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: 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) 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 References 1. Khorana, H.G. (1992) Rhodopsin, photoreceptor of the rod cell. An emerging pattern for structure and function. J. Biol. Chem. 267, 1–4. 2. 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(1997) Structural mapping of the glycans from the egg glyco- proteins of Schistosoma mansoni and Schistosoma japonicum: identification of novel core structures and terminal sequences. Glycobiology 7, 663–677. 33. Tretter,V.,Altmann,F.&Marz,L.(1991)Peptide–N4-(N-acetyl- b-glucosaminyl) asparagine amidase F cannot release glycans with fucose attached a1–3 to the asparagine-linked N-acetylglucosa- mine residue. Eur. J. Biochem. 199, 647–652. 34. Hollander, T., Aeed, P.A. & Elhammer, A.P. (1993) Character- ization of the oligosaccharide structures on bee venom phospho- lipase A2. Carbohydr. Res. 247, 291–297. 2632 N. Takahashi et al.(Eur. J. Biochem. 270) Ó FEBS 2003 . 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 ,. 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,