Báo cáo khoa học: N-Glycan structures of squid rhodopsin Existence of the a1–3 and a1–6 difucosylated innermost GlcNAc residue in a molluscan glycoprotein pot

N-Glycan structures of squid rhodopsin
Existence of the a1–3 and a1–6 difucosylated innermost GlcNAc residue
in a molluscan glycoprotein
Noriko Takahashi
1
, Katsuyoshi Masuda
2
, Kenji Hiraki
2
, Kazuo Yoshihara
2
, Hung-Hsiang Huang
3
,
Kay-Hooi Khoo
3
and Koichi Kato
1
1
Graduate School of Pharmaceutical Sciences, Nagoya City University, Japan;
2
Suntory Institute for Bioorganic Research,
Shimamoto-cho, Mishima-gun, Osaka, Japan;
3
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
To determine the glycoforms of squid rhodopsin, N-glycans
were released by glycoamidase A digestion, reductively
aminated with 2-aminopyridine, and then subjected to 2D
HPLC analysis [Takahashi, N., Nakagawa, H., Fujikawa,
K., Kawamura, Y. & Tomiya, N. (1995) Anal. Biochem. 226,
139–146]. The major glycans of squid rhodopsin were shown

to possess the a1–3 and a1–6 difucosylated innermost
GlcNAc residue found in glycoproteins produced by insects
and helminths. By combined use of 2D HPLC, electrospray
ionization-mass spectrometry and permethylation and
gas chromatography-electron ionization mass spectrometry
analyses, it was revealed that most (85%) of the N-glycans
exhibit the novel structure Mana1–6(Mana1–3)Manb1–
4GlcNAcb1–4(Galb1–4Fuca1–6)(Fuca1–3)GlcNAc.
Keywords: 2D HPLC mapping; mass spectrometry; N-gly-
can structures; rhodopsin; squid.
Rhodopsin, the visual pigment in the photoreceptor cells, is a
typical seven transmembrane receptor and has been widely
studied to elucidate the mechanisms of a visual transduction
cascade [1,2]. The N-terminal segment of rhodopsin is
N-glycosylated. It has been reported that the carbohydrate
moieties contribute to the integrity of rhodopsin functions,
and abnormalities in the N-glycosylation of rhodopsin are
associated with autosomal dominant retinitis pigmentosa
[3–8]. The rhodopsin N-glycan structures have so far been
determined for bovine [9,10], frog [11], human [12], rat [13]
and octopus [14]. Mammalian and frog rhodopsins, which
conserve two potential glycosylation sites, Asn2 and Asn15,
predominantly express the structure Mana1–6(GlcNAc
b1–2Mana1–3)Manb1–4GlcNAcb1–4GlcNAc. In the case
of bovine, human and frog rhodopsin, both of these sites are
glycosylated. On the other hand, a major glycoform of
octopus rhodopsin, which possesses only one N-glycosyla-
tion site atAsn9, is Mana1–6(Galb1–3GlcNAcb1–2Mana1–
3)Manb1–4GlcNAcb1–4(Galb1–4Fuca1–6)GlcNAc. Thus,
there is asignificant difference between octopus andthe other

species with respect to the N-glycosylation of rhodopsin
in terms of terminal fucosylation and galactosylation.
Here, in the quest for the Ômissing linkÕ in rhodopsin
glycosylation, we attempt to elucidate the detailed structures
of the N-glycans released from rhodopsin of a squid
(Todarodes pacificus), which possesses one glycosylation site
at Asn8 [15] (corresponding to Asn9 in octopus rhodopsin).
As far as we know, this is the first description of the
carbohydrate structure of squid glycoproteins.
Materials and methods
Enzymes
Glycoamidase A (also known as glycopeptidase A, EC
3.5.1.52) from sweet almond [16] and b-galactosidase and
a-mannosidase from jack bean were purchased from
Seikagaku Kogyo (Tokyo, Japan). Trypsin, chymotrypsin
and Pronase were from Sigma Chemical Co. (St Louis, MO,
USA). a-
L
-Fucosidase from bovine kidney was purchased
from Boehringer-Mannheim (Mannheim, Germany).
Reference N-glycans
The pyridylamino (PA) derivatives of isomalto-oligosac-
charides 4–20 (degree of polymerization of glucose residues)
were from Seikagaku Kogyo. PA-oligosaccharide 010.1F
was obtained from neuropsin (murine hippocampus serine
protease) produced in Trichoplusia ni cells [17].
Preparation of rhodopsin from squid
Rhodopsin was prepared from Japanese flying squid,
Todarodes pacificus, caught in the Sea of Japan in autumn
as described previously [18,19]. Briefly, rhabdomeric

Correspondence to N. Takahashi, Graduate School of
Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori,
Mizuho-ku, Nagoya 467-8603 Japan. Tel./Fax: + 81 52 836 3450,
E-mail:
Abbreviations: CID-MS/MS, collision-induced dissociation mass
spectrometry/mass spectrometry; ESI-MS, electrospray ionization-
mass spectrometry; GC-EI-MS, gas chromatography-electron
ionization MS; GU, glucose unit; GU(amide), GU value on the amide
column; GU(ODS), GU value on the octadecyl silica column; ODS,
octadecyl silica; PA, pyridylamino; Q-TOF, quadrupole time-of-flight.
Enzyme: Glycoamidase A (glycopeptidase A, EC 3.5.1.52).
Note: For the code numbers and structures of the PA-oligosaccharides,
please refer to the FCCA web site ( />(Received 28 February 2003, revised 19 April 2003,
accepted 25 April 2003)
Eur. J. Biochem. 270, 2627–2632 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03636.x
membranes were isolated from squid retinae by repetitive
sucrose flotation. Rhodopsin was extracted in 2.5% (w/v)
sucrose monododecanoate (Dojin Kagaku, Kumamoto,
Japan) and purified using DEAE-cellulose (Whatman,
Maidstone, Kent, UK) and concanavalin A–Sepharose 4B
(Amersham Biosciences, Piscataway, NJ, USA) column
chromatography. a-Methyl mannoside in the specimen
eluted from the concanavalin A–Sepharose 4B column was
removed by dialysis.
Preparation of pyridylaminated N-glycans from squid
rhodopsin and characterization by 2D mapping
Rhodopsin protein (1 mg), corresponding to 20 nmol
oligosaccharides, was used as the starting material. All
experimental procedures used, including chromatographic
conditions, have been detailed previously [20,21]. Briefly, the

rhodopsin glycoprotein was proteolysed with a mixture of
trypsin and chymotrypsin, and the proteolysate was further
digested with glycoamidase A to release N-glycans. After
removal of the peptide materials, the reducing ends of the
N-glycans were derivatized with 2-aminopyridine [22]. The
mixture of PA-oligosaccharides was applied to an octadecyl
silica (ODS) HPLC column, and the elution times of the
individual peaks were normalized with reference to the
PA-derivatized isomalto-oligosaccharides of polymerization
degree 4–20 and represented by GU(ODS). Then, individual
fractions separated on the ODS column were applied to the
amide-silica column. In a similar way, the retention times
of the individual peaks on the amide-silica column were
represented by GU(amide). Thus, a given compound from
these two columns provided a unique set of GU(ODS) and
GU(amide) values, which corresponded to co-ordinates
of the 2D HPLC map [20,21]. By comparison with the
co-ordinates of  500 reference PA-oligosaccharides col-
lected so far, the N-glycans from squid rhodopsin were
identified. Identification was confirmed by cochromato-
graphy with a candidate reference on the columns.
Exoglycosidase digestion procedure
a-
L
-Fucosidase. To eliminate a1–3 fucose residues, the
reaction mixture (final 20 lL) containing PA-glycan
(5–50 pmol), a-
L
-fucosidase from bovine kidney (200 mU)
and 0.4

M
acetate buffer (pH 4.5) was incubated for
1–2 days at 37 °C. The reaction products were analysed
by the 2D mapping technique.
b-Galactosidase. The reaction mixture (final 20 lL) con-
taining purified PA-glycan (5–50 pmol), b-galactosidase
from jack bean (5 mU) and 0.1
M
citrate/phosphate buffer
(pH 4.0) was incubated overnight at 37 °C. The reaction
products were analysed by the 2D mapping technique.
Nanoflow ESI-MS analyses
ESI (electrospray ionization)-MS spectra were acquired
using a quadrupole time-of-flight (Q-TOF) instrument
(Micromass, Manchester, UK) and
MASSLYNX
data acqui-
sition. This instrument is a hybrid quadrupole orthogonal
acceleration time-of-flight mass spectrometer, with a
Z-spray nanoflow electrospray ion source. It was operated
in the positive-ion mode. Purified samples were dissolved in
50% aqueous methanol solution containing 0.2% formic
acid, and loaded into a nanoflow tip. A high voltage
(1.0 kV) was applied to the nanoflow tip of the capillary.
MALDI-QTOF MS/MS sequencing and gas
chromatography-electron ionization MS (GC-EI-MS)
methylation analysis
Glycans were permethylated using the NaOH/dimethyl
sulfoxide slurry method as described by Dell et al.[23].
Permethylated glycans were first examined for purity and

subjected to collision-induced dissociation (CID) MS/MS
sequencing using a dedicated MALDI-QTOF Ultima
instrument (Micromass). Samples in acetonitrile were mixed
1:1witha-cyano-4-cinnamic acid matrix (in acetonitrile/
0.1% trifluoroacetic acid, 99 : 1, v/v) and spotted on the
target plate. The nitrogen UV laser (337 nm wavelength)
was operated at a repetition rate of 10 Hz under full power
(300 lJ per pulse). For CID-MS/MS, argon was used as the
collision gas with a collision energy manually adjusted
(between 50 and 200 V) to achieve the optimum degree of
fragmentation for the parent ions under investigation. For
GC-EI-MS linkage analysis, partially methylated alditol
acetates were prepared from permethyl derivatives by
hydrolysis (2
M
trifluoroacetic acid, 121 °C, 2 h), reduction
(10 mgÆmL
)1
NaBH
4
,25°C, 2 h), and acetylation (acetic
anhydride, 100 °C, 1 h). GC-EI-MS was carried out using a
Hewlett-Packard Gas Chromatograph 6890 connected to a
HP 5973 Mass Selective Detector. Sample was dissolved in
hexane before splitless injection into an HP-5MS fused silica
capillary column (30 m · 0.25 mm internal diameter,
Hewlett-Packard)
1
. The column head pressure was main-
tained at  56.6 kPa to give a constant flow rate of

1mLÆmin
)1
using helium as carrier gas. The initial oven
temperature was held at 60 °C for 1 min, increased to 90 °C
for1min,andthento290°C for 25 min.
Results
HPLC profile of PA-oligosaccharide derived
from squid rhodopsin
N-Glycans were released from squid rhodopsin by glyco-
amidase A, derivatized with 2-aminopyridine, and then
subjected to ODS column chromatography. Most (90%) of
the PA-oligosaccharides were eluted apparently as a single
fraction at 14.6 min, which corresponds to a GU(ODS) of
9.8 under the experimental conditions (Fig. 1). This fraction
(tentatively named glycan B) was further chromatographed
on the amide-silica column and separated into two fractions,
glycan B1 and glycan B2, with a molar ratio of 17 : 1 (data
not shown). Each of the two minor fractions with
GU(ODS) of 8.8 and 11.3 gave a single peak on the
amide-silica column, and hereafter are designated glycan A
and glycan C, respectively. The GU(ODS) and GU(amide)
of these four glycans are summarized in Table 1.
On the basis of the GU data of glycan A, i.e. GU(ODS)
of 8.8 and GU(amide) of 5.6, the reference compound
010.1F, which has been reported to exhibit GU(ODS) of 8.6
and GU(amide) of 5.5 [17], was chosen as a candidate
for identification by cochromatography. Glycan A was
2628 N. Takahashi et al.(Eur. J. Biochem. 270) Ó FEBS 2003
coeluted with the reference compound 010.1F from the
ODS column as well as the amide-silica column. Hence,

glycan A was concluded to be 010.1F, Mana1–6(Mana1–
3)Manb1–4GlcNAcb1–4(Fuca1–6)(Fuca1–3)GlcNAc. This
conclusion was confirmed by the fucosidase digestion,
which gave rise to the trimannosyl core, and by ESI-MS
analysis (data not shown). The GU(ODS) and GU(amide)
of the other three glycans, including the most predominant
glycan (glycan B1), did not coincide with any reference
co-ordinate on the 2D map reported so far, indicating that
squid rhodopsin exhibits novel N-glycans, which are differ-
ent from those expressed by rhodopsins of other species.
Identification of the glycan B1 structure
The molecular mass of glycan B1 measured by ESI-MS
analysis was 1442.4 Da, which corresponds to Hex
4
Hex-
NAc
2
DeoxyHex
2
(Fig. 2). After b-galactosidase digestion,
glycan B1 was converted into glycan A, i.e. Hex
3
Hex-
NAc
2
DeoxyHex
2
, indicating that glycan B1 is a b-galactosyl
derivative of glycan A (Fig. 3). Glycan B1 could not be
digested with a-fucosidase under conditions in which an

a1–6-linked fucosyl group was released from glycan A (data
not shown), suggesting that the a1–6-linked fucose residue
may be blocked with a b-galactose residue in glycan B1.
To determine the location and linkage of the b-galactose
residue unambiguously, MALDI-MS/MS and GC-EI-MS
linkage analyses were carried out (Fig. 4). After permethy-
lation, the PA-tagged glycan B1 afforded an [M + Na]
+
molecular ion at m/z 1815, which was selected as parent ion
for CID-MS/MS analysis on a MALDI-QTOF instrument.
As shown in Fig. 4A, the predominant fragment ion pair
(m/z 944 and 894) from cleavage at the chitobiose core
firmly shows the existence of the extra Gal residue on the
reducing end GlcNAc. A fragment ion at m/z 433 provides
direct evidence of a Gal-Fuc unit whereas the ion at m/z 519
can be rationalized as arising from multiple cleavages
consistent with the location of this unusual disaccharide unit
at the C6 position of the reducing end GlcNAc. When
subjected to linkage analysis, glycan B1 gave terminal Fuc,
terminal Man, terminal Gal, 3,6-linked Man, 4-linked
GlcNAc and, importantly, a peak that can be assigned as
4-linked Fuc on the basis of the EI-MS pattern (Fig. 4B).
Taken together, the results unambiguously establish that the
extra b-Gal residue is 4-linked to the Fuc on the 6 arm of a
difucosylated trimannosyl core structure. These results
indicate that the structure of the major N-glycan of squid
rhodopsin is unique: Mana1–6(Mana1–3)Manb1–4Glc-
NAcb1–4(Galb1–4Fuca1–6)(Fuca1–3)GlcNAc.
Identification of glycan B2 and C structures
The molecular mass of glycan C determined by ESI-MS

analysis was 1296.3 Da, which corresponds to Hex
4
Hex-
NAc
2
DeoxyHex
1
. On inspection of these data, we specula-
ted that glycan C is an analog of glycan B1 lacking
one fucosyl group. To examine this, we carried out an
a-fucosidase digestion of glycan B1. Although the digestion
under the milder reaction condition resulted in no defuco-
sylation of glycan B1 (vide supra), it was converted into
glycan C after incubation for 2 days at a higher enzyme to
substrate concentration, which was confirmed by cochro-
matography of the digestion product of glycan B1 with
glycan C on the ODS and amide-silica columns. On the
basis of these data, we conclude that glycan C is an analog
of glycan B1 that lacks only the a1–3-linked fucose residue.
ESI-MS analysis showed that the molecular mass of
glycan B2 was 1280.3 Da, which corresponds to Hex
3
Hex-
NAc
2
DeoxyHex
2
. This suggests that glycan B2 is an analog
of glycan B1 that lacks one of the two nonreducing terminal
Fig. 1. Elution profile on the ODS column of the PA-oligosaccharide

mixture obtained from squid rhodopsin.
Ó FEBS 2003 N-glycan structures of squid rhodopsin (Eur. J. Biochem. 270) 2629
mannose residues. As reference compounds corresponding
to these candidates were not available, we determined effect
of the demannosylation on the GU co-ordinates in the 2D
map based on the diagram of the partial unit contribution
(UC)values,whichwerecalculatedonthebasisof
accumulated GU data from the 2D map by multiple
regression [24]. We have demonstrated that the GU(ODS)
and GU(amide) of a given PA-glycan can be represented by
the sum of the contribution of each component monosac-
charide unit. The UC values of the a1–6-linked and a1–3-
linked mannose residues on GU(ODS) and GU(amide)
values have been reported as + 0.80 and + 1.29, respect-
ively, for a1–6-linked mannose, and – 0.01 and + 1.03,
respectively, for a1–3-linked mannose [24]. The fact that the
differences in the GU(ODS) and GU(amide) between
glycan B2 (9.8, 5.7) and glycan B1 (9.8, 6.7) were 0.0 and
1.0, respectively, strongly suggests that glycan B2 lacks the
a(1,3)-linked but not the a1–6-linked mannose residue.
The structures of the N-glycans of squid rhodopsin are
summarized in Table 1.
Discussion
The N-glycosylation profiles of rhodopsin in human [12],
bovine [9,10], rat [13] and frog [11] have been reported.
The N-glycans expressed on rhodopsin of these animals
possess a major common structure GlcNAcb1–2 Mana1–
3(Mana1–6)Manb1–4GlcNAcb1–4GlcNAc. In contrast,
octopus rhodopsin [14] expresses a unique N-glycan struc-
ture which contains a characteristic Galb1–4Fuca1–6

branch attached to the reducing terminal GlcNAc. Most
of the N-glycans of squid rhodopsin determined in this
study also exhibit this branch. However, there is a significant
difference in N-glycan structures between squid and octopus
rhodopsin molecules. Squid rhodopsin lacks the terminal
Galb1–3GlcNAcb1–2 sequence. Moreover, most (93.4%)
of the N-glycans in squid rhodopsin possess the a1–3 and
a1–6 difucosylated innermost GlcNAc residue, which has
not been reported for octopus rhodopsin or glycoproteins
from other molluscs. It has been proposed that N-glycosy-
lation blocks reorientation of a polypeptide chain within the
translocon and therefore can influence topogenesis of
membrane glycoproteins [25]. Molluscan rhodopsin posses-
ses only one N-glycosylation site, whereas frog, bovine, and
human (and possibly other mammalian) rhodopsin mole-
cules have conserved two N-glycosylation sites at their
N-terminal segments. We speculate that the bulky branches
Fig. 2. Electrospray ionization mass spectrum of PA-glycan B1.
Fig. 3. Relationship of coordinates of PA-oligosaccharides glycans A,
B1, B2 and C, on the 2D map. The starting material, glycan B1, was
converted into glycans A, B2 and C after treatment with b-galacto-
sidase, a-mannosidase and a-
L
-fucosidase, respectively.
2630 N. Takahashi et al.(Eur. J. Biochem. 270) Ó FEBS 2003
attached to the innermost GlcNAc residue, i.e. Galb1–
4Fuca1–6 and/or the Fuca1–3 residues, act as a stopper, by
which the only glycan can prevent the nascent polypeptide
chain from reorienting within the translocon. It is also
possible that difucosylation of the innermost GlcNAc

affects rhodopsin function by a local conformational change
in the polypeptide chain. Examination of the N-glycan
structures provides insight into the processing of sugar
chains in molluscs (Fig. 5) In this context, the question of
whether the unique N-glycan structures of rhodopsin are
common to other squid glycoproteins is of great import-
ance. The difucosyl trimannosyl core structure has so far
been found in glycoproteins from insects [26–30] and
helminths [31,32]. Therefore, it would be of interest to
investigate the universality of this core structure in glyco-
proteins from animals. For this purpose, glycoami-
dase A could be a useful tool because N-glycans with an
a1–3-fucosylated reducing end cannot be released effectively
by treatment with peptide–N4-(N-acetyl-b-glucosaminyl)
asparagine amidase F [33] or hydrazinolysis [34].
Acknowledgements
We thank the Core Facilities for Proteomic research at the Academia
Sinica, Taiwan, for the use of the MALDI-QTOF instrument. This
work was supported by Grant-in-Aid for Scientific Research from
the Ministry of Education, Culture, Sports, Science and Technology,
CREST of the Japan Science and Technology Corporation, the
Yamada Science Foundation, and the Mizutani Foundation for
Glycoscience. K.K.H. and H.H.H. are supported financially by the
Academia Sinica, Taiwan.
Fig. 5. Proposed N-glycan-processing pathway in molluscs.
Fig. 4. MALDI-CID-MS/MS sequencing of
permethylated PA-glycan B1 (A) and further
identification of linkage position by GC-EI-MS
analysis (B). The MS/MS fragment ions were
assigned as shown schematically. The EI-mass

spectrum for the 4-linked Fuc peak is shown in
(B) together with the fragmentation scheme
for all three possible singly linked Fuc residues
(a–c). No other peak corresponding to other
singly linked deoxyhexose could be detected
when the chromatogram was extracted for
ions at m/z 118 and 189. Other peaks in the gas
chromatogram were identified by referring to
their retention time and EI spectra, compared
against authentic standards.
Ó FEBS 2003 N-glycan structures of squid rhodopsin (Eur. J. Biochem. 270) 2631
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