Structuralanalysisof N-glycans ofthe planarian
Dugesia japonica
Shunji Natsuka
1,2
, Yukiko Hirohata
2
, Shin-ichi Nakakita
3
, Wataru Sumiyoshi
3
and Sumihiro Hase
2
1 Department of Biology, Faculty of Science, Niigata University, Japan
2 Department of Chemistry, Graduate School of Science, Osaka University, Japan
3 Life Science Research Center, Kagawa University, Japan
Introduction
The structures of N-glycans are generally diverse, but
the variation in diversity across the phylogeny is still
unknown. In particular, structural information on
invertebrate glycans is very limited. The nematode
Caenorhabditis elegans has various unique N-glycans
[1–4], even though it has a very simple body construc-
tion. The major N-glycans in the cephalopods squid
[5,6] and octopus [7] are similar to the complex glycans
of vertebrates. In contrast, gastropods, which, like
cephalopods, also belong to the mollusk family, have
quite different N-glycans that are modified with methyl
residues [8,9]. This suggests that phylogenic closeness
and N-glycan similarity are not correlated in a simple
way. In this study, we analyzed the N-glycan structures
of planarians, which are platyhelminths, in order to
verify this proposal. Planarians are lower animals com-
pared with mollusks, i.e. squids, octopuses and snails,
whose N-glycan structures have already been studied.
If N-glycans become more complex along the phylog-
eny, the N-glycans of planarians will less complicated
than those of higher animals. This prediction arises
from the simple logic that an organism with a complex
system will have complex and diverse molecules. In
contrast, if theplanarian has unique and complex
N-glycans, the view that the diversity of N-glycans
does not correlate simply with the phylogenic process
is supported. The species ofplanarian used in this
study, Dugesia japonica, is a free-living platyhelminth.
Keywords
diversity; methylation; N-glycans; phylogeny;
planarian
Correspondence
S. Natsuka, Department of Biology, Faculty
of Science, Niigata University, Niigata
950-2181, Japan
Tel ⁄ Fax: +81 25 262 6174
E-mail: natsuka@bio.sc.niigata-u.ac.jp
(Received 13 August 2010, revised 15
November 2010, accepted 17 November
2010)
doi:10.1111/j.1742-4658.2010.07966.x
To investigate the relationship between phylogeny and glycan structures,
we analyzed the structure ofplanarian N-glycans. Theplanarian Duge-
sia japonica, a member ofthe flatworm family, is a lower metazoan. N-gly-
cans were prepared from whole worms by hydrazinolysis, followed by
tagging with the fluorophore 2-aminopyridine at their reducing end. The
labeled N-glycans were purified, and separated by three HPLC steps. By
comparison with standard pyridylaminated N-glycans, it was shown that
the N-glycans ofplanarian include high mannose-type and pauci-mannose-
type glycans. However, many ofthe major N-glycans from planarians have
novel structures, as their elution positions did not match those ofthe stan-
dard glycans. The results of mass spectrometry and sugar component anal-
yses indicated that these glycans include methyl mannoses, and that the
most probable linkage was 3-O-methylation. Furthermore, the methyl resi-
dues on the most abundant glycan may be attached to the non-reducing-
end mannose, as the glycans were resistant to a-mannosidase digestion.
These results indicate that methylated high-mannose-type glycans are the
most abundant structure in planarians.
Abbreviations
PA, pyridylamino.
452 FEBS Journal 278 (2011) 452–460 ª 2011 The Authors Journal compilation ª 2011 FEBS
Platyhelminths also include Schistosoma species, which
are parasitic flatworms. The N-glycan structures of
parasitic flatworms have been reported previously [10]
and include multi-fucosylated complex-type structures.
However, the glycan structure of parasites may be dif-
ferent from their ancestral forms because of high selec-
tion pressure due to competing with a host immune
system. For that reason, we chose to use a free-living
platyhelminth.
Results
N-glycans were liberated from lyophilized planarians
and tagged with 2-aminopyridine at their reducing ter-
minus. These pyridylaminated (PA-) N-glycans were
separated by anion-exchange chromatography into
neutral (N) and acidic (A) fractions (Fig. 1A).
Reducing-end analysis indicated that the neutral frac-
tion included over 95% ofthe total amount of PA-N-
glycans (Fig. 1B). The neutral fraction was further sep-
arated by size-fractionation HPLC (Fig. 2). Thirteen
fractions (A–M) with molecular sizes indicating the
presence of more than three glucose units were col-
lected, and further separated by reversed-phase HPLC
(Fig. 3). Peaks that were > 5% in area ofthe major
PA-glycan (pN4) were collected and named pN1–
pN18. A two-dimensional glycan map was prepared
based on the retention times ofthe PA-glycans on size-
fractionation and reversed-phase HPLC (Fig. 4). To
reduce fluctuations in retention time, the retention time
on size-fractionation HPLC was normalized against
glucose unit, and that on reversed-phase HPLC was
converted to the reversed-phase scale, as described in
Experimental procedures. The positions of pN1, pN11,
pN13, pN15, pN16, pN17 and pN18 on the map
coincided with those ofthe standard PA-glycans M2B,
M5A, M6B, M7B, M8A, M9A and G1M9A, respec-
tively (Table 1). As the result of MS analysis sup-
ported these assignments (Table 2), we concluded that
the planarian N-glycans pN1, pN11, pN13, pN15,
pN16, pN17 and pN18 did indeed match these
standard structures. The results of MS analysis
of other PA-glycans suggested multi-methylation of
Fig. 1. Anion-exchange HPLC. (A) PA-N-glycans from planarians
were separated into neutral (N) and acidic (A) fractions by anion-
exchange chromatography. The thick bar indicates the collected
fractions. X indicates a peak derived from reagents. The fluores-
cence scale in the inset chromatogram is magnified 30-fold.
Arrowheads labeled S0–S4 indicate elution positions of standard
PA-N-glycans with 0–4 sialic acids, respectively. (B) Contents of
PA-glycans that have PA-GlcNAc at their reducing end in the
fractions from anion-exchange chromatography.
Fig. 2. Size-fractionation HPLC. The neutral PA-glycans were sepa-
rated by size-fractionation HPLC. Thirteen major peaks (A–M), the
molecular sizes of which were larger than that of isomaltotriose
trisaccharide, were isolated. Arrowheads indicate the elution posi-
tions of PA-isomalto-oligosaccharides with degrees of polymeriza-
tion from 1 to 15.
S. Natsuka et al. N-glycans of planarian
FEBS Journal 278 (2011) 452–460 ª 2011 The Authors Journal compilation ª 2011 FEBS 453
oligomannosidic N-glycans (Fig. 5 and Table 2). We
subsequently analyzed the sugar composition of pN4
(Fig. 6). As methyl residues were not released from
sugars under the hydrolyzing conditions ofthe analy-
sis, methylated monosaccharide(s) could be detected.
Indeed, a peak that had an elution position that
coincided with that of pyridylaminated 3-O-methyl
mannose was detected on both reversed-phase and
borate-chelating anion-exchange HPLC. Mannose and
N-acetylglucosamine were also detected by the HPLC
analyses. Based on the results ofthe MS and sugar
component analyses, pN4 was estimated to comprise
three methyl mannoses, two mannoses and two
N-acetylglucosamines, and the linkage position of the
O-methyl residues was tentatively assigned as the
third position ofthe mannoses. Furthermore, the 3-O-
methyl mannoses may be located at the non-reducing
end, as pN4 was resistant to a-mannosidase digestion
(data not shown). We then investigated the core struc-
ture of pN4 by partial acid hydrolysis. pN4 was par-
tially hydrolyzed to form fragments of various lengths.
The reducing-end fragments with pyridylamino resi-
dues were analyzed by size-fractionation and reversed-
phase HPLC, and plotted as a two-dimensional
map (Fig. 7). Fragments with elution positions that
coincided with GlcNAcb1-4GlcNAc-PA, Manb1-4Glc-
NAcb1-4GlcNAc-PA and Mana1-6Manb1-4GlcNAcb1-
4GlcNAc-PA were formed from pN4. These results
indicate that the structure of pN4 was Mana1-
6Manb1-4GlcNAcb1-4GlcNAc-PA, modified with three
3-O-methyl mannoses. Furthermore, based on the
results ofthe MS analysis, pN6 and pN10 may contain
fucose, which is a common deoxyhexose in animals.
However, further analysis was not performed because
the amounts of material available were too small.
Discussion
The results of this study show that theplanarian has
unique variation in its N-glycans, which are modified
by methyl residues at the 3-position of mannose. It has
been reported that certain snails and slugs have methy-
lated pauci-mannose-type glycans [8,9]. The linkage
position ofthe methyl residue was also reported to be
the 3-position of mannose. However, planarians and
snails are not closely related from the phylogenic point
of view: planarians are platyhelminths while snails are
mollusks. In a previous paper, we described the N-gly-
cans of squid, another mollusk [6]. However, the char-
acteristic structures of squid N-glycans are quite
Fig. 3. Reversed-phase HPLC of fractions (A)–(M). Fractions (A)–(M) obtained on size-fractionation HPLC were further separated by
reversed-phase HPLC, and the major PA-glycans isolated were named pN1–pN18.
N-glycans ofplanarian S. Natsuka et al.
454 FEBS Journal 278 (2011) 452–460 ª 2011 The Authors Journal compilation ª 2011 FEBS
different from those of snails. These results suggest
that phylogenic closeness and N-glycan similarity are
not correlated in a simple way.
Each metazoan has characteristic N-glycans. For
example, C. elegans has highly fucosylated core struc-
tures [4], squid has a complex type with type 1
lactosamine [5,6], snails [8,9] and planarians have a
3-O-methylated oligomannosidic type, and vertebrates
have a complex type with type 2 lactosamine. Even
the protozoan Dictyostelium discoideum has unique
N-glycan structures, with intersecting GlcNAc [11].
However, many genes encoding the enzymes that syn-
thesize these specific glycans of each organism have
not been identified. For example, the existence of an
enzyme that catalyzes mannose methylation was sug-
gested by the results of this study. Methylated N-gly-
cans have been discovered in various invertebrates,
for example snails [8,12], slugs [9] and nematodes [1].
However, a gene encoding an enzyme capable of syn-
thesizing methylated glycans has not been reported so
far. In future studies, research to identify the genes
involved in biosynthesis ofthe methylated glycan will
be required.
Why does each organism have unique N-glycan
structures? One possible answer is that primitive multi-
cellular organisms may already have the genetic poten-
tial in their primitive genome for synthesizing the vari-
ous N-glycan structures. From many possible
variations, each organism has ‘chosen’ particular struc-
tures during evolution. To verify this hypothesis, it is
necessary to compare the genes involved in the synthe-
sis of N-glycans in many organisms. The hypothesis
would gain strong support if lower metazoans were
found to have the genetic potential to synthesize the
N-glycans found in higher metazoans. Furthermore, it
is possible that particular glycan structures are not
inevitable, and that micro-heterogeneity is essential for
each organism, i.e. alternative sets of glycans may be
able to form similar systems in organisms. The study
of glycans from the phylogenic viewpoint remains
important in order to be able to answer this question.
Experimental procedures
Preparation of PA-glycans from planarian
The planarians (Dugesia japonica) were purchased from a
local animal vendor and kept in water at 14 °C in the dark.
They were fed with chicken liver. After starvation for
5 days, the planarians were washed with water and lyophi-
lized. N-linked glycans were liberated from the glycopro-
teins of lyophilized planarians by hydrazinolysis as
described previously [13,14]. Briefly, 2 mg ofthe sample
was heated at 100 °C for 10 h with 0.2 mL of anhydrous
hydrazine. After removal of hydrazine by repeated evapora-
tions, the glycans were re-N-acetylated with acetic anhy-
dride in a saturated sodium bicarbonate solution, and then
passed through a Dowex 50Wx2 (H
+
) cation exchanger
(Dow Chemicals, Midland, MI, USA) to remove sodium
ions. The reducing ends ofthe liberated glycans were
tagged with the fluorophore 2-aminopyridine. Lyophilized
samples were heated at 90 °C for 60 min with 20 lL of pyr-
idylamination reagent, and then heated at 80 °C for 35 min
after the addition of 70 lL of reducing reagent, as
described previously [14].
Purification of PA-glycans
The pyridylamination reaction mixture was diluted with
150 lL of water and extracted twice using 200 lL of phe-
nol ⁄ chloroform (1 : 1 v ⁄ v) to remove the excess reagents
[15]. The water layer that contained the PA-glycans was
purified by gel filtration on a mini-column (1 · 8 cm, TSK-
gel Toyopearl HW-40F, Tosoh, Tokyo, Japan). After load-
ing the sample, the eluate between 2 and 6 mL was
collected as the PA-glycan fraction and lyophilized. The
PA-glycans were further purified using a graphite carbon
Fig. 4. Two-dimensional HPLC map ofthe PA-glycans from planar-
ian. The retention times on size-fractionation and reversed-phase
HPLC were converted to glucose units and the reversed-phase
scale, respectively, as described in Experimental procedures. pN1–
pN18 were plotted as circles on a two-dimensional map. The diam-
eter ofthe circles is proportional to the cube root ofthe detected
amount of PA-glycans. The positions of standard PA-glycans are
indicated by crosses labeled (a)–(g). The standards were: (a) M2B,
(b) M5A, (c) M6B, (d) M7B, (e) M8A, (f) M9A and (g) G1M9A. The
structures ofthe standard PA-glycans are shown in Table 1.
S. Natsuka et al. N-glycans of planarian
FEBS Journal 278 (2011) 452–460 ª 2011 The Authors Journal compilation ª 2011 FEBS 455
Table 1. Estimated structures of PA-N-glycans prepared from planarian. Ratios indicate the relative amounts ofthe glycans, with the amount
of the most abundant glycan, pN4, taken as 100.
pN1
Peak #
pN2
pN3
pN4
pN5
pN6
pN7
pN8
pN9
pN10
pN11
pN12
pN13
pN14
pN15
pN16
pN17
pN18
Ratio
5.2
14.5
6.0
100
20.2
20.4
22.4
14.3
6.4
5.9
5.6
27.0
19.7
10.6
8.8
17.3
32.9
5.5
Abbreviation
M2B
Me
3
-M5
M5A
M6B
M7B
M8A
M9A
G1M9A
Estimated Structure
Manα1
Manα1
Manα1
Manα1
Manα1
Manα1
Manα1
Manα1
Manα1
Manα1
Manα1
Manα1-2Manα1
Manα1-2Manα1-2Manα1
Manα1-2Manα1-2Manα1
Manα1-2Manα1-2Manα1
Glcα1-3Manα1-2Manα1-2Manα1
Manα1-2Manα1
Manα1-2Manα1
Manα1-2Manα1
Manα1-2Manα1
Manα1-2Manα1
Manα1
Manα
1
Manα1
Manα1
6
6
3
6
6
6
6
6
3
3
3
6
6
6
6
3
3
3
3
3
6
3
3
3
Manα1
(Me-Hex-)
2
Manβ1-4G1cNAcβ1-4G1cNAc-PA
(Me-Hex-)
2
Hex-Manβ1-4G1cNAcβ1-4G1cNAc-PA
(Me-Hex-)
3
(Hex-)
2
Manβ1-4G1cNAcβ1-4G1cNAc-PA
(Me-Hex-)
3
(Hex-)
3
Manβ1-4G1cNAcβ1-4G1cNAc-PA
Manβ1-4G1cNAcβ1-4G1cNAc-PA
Manβ1-4G1cNAcβ1-4G1cNAc-PA
Manβ1-4G1cNAcβ1-4G1cNAc-PA
Manβ1-4G1cNAcβ1-4G1cNAc-PA
Manβ1-4G1cNAcβ1-4G1cNAc-PA
Manβ1-4G1cNAcβ1-4G1cNAc-PA
(Me-Hex-)
3
(Hex-)
3
Manβ1-4G1cNAcβ1-4G1cNAc-PA
(Me-Hex-)
3
(Hex-)
4
Manβ1-4G1cNAcβ1-4G1cNAc-PA
(Me-Hex-)
3
(Hex-)
5
Manβ1-4G1cNAcβ1-4G1cNAc-PA
(Me-Hex-)
3
Manβ1-4G1cNAcβ1-4G1cNAc-PA
(Me-)
3
(Hex1-)
3
(dHex1-) Manβ1-4G1cNAcβ1-4G1cNAc-PA
(Me-)
3
(Hex-)
4
(dHex)(Pen-)Manβ1-4G1cNAcβ1-4G1cNAc-PA
(Me-)
2
(Hex-)
3
(dHex)
2
Manβ1-4GleNAeβ1-4GleNAe-PA
or
Manβ1-4G1cNAcβ1-4G1cNAc-PA
(Me-3Man1-)
3
Manβ1-4GlcNAcβ1-4GlcNAc-PA
6
6
{
N-glycans ofplanarian S. Natsuka et al.
456 FEBS Journal 278 (2011) 452–460 ª 2011 The Authors Journal compilation ª 2011 FEBS
cartridge (GL-Pak Carbograph 300 mg; GL Sciences Ltd,
Tokyo, Japan). The glycan mixture was dissolved in 1 mL
of 50 mm ammonium acetate, pH 6.0, and loaded onto the
cartridge. After washing with 5 mL of 50 mm ammonium
acetate, pH 6.0, the glycans were eluted using 5 mL of 60%
acetonitrile in the ammonium acetate buffer.
High-performance liquid chromatography for
PA-glycan separation
HPLC was performed using a Waters Alliance HPLC sys-
tem with a Waters 2475 fluorescence spectrophotometer
(Milford, MA, USA) unless otherwise mentioned. Anion-
exchange HPLC was performed using a TSKgel DEAE-
5PW column (0.75 · 7.5 cm; Tosoh) at a flow rate of
1.0 mLÆmin
)1
. The column was equilibrated with diluted
ammonium solution, pH 9.0. Five minutes after the sample
had been injected, the concentration of ammonium acetate,
pH 9.0, was increased linearly to 0.2 m in the first 20 min,
Fig. 6. Sugar component analysisof pN4. The pyridylaminated
monosaccharides prepared from pN4 were analyzed by reversed-
phase HPLC (A) and borate-chelating anion-exchange HPLC (B).
Arrows (a)–(f) indicate the elution positions of PA-Man, PA-4-O-
methyl mannose, PA-GlcNAc, PA-2-O-methyl mannose, PA-6-O-
methyl mannose and PA-3-O-methyl mannose, respectively.
Fig. 5. MALDI-TOF mass spectrum of pN4. Experimental details
are given in Experimental procedures.
Table 2. Results of mass analysis. Me, methyl residue; Hex, hexose; dHex, deoxyhexose; Pen, pentose; HexNAc, N-acetylhexosamine; PA,
pyridylamino residue.
Code Mass number (observed) Estimated composition (Na
+
adduct) Mass number (calculated)
pN1 849.509 Hex
2
HexNAc
2
PA 849.323
pN2 1039.266 Me
2
Hex
3
HexNAc
2
PA 1039.407
pN3 1215.788 Me
3
Hex
4
HexNAc
2
PA 1215.475
pN4 1377.866 Me
3
Hex
5
HexNAc
2
PA 1377.528
pN5 1201.577 Me
2
Hex
4
HexNAc
2
PA 1201.460
pN6 1347.550 Me
2
Hex
4
dHexHexNAc
2
PA 1347.518
pN7 1539.895 Me
3
Hex
6
HexNAc
2
PA 1539.581
pN8 1702.141 Me
3
Hex
7
HexNAc
2
PA 1701.634
pN9 1702.017 Me
3
Hex
7
HexNAc
2
PA 1701.634
pN10 1656.007 Me
3
Hex
5
dHexPenHexNAc
2
PA
or Me
2
Hex
5
dHex
2
HexNAc
2
PA
1655.628
pN11 1335.957 Hex
5
HexNAc
2
PA 1335.481
pN12 1864.035 Me
3
Hex
8
HexNAc
2
PA 1863.686
pN13 1497.876 Hex
6
HexNAc
2
PA 1497.534
pN14 2025.914 Me
3
Hex
9
HexNAc
2
PA 2025.739
pN15 1659.802 Hex
7
HexNAc
2
PA 1659.587
pN16 1821.713 Hex
8
HexNAc
2
PA 1821.640
pN17 1983.933 Hex
9
HexNAc
2
PA 1983.692
pN18 2146.133 Hex
10
HexNAc
2
PA 2145.745
S. Natsuka et al. N-glycans of planarian
FEBS Journal 278 (2011) 452–460 ª 2011 The Authors Journal compilation ª 2011 FEBS 457
and then to 0.5 m over the next 10 min. The PA-glycans
were detected using a fluorescence spectrophotometer with
an excitation wavelength of 310 nm and an emission wave-
length of 380 nm. Size-fractionation HPLC was performed
using a TSK gel Amide 80 column (0.46 · 7.5 cm; Tosoh)
at a flow rate of 0.5 mLÆmin
)1
. The column was equili-
brated with 50 mm ammonium formate, pH 4.4, containing
80% acetonitrile. After the sample had been injected, the
acetonitrile concentration was decreased linearly from 80%
to 65% over the first 5 min, 65% to 55% over the second
5 min, and then 55% to 30% over the next 25 min. The
PA-glycans were detected using a fluorescence spectropho-
tometer with an excitation wavelength of 315 nm and an
emission wavelength of 400 nm. The molecular size of each
PA-glycan is given in terms of glucose units based on the
elution times of PA-isomalto-oligosaccharides [14].
Reversed-phase HPLC was performed on a Cosmosil 5C18-
P column (0.2 · 25 cm; Nacalai Tesque, Kyoto, Japan) at a
flow rate of 0.2 mLÆmin
)1
. The column was equilibrated
with 100 mm triethylamine acetate, pH 4.0. After injection
of the sample, the 1-butanol concentration was increased
linearly from 0.075% to 0.5% over 105 min. The PA-gly-
cans were detected using an excitation wavelength of
315 nm and an emission wavelength of 400 nm. The reten-
tion time of each PA-glycan was converted to the reversed-
phase scale as described previously [16]. Reversed-phase
HPLC for PA-monosaccharides was performed on an Ul-
trasphere-ODS column (1.0 · 25 cm; Beckman Coulter,
Brea, CA, USA) at a flow rate of 1.5 mLÆmin
)1
. The col-
umn was equilibrated with 1% acetonitrile in 0.25 m
sodium citrate, pH 4.0. The sample was separated by iso-
cratic elution, and detected using a fluorescence spectropho-
tometer with an excitation wavelength of 315 nm and an
emission wavelength of 400 nm. Borate-chelating anion-
exchange HPLC was performed on a TSKgel Sugar AX-I
column (0.46 · 15 cm; Tosoh) at a flow rate of 0.3
mLÆmin
)1
using a Waters 515 HPLC pump and a Hitachi
L-7485 fluorescence spectrophotometer (Tokyo, Japan).
The column was equilibrated with 10% acetonitrile in 0.8 m
potassium borate, pH 9.0, at a temperature of 74 °Cina
column oven (CTO-10ASvp; Shimadzu, Kyoto, Japan). The
sample was separated by isocratic elution, and detected
using a fluorescence spectrophotometer with an excitation
wavelength of 310 nm and an emission wavelength of
380 nm.
Reducing-end analysis
The reducing-end analysis was performed as reported previ-
ously [17]. The PA-glycans were hydrolyzed with 4 m
hydrochloric acid at 100 °C for 4 h. After complete evapo-
ration ofthe hydrochloric acid, the liberated monosaccha-
rides were re-N-acetylated with acetic anhydride in a
saturated sodium bicarbonate solution. PA-monosaccha-
rides derived from reducing ends were purified on a small
cation-exchange column, as described previously [18].
Briefly, a sample was applied to a Dowex 50Wx2 (H
+
)
column (0.5 · 3 cm). After the column had been washed
with 2 mL of water, PA-sugars were eluted using 3 mL of
2.5% ammonia solution. The eluate was lyophilized to
remove ammonia, and then analyzed by HPLC. PA-sugars
were separated using a TSKgel Sugar AX-I column
(0.46 · 15 cm; Tosoh) equilibrated with 10% acetonitrile
in 0.8 m potassium borate, pH 9.0, at a flow rate of
0.3 mLÆmin
)1
at 74 °C, and detected using a fluorescence
spectrophotometer at an excitation wavelength of 310 nm
and an emission wavelength of 380 nm. The molar ratio of
GlcNAc detected in the fractions was taken as the molar
ratio of N-glycans.
Sugar component analysis
The PA-glycans were hydrolyzed with 4 m trifluoroacetic
acid at 100 °C for 4 h. After complete evaporation of triflu-
oroacetic acid, the liberated monosaccharides were re-N-
acetylated with acetic anhydride in a mixture of methanol
and pyridine (4 : 1 v ⁄ v) for 30 min at room temperature.
The solvent was evaporated, and then the monosaccharides
Fig. 7. Two-dimensional HPLC map ofthe partially hydrolyzed frag-
ments from pN4. pN4 was partially hydrolyzed, and glycan frag-
ments from the pyridylaminated reducing end were separated
using two types of HPLC, as described in Experimental procedures.
The retention times on size-fractionation and reversed-phase
HPLC were converted to glucose units and the reversed-phase
scale, respectively. The positions ofthe standard PA-glycans
GlcNAcb1-4GlcNAc-PA, Manb1-4GlcNAcb1-4GlcNAc-PA, Mana1-
3Manb1-4GlcNAcb1-4GlcNAc-PA (M2A) and Mana1-6Manb1-4Glc-
NAcb1-4GlcNAc-PA (M2B) are indicated by crosses labeled (a)–(d),
respectively.
N-glycans ofplanarian S. Natsuka et al.
458 FEBS Journal 278 (2011) 452–460 ª 2011 The Authors Journal compilation ª 2011 FEBS
were tagged with 2-aminopyridine by a pyridylamination
method modified for monosaccharides [18]. The excess
reagents were removed by repeated evaporation with tolu-
ene and methanol. The PA-monosaccharides were analyzed
by reversed-phase and borate-chelating anion-exchange
HPLC. Standard methylated monosaccharides were
prepared as described previously [19].
Enzyme digestion
Approximately 10 pmol of PA-glycans were digested with
10 unitsÆmL
)1
of jack bean (Canavaria gladiata) a-mannosi-
dase (Seikagaku Kogyo, Tokyo, Japan) in 20 lLof50mm
sodium citrate buffer, pH 4.5, for 20 h at 37 °C.
Mass spectrometric analysis
PA-glycans separated by HPLC were lyophilized to remove
volatile salts in the buffer, and 1–10 pmol were co-crystal-
lized with 1 mgÆmL
)1
of 2,5-dihydroxybenzoic acid in ace-
tonitrile on an AnchorChip target plate (Bruker Daltonics,
Billerica, MA, USA) according to the manufacturer’s
protocol. MALDI-TOF mass spectra were recorded using
an Autoflex II (Bruker Daltonics) in reflector mode.
Partial acid hydrolysis
The PA-glycans were hydrolyzed with 1 m trifluoroacetic
acid at 100 ° C for 30 min [20]. After complete evaporation
of trifluoroacetic acid, the hydrolyzed PA-glycans were
re-N-acetylated with acetic anhydride in a saturated sodium
bicarbonate solution as described above. The PA-glycans
were purified on a small cation-exchange column, as
described previously [18], and analyzed by size-fractionation
and reversed-phase HPLC to produce a glycan map.
References
1 Altmann F, Fabini G, Ahorn H & Wilson IB (2001)
Genetic model organisms in the study of N-glycans.
Biochimie 83, 703–712.
2 Natsuka S, Adachi J, Kawaguchi M, Nakakita S, Hase
S, Ichikawa A & Ikura K (2002) Structuralanalysis of
N-linked glycans in Caenorhabditis elegans. J Biochem
131, 807–813.
3 Cipollo JF, Costello CE & Hirschberg CB (2002) The
fine structure of Caenorhabditis elegans N-glycans.
J Biol Chem 277, 49143–49157.
4 Hanneman AJ, Rosa JC, Ashline D & Reinhold VN
(2006) Isomer and glycomer complexities of core
GlcNAcs in Caenorhabditis elegans. Glycobiology 16,
874–890.
5 Takahashi N, Masuda K, Hiraki K, Yoshihara K,
Huang HH, Khoo KH & Kato K (2003) N-Glycan
structures of squid rhodopsin. Eur J Biochem 270,
2627–2632.
6 Natsuka S, Ishida M, Ichikawa A, Ikura K & Hase S
(2006) Comparative biochemical study of N-linked gly-
cans from skin of a squid, Todarodes pacifics. J Biochem
140, 87–93.
7 Zhang Y, Iwasa T, Tsuda M, Kobata A & Takasaki S
(1997) A novel monoantennary complex-type sugar
chain found in octopus rhodopsin: occurrence of the
Galb1 fi 4Fuc group linked to the proximal N-acetyl-
glucosamine residue ofthe trimannosyl core. Glycobiolo-
gy 7, 1153–1158.
8 van Kuik JA, van Halbeek H, Kamerling JP &
Vliegenthart JF (1985) Primary structure of the
low-molecular-weight carbohydrate chains of
Helix pomatia a-hemocyanin. Xylose as a constituent of
N-linked oligosaccharides in an animal glycoprotein.
J Biol Chem 260, 13984–13988.
9 Gutternigg M, Ahrer K, Grabher-Meier H, Bu
¨
rgmayr S
& Staudacher E (2004) Neutral N-glycans ofthe gastro-
pod Arion lusitanicus. Eur J Biochem 271, 1348–1356.
10 Khoo KH, Chatterjee D, Caulfield JP, Morris HR &
Dell A (1997) Structural mapping ofthe glycans from
the egg glycoproteins of Schistosoma mansoni and Shis-
tosoma japonicum: identification of novel core structures
and terminal sequences. Glycobiology 7, 663–677.
11 Sharkey DJ & Kornfeld R (1989) Identification of an
N-acetylglucosaminyltransferase in Dictyostelium dis-
coideum that transfers an ‘intersecting’ N-acetylglucos-
amine residue to high mannose oligosaccharides. J Biol
Chem
264, 10411–10419.
12 Lehr T, Geyer H, Maass K, Doenhoff MJ & Geyer R
(2006) Structural characterization of N-glycans from the
freshwater snail Biomphalaria glabrata cross-reacting
with Schistosoma mansoni glycoconjugates. Glycobiology
17, 82–103.
13 Yosizawa Z, Sato T & Schmid K (1966) Hydrazinolysis
of a-1-acid glycoprotein. Biochim Biophys Acta 121,
417–420.
14 Natsuka S & Hase S (1998) Analysisof N- and O-gly-
cans by pyridylamination. Methods Mol Biol 76, 101–
113.
15 Yanagida K, Natsuka S & Hase S (1999) A pyridylam-
ination method aimed at automatic oligosaccharide
analysis of N-linked sugar chains. Anal Biochem 274,
229–234.
16 Yanagida K, Ogawa H, Omichi K & Hase S (1998)
Introduction of a new scale into reversed-phase high-
performance liquid chromatography of pyridylamino
sugar chains for structural assignment. J Chromatogr A
800, 187–198.
17 Makino Y, Kuraya N, Omichi K & Hase S (1996) Clas-
sification of sugar chains of glycoproteins by analyzing
reducing end oligosaccharides obtained by partial acid
hydrolysis. Anal Biochem 238, 54–59.
S. Natsuka et al. N-glycans of planarian
FEBS Journal 278 (2011) 452–460 ª 2011 The Authors Journal compilation ª 2011 FEBS 459
18 Suzuki J, Kondo A, Kato I, Hase S & Ikenaka T
(1991) Analysis by high-performance anion-exchange
chromatography of component sugars as their fluores-
cent pyridylamino derivatives. Agric Biol Chem 55,
283–284.
19 Mega T, Nishikawa A & Ikenaka T (1983) A conve-
nient method for the preparation of all the partially
methylated derivatives of methyl a-d-mannopyranoside
and a-d-galactopyranoside. Carbohydr Res 112,
313–319.
20 Makino Y, Omichi K & Hase S (1998) Analysisof oli-
gosaccharide structures from the reducing end terminal
by combining partial acid hydrolysis and a two-dimen-
sional sugar map. Anal Biochem 264, 172–179.
N-glycans ofplanarian S. Natsuka et al.
460 FEBS Journal 278 (2011) 452–460 ª 2011 The Authors Journal compilation ª 2011 FEBS
. map. The diam- eter of the circles is proportional to the cube root of the detected amount of PA-glycans. The positions of standard PA-glycans are indicated by crosses labeled (a)–(g). The standards. In contrast, if the planarian has unique and complex N-glycans, the view that the diversity of N-glycans does not correlate simply with the phylogenic process is supported. The species of planarian. November 2010) doi:10.1111/j.1742-4658.2010.07966.x To investigate the relationship between phylogeny and glycan structures, we analyzed the structure of planarian N-glycans. The planarian Duge- sia japonica, a member of the flatworm family,