantennary
PA-asialobiantennary
10
7.9
19
21
PA-sialobiantennary
17
24
PA-fucosyl trimannosyl core (M3F)
PA-fucosyl agalactosylbiantennary
PA-fucosyl asialobiantennary
< 0.1
< 0.1
< 0.1
18
8.8
9.2
Various PA-oligosaccharides were used as the substrate for these assays.
aThe relative activity of each enzyme for a given PA-oligosaccharide was determined using a single assay
and calculated with respect to the activity of M3, which was set at 100.
25

Endo-M Variant Acting on Core-fucosylated N-Glycans
Table 4. Conservation (and variance) of the amino acid residue at position 251 of Endo-M among
GH85 members and their preference for core-fucosylated glycan substrates.
Preference for core
Enzyme
Alignmenta
Reference
fucosylated glycans
Trp 251 of Endo-M
A D N F F A N F N W D K A K N D Y
A D E M F L N F W W T E D K L A G
4
3
2
2
2
2
2
2
2
2
3
3
0
1
7
7
6
6
5
7
6
3
1
0
8
5
4
5
1
7
9
4
7
0
3
5
Endo-D
Yes
No
No
No
(14)
(52)
(13)
(51)
(16)
(16)
(26, 27)
(15)
(53), pcc
(50)
(2)
EndoBB
A D S M F L N F W W R D Q R Q
A D S M F L N F W W W N H S Q E R
S S G F T N Y W W Y N D A P Q K
S S G L F T N Y A W Y N H F P Q R
T D G F L N Y W W K K E Y P E M
S D A F F S N Y W W N K N L Q E
C D G L F S N Y T W K A K Y P Q E
C D A Y L N Y N W K D K E L L R
-
-
Endo-A
Endo-BH
Endo-CC1
Endo-CC2
Endo-M
b
I
-
b
-
I
No
No
No
No
I
Endo-Om
Endo-Os
Endo-CE
HsENGase
MmENGase
I
b
C D G F F T N Y N W R E E H L E R
C D G F F T N Y N W R E D H L Q R
-
b
-
-
aGenBank accession number of amino acid sequences used for alignment are as follows: BAB62042.1
(Endo-D), AAN25135.1 (EndoBB), AAD10851.1 (Endo-A), BAB04504.1 (Endo-BH), XP_001839402
(Endo-CC1), XP_002911817 (Endo-CC2), BAB43869.1 (Endo-M), BAN20080.1 (Endo-Om),
BAF17177.1 (Endo-Os), BAB84821.1 (Endo-CE), AAM80487.1 (human ENGase), and BAC33415.1
(Mus musculus ENGase).
bno available information
cpersonal communication
26

A
W244
W216
C
P128
Y131
B
D
N413
H384
W292
S295
125128
Endo-M RNGVKCLGTFLVEGNNQMHEMEALLHGPPLLNNTDDPMRLWSPYYADQLVAIAKHYGFDG 171
Endo-A RNGVPILGNVFFPPTVYGGQLEWLEQMLEQEEDGSFPL-------ADKLLEVADYYGFDG 167
Endo-D RNGVPVYGTLFFNWSNSIADQERFAEALKQDADGSFPI-------ARKLVDMAKYYGYDG 318
**** *..:. .
: * : .
: . *:
* :*: :*.:**:**
175177
228
Endo-M WLFNIECEFFPFPTNPKFKAEELAKFLHYFKEKLHNEIPGSQLIWYDSMTNEGEIHWQNQ 231
Endo-A WFINQETEG-----ADEGTAEAMQAFLVYLQEQKPEG---MHIMWYDSMIDTGAIAWQNH 219
Endo-D YFINQETTGD----LVKPLGEKMRQFMLYSKEYAAKVNHPIKYSWYDAMTYNYGRYHQDG 374
:::* *
: .* : *: * :* :
: ***:*
*:
251
Endo-M LTWKNELFFK------NTDGIFLNYWWKKEYPEMARRVAEGIGRSGLEVYFGTDVWGRHT 285
Endo-A LTDRNKMYLQ-NGSTRVADSMFLNFWWRDQRQSNELAQA--LGRSPYDLYAGVDVEARG- 275
Endo-D LGEYNYQFMQPEGDKVPADNFFANFNWDKAKNDYTIATANWIGRNPYDVFAGLELQQGGS 434
* * :::
:*.:* *: * . .
* :**. ::: * ::
Figure 1. Katoh et al.

A
OH
O
O
S
OH
O
O
S
HO
HO
HO
HO
HO
HO
M3-biotin
M GN
3 1
GN-biotin
NH
HN
NH
HN
OH
O
OH
OH
OH
O
O
O
H
N
OH
O
OH
O
H
N
O
O
O
HO
HO
HO
HO
O
O
O
O
OH
NHAc
+
HO
HO
HO
NHAc
NHAc
O
O
O
NHAc
O
Endo-M WT
HO
HO
HO
HO
O
O
HO
HO
OH
OH
M3F-biotin
OH
OH
OH
O
Fα1-6GN-biotin
OH
O
O
S
HO
HO
HO
OH
HO
HO
HO
HO
O
S
OH
OH
O
M GN
3
OH
1
NH
O
HN
NH
HN
OH
OH
O
O
O
OH
O
OH
O
O
H
N
O
O
H
N
O
O
HO
HO
O
O
O
O
O
OH
NHAc
+
HO
HO
HO
NHAc
HO
O
O
NHAc
O
NHAc
O
Endo-M W251
mutants
HO
HO
HO
HO
HO
O
O
HO
OH
OH
1363.6
C
B
Fα1-6GN-biotin
M3GN1
biotin
[M+Na]+
177.0
449.5
M3F-biotin
673.6
biotin
[M+Na]+
177.0
200
[M+Na]+
1000
730.6
800
449.5
302.2
M3F-biotin
M3-biotin
400
600
1200
1400
1600
1800
2000
m/z
Figure 2. Katoh et al.

M3F-biotin
M3-biotin
100
80
60
40
20
0
WT A
(W)
G
V
L
I
S
T
M
P
F
Y
D
E
N
Q
K
R
H
Endo-M W251 mutants
Figure 3. Katoh et al.

A
B
2041.1
1836.0
2245.2
2250
2606.3
500
500
500
500
750
750
750
750
1000
1000
1000
1000
1250
1500
1750
2000
2500
2750
2750
2750
2750
3000
3000
3000
3000
1416.8
1250
1500
1750
2000
2250
2500
C
D
1620.9
1416.8
1825.0
1171.7
1250
2187.1
2250
2548.3
2500
1500
1750
2000
1416.8
1620.9
1825.0
2187.1
2250
1171.7
1250
2548.3
2500
1982.0
2000
1500
1750
m/z
Figure 4. Katoh et al.