N-Glycan structures of squid rhodopsinExistence of the a1–3 and a1–6 difucosylated innermost GlcNAc residue in a molluscan glycoprotein Noriko Takahashi1, Katsuyoshi Masuda2, Kenji Hirak
Trang 1N-Glycan structures of squid rhodopsin
Existence of the a1–3 and a1–6 difucosylated innermost GlcNAc residue
in a molluscan glycoprotein
Noriko Takahashi1, Katsuyoshi Masuda2, Kenji Hiraki2, Kazuo Yoshihara2, Hung-Hsiang Huang3,
Kay-Hooi Khoo3and Koichi Kato1
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 2DHPLC, 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: 2DHPLC 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 at Asn9, is Mana1–6(Galb1–3GlcNAcb1–2Mana1–
3)Manb1–4GlcNAcb1–4(Galb1–4Fuca1–6)GlcNAc Thus,
there is a significant difference between octopus and the other species with respect to the N-glycosylation of rhodopsin
in terms of terminal fucosylation and galactosylation Here, in the quest for the missing link 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: ntakahas@phar.nagoya-cu.ac.jp
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 (http://www.gak.co.jp/FCCA).
(Received 28 February 2003, revised 19 April 2003,
accepted 25 April 2003)
Trang 2membranes 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 2DHPLC 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 0.4M acetate buffer (pH 4.5) was incubated for
1–2 days at 37C The reaction products were analysed
by the 2Dmapping 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 0.1Mcitrate/phosphate buffer
(pH 4.0) was incubated overnight at 37C The reaction
products were analysed by the 2Dmapping technique
Nanoflow ESI-MS analyses
ESI (electrospray ionization)-MS spectra were acquired
using a quadrupole time-of-flight (Q-TOF) instrument
(Micromass, Manchester, UK) andMASSLYNXdata
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 : 1 with a-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 (2Mtrifluoroacetic acid, 121C, 2 h), reduction (10 mgÆmL)1NaBH4, 25C, 2 h), and acetylation (acetic anhydride, 100C, 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
1 mLÆmin)1using helium as carrier gas The initial oven temperature was held at 60C for 1 min, increased to 90 C for 1 min, and then to 290C 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
Trang 3coeluted 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 2Dmap 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 Hex4 Hex-NAc2DeoxyHex2(Fig 2) After b-galactosidase digestion, glycan B1 was converted into glycan A, i.e Hex3 Hex-NAc2DeoxyHex2, 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 Hex4 Hex-NAc2DeoxyHex1 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 Hex3 Hex-NAc2DeoxyHex2 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 obtainedfrom squidrhodopsin.
Trang 4mannose 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, which were calculated on the basis of
accumulated GU data from the 2Dmap 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 andC, 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.
Trang 5attached 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 ProposedN-glycan-processing pathway in molluscs.
Fig 4 MALDI-CID-MS/MS sequencing of
permethylatedPA-glycan B1 (A) andfurther
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.
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