Identification and characterization of UDP-mannose in human cell lines and mouse organs: Differential distribution across brain regions and organs

Article abstract of DOI:10.1016/j.bbrc.2017.10.173

Mannosylation in the endoplasmic reticulum is a key process for synthesizing various glycans. Guanosine diphosphate mannose (GDP-Man) and dolichol phosphate-mannose serve as donor substrates for mannosylation in mammals and are used in N-glycosylation, O-mannosylation, C-mannosylation, and the synthesis of glycosylphosphatidylinositol-anchor (GPI-anchor). Here, we report for the first time that low-abundant uridine diphosphate-mannose (UDP-Man), which can serve as potential donor substrate, exists in mammals. Liquid chromatography-mass spectrometry (LC-MS) analyses showed that mouse brain, especially hypothalamus and neocortex, contains higher concentrations of UDP-Man compared to other organs. In cultured human cell lines, addition of mannose in media increased UDP-Man concentrations in a dose-dependent manner. These findings indicate that in mammals the minor nucleotide sugar UDP-Man regulates glycosylation, especially mannosylation in specific organs or conditions.

Full text of DOI:10.1016/j.bbrc.2017.10.173

Biochemical and Biophysical Research Communications xxx (2017) 1e7
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Identification and characterization of UDP-mannose in human cell
lines and mouse organs: Differential distribution across brain regions
and organs
,
Kazuki Nakajima a b, Yasuhiko Kizuka b, Yoshiki Yamaguchi c, Yoshio Hirabayashi d,
,
*
Kazuo Takahashi e, Yukio Yuzawa e, Naoyuki Taniguchi b
a Department of Academic Research Support Promotion Facility, Center for Research Promotion and Support, Fujita Health University, Toyoake, Aichi 470-
1192, Japan
b Disease Glycomics Team, Systems Glycobiology Research Group, RIKEN-Max Planck Joint Research Center, RIKEN Global Research Cluster, 2-1 Hirosawa,
Wako, Saitama 351-0198, Japan
c Structural Glycobiology Team, Systems Glycobiology Research Group, RIKEN-Max Planck Joint Research Center, RIKEN Global Research Cluster, 2-1
Hirosawa, Wako, Saitama 351-0198, Japan
d Molecular Membrane Neuroscience, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
e Department of Nephrology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Mannosylation in the endoplasmic reticulum is a key process for synthesizing various glycans. Guanosine
diphosphate mannose (GDP-Man) and dolichol phosphate-mannose serve as donor substrates for
mannosylation in mammals and are used in N-glycosylation, O-mannosylation, C-mannosylation, and the
synthesis of glycosylphosphatidylinositol-anchor (GPI-anchor). Here, we report for the first time that
low-abundant uridine diphosphate-mannose (UDP-Man), which can serve as potential donor substrate,
exists in mammals. Liquid chromatography-mass spectrometry (LC-MS) analyses showed that mouse
brain, especially hypothalamus and neocortex, contains higher concentrations of UDP-Man compared to
other organs. In cultured human cell lines, addition of mannose in media increased UDP-Man concen-
trations in a dose-dependent manner. These findings indicate that in mammals the minor nucleotide
sugar UDP-Man regulates glycosylation, especially mannosylation in specific organs or conditions.
© 2017 Elsevier Inc. All rights reserved.
Received 19 October 2017
Accepted 30 October 2017
Available online xxx
Keywords:
Nucleotide sugar
UDP-mannose
Mannosylation
LC-ESI-MS/MS
Hypothalamus
Neocortex
1. Introduction
With N-glycosylation Dol-P-Man and GDP-Man concentrations
regulate mannosylation in the highly organized biosynthesis of N-
Glycosylation of proteins and lipids is vitally important in many
biological processes. Dysregulation of glycosylation is associated
with various diseases, such as diabetes mellitus, cancer, and
degenerative neuromuscular disease [1e4]. Mannosylation occur-
ring in the endoplasmic reticulum (ER) plays a key role in the
synthesis of multiple types of glycans. Dolichol-linked mannose
(Dol-P-Man) and GDP-mannose (GDP-Man) are donor substrates
for mannosylation. Dol-P-Man is synthesized from mannose-6-
phosphate via several intermediates (e.g., GDP-Man) of mannose
metabolism [5]. These intermediates are also used in N-glycosyla-
tion, synthesis of GPI-anchor, O-mannosylation, and C-
mannosylation.
glycan precursors, dolichol-linked oligosaccharides (DLOs). DLO
biosynthesis is sensitive to the cellular metabolic states of mono-
saccharides. For example, glucose deprivation reduces GDP-Man
concentrations, which in turn leads to the biosynthetic arrest of
DLOs [6,7]. This facilitates the premature degradation of DLOs by
pyrophosphatase [8]. This serves as a quality control system that
prevents abnormal N-glycosylation [8].
Dol-P-Man deficiencies related to genetic factors cause
congenital disorders of glycosylation (CDG), resulting in severe
clinical symptoms [9]. For example, PMI-CDG and PMM2-CDG
subtypes are caused by genetic mutations in phosphomannose
isomerase and phosphomannomutase 2. Both of these enzymes are
* Corresponding author.
E-mail addresses: dglycotani@riken.jp, rvw.tani@sanken.osaka-u.ac.jp (N. Taniguchi).
https://doi.org/10.1016/j.bbrc.2017.10.173
0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: K. Nakajima, et al., Identification and characterization of UDP-mannose in human cell lines and mouse organs:
Differential distribution across brain regions and organs, Biochemical and Biophysical Research Communications (2017), https://doi.org/
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2
K. Nakajima et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
2. Materials and methods
The abbreviations used
2.1. Materials
CDG
congenital disorders of glycosylation
Uridine-5-diphospho-a-D-mannopyranoside (UDP-Man) was
CMP-NeuAc cytidine monophospho N-acetyl-
acid
Dol-P-Man dolichol-phosphate-mannose
D-neuraminic
purchased from Sigma Aldrich Japan (Tokyo, Japan). Ammonium
bicarbonate, acetonitrile, and distilled water were of LC-MS grade
(Thermo Fisher Scientific, Waltham, MA). Triethylamine and formic
acid of LC-MS grade were purchased from Wako Chemicals (Osaka,
Japan). The sources of other materials were as follows: high-glucose
DLO
dolichol-linked oligosaccharide
ESI-MS/MS electrospray ionization-tandem mass
spectrometry
GDP-Man guanosine diphosphate mannose
GDP-Fuc guanosine diphosphate fucose
GPI-anchor glycosylphosphatidylinositol-anchor
LC-MS liquid chromatography-mass spectrometry
ꢀ
DMEM (Sigma Aldrich, Japan); fetal bovine serum (Biowest, Nuaille,
France); glucose-free DMEM, penicillin, and streptomycin sodium
salt (Life Technologies, Carlsbad, CA); all other chemicals (Wako
Chemicals, Osaka, Japan).
MEF
mouse embryonic fibroblast
2.2. Cell lines and animals
NMR
nuclear magnetic resonance
UDP-Gal uridine diphosphate galactose
The human hepatoma cell line Hep3B, human breast cancer cell
line MCF7, and the human lung adenocarcinoma epithelial cell line
A549 were obtained from the ATCC (Manassas, VA). Human
pancreatic cancer cell line PK8 was obtained from the Cell Resource
Center for Biomedical Research Institute of Development, Aging,
and Cancer (Tohoku University, Miyagi, Japan). Mouse embryonic
fibroblast (MEF) cells were cultured and maintained as described
previously [26]. Inadvertent mycoplasma infection of the MEF cell
line was detected using e-MycoTM plus Mycoplasma PCR detection
kit (iNtRON Biotechnology Inc, Jungwong-gu, Seongnam, Korea).
Genomic DNA in harvested MEF cells was extracted using the i-
genomic CTB DNA Extraction Mini Kit (iNtRON Biotechnology Inc.,
Korea).
UDP-Glc uridine diphosphate glucose
UDP-GlcA uridine diphosphate glucuronic acid
UDP-HexNAc uridine diphosphate N-acetyl hexosamine
UDP-Man uridine diphosphate mannose
UDP
uridine diphosphate
UMP
uridine monophosphate
involved in GDP-Man biosynthesis [10,11]. In PMI-CDG patients,
oral mannose supplementation restores normal glycosylation.
Symptoms are relieved, because mannose can be converted to GDP-
Man [10]. In mouse models of autoimmune diabetes and airway
inflammation, oral mannose supplementation induces regulatory T
cells and suppresses the immunopathology [12]. Genetic mutations
associated with Dol-P-Man biosynthesis give rise to a high inci-
Cell lines were cultured in high-glucose DMEM supplemented
with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL
streptomycin until they reached approximately 60e70% conflu-
ence. To produce nucleotide sugars in the cell cultures, the culture
medium was replaced with low glucose DMEM supplemented with
mannose at various concentrations, ranging up to 20 mM. Then, the
cells were maintained in culture for 3, 6,12, or 24 h, followed by cell
extraction and analysis (below).
All animal experiments were performed in compliance with the
Institutional Guidelines for Animal Experiments of RIKEN. RIKEN
institutional policies are consistent with ARRIVE Guidelines and
follow international standards. To assess the regional distribution
of UDP-Man in different organs, we used four 10-week-old male
C57BL/6N mice (Charles River, MA). Mice were deeply anesthetized,
and the liver, lung, brain, and kidney were extirpated immediately,
and then placed into liquid nitrogen. The brain was further divided
into seven parts according to standard anatomical regions: olfac-
tory bulb, hypothalamus, cerebellum, medulla oblongata, hippo-
campus, neocortex, and “other” brain regions not included in the
first six.
dence of
a-dystroglycanopathy [13e15]. This is caused by a defi-
ciency of O-mannosyl glycans on
a
-dystroglycan in brain,
peripheral nerves, and skeletal muscle [16,17]. Indeed, man-
nosylation is vital for mammalian development and cellular ho-
meostasis. However, the precise roles of nucleotide sugar
metabolism in regulating mannosylation are still not fully under-
stood, particularly in O-mannosylation, GPI-anchor synthesis, and
C-mannosylation.
In mammals, at least 12 types of nucleotide sugars are known. In
contrast many more nucleotide sugars have been identified in
plants and bacteria. At least 30 types of low-abundant nucleotide
sugars exist in plants, and 70 nucleotide sugars have been identified
in bacteria [18e21]. This disparity between mammals and plants
and bacteria raises the possibility that minor nucleotide sugars
remain to be identified in mammals. Indeed, low-abundant
nucleotide sugars like UDP oligosaccharides are found in human
milk [22,23].
We previously developed two methods for monitoring nucleo-
tide sugar metabolism by using ion-pair reversed-phase LC and LC-
electrospray ionization-tandem mass spectrometry (ESI-MS/MS)
[24,25]. With these methods, cellular concentrations of abundant
nucleotide sugars in mammalian cell lines were determined
simultaneously [24]. In the present study, we sought to identify and
measure low-abundant nucleotide sugars using these methods. We
detected a nucleotide sugar that increases in concentration in
mannose-rich media and found this nucleotide to be UDP-Man. We
also determined UDP-Man concentrations in several human cell
lines and mouse tissues, which led to the revelation that UDP-Man
likely plays a unique role in glycosylation in specific mammalian
organs, including brain.
2.3. Preparation of cellular extracts from cultured cells and mouse
tissues
Nucleotide sugars were prepared from the cultured cells (6-cm
diameter dish), according to a previous report [24]. Cells were
collected in ice-cold 70% ethanol (2 ml); and GDP-Glc (500 pmol)
was added as an internal standard. The extract was centrifuged at
16,000 g for 15 min at 4 ꢀC, and the supernatant was lyophilized.
The freeze-dried samples were subjected to solid-phase extraction
using an Envi-Carb column (100 mg; Supelco Inc, Bellafonte, PA).
For mouse tissue samples, blocks of same-organ samples
(30e90 mg) were manually homogenized in ice-cold 75% ethanol,
and the tissue homogenates were prepared similarly to that
described for cells. An Envi-Carb column (250 mg) was used also for
Please cite this article in press as: K. Nakajima, et al., Identification and characterization of UDP-mannose in human cell lines and mouse organs:
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K. Nakajima et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
3
tissue samples.
sensitivity of nucleotide sugars at a level of 10 femtomoles and
better separation based on the sugar motif (UDP-Gal and UDP-Glc).
Furthermore, the MS/MS analysis of the deprotonated ion [M-H]- of
UDP-hexose in the negative ion mode identifies molecules based on
their characteristic fragment ions. This is because the fragment
pattern in UDP-hexose is dependent on the cis/trans anomeric
configuration of the phosphate group and 2-hydroxyl group of the
hexose motif [28]. Because UDP-Glc and UDP-Gal produce one
major fragment ion of [UMP]- at m/z 323, we were able to trace
UDP-Glc and UDP-Gal with the cis-configuration by monitoring
mass chromatograms of m/z 565/m/z 323 (Fig. S1). In contrast, UDP-
Man (commercially available standard) with the trans-configura-
tion was detected by the characteristic fragment ion of [UDP]- at m/
z 403 (Fig. S1).
We analyzed cellular extracts from a hepatoma cell line (Hep3B)
and MEFs after mannose supplementation, because these cells
likely incorporate mannose efficiently via mannose-specific trans-
porters [29]. Other cancer cell lines were also analyzed to test
whether UDP-Man is distributed ubiquitously in other species and
tissues (Fig. S2). Interestingly, in the Hep3B cell line cultured in the
presence of 20 mM mannose, an unexpected additional peak was
reliably detected at m/z 565/m/z 403 after ~8 min. This peak was
barely detectable in mannose-free media (Fig. 1), implying that this
peak reflects UDP-Man. The unexpected additional peak at m/z 565/
m/z 323 was weakly detected because of the presence of abundant
UDP-Gal and UDP-Glc, even though the molecule was increased in
mannose-rich media.
2.4. Ion-pair reversed-phase LC-ESI-MS/MS
Ion-pair reversed-phase LC-ESI-MS/MS was performed using an
Agilent 1100 series HPLC system (Agilent Technologies, Santa Clara,
CA) coupled to an Esquire HCT ion trap mass spectrometer (Bruker
Daltonics, Germany), according to a previously published method
[25,27]. Cellular extracts were separated on an Inertsil ODS-3 col-
umn (3
mm, 150 ꢁ 2.1 mm, GL Science, Fukushima, Japan). Elution
buffers were: (A) 20 mM triethylammonium acetate (TEAA) buffer,
pH 6.0; and (B) buffer A containing 20% acetonitrile [25]. Portions of
cellular extracts were analyzed by MS/MS analysis. For quantifica-
tion, we calculated the peak area of extracted ion chromatograms of
the major product ions. MS/MS transition for CMP-NeuAc was
defined as m/z 613 to m/z 322, corresponding to a [CMP]- ion. Other
nucleotide sugars were as follows: m/z 565/m/z 323 for UDP-Gal
and UDP-Glc; m/z 606/m/z 385 for UDP-HexNAc; m/z 604/m/z 442
for GDP-Man; m/z 604/m/z 362 for GDP-Glc; m/z 579/m/z 323 for
UDP-GlcA; and m/z 588/m/z 442 for GDP-Fuc. UDP-Man was
monitored based on the characteristic fragment ion of m/z 565/m/z
403. Nucleotide sugar concentrations were normalized from the
recovery of exogenous GDP-Glc and then expressed as pmol/mg
protein.
2.5. Isolation of UDP-Man in mammalian cell lines
We prepared a mixture of nucleotide sugars from MEFs. For
starting material, we cultured large amounts (5 ꢁ 15-cm diameter
dishes) of MEFs in low glucose DMEM supplemented with 20 mM
mannose. Extracts from the cells were then subjected to prepara-
tive HPLC under slightly modified conditions as those in Ref. [27].
After cell extraction, the extracts were fractionated by ion-pair
reversed-phase LC by monitoring the ultraviolet absorption at
254 nm. To separate UDP-Man from CMP-NeuAc, we used the
following: (A) elution buffer (20 mM TEAA buffer, pH 5.2); and (B)
buffer A containing 20% acetonitrile. The fractionated solutions
were lyophilized and analyzed by ion-pair reversed-phase LC, as
reported previously [24].
3.2. Isolation and characterization of UDP-Man
The additional prominent peak detected at m/z 565/m/z 403 was
very clear in several cell lines cultured in mannose-rich conditions
(Fig. S2). Thus, we characterized the structure of the molecule by
purifying the nucleotide sugars from extracts of MEFs. The purified
nucleotide sugar showed a single peak in the HPLC chromatogram
Isolated UDP-Man (500 pmol) was hydrolyzed under acidic
conditions with 0.02 M HCl (20 m
L) at 100 ꢀC for 20 min, and the pH
of the samples was neutralized with 0.02 M NaOH [22]. A portion of
the hydrolysate was analyzed by ion-pair reversed-phase LC [24].
2.6. NMR
NMR spectra were recorded with a 600-MHz NMR spectrometer
(BrukerBioSpin, Osaka, Japan) equipped with a 5-mm TXI probe
(Bruker). The probe temperature was set at 25 ꢀC. The sample
(200e500 nmol) was dissolved in 600 mL of D2O (99.99 atom %D).
One-dimensional 1H-NMR spectra were collected with presatura-
tion of the HDO signal. 1H chemical shifts were reported relative to
the external standard 4,4-dimethyl-4-silapentane-1-sulfonic acid.
The NMR data were processed with TopSpin (version 2.1; Bruker),
and the spectra were displayed using XWIN-PLOT (version 3.5;
Bruker).
3. Results
3.1. LC-MS detection of UDP-Man induced in cells cultured in
mannose-rich media
Fig. 1. Detection of UDP-Man in a mammalian hepatoma cell line cultured in
mannose-rich media. Mass chromatograms of UDP-Man, UDP-Glc, and UDP-Gal. The
concentrations of these compounds were measured at m/z 565/m/z 403 for UDP-Man,
and at m/z 565/m/z 323 for UDP-Glc and UDP-Gal. Hep3B cells were cultured in low-
glucose media supplemented with 20 mM mannose media or in low-glucose media
lacking mannose for 24 h.
We first searched for the existence of low-abundant nucleotide
sugars using ion-pair reversed-phase ESI-MS/MS using procedures
in previous reports [24,25,27]. This system provides good
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K. Nakajima et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
Table 1
(Fig. 2A, bottom line), corresponding to the reference standard of
UDP-Man (Fig. 2A, top line). We then subjected the fraction to 1H-
NMR analysis. The spectral pattern and chemical shift of the sample
Nucleotide sugar concentrations in Hep3B cultured in mannose-free or mannose-
rich media.
Nucleotide sugar concentrations (pmol/mg protein)
are almost identical to those of standard UDP-a-D-mannose (Fig. 2B,
and Table S1), suggesting that the fraction contains UDP-
a-D-
Mannose-free media
20 mM Mannose-rich media
mannose.
CMP-NeuAc
UDP-Man
UDP-Gal
137 24
0.76 0.28
111 40
515 26
To further investigate the nucleotide motif, the purified nucle-
otide sugar was hydrolyzed under acidic conditions. Elution times
of the major hydrolysates corresponded to UMP and UDP (Fig. 2A,
middle line). These results clearly demonstrate that the additional
peak found in mannose-treated cells was UDP-Man.
739
9
1417 72
4132 551
1119 132
33.7 1.0
695 143
363 23
UDP-Glc
2449 100
2110 44
16.2 7.1
423 43
UDP-HexNAc
GDP-Man
UDP-GlcA
GDP-Fuc
263
5
3.3. Nucleotide sugar concentrations in cells cultured in mannose-
free or mannose-rich media
quantified based on available reference standards as well as on MS/
MS analysis (Fig. S3). With 20 mM mannose, the concentration of
UDP-Man increased in all cell lines, although this increase differed
among cell lines (Fig. S2). UDP-Man concentrations were remark-
ably elevated within 3 h of culturing the cells with mannose
To test whether there is a dose-dependent relationship between
increases in UDP-Man and mannose concentrations, we quantified
nucleotide sugar concentrations in cells cultured in mannose-rich
media (Table 1). The other nucleotide sugars, CMP-NeuAc, UDP-
HexNAc, GDP-Man, UDP-GlcA, and GDP-Fuc, were assigned and
Fig. 2. Identification of purified UDP-Man. (A) Ion-pair reversed-phase LC profiles of purified UDP-Man and hydrolysates produced through acid hydrolysis. The upper profile
represents the result of additional experiments involving mixtures of 20 different types of nucleotide sugars and nucleotides. Arrows indicate the elution positions of authentic
compounds (UDP-Man, UMP, and UDP). (B) The 600 MHz 1H-NMR spectra of UDP-Man (standard, 200e400 nmol) and purified UDP-Man (500 nmol) dissolved in D2O. Probe
temperature was set at 298 K. The residual HDO signal was suppressed using presaturation.
Please cite this article in press as: K. Nakajima, et al., Identification and characterization of UDP-mannose in human cell lines and mouse organs:
Differential distribution across brain regions and organs, Biochemical and Biophysical Research Communications (2017), https://doi.org/
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K. Nakajima et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
5
concentrations ranging from 0.2 to 20 mM (Fig. S4). With mannose-
free media, the fragment ion at m/z 403, representing UDP-Man,
was barely detectable (Fig. S5). For Hep3B cells cultured with
20 mM mannose, UDP-Man concentration increased by 650-fold
compared to that in mannose-free media (Table 1). GDP-Man
We further tested whether UDP-Man distribution differed
depending on brain region. UDP-Man concentration was quantified
in olfactory bulb, hypothalamus, cerebellum, medulla oblongata,
hippocampus, neocortex, and other regions (i.e., other combined
brain regions not in the six listed) of adult mice. UDP-Man con-
centrations was distinctly different across regions. Hypothalamus
and neocortex were especially rich in UDP-Man, but olfactory bulb
and cerebellum were poor. In contrast, GDP-Man concentration was
similar across regions. The UDP-Man:GDP-Man ratios were highest
in hypothalamus (1:7), implying that high ratios of UDP-Man to
GDP-Man may reflect a potential donor substrate function for
mannosyltransferases in hypothalamus.
concentration increased only slightly in
a mannose dose-
dependent manner. For Hep3B cells cultured in mannose-free
media, the UDP-Man concentration was 20- to 50-fold lower than
GDP-Man concentration. Other nucleotide sugars showed moder-
ate changes. UDP-GlcNAc concentration, for example, decreased by
50% when the cells were cultured in the presence of 20 mM
mannose, while UDP-Glc, UDP-Gal, and UDP-GlcA concentrations
increased (Table 1). The overall changes in nucleotide sugar con-
centrations are consistent with those reported previously [30].
4. Discussion
3.4. Distribution in different organs
We have demonstrated for the first time the presence of a minor
nucleotide sugar, UDP-Man, in mammals. Ion-pair reversed-phase
LC-ESI-MS/MS identified UDP-Man in mouse brain, liver, and kid-
ney. NMR analysis identified the structure of purified UDP-Man
from a cell line cultured in mannose-rich media. The MS/MS
analysis of the deprotonated ion [M-H]e at m/z 565 and the
monitoring of the characteristic fragment ions of [UDP]e at m/z 403
facilitated the detection of the UDP-Man peak in mammalian
sources (Fig. 1). The ion-pair reversed-phase mode also enabled the
separation of UDP-Man from other nucleotide sugars, which facil-
itated the identification and purification of UDP-Man.
UDP-Man was detected in Hep3B, MCF7, A549, and PK8 cell lines
in very small amounts, and its concentrations were elevated in cells
grown in mannose-rich media (Table 1). While our study is the first
to identify UDP-Man in mammals, “UDP-Man” has been reported in
a few microbes. Singh and Hogan reported that UDP-Man in
We next quantified UDP-Man concentrations in mouse liver,
brain, lung, and kidney. For each tissue sample, three separate
preparations were analyzed. The concentration of UDP-Man was
markedly different in different organs (Fig. 3A). Brain, for example,
had ~10-fold higher concentration of UDP-Man compared to other
organs. The GDP-Man concentration in brain was 3-fold higher
compared to that in other organs. GDP-Fuc and UDP-HexNAc con-
centrations were slightly higher than that of UDP-Man across the
tissues analyzed, and quite similar to those reported previously
[31]. The UDP-GlcNAc:UDP-GalNAc ratio is known to be constant
(2.3e2.7) across different tissues [24]. However, we found that
UDP-Man:GDP-Man ratios in the liver, brain, lung, and kidney
varied, showing ratios of 1:71.3, 1:20.4, 1:30.4, and 1:9.0,
respectively.
Fig. 3. UDP-Man, GDP-Man, GDP-Fuc, and UDP-HexNAc concentrations in different mouse organs. (A) Liver, brain, lung, and kidney. (B) Different regions of mouse brain.
Independent samples were analyzed (n ¼ 4), and the data were normalized to units of pmol/mg protein.
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K. Nakajima et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
Mycobacterium smegmatis may be involved in the synthesis of
mycobacterial polysaccharides [32]. We do not believe the UDP-
Man we identified in our cell cultures spuriously derives from
inadvertent bacterial contamination for several reasons. First, the
tissue samples were taken from mice under SPF sterile conditions
and immediately frozen before use. Second, cell lines were cultured
in the presence of antibiotics. Third, we proactively tested for my-
coplasma infection, and none could be detected in the MEF cell line
(Fig. S6). Although there remains a slight possibility that the UDP-
Man we identified derived from undetected bacterial contamina-
tion, the possibility is negligible.
In mammals, the metabolism of major nucleotide sugars
metabolism has been established [18,20,21]. However, relatively
small numbers of nucleotide sugars are involved in glycosylation in
mammals compared to that in other organisms. Therefore, this
newly detected UDP-Man could also serve as a potential donor
substrate for mannosyltransferases and, as such, be a novel regu-
lator of mannosylation.
the animal experiments. This work was supported by Grant-in-Aid
for Young Scientists (B), 24790301 (to KN) and Grant-in-Aid for
Scientific Research (B), 15H04700 (to NT) from the Ministry of Ed-
ucation, Culture, Sports, Science and Technology of Japan.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.bbrc.2017.10.173.
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tified UDP-a-D-Man is linked via a-linkage, like UDP-Glc and UDP-
Gal, UDP-Man might be synthesized in a similar alternate pathway
via UDP-glucose pyrophosphorylase or UDP-galactose pyrophos-
phorylase. Determining the key enzymes involved in UDP-Man
synthesis is an important future step.
How does glycosylation via UDP-Man occur? Adamany et al.
assessed the donor specificities of a thyroid mannosyltransferase in
an in vitro enzymatic reaction and found that the relative activity
toward UDP-Man is 45% of that toward GDP-Man in glycoprotein
synthesis [33]. Although the UDP-Man concentration was quite low
when compared to GDP-Man concentration (Fig. 3 and Table 1),
UDP-Man is possibly involved not only in N-glycosylation but also
in other glycosylations (e.g., synthesis of GPI-anchor, and O- and C-
mannosylation).
Is glycosylation via UDP-Man more important in particular tis-
sues? Interestingly, we observed characteristic differential distri-
bution of UDP-Man. UDP-Man concentrations are greater in the
hypothalamus and neocortex compared to other brain regions
(Fig. 3). Thus, this pattern may reflect functional differences in
glycosylation occurring through UDP-Man in the brain. For
example, hypothalamus-specific function might be relevant to
metabolic regulation due to active glucose metabolism in the brain.
Thus, investigating the biological significance and functions of
UDP-Man in mammals may pose many challenges because of its
low abundance. However, given its roles in quality control of
glycosylation [8], and immune system function [12], a better un-
derstanding of glycosylation via UDP-Man in humans may open up
new avenues for treating neurological disease.
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In conclusion, the detection of UDP-Man in mammalian tissues
provides new insights into understanding the regulatory mecha-
nisms of glycosylation.
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgments
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We thank Dr. Takashi Angata (Academia Sinica, Taiwan) and Dr.
Kazuaki Ohtsubo (Kumamoto University) for helpful suggestions on
research designs, and Dr. Masanori Kugita (Fujita Health University)
and Ms. Mizuki Kurosawa (RIKEN, BSI) for technical support with
Please cite this article in press as: K. Nakajima, et al., Identification and characterization of UDP-mannose in human cell lines and mouse organs:
Differential distribution across brain regions and organs, Biochemical and Biophysical Research Communications (2017), https://doi.org/
10.1016/j.bbrc.2017.10.173

K. Nakajima et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e7
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Please cite this article in press as: K. Nakajima, et al., Identification and characterization of UDP-mannose in human cell lines and mouse organs:
Differential distribution across brain regions and organs, Biochemical and Biophysical Research Communications (2017), https://doi.org/
10.1016/j.bbrc.2017.10.173