Carbohydrate Binding Specificity of a Fucose-specific Lectin from Aspergillus oryzae
2007; Elsevier BV; Volume: 282; Issue: 21 Linguagem: Inglês
10.1074/jbc.m701195200
ISSN1083-351X
AutoresKengo Matsumura, Katsuya Higashida, Hiroki Ishida, Yoji Hata, Kenji Yamamoto, Masaki Shigeta, Yoko Mizuno-Horikawa, Xiangchun Wang, Eiji Miyoshi, Jianguo Gu, Naoyuki Taniguchi,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoThe α1,6-fucosyl residue (core fucose) of glycoproteins is widely distributed in mammalian tissues and is altered under pathological conditions. A probe that specifically detects core fucose is important for understanding the role of this oligosaccharide structure. Aleuria aurantia lectin (AAL) and Lens culimaris agglutinin-A (LCA) have been often used as carbohydrate probes for core fucose in glycoproteins. Here we show, by using surface plasmon resonance (SPR) analysis, that Aspergillus oryzae l-fucose-specific lectin (AOL) has strongest preference for the α1,6-fucosylated chain among α1,2-, α1,3-, α1,4-, and α1,6-fucosylated pyridylaminated (PA)-sugar chains. These results suggest that AOL is a novel probe for detecting core fucose in glycoproteins on the surface of animal cells. A comparison of the carbohydrate-binding specificity of AOL, AAL, and LCA by SPR showed that the irreversible binding of AOL to the α1,2-fucosylated PA-sugar chain (H antigen) relative to the α1,6-fucosylated chain was weaker than that of AAL, and that the interactions of AOL and AAL with α1,6-fucosylated glycopeptide (FGP), which is considered more similar to in vivo glycoproteins than PA-sugar chains, were similar to their interactions with the α1,6-fucosylated PA-sugar chain. Furthermore, positive staining of AOL, but not AAL, was completely abolished in the cultured embryo fibroblast (MEF) cells obtained from α1,6-fucosyltransferase (Fut8) knock-out mice, as assessed by cytological staining. Taken together, these results suggest that AOL is more suitable for detecting core fucose than AAL or LCA. The α1,6-fucosyl residue (core fucose) of glycoproteins is widely distributed in mammalian tissues and is altered under pathological conditions. A probe that specifically detects core fucose is important for understanding the role of this oligosaccharide structure. Aleuria aurantia lectin (AAL) and Lens culimaris agglutinin-A (LCA) have been often used as carbohydrate probes for core fucose in glycoproteins. Here we show, by using surface plasmon resonance (SPR) analysis, that Aspergillus oryzae l-fucose-specific lectin (AOL) has strongest preference for the α1,6-fucosylated chain among α1,2-, α1,3-, α1,4-, and α1,6-fucosylated pyridylaminated (PA)-sugar chains. These results suggest that AOL is a novel probe for detecting core fucose in glycoproteins on the surface of animal cells. A comparison of the carbohydrate-binding specificity of AOL, AAL, and LCA by SPR showed that the irreversible binding of AOL to the α1,2-fucosylated PA-sugar chain (H antigen) relative to the α1,6-fucosylated chain was weaker than that of AAL, and that the interactions of AOL and AAL with α1,6-fucosylated glycopeptide (FGP), which is considered more similar to in vivo glycoproteins than PA-sugar chains, were similar to their interactions with the α1,6-fucosylated PA-sugar chain. Furthermore, positive staining of AOL, but not AAL, was completely abolished in the cultured embryo fibroblast (MEF) cells obtained from α1,6-fucosyltransferase (Fut8) knock-out mice, as assessed by cytological staining. Taken together, these results suggest that AOL is more suitable for detecting core fucose than AAL or LCA. Lectins are specific carbohydrate-binding or carbohydrate-cross-linking proteins. Many studies have isolated and investigated lectins from a wide range of species including plants, animals and microorganisms. The cell surfaces of organisms are covered with abundant and diverse carbohydrates. Because of structural diversity, the set of carbohydrates that is expressed on a cell surface has a role in various biological recognition phenomena, including cell-cell interactions, cell-substratum interactions, and metastasis of tumor cells, among others (1Drickamer K. Taylor M.E. Annu. Rev. Cell Biol. 1993; 9: 237-264Crossref PubMed Google Scholar). Therefore, some lectins have particular value as specific probes for investigating the distribution, structure and biological function of carbohydrate chains on the cell surface of animal, plant, and microorganism because of their specificity for defined carbohydrate structures (2Vijayan M. Chandra N. Curr. Opin. Struct. Biol. 1999; 9: 707-714Crossref PubMed Scopus (232) Google Scholar). α-l-Fucopyranosyl residues are widely distributed in cell-surface sugar chains and often play important roles in biological phenomena. These residues constitute a part of important antigens, such as the blood group antigen H (3Pereira M.E.A. Kabat E.A. Biochemistry. 1974; 13: 3184-3192Crossref PubMed Google Scholar) and stage-specific embryonic antigens (4Stelck S. Robitzki A. Willbold E. Layer P.G. Glycobiology. 1999; 9: 1171-1179Crossref PubMed Google Scholar). Increased levels of fucosyl residues and changes in fucosylation patterns, as a result of different expression levels of various fucosyltransferases, act as specific markers for developmental antigens, particularly in inflammatory processes and in various cancers (5Kolanus W. Bevilacqua M. Seed B. Science. 1990; 250: 1132-1135Crossref PubMed Scopus (887) Google Scholar, 6Noda K. Miyoshi E. Gu J. Gao C. Nakahara S Kitada T. Honke K. Suzuki K. Yoshihara H. Yoshikawa K. Kawano K. Tonetti M. Kasahara A. Hori M. Hayashi N. Taniguchi N. Cancer Res. 2003; 63: 6282-6289PubMed Google Scholar). Furthermore, the α1,6-fucosylated oligosaccharide content of both liver and serum glycoproteins is elevated during the development of malignant liver diseases because the activity of Fut8 is increased (7Hutchinson W.L. Du M.Q. Johnson P.J. Williams R. Hepatology. 1991; 13: 683-688Crossref PubMed Google Scholar). In particular, the sugar chains of α-fetoprotein in serum, a well established tumor marker that is produced by hepatocellular carcinomas, have an abundance of core fucose (8Ohno M. Nishikawa A. Kouketsu M. Taga H. Endo Y. Hada T. Higashino K. Taniguchi N. Int. J. Cancer. 1992; 51: 315-317Crossref PubMed Scopus (55) Google Scholar). To date, some lectins have been identified as fucose-specific including Lotus tetragonolobus (3Pereira M.E.A. Kabat E.A. Biochemistry. 1974; 13: 3184-3192Crossref PubMed Google Scholar) and Ulex europaeus (9Matsumoto I. Osawa T. Biochim. Biophys. Acta. 1969; 194: 180-189Crossref PubMed Google Scholar) lectins from plants, Anguilla lectin from eel (10Watkins W.M. Morgan W.T.J. Nature. 1952; 169: 825-826Crossref PubMed Scopus (114) Google Scholar), Aleuria aurantia lectin (AAL) 2The abbreviations used are: AAL, Aleuria aurantica lectin; LCA, Lens culinaris agglutinin; AOL, Aspergillus oryzae lectin; PA, pyridylaminated; SPR, surface plasmon resonance; FGP, α1,6-fucosylated glycopeptide; MEF, mouse embryo fibroblasts; FUT8, α1,6-fucosyltransferase; SGP, sialylglycopeptide; PBS, phosphate-buffered saline; RU, resonance units; GlcNAc, N-acetylglucosamine; MES, 4-morpholineethanesulfonic acid. from mushroom (11Kochibe N. Furukawa K. Biochemistry. 1980; 19: 2841-2846Crossref PubMed Google Scholar), Rhizopus stolonifer lectin from fungi (12Oda Y. Senaha T. Matsuno Y. Nakajima K. Naka R. Kinoshita M. Honda E. Furuta I. Kakehi K. J. Biol. Chem. 2003; 278: 32439-32447Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), and Ralstonia solanacearum lectin from bacteria (13Sudakevitz D. Imbertyu A. Gilboa-Garber N. J. Biochem. (Tokyo). 2002; 132: 353-358Crossref PubMed Google Scholar). Among these lectins, AAL and R. stolonifer lectin preferentially bind to α1,6-fucosylated oligosaccharides, whereas Ulex europaeus and Lotus tetragonolobus lectins prefer α1,2-linked fucose residues (14Stephan E.B. Juergen T. Young-Ok P. Franz-George H. Jacques B. Robert F. Glycoconj. J. 1996; 13: 585-590Crossref PubMed Scopus (63) Google Scholar). AAL is a commercially available lectin that is known for its high affinity for α1,6-fucosylated oligosaccharides (15Fukumori F. Takeuchi N. Hagiwara T. Ohbayashi H. Endo T. Kochibe N. Nagata Y. Kobata A. J. Biochem. 1990; 107: 190-196Crossref PubMed Google Scholar), and it is widely used to estimate the extent of α1,6-fucosylation (core fucosylation) on glycoproteins and to fractionate glycoproteins (16Yamashita K. Kochibe N. Ohkura T. Ueda I. Kobata A. J. Biol. Chem. 1985; 260: 4688-4693Abstract Full Text PDF PubMed Google Scholar). Another lectin that recognizes oligosaccharides containing core fucose would be a valuable tool in glycobiological research because only a few lectins have been identified as specific for core fucose, and AAL itself exhibits broad specificity for α1,2-, α1,3-, and α1,4-fucose-containing oligosaccharides (17Wimmerova M. Mitchell E. Sanchez J.F. Gautier C. Imberty A. J. Biol. Chem. 2003; 278: 27059-27067Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). We previously identified a novel lectin, AOL, in iron-deficient cultures of the filamentous fungus A. oryzae; this lectin turned out to be l-fucose-specific from a hemagglutination inhibition assay using several monosaccharides and the encoding gene, fleA, was found to share 26% homology with AAL in primary structure (18Ishida H. Moritani T. Hata Y. Kawato A. Suginami K. Abe Y. Imayasu S. Biosci. Biotechnol. Biochem. 2002; 66: 1002-1008Crossref PubMed Google Scholar). Here, to verify whether AOL might be a valuable tool in glycobiological studies involving immunochemical, biochemical, and functional techniques for characterizing, we have examined the carbohydrate binding specificity of AOL by using SPR analysis, lectin affinity chromatography, lectin blot analysis, and immunocytochemical staining as compared with AAL. Our results indicate that AOL is, to our knowledge, the most specific probe for core fucose identified so far. Materials—AAL and LCA were purchased from Seikagaku Kogyo (Tokyo, Japan). The seven PA-sugar chains were purchased from Takara Bio. (Kyoto, Japan). The Lewis x trisaccharide was purchased from Dextra Laboratories, Ltd. (Reading, UK). The nonlabeled sugar was pyridylaminated with GlycoTag (Takara Bio) and then was subjected to a Cellulose Cartridge Glycan preparation kit (Takara Bio) to obtain α1,3-fucosylated (tri Le-x) PA-sugar chain. Structures of the differently fucosylated oligosaccharides used are shown in Table 1 and Fig. 6. Neuraminidase, β-galactosidase, and α1,6-fucosyltransferase were purchased from Nakarai Tesque (Kyoto, Japan), Seikagaku Kogyo and TOYOBO (Osaka, Japan), respectively.TABLE 1Structure of PA-sugar chains and FGP Open table in a new tab FIGURE 6Comparison of fractionation of fucosylated oligosaccharides by AOL and AAL lectin affinity chromatography. α1,6-fucosylated (biantennary) and α1,3-fucosylated (tri Le-x) PA-sugar chains were applied to an AOL (A) or AAL (B)-immobilized column (bed volume, 1 ml) equilibrated with 50 mm sodium phosphate buffer, pH7.4, containing 0.15 m NaCl, at room temperature. Fractions of 0.5 ml were collected throughout, and each PA-sugar chain was separated by reversed-phase HPLC and quantified by fluorescence. Initially, four volumes of equilibration buffer were applied to elute the non-bound fraction, and then a linear gradient of 0.05–0.55 mm fucose in the same buffer was applied over five volumes, followed by a volume of 1 mm fucose, a volume of 10 mm fucose, and four volumes of equilibration buffer. Closed circles, α1,6-fucosylated (biantennary); open circles, α1,3-fucosylated (tri Le-x) PA-sugar chains.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Preparation of FGP—FGP was prepared from a sialylglycopeptide (SGP) obtained from egg yolk as described by Seko et al. (19Seko A. Koketsu M. Nishizono M. Enoki Y. Ibrahim H.R. Juneja L.R. Kim M. Yamamoto T. Biochim. Biophys. Acta. 1997; 1335: 23-32Crossref PubMed Scopus (206) Google Scholar). In brief, the SGP (2.5 mg) was dissolved in 0.2 ml of 50 mm sodium citrate buffer, pH 5.0, and incubated with neuraminidase (3 units) at 37 °C for 24 h. The mixture was heated in a boiling water bath for 5 min and centrifuged at 10,000 × g, and then the supernatant was subjected to a Cellulose Cartridge Glycan preparation kit (Takara Bio) to obtain the asialo-glycopeptide. This asialo-glycopeptide (1.28 mg) was dissolved in 200 μl of 50 mm sodium citrate buffer, pH 5.0, and digested with β-galactosidase (5 units) at 37 °C for 48 h. After purification with the Cellulose Cartridge, the glycopeptide was dissolved in 0.2 ml of 200 mm MES buffer, pH 7.0, and then incubated with α1,6-fucosyltransferase (5 mU) at 37 °C for 24 h. The products were fluorescently labeled with N-[2-(2-pyridylamino)ethyl] succinamic acid 5-norbornene-2,3-dicarboxyimide ester (WAKO) and purified by HPLC as described previously (20Inamori K. Endo T. Gu J. Matsuo I. Ito Y. Fujii S. Iwasaki H. Narimatsu H. Miyoshi E. Honke K. Taniguchi N. J. Biol. Chem. 2004; 279: 2337-2340Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The structure of the FGP was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Preparation of AOL—AOL was prepared by expressing the encoding gene, fleA, in the homologous hyperexpression system of A. oryzae as we described previously (18Ishida H. Moritani T. Hata Y. Kawato A. Suginami K. Abe Y. Imayasu S. Biosci. Biotechnol. Biochem. 2002; 66: 1002-1008Crossref PubMed Google Scholar). The A. oryzae mycelium was used to prepare intracellular protein. A transformant harboring the fleA gene under the control of the melO promoter was cultured in 100 ml of modified Czapek-Dox medium (0.3% NaNO3, 0.2% KCl, 0.1% KH2PO4, 0.05% MgSO4·7H2O, 0.002% FeSO4·7H2O together with 6% glucose, pH 6.0) at 30 °C for 7 days. After collecting the mycelia, a cell-free extract was prepared by disruption with sea sand in 20 mm sodium phosphate buffer (pH 7.0) containing 1.0 mm phenylmethylsulfonyl fluoride. The resultant homogenate was centrifuged (10,000 × g, 10 min), and the supernatant was used for further purification. All purification steps were performed at 4 °C. After salt precipitation with ammonium sulfate, the active hemagglutinating fraction (precipitated at 0.30–0.75 saturation) of the cell-free extract was suspended in 20 mm sodium phosphate buffer (pH 6.0). The suspension was dialyzed overnight against the same buffer and then applied to a column of CM Toyoperl 650 m, 1.6 cm × 10 cm (TOSO, Tokyo, Japan), equilibrated with 50 mm sodium phosphate buffer (pH 6.0). AOL was eluted with a 0–500 mm NaCl linear gradient in the same buffer. The peak fractions with hemagglutinating activity were pooled and dialyzed overnight against the same buffer. Purified AOL was stored at 4 °C because freezing causes 15–20% loss of the protein. SPR Measurements—The Biacore 2000 instrument, BIA evaluation software 3.0, sensor chip CM5 and the amino coupling kit were obtained from Biacore AB (Uppsala, Sweden). The surface of a research grade CM5 sensor chip was activated at a flow rate of 5 μl/min with 1:1 mixture of N-hydroxysuccinimide and N-ethyl-N′-(dimethylaminopropyl) carbodiimide solution for 20 min. Each lectin, at a concentration of 100 μg/ml in 10 mm sodium acetate buffer, pH 5.0, was injected for 20 min, and the remaining N-hydroxysuccinimide esters were blocked by the addition of 1 m ethanolamine, for 20 min. AOL, AAL, and LCA were immobilized to flow cells on the CM5 sensor chip at 322, 413, and 541 fmol, respectively. Measurements were carried out simultaneously on all four measuring channels of which three were immobilized AOL, AAL, and LCA, and the fourth channel was the reference flow cell. The fourth channel was treated identically except for the injection of lectin. All analyses were performed at 25 °C in HBS buffer (10 mm Hepes buffer, pH 7.4 containing 0.01% Tween 20) at a flow rate of 50 μl/min. The concentrations of PA-sugar chains and FGP solutions were, respectively, 1.0 and 7.0 nmol/ml in 100 μl of HBS buffer. The analytes were injected for 2 min, and after a dissociation period of 3.5 min, the surface was regenerated with a 1-min pulse of 100 mm glycine buffer, pH 3.0. All binding experiments were repeated three times, and similar results were obtained. Fractionation of PA-Sugar Chains by AOL or AAL Lectin Affinity Chromatography—Purified AOL (2.5 mg) and AAL (2.7 mg) were each dissolved in 200 mm sodium acid carbonate buffer (pH 8.3) containing 0.5 m NaCl and coupled to an NHS-activated HiTrap column (1 ml; GE Healthcare, Buckinghamshire, UK) according to the manufacturer's instructions. The amount of protein immobilized was determined by measuring the amount of uncoupled protein in the wash fraction. Lectin affinity chromatography was performed at room temperature. The AOL- or AAL-immobilized column was equilibrated with 50 mm sodium phosphate buffer (pH 7.4), containing 0.15 m NaCl. α1,6-fucosylated (biantennary), and α1,3-fucosylated (tri Le-x) PA-sugar chains (80 pmol each) were applied to each column. The non-bound fraction was eluted with four volumes of equilibration buffer. The bound fraction was eluted with a linear gradient of 0.05–0.55 mm fucose in equilibration buffer applied over five volumes, and the column was then washed with a volume of 1 mm fucose, a volume of 10 mm fucose, and four volumes of equilibration buffer. Fractions of 0.5 ml were collected throughout. To separate PA-sugar chains, a 20-μl aliquot of each fraction was applied to reversed-phase HPLC using 20 mm phosphate buffer, pH 4.0, containing 1.0% (v/v) nBtOH, at 40 °C. Each PA-sugar chain was quantified by fluorescence. Biotinylation of Lectins—AOL, AAL, and LCA were biotinylated using a biotin labeling kit from Roche Applied Science (Tokyo, Japan). The d-biotinoyl-ϵ-aminocaproic acid-N-hydroxysuccinimide ester (15 μl; 2 mg/ml in dimethylformamide) was added dropwise to a solution of each lectin (1.0 ml; 1.0 mg/ml in phosphate-buffered saline (PBS)) while being agitated with a vortex mixer. After incubation for 2 h at room temperature with gentle stirring, the labeled lectin was collected by Sephadex G-25 column chromatography and stored at 4 °C until use. Lectin Blot Analysis—Lectin blot analyses were performed as described previously (21Miyoshi E. Ihara Y. Hayashi N. Fusamoto H. Kamada T. Taniguchi N. J. Biol. Chem. 1995; 270: 28311-28315Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In brief, 10 μg of protein was subjected to 8% SDS-PAGE. After electrophoresis, the gels were blotted onto nitrocellulose membranes. The membranes were incubated overnight with 3% bovine serum albumin in Tris-buffered saline (20 mm Tris, 0.5 m NaCl, pH 7.5; TBS), and then for 1 h with 1.9 μg/ml of biotinylated AOL lectins or 0.5 μg/ml of biotinylated AAL in TBST (TBS containing 0.05% Tween 20). After being washed with TBST, the membranes were incubated with horseradish peroxidase-conjugated avidin (VECTASTAIN ABC kit; Vector Laboratories, Burlingame, CA) for 30 min and then washed with TBST. Staining was performed with ECL Western blot detection reagents (GE Healthcare). Cytochemical Staining with Lectins—MEF cell cultures were prepared as previously described (22Wang X. Inoue S. Gu J. Miyoshi E. Noda K. Li W. Mizuno-Horikawa Y. Nakano M. Asahi M. Takahashi M. Uozumi N. Ihara S. Lee S.H. Ikeda Y. Yamaguchi Y. Aze Y. Tomiyama Y. Fujii J. Suzuki K. Kondo A. Shapiro S.D. Lopez-Otin C. Kuwaki T. Okabe M. Honke K. Taniguchi N. Proc. Natl. Acad. Sci. 2005; 102: 15791-15796Crossref PubMed Scopus (351) Google Scholar). In brief, cultures of fibroblasts from wild-type or Fut8 knock-out mice (22Wang X. Inoue S. Gu J. Miyoshi E. Noda K. Li W. Mizuno-Horikawa Y. Nakano M. Asahi M. Takahashi M. Uozumi N. Ihara S. Lee S.H. Ikeda Y. Yamaguchi Y. Aze Y. Tomiyama Y. Fujii J. Suzuki K. Kondo A. Shapiro S.D. Lopez-Otin C. Kuwaki T. Okabe M. Honke K. Taniguchi N. Proc. Natl. Acad. Sci. 2005; 102: 15791-15796Crossref PubMed Scopus (351) Google Scholar) were prepared by trypsinization of 14–15 day embryos. The cells were grown in Dulbecco's modified Eagle's medium (Sigma) at 37 °C in a humidified incubator supplied with 5% CO2 in air. The cell solution (0.3 ml) was plated at an initial density of 1.0 × 104 cells/ml on coverslips (Fisher Scientific Waltham, MA), and placed in current 24-well cell-culture plates (IWAKI Chiba Japan). The cells were incubated for 24 h before examination and were washed three times with PBS buffer at 0.5 ml/well (washing step). The cells were fixed in 0.1 m phosphate buffer containing 4% paraformaldehyde for 20 min at room temperature and dehydrated in ice-cold ethanol for 10 min after the washing step. Unbound sites were blocked by incubation with 200 μl of 1.0% (w/v) bovine serum albumin in washing solution for 30 min. After the blocking solution was removed, the wells were incubated with 100 μl of biotinylated AOL (5.0 or 50 μg/ml) or AAL (2.5 or 5.0 μg/ml) in blocking solution for 2 h at room temperature. Localization of lectin was visualized by an avidin-biotin coupling immunofluorescence technique. The cultured cells were incubated with streptavidin, Alexa Fluor 546 conjugate (Molecular Probes S11225, Eugene, OR) at room temperature for 1 h, then washed and mounted with aqueous mounting medium (Permaflour, Beckman-Coulter, Paris, France). Fluorescent images were analyzed with an Olympus fluorescence microscope BXF50–3 (Olympus, Tokyo, Japan). Preparation and Protein Characteristics of AOL—To clarify the function of AOL, its encoding gene fleA was overexpressed in A. oryzae, resulting in a maximum yield of 1.0 g/liter-broth/7 d of AOL. The intracellular recombinant AOL was purified to such an extent that it resulted in the agglutination of rabbit red blood cells at a concentration of 3.9 μg/ml of lectin in PBS (pH 7.2), as described under “Experimental Procedures.” The purified AOL gave a single band on SDS-PAGE (Fig. 1 lane 1). As we described previously, AOL shares 26% homology with AAL in primary structure (18Ishida H. Moritani T. Hata Y. Kawato A. Suginami K. Abe Y. Imayasu S. Biosci. Biotechnol. Biochem. 2002; 66: 1002-1008Crossref PubMed Google Scholar). As shown in Fig. 1, the molecular weight of AOL on SDS-PAGE was 35,000, a little higher than that of AAL. By contrast, LCA appeared as two bands corresponding to molecular weights of 6000 and 18,000. Previous studies have reported the molecular weight of AAL as 66,796, consisting of identical subunits of 33,398 (15Fukumori F. Takeuchi N. Hagiwara T. Ohbayashi H. Endo T. Kochibe N. Nagata Y. Kobata A. J. Biochem. 1990; 107: 190-196Crossref PubMed Google Scholar), and that of LCA as 46,000, consisting of four subunits (α2β2): two of 5,710 and two of 17,572 (24Toyoshima S. Osawa T. Tonomura A. Biochim. Biophys. Acta. 1970; 221: 514-521Crossref PubMed Scopus (94) Google Scholar). In SPR analysis, HCl injection (100 mm, 100 μl) into a CM5 sensor chip flow cell immobilized with purified AOL resulted in a decrease in resonance units (RU), corresponding to approximately half the amount of immobilized ligand (data not shown), which suggests that AOL consists of two identical subunits. In addition to the three-dimensional structure, AOL and AAL were found to have almost the same isoelectric point, pI 9.0 (data not shown). These results suggest that AOL is similar to AAL not only in amino acid sequence but also in protein characteristics. The Carbohydrate Binding Specificity of AOL—An oligosaccharide binding study has shown that AAL is highly specific for α1,6-fucosylated oligosaccharides (16Yamashita K. Kochibe N. Ohkura T. Ueda I. Kobata A. J. Biol. Chem. 1985; 260: 4688-4693Abstract Full Text PDF PubMed Google Scholar). To elucidate whether AOL is equally specific for α1,6-fucosylated oligosaccharides, we investigated the interaction of six differently fucosylated PA-sugar chains with AOL immobilized to a CM5 sensor chip flow cell. The molar amount of each PA-sugar chain that interacts with AOL can be deduced from the differences in the RU values of an overlay plot of the sensorgrams (Fig. 2A), which are equivalent to mass change. From the molar amount of each PA-sugar chain that interacted with immobilized AOL (Fig. 3A, solid bar), AOL has the strongest preference for the α1,6-fucosylated PA-sugar chain among the α1,2-, α1,3-, α1,4-, and α1,6-fucosylated chains tested. These results suggest that, like AAL, AOL is highly specific for α1,6-fucosylated oligosaccharides.FIGURE 3The molar amount of each PA-sugar chain that interacts with AOL or AAL. The molar amount of each PA-sugar chain that interacts with AOL (A) or AAL (B) was deduced from the differences in the RU values of an overlay plot of the sensorgrams (Figs. 2 and 4), which are equivalent to mass change. Total binding (solid bar) indicates differences in the 10-s average RU values before the start of injection (90–100 s) and before the end of injection (210–220 s); irreversible binding (slashed bar) indicates differences before the start of injection (90–100 s) and at equilibrium after the injection had stopped and had been replaced by a buffer flow (420–430 s).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Comparison of the Carbohydrate Binding Specificity of AOL, AAL, and LCA—AAL and LCA are widely used to estimate the extent of core fucosylation on glycoproteins and to fractionate glycoproteins. To compare the carbohydrate binding specificity of AOL with that of AAL and LCA, we investigated the interaction of the six PA-sugar chains with these lectins immobilized to the CM5 sensor chip flow cells by using BIAcore. As shown in Fig. 2, AOL and AAL showed binding to the PA-sugar chains tested, whereas LCA showed no binding to them (data not shown). From the molar amount of each PA-sugar chain that interacted with each immobilized lectin (Fig. 3, solid bar), both AOL and AAL apparently displayed similar specificities for the α1,6-fucosylated PA-sugar chain. As shown in the overlay plot of sensorgrams (Fig. 2B), however, the rate of the rise in the RU value over the injection time (100–220 s) and the fall over the dissociation time (220–430 s) indicated that the binding to AAL occurs in a biphasic fashion in a series of fast and slow interactions, unlike the binding to AOL (Fig. 2A). Furthermore, the RU value at equilibrium during the dissociation time (420–430 s) in the overlay plot of sensorgrams (Fig. 2) indicated that, respectively, 1.5 times and 5.1 times as many α1,6 (N) and α1,2 (H antigen) fucosylated PA-sugar chains remained bound to AAL without dissociation as remained bound to AOL. These results suggest that the irreversible binding of these PA-sugar chains is much stronger to AAL than to AOL. Consequently, we compared the carbohydrate-binding specificity of AOL with that of AAL for two different binding properties: namely, total binding and irreversible binding, as shown in Fig. 3 (solid bar and slashed bar, respectively). Comparison of Specificity for Core Fucosylation of AOL with That of AAL—To elucidate which is the more specific lectin for core fucosylation, we determined whether AOL or AAL showed quantitatively lower binding to the other fucosylated PA-sugar chains relative to α1,6-fucosylated PA-sugar chains. From the results of total binding shown in Fig. 4A, AAL displayed quantitatively lower binding to other fucosyl PA-sugar chains relative to the α1,6-fucosylated PA-sugar chain than did AOL. From the results of the irreversible binding shown in Fig. 4B, however, the amount of AAL binding to α1,2 (H antigen) relative to α1,6 (N) PA-sugar chains is higher than that of AOL. Taken together, we conclude that AOL is more specific for core fucosylation than is AAL in terms of irreversible binding. Comparison of the Specificity of AOL for FGP with That of AAL and LCA—As described above, we investigated the interactions of lectins with PA-sugar chains to determine their specificities for fucosylated oligosaccharides. However, PA-sugar chains are artificial oligosaccharides and may not be representative of in vivo fucosylated oligosaccharides owing to the decyclization of the reducing terminal N-acetylglucosamine (GlcNAc) of these sugar chains with pyridylamine (Table 1). Then, to compare the interactions of the lectins with an oligosaccharide of in vivo glycoproteins, we purified FGP from hen's egg yolk as described under “Experimental Procedures.” FGP has N-glycans with α1,6-fucosyl residue bound to the core GlcNAc linked to peptide as shown in Table 1. The linked peptide chain has three amino groups (two lysine residues and the N-terminal group) and, after fluorescent labeling, an average of 2.0 of these three amino groups were labeled with N-[2-(2-pyridylamino) ethyl]succinamic acid 5-norbornene-2,3-dicarboxyimide ester (data not shown). As shown in Fig. 5, the rate of the rise in the RU value over the injection time (100–220 s) and the rate of the fall over the dissociation time (220–430 s) indicated that binding to AAL occurs in a biphasic fashion in a series of fast and slow interactions, and the RU value at equilibrium during the dissociation time (420–430 s) indicated that 2.6 times as much FGP remains bound to AAL without dissociation as remains bound to AOL. These results were similar to the plots for binding to the α1,6 (N) PA-sugar chains shown in Fig. 2. From the irreversible binding (Fig. 3, slashed bar), however, the interactions of FGP with immobilized AOL and AAL were weaker than those of the α1,6 (N) PA-sugar chains, respectively. This weaker binding must result from a stronger electrostatic repulsion from FGP than from the PA-sugar chains because both AOL and AAL have positive charges under the conditions used for SPR analysis at pH 7.4 and FGP has more p
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