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

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 Katayama^1,2, 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

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.caz y .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

Man₃GlcNAc₁and 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 K_mvalues

for Man₃GlcNAc₂-biotin between the two

enzymes. However, the K_mvalue of W251N for

the core-fucosylated substrate is more than

90-fold higher than that for the afucosylated one.

The k_catvalues 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 K_mvalues—

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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oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J.

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Fan, S. Q., Huang, W., and Wang, L. X. (2012) Remarkable transglycosylation activity of

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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

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Huang, W., Giddens, J., Fan, S. Q., Toonstra, C., and Wang, L. X. (2012) Chemoenzymatic

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and Shirai, T. (2015) Glycoengineered monoclonal antibodies with homogeneous glycan (M3, G0,

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antibody-dependent cellular cytotoxicity activities. PLoS One. 10, 1–24

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Endoglycosidase S2 Show Potent Transglycosylation Activity and Remarkably Relaxed Substrate

Specificity for Antibody Glycosylation Remodeling. J. Biol. Chem. 291, 16508–16518

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Duffy, T., Porter, T. J., and Rouse, J. C. (2014) Development and application of a robust N-glycan

profiling method for heightened characterization of monoclonal antibodies and related

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Caenorhabditis elegans and its expression in Escherichia coli. Glycobiology. 12, 581–587

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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

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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 activity^aRelative activity^b

μmol min^-1mg^-1

%

100

2.4

13

μmol min^-1mg^-1

<0.01

nd^c

%

<0.2

-

Wild type

G125W

Q128A

Q128S

6.49

0.15

0.82

1.3

<0.01

0.13

<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

k_cat

K_m

k_cat/K_m

mM^-1s^-1

46

k_cat

s^-1

K_m

mM

k_cat/K_m

s^-1

mM

mM^-1s^-1

WT

17.5 ± 2.5^a

0.39 ± 0.11

0.21 ± 0.05

nd^b

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

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

Alignment^a

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

3

0

1

7

6

5

7

6

3

1

0

8

5

4

5

1

7

9

4

7

0

3

5

Endo-D

Yes

No

(14)

(52)

(13)

(51)

(16)

(26, 27)

(15)

(53), pc^c

(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

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

S

OH

O

S

HO

M3-biotin

M GN

3 1

GN-biotin

NH

HN

NH

HN

OH

O

OH

O

H

N

OH

O

OH

O

H

N

O

HO

O

OH

NHAc

+

HO

NHAc

O

NHAc

O

Endo-M WT

HO

O

HO

OH

M3F-biotin

OH

O

Fα1-6GN-biotin

OH

O

S

HO

OH

HO

O

S

OH

O

M GN

3

OH

1

NH

O

HN

NH

HN

OH

O

OH

O

OH

O

H

N

O

H

N

O

HO

O

OH

NHAc

+

HO

NHAc

HO

O

NHAc

O

NHAc

O

Endo-M W251

mutants

HO

O

HO

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

750

1000

1250

1500

1750

2000

2500

2750

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.

Article Doi

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

DOI: 10.1074/jbc.M116.737395

Source and publish data:

Authors:

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

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

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