Synthetic trisaccharides reveal discrimination of: Endo -glycosidic linkages by exo -acting α-1,2-mannosidases in the endoplasmic reticulum

Article abstract of DOI:10.1039/d1ob00428j

A tri-antennary Man9GlcNAc2 glycan on the surface of endoplasmic reticulum (ER) glycoproteins functions as a glycoprotein secretion or degradation signal after regioselective cleavage of the terminal α-1,2-mannose residue of each branch. Four α-1,2-mannos

Full text of DOI:10.1039/d1ob00428j

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Synthetic trisaccharides reveal discrimination of
endo-glycosidic linkages by exo-acting α-1,2-
mannosidases in the endoplasmic reticulum†
Cite this: Org. Biomol. Chem., 2021,
19, 4137
Kyohei Nitta, Taiki Kuribara and Kiichiro Totani
*
A tri-antennary Man9GlcNAc2 glycan on the surface of endoplasmic reticulum (ER) glycoproteins func-
tions as a glycoprotein secretion or degradation signal after regioselective cleavage of the terminal α-1,2-
mannose residue of each branch. Four α-1,2-mannosidases—ER mannosidase I, ER degradation-enhan-
cing α-mannosidase-like protein 1 (EDEM1), EDEM2, and EDEM3—are involved in the production of these
signal glycans. Although selective production of signal glycans is important in determining the fate of gly-
coproteins, the branch-discrimination abilities of the α-1,2-mannosidases are not well understood. A
structural feature of the Man9GlcNAc2 glycan is that all terminal glycosidic linkages of the three branches
are of the α-1,2 type, while the adjacent inner glycosidic linkages are different. In this study, we examined
whether the α-1,2-mannosidases showed branch specificity by discriminating between different inner gly-
cosides. Four trisaccharides with different glycosidic linkages [Manα1-2Manα1-2Man (natural A-branch),
Manα1-2Manα1-3Man (natural B-branch), Manα1-2Manα1-6Man (natural C-branch), and Manα1-2Manα1-
4Man (unnatural D-branch)] were synthesized and used to evaluate the hypothesis. When synthesizing
these oligosaccharides, highly stereoselective glycosylation was achieved with a high yield in each case by
adding a weak base or tuning the polarity of the mixed solvent. Enzymatic hydrolysis of the synthetic tri-
saccharides by a mouse liver ER fraction containing the target enzymes showed that the ER α-1,2-manno-
sidases had clear specificity for the trisaccharides in the order of A-branch > B-branch > C-branch ≈
D-branch. Various competitive experiments have revealed for the first time that α-1,2-mannosidase with
inner glycoside specificity is present in the ER. Our findings suggest that exo-acting ER α-1,2-mannosi-
dases can discriminate between endo-glycosidic linkages.
Received 5th March 2021,
Accepted 13th April 2021
DOI: 10.1039/d1ob00428j
rsc.li/obc
protein.7 The M9-glycoprotein is recognized by the folding
sensor enzyme UDP-glucose:glycoprotein glucosyltransferase 1
Introduction
A Glc3Man9GlcNAc2 glycan is transferred to the nascent poly- (UGGT1),8–10 which checks the folding state. The partially
peptide in the side chain of the Asn residue of the consensus folded glycoprotein is glucosylated by UGGT1 before accelera-
Asn-Xaa-Ser/Thr sequence (Xaa is any amino acid other than tion of G1M9-glycoprotein folding by CNX/CRT. The folding
Pro) by the action of oligosaccharyl transferase1 in the endo- acceleration/folding check cycle is called the CNX/CRT cycle11
plasmic reticulum (ER) (Fig. 1). The Glc3Man9GlcNAc2-glyco- and it plays a pivotal role in the folding mechanism in glyco-
protein is converted to the Glc1Man9GlcNAc2 (G1M9)-glyco- protein quality control.12,13
protein by glucosidase I (GI)2 and glucosidase II (GII)3,4 and it
The completely folded M9-glycoprotein and misfolded M9-
interacts with lectin-like molecular chaperones calnexin glycoprotein have their outermost mannose trimmed by ER
(CNX)5 and calreticulin (CRT).6 Upon approaching the correct α-1,2-mannosidases [ER mannosidase I (ERManI),14 ER degra-
folding state, the binding between the G1M9-glycoprotein and dation-enhancing α-mannosidase-like protein 1 (EDEM1),15
CNX/CRT is weakened, and subsequent cleavage of the term- EDEM2,16 and EDEM317] to produce glycoproteins with the
inal glucose by GII produces the Man9GlcNAc2 (M9)-glyco- Man8AGlcNAc2 (M8A), Man8BGlcNAc2 (M8B) or Man8CGlcNAc2
(M8C) glycan.18,19 The M8B-glycoprotein is recognized by the
cargo-receptor ER-Golgi intermediate compartment 53 kDa
protein (ERGIC53),20 which transports completely folded glyco-
Department of Materials and Life Science, Seikei University, Musashino-shi, Tokyo,
180-8633, Japan. E-mail: ktotani@st.seikei.ac.jp
proteins from the ER to the Golgi apparatus. Thus, the M8B
†Electronic supplementary information (ESI) available: Additional figures
glycan functions as a secretion signal of properly folded glyco-
proteins. The M8A- or M8C-glycoproteins are further trimmed
(Fig. S1–S3), details of synthesis, characterization of new compounds, and
methods for enzyme assays. See DOI: 10.1039/d1ob00428j
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Fig. 1 Glycan trimming in ER glycoprotein quality control.
Fig. 2 Design of trisaccharide substrates for glycan specificity analysis
of ER α-1,2-mannosidases.
by ER α-1,2-mannosidases to produce the Man5GlcNAc2 (M5)-
glycoprotein. The M5-glycoprotein is excreted from the ER to
the cytosol and passed on to the proteasomal degradation
system through collaboration with various lectins represented
by osteosarcoma-amplified 9 (OS-9).21 Both M8A and M8C are
precursors for the degradation signals of misfolded glyco-
proteins. In this way, ER α-1,2-mannosidases produce different
glycan structures that serve as secretion or degradation signals
after regioselective cleavage of the terminal mannose of the tri-
antennary M9 glycan.
ER α-1,2-mannosidases are exo-acting enzymes belonging to
the glycoside hydrolase 47 family. ERManI reportedly cleaves
Manα-1,2 linkages at the B-branch of the M9 glycan to produce
the M8B glycan in vitro.14 This enzyme preferentially acts on
denatured glycoproteins and can cleave all Manα-1,2-linkages
of the M9-glycoprotein under high enzyme concentrations.22
According to a comparison of glycan profiles among single
knockout cells of various EDEMs, EDEM1, EDEM2, and
EDEM3 show mannosidase activity in vivo, but the activity of
each EDEM alone has not been detected in vitro.23 Recently, it
has been reported that EDEMs show mannosidase activity by
were responsible for the regioselectivity of ER α-1,2-
mannosidases.
In this study, we synthesized a series of trisaccharides
(A3,27 B3,28 C3, and D3) with different inner glycosidic lin-
kages (Fig. 2), and conducted glycan specificity analysis and
related competitive analysis of ER α-1,2-mannosidases using
these trisaccharides. The results clearly showed that the reac-
tivities of α-1,2-mannosidases in the mouse liver ER fraction
were in the order A-branch > B-branch > C-branch ≈ D-branch.
These findings suggest that the ER fraction contains exo-acting
α-1,2-mannosidases with the ability to discriminate endo-glyco-
sidic linkages.
Results and discussion
Synthesis of the trisaccharide substrates
forming a complex with ER-resident protein 46, protein di- First, we synthesized the monosaccharide units required for
sulfide isomerase, or TXNDC11 via their cysteine residue.24–26 the synthesis of the target trisaccharides (Scheme 1). DAST-
It has been suggested that EDEMs recognize the aglycone site mediated ring-opening fluorination29 was conducted with the
of glycoproteins through complex formation.
starting material 1 to give mannosyl fluoride 2 as a common
Most studies on the use of ER α-1,2-mannosidases have glycosyl donor for use in this study. We also used 1 to syn-
focused on discriminating the aglycone state of substrate gly- thesize glycosyl acceptors 5, 8, 10, and 11 for the formation of
coproteins because these enzymes are responsible for produ- α-1,2-, α-1,3-, α-1,6-, and α-1,4-linkages.
cing secretion and degradation signals. However, the regio-
Next, we examined the synthesis of the target trisaccharides
selectivity of ER α-1,2-mannosidases according to the struc- using the common glycosyl donor 2 and the relevant glycosyl
tural features of each branch has not been verified. To address acceptor (5, 8, 10, or 11) (Scheme 2 and Table 1). In the syn-
this issue, we focused on the differences in the inner glycosi- thesis of disaccharide 12, when glycosylation was performed in
dic linkages of each branch (Fig. 2). The A-, B- and C-branches toluene using AgOTf-Cp2HfCl2 as a promoter, disaccharide 12
of the M9 glycan have different inner glycosidic linkages of was obtained in 98% yield with 3% of the undesired β-product
Manα-1,2, Manα-1,3, and Manα-1,6, respectively. We hypoth- (entry 1). Because purification of the desired α-product from
esized that these differences in the inner glycosidic linkages the α/β-mixture was difficult, we explored the reaction con-
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Scheme 1 Synthesis of monosaccharide units.
ditions to improve the stereoselectivity. The α-selectivity in this
glycosylation is attributed to “neighboring group partici-
pation”30 in the cation intermediate (major pathway in Fig. 3).
In contrast, TfO− derived from AgOTf could coordinate from
the α-face to the oxocarbenium cation, and the subsequent
SN2-like attack of the glycosyl acceptor should result in the
undesired β-product (minor pathway in Fig. 3). Therefore, we
investigated the reaction conditions to neutralize TfOH gener-
ated in the reaction mixture by adding 2,6-di-tert-butyl-4-
methylpyridine (DTBMP)31 as an acid scavenger, and suc-
ceeded in synthesizing disaccharide 12 in 93% yield with
perfect α-selectivity (entry 2). The DTBMP-H+ ion produced in
the reaction mixture might form an ion pair with the triflate
anion, enable the suppression of the formation of α-contact
ion pairs in the minor pathway, and provide excellent stereo-
selectivity via the major pathway. Similar reaction conditions
were applied for subsequent glycosylation to give trisaccharide
14 in 84% yield with excellent α-selectivity (entry 3). To further
improve the product yield, the solvent was changed from
Scheme 2 Synthesis of the target trisaccharides. Glycosylation con-
ditions are listed in Table 1. (a) NaOMe, MeOH/THF = 1 : 1, v/v. (b) 20%
Pd(OH)2/C, H2, MeOH/THF = 1 : 1, v/v. (c) LiOH, H2O2 aq., THF.
toluene to CH2Cl2, which had higher polarity to stabilize the the addition of the base and adjustment of the ratio of sol-
intermediate cation, and a 95% yield was achieved but with vents with different polarities.
lower stereoselectivity (α/β = 67 : 33) (entry 4). The higher reac-
For the synthesis of disaccharide 17, we performed glycosy-
tivity under these reaction conditions might yield more of the lation using a mixture of CH2Cl2 and toluene because acceptor
β-product by decreasing neighboring group participation. 8 is insoluble in toluene. In this case, we were concerned
Although we tried to achieve both high yield and high stereo- about β-product formation not because of neighboring group
selectivity with a mixture of these two solvents (entries 5 and participation but because of the improved reactivity. When the
6), we could not improve on the results in entry 3. Therefore, reaction temperature was decreased to −40 °C, disaccharide 17
we investigated the reaction without DTBMP, which gave a was obtained in high yield with high α-selectivity (entry 8). In
high yield in entry 1 under non-neutralizing acidic glycosyla- the subsequent synthesis of trisaccharide 19, the optimum
tion conditions. We succeeded in synthesizing trisaccharide 14 conditions for synthesizing trisaccharide 14 (entry 7) were
with a 99% yield and α/β > 95 : 5 (entry 7). Because 14α and different, and although the resulting stereoselectivity was
14β could be separated and purified by silica gel column perfect, the yield was moderate at 62% (entry 9). Therefore, in
chromatography, these reaction conditions were identified as our experiments investigating the improvement of the yield
the optimum conditions. Through this series of experiments, using a solvent mixture to stabilize the cation intermediate, we
we found that the yield and stereoselectivity fluctuated with succeeded in synthesizing trisaccharide 19 quantitatively with
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Table 1 Examination of glycosylation reactions
Entry
Acceptor
Solvent
Reagents
Time (min)
Product (yield)
α/βd,e
1a
5
5
Toluene
Toluene
Toluene
CH2Cl2
Toluene/CH2Cl2 = 1 : 1
Toluene/CH2Cl2 = 2 : 1
Toluene
Toluene/CH2Cl2 = 1 : 1
Toluene
Toluene/CH2Cl2 = 1 : 1
Toluene
Toluene
AgOTf, Cp2HfCl2, MS AW-300
40
70
80
75
60
60
60
70
90
50
90
40
40
70
50
12 (98%)
12 (93%)
14 (84%)
14 (95%)
14 (93%)
14 (81%)
14 (99%)
17 (99%)
19 (62%)c
19 (quant.)
22 (90%)
24 (92%)
24 (quant.)
27 (90%)
29 (84%)
>95 : 5 f
α onlyg
α onlyg
67 : 33
84 : 16
89 : 11
>95 : 5h
α onlyg
α onlyg
>95 : 5h
α onlyg
>95 : 5h
α onlyg
α onlyg
α onlyg
2a
AgOTf, Cp2HfCl2, MS AW-300, DTBMP
AgOTf, Cp2HfCl2, MS AW-300, DTBMP
AgOTf, Cp2HfCl2, MS AW-300, DTBMP
AgOTf, Cp2HfCl2, MS AW-300, DTBMP
AgOTf, Cp2HfCl2, MS AW-300, DTBMP
AgOTf, Cp2HfCl2, MS AW-300
AgOTf, Cp2HfCl2, MS AW-300, DTBMP
AgOTf, Cp2HfCl2, MS AW-300
AgOTf, Cp2HfCl2, MS AW-300
AgOTf, Cp2HfCl2, MS AW-300, DTBMP
AgOTf, Cp2HfCl2, MS AW-300
AgOTf, Cp2HfCl2, MS AW-300, DTBMP
AgOTf, Cp2HfCl2, MS AW-300, DTBMP
AgOTf, Cp2HfCl2, MS AW-300
3a
13
13
13
13
13
8
18
18
10
23
23
11
28
4a
5a
6a
7a
8b
9a
10a
11a
12a
13a
14a
15a
Toluene
Toluene
Toluene
a Reaction performed at −20 to 0 °C. b Reaction performed at −40 °C. c Acceptor 18 was recovered in 27% yield. d The ratio of α/β was determined
1
by 1H NMR spectroscopy. e Stereochemistry was determined by JC–H coupling measurements32 using non-decoupling HMQC. f β detected: 3%
(1H NMR). g No β detected. h β detected: 1% (1H NMR).
14, 19, 24, and 29 were deprotected to convert them to the
target trisaccharides A3, B3, C3, and D3, respectively.
Glycan-specificity analysis of ER α-1,2-mannosidases
Next, we analyzed the glycan specificity of ER α-1,2-mannosi-
dases using the four types of synthesized trisaccharides.
Because the detection of the activity of the enzyme alone has
not been reported in vitro for EDEM1, EDEM2, and EDEM3, it
is difficult to analyze individual glycan specificity using the
recombinant enzyme. Therefore, we decided to use an ER frac-
tion extracted from biological samples as an enzyme source
containing the α-1,2-mannosidases. As a tissue for the extrac-
tion of the ER fraction, we selected the senescence-accelerated
mouse prone 6 (SAMP6)33 liver, which we have previously used
for the analysis of the activity of ER α-1,2-mannosidases using
an M9-type synthetic glycan substrate.18,34
Extraction of the ER fraction from the SAMP6 liver was per-
formed
by
centrifugal
fractionation.18,34
Because
α-mannosidase is also present in the Golgi apparatus,35 the
ER/Golgi content of each centrifugal fraction was verified by
western blotting using both ER and Golgi marker proteins as
indicators. This proved that the extracted ER fraction had no
Golgi contamination (Fig. S1†).
Fig. 3 Plausible mechanism of the glycosylation reaction.
Synthetic trisaccharides A3, B3, C3, and D3 were incubated
α/β > 95 : 5 (entry 10). Disaccharides 22 and 27 were both syn- with the ER fractions at 37 °C, and the time courses of the
thesized in high yields with high stereoselectivity (entries 11 yields of disaccharides A2, B2, C2, and D2 in the reaction mix-
and 14) using the same reaction conditions as in the synthesis tures were analyzed quantitatively by HPLC (Fig. 4a). The
of disaccharide 12 (entry 2). In the preparation of trisaccharide hydrolysis yields for A3, B3, C3, and D3 at 8 h were 66%, 69%,
24, we synthesized the target product with complete 35%, and 35%, respectively (Fig. 4b). These results suggest
α-selectivity using the reaction conditions with DTBMP (entry that the reactivities of ER α-1,2-mannosidases change accord-
13) because it was difficult to separate and purify trace ing to differences in the inner glycosidic linkage of the sub-
amounts of the undesired β-product generated under the reac- strate trisaccharide, as we showed in the working hypothesis.
tion conditions without DTBMP (entry 12). Trisaccharide 29 D3 has an inner α-1,4-linkage that does not exist in the M9-
was synthesized in high yield with high stereoselectivity (entry type glycan, which is a substrate for ER α-1,2-mannosidases
15) using the optimum conditions used for the synthesis of tri- in vivo. We proposed two hypotheses for the hydrolysis of D3.
saccharide 14 (entry 7). Finally, the protected trisaccharides The first hypothesis was that ER α-1,2-mannosidase was acti-
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The hydrolysis profiles of A3 and B3 were similar (Fig. 4b).
However, considering that the cleavage of the terminal Man at
the A and B branches is responsible for the production of gly-
coprotein secretion and degradation signals, respectively,12,13
it is unlikely that the cleavage at each branch is caused by the
same ER α-1,2-mannosidase. In this experiment, the possi-
bility of further cleavage of the product A2 was not considered
because it was undetectable as the HPLC retention time of A1
coincided with those of components derived from the ER frac-
tion. Therefore, differences in the reactivity of ER α-1,2-manno-
sidases with A3 and B3 were evaluated using further experi-
ments, which are described later in the text. At this stage, the
cleavage specificity of ER α-1,2-mannosidases inferred from
the results in Fig. 4 was A3 ≈ B3 > C3 ≈ D3.
Because each branch trisaccharide is present in the M9-type
glycan structure, we then examined the competition in
mannose trimming when the branch trisaccharides A3, B3,
and C3 were added to the same reaction mixture. For the com-
petitive experiment, an internal standard was necessary to cal-
culate the hydrolysis yields from the HPLC chromatograms
because the retention times of the mannose trimming pro-
ducts A2, B2, and C2 were identical (Fig. S2†). Because D2 had
a different retention time from the other mannose trimming
products, we decided to use this compound as an internal
standard after confirming that the addition of D2 did not
affect the hydrolysis reactions of A3, B3, and C3 (Fig. S3†).
The competitive mannose trimming experiments were con-
ducted in the ER fraction with branch trisaccharides A3, B3,
and C3 and the internal standard D2 (Fig. 5). In the competi-
tive experiments, remarkable decreases in the activity were
observed for B3 and C3 compared with the individual experi-
Fig. 4 Hydrolysis assay of A3, B3, C3 or D3 in the ER fraction. (a)
Typical HPLC profiles of the glycan trimming reaction. Conditions for
HPLC: a TSK-GEL Amide-80 column (25 cm × 4.6 mm i.d.), mobile
phase CH3CN/H2O, gradient from 98 : 2 v/v to 90 : 10 v/v over 10 min
followed by that from 90 : 10 v/v to 65 : 35 v/v over 10 min, and a mobile
phase flow rate of 1.0 mL min−1 at 40 °C. (b) Hydrolysis yields of branch
trisaccharides A3, B3, C3, and D3 (250 μM) treated with the ER fraction
(3 mg protein per mL). Each data point represents the mean value with
the standard deviation (n = 9).
vated by the recognition of the inner Manα1-4Man linkage,
and the second hypothesis was that ER α-1,2-mannosidase had
weak recognition of the inner glycosidic linkage. Considering
that there is limited evidence of the existence of enzymes that
recognize the non-ER type glycosidic linkage, the former
hypothesis is unlikely. On the other hand, it has been reported
that ERManI primarily cleaves the B-branch in vitro, and also
cleaves other branches secondarily.22 Considering these infer-
ences and findings, we conclude that the latter hypothesis is
plausible at this stage. Because the cleavage profiles of C3 and
D3 were almost identical, it is possible that C3 was also
cleaved by ER α-1,2-mannosidases with weak recognition of
the inner glycosidic linkage. However, the enzyme involved in
the cleavage of C3 may simply be less active than the enzyme
involved in the cleavage of A3 and B3, so these issues were re-
addressed using the results of competitive experiments with
C3 and D3, which are described later in the text.
Fig. 5 Hydrolysis yields after 6 h from the competitive assay of A3/B3/
C3 mixtures (250 μM each) in the ER fraction (3 mg protein per mL). The
diagonal bars represent the individual hydrolysis yields of A3, B3, and
C3. Each data point represents the mean value with the standard devi-
ation (n = 3).
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ments (Fig. 4), which gave a glycan specificity order of A3 > B3
> C3. We compared the resulting glycan specificity with the
glycan trimming profile of ER α-1,2-mannosidases for M9-Gly-
BODIPY reported in our previous study,18 which was A-branch
≈ B-branch > C-branch (Fig. 6). The reported specificity
(Fig. 6c) was similar to the specificity obtained in the individ-
ual experiments (Fig. 4b). These results indicate that the
branches of the M9 glycan do not interact with each other. In
fact, a foldback conformation in which the B-branch interacts
with the core structure (Manβ1-4GlcNAcβ1-4GlcNAc) has been
reported for the M9 glycan.36,37 In the competitive experiment
shown in Fig. 5, free diffusion of each branch trisaccharide
substrate may have caused competition among A3, B3, and C3,
leading to a decrease in the cleavage activity for B3 and C3.
The results in Fig. 5 suggest that the A-branch structure
with the inner α-1,2-linkage would be most reactive for ER
α-1,2-mannosidases. To confirm this, it is necessary to discuss
the reactivity of ER α-1,2-mannosidases with the product A2.
As mentioned above, the hydrolysis product A2 can be further
cleaved by the enzymes. In fact, ERManI reportedly finally
trims Manα1-2Manα1-2Man to a monosaccharide.22 If A2 is
recognized by the ER α-1,2-mannosidases responsible for the
cleavage of B3 and C3, the apparent cleavage activity of B3 and
C3 will be lower than the original activity. These phenomena
were not consistent with our working hypothesis. Therefore,
we investigated the effect of A2 on the production of disacchar-
ides from each branch trisaccharide.
Fig. 7 Hydrolysis yield of A2 (250 μM) in the ER fraction (3 mg protein
per mL). The dashed lines represent the individual hydrolysis profiles of
A3, B3, and C3. Each data point represents the mean value with the
standard deviation (n = 3).
hydrolysis yields of A2 and A1 from A3 were analyzed (Fig. 8).
In the hydrolysis using A2 as a substrate, the product A1 was
observed immediately after the reaction started (Fig. 7). In con-
trast, when A3 was used as a substrate, the formation of A1
was not observed until 4 h after the reaction started, and
approximately 10% was formed after 8 h (Fig. 8). Thus, the
cleavage of B3 and C3 shown in Fig. 5 would not be affected by
A2. Although the specificities of the ER α-1,2-mannosidases
were in the order A3 ≈ B3 > C3 ≈ D3 (Fig. 4), the true speci-
ficity seemed to be A3 > B3 > C3 ≈ D3 considering the further
cleavage of generated A2.
In a system with D2 added as an internal standard, the
hydrolysis yield of A2 can be calculated even if the retention
time of the product A1 and the retention time of the com-
ponent derived from the ER fraction are identical in the HPLC
analysis. Using this experimental procedure, we examined
whether A2 was cleaved into A1 by ER α-1,2-mannosidases,
and found that the trimming activity for A2 was similar to
those for A3 and B3 (Fig. 7). Next, the time courses of the
Next, to clarify the competitive relationship between each
branch, we conducted competitive experiments in which two
types of synthetic trisaccharides were systematically added to
the reaction mixture (Fig. 9). A competitive experiment with A3
Fig. 6 Mannose trimming reaction of M9-Gly-BODIPY in the ER frac-
tion. (a) Assay conditions. (b) Structure of M9-Gly-BODIPY. (c) HPLC
chromatogram of the reaction mixture showing the mannose profile
obtained from ref. 18.
Fig. 8 Time course of hydrolysis yields from A3 (250 μM) to A2 and A2
to A1 in the ER fraction (3 mg protein per mL). Each data point rep-
resents the mean value with the standard deviation (n = 3).
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Fig. 9 Hydrolysis yields after 6 h from competitive assays with two-branch trisaccharides. The diagonal bars represent the individual hydrolysis
yields of the corresponding trisaccharides. Results of competitive assays with A3 (250 μM) and B3 (250 μM) (a), A3 (250 μM) and C3 (250 μM) (b), and
B3 (250 μM) and C3 (250 μM) (c) in the ER fraction (3 mg protein per mL). Each data point represents the mean value with the standard deviation
(n = 3).
and B3 showed relatively reduced activity for B3 (Fig. 9a), and for A3 and B3 greatly improved compared with that for A3 and
a competitive experiment with A3 and C3 showed reduced B3 without D3 (Fig. 10a and b). This phenomenon can be
activity for C3 (Fig. 9b). In addition, a competitive experiment explained by the fact that D3 acts as a pharmacological chaper-
with B3 and C3 showed relatively reduced activity for C3 one38 to contribute to the restoration of the activity of the par-
(Fig. 9c). According to these results, we concluded that the tially unfolded ER α-1,2-mannosidases. Pharmacological cha-
branch specificities of the ER α-1,2-mannosidases under com- perone is a general term for compounds that weakly interact
petitive conditions were in the order A3 > B3 > C3. We further with enzymes that have been denatured by diseases to
examined the possibility that product inhibition by A2 affected promote their refolding and restore enzyme activity. As men-
the cleavage activity for B3 and C3 (Fig. 5). When A2 was tioned above, the ER fraction used in this study was extracted
added to the reaction mixture as shown in Fig. 9c, the cleavage from the senescence-accelerated mouse liver. Because the
activity for B3 and C3 was not significantly affected (Fig. S4†). mouse model showed aging symptoms such as osteoporosis,
This finding shows that the difference in the cleavage activity the activity of the corresponding ER α-1,2-mannosidases might
for each branch observed in this study is not caused by the be somewhat reduced. Specifically, these results indicate that
effect of product inhibition by A2 but by differences in the D3 might be a lead compound in the development of pharma-
inner glycosidic linkages. This series of experiments revealed cological chaperones for ER α-1,2-mannosidases. Conversely,
that ER α-1,2-mannosidases could discriminate between endo- no significant changes in activity were detected in the competi-
glycosidic linkages.
tive assay of C3 with D3. This suggests that the enzymes
Finally, we examined the competitive experiments of responsible for the cleavage of C3 and D3 are different.
natural trisaccharides A3, B3, and C3 with unnatural tri- Although C3 and D3 may have been cleaved by the same ER
saccharide D3 (Fig. 10). On addition of D3, the cleavage activity α-1,2-mannosidase, as shown in Fig. 4, the result in Fig. 10c
Fig. 10 Hydrolysis yields after 6 h from competitive assays of natural A3, B3, or C3 branches (250 μM) with the unnatural D3 branch (250 μM) in the
ER faction (3 mg protein per mL). The diagonal bars represent the individual hydrolysis yields of the corresponding trisaccharides. (a) Competitive
assay of A3 with D3. (b) Competitive assay of B3 with D3. (c) Competitive assay of C3 with D3. Each data point represents the mean value with the
standard deviation (n = 3).
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revealed that both trisaccharides were cleaved by different
enzymes. Overall, we speculate that there are two categories of
Acknowledgements
α-1,2-mannosidases in the ER: (a) the enzymes responsible for This work was supported by the Japan Society for the
the cleavage of A3 and B3 are sensitive to the enhancement by Promotion of Science (JSPS) Grants-in-Aid for Scientific
D3 and (b) the enzyme responsible for the cleavage of C3 is Research (JSPS KAKENHI) (grant numbers JP16H06290 and
barely affected by D3.
JP16K01938). We thank Dr Yukishige Ito for helpful
discussions.
Conclusions
Notes and references
In this study, we synthesized a series of trimannosides with
different inner glycosidic linkages, which corresponded to the
three branched chains of a high-mannose type glycan. The
resulting trimannosides were applied to analyze the branch
discrimination abilities of ER α-1,2-mannosidases. Various
competitive experiments using synthetic trimannosides and
the mouse liver ER fraction revealed that some ER α-1,2-man-
nosidases could discriminate endo-glycosidic linkages. In the
synthesis of trimannoside substrates A3, B3, C3, and D3, glyco-
syl donors and acceptors were efficiently prepared from a
common starting material. Moreover, the stereoselectivities of
glycosylation reactions were optimized by tuning the solvent
polarity and/or adding a mild base. In glycan specificity ana-
lysis of the ER α-1,2-mannosidases, a branch specificity order
of A3 > B3 > C3 was found by a combination of the results
from individual cleavage assays of A3, B3, and C3, competitive
cleavage assays of a mixture of A3 and B3 with C3, and com-
petitive cleavage assays of A3 with B3, A3 with C3, and B3 with
C3. This is the first experimental demonstration that ER α-1,2-
mannosidases can discriminate inner glycosidic linkages in
the cleaved outer Manα1-2Man linkage. Competitive assays of
D3 with A3, B3, or C3 provided preliminary insight into the
pharmacological chaperone activity of D3 trisaccharide.
Although the enzyme responsible for the cleavage of each
branch has not been identified in this study, the discovery of
the ability of ER α-1,2-mannonsidases to discriminate inner
glycosidic linkages will enable the identification of the respon-
sible enzyme in future research. For instance, the responsible
enzyme may be identified by comparing the mannose trim-
ming of trisaccharides synthesized in this study by ER frac-
tions extracted from single knockout cells of each ER α-1,2-
mannosidase. Our findings will also open the door for the
manipulation of glycoprotein secretion/degradation signals
with selective inhibitors based on the synthetic trisaccharides
A3, B3 and C3 as lead compounds. Thus, the results of this
study are expected to lead to advanced research to obtain a
precise understanding of the complementary functions of ER
α-1,2-mannosidases in glycoprotein quality control and the
related protein-folding diseases.39 Further studies are in pro-
gress along this line and will be reported in due course.
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