Glycobiology Advance Access originally published online on September 21, 2005
Glycobiology 2006 16(1):46-53; doi:10.1093/glycob/cwj038
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Elucidation of binding specificity of Jacalin toward O-glycosylated peptides: quantitative analysis by frontal affinity chromatography
2 Glycogene Function Team and 3 Gzlycostructure Analysis Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Central-2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan; and 4 Amersham Biosciences KK, 3-25-1 Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan
1 To whom correspondence should be addressed; e-mail: h.narimatsu{at}aist.go.jp
Received on June 3, 2005; revised on August 15, 2005; accepted on September 12, 2005
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Abstract |
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Jacalin, a lectin from the jackfruit Artocarpus integrifolia, has been known as a valuable tool for specific capturing of O-glycoproteins such as mucins and IgA1. Though its sugar-binding preference for T/Tn-antigens is well established, its detailed specificity has not been elucidated. In this study, we prepared a series of mucin-type glycopeptides using human glycosyltransferases, that is, ST6GalNAc1, Core1Gal-T1 and -T2, ß3Gn-T6, and Core2GnT1, and investigated their binding to immobilized Jacalin by frontal affinity chromatography (FAC). As a result, consistent with the previous observation, Jacalin showed high affinity for T-antigen (Core1) and Tn-antigen (alpha N-acetylgalactosamine)-attached peptides. Furthermore, we here show as novel findings that (1) Jacalin also showed significant affinity for Core3 and sialyl-T (ST)-attached peptides, but (2) Jacalin could not bind to Core2, Core6, and sialyl-Tn (STn)-attached peptides. The results were also confirmed by FAC using p-nitrophenyl (pNP)-derivatized saccharides. In conclusion, Jacalin binds to a GalNAc
1-peptide, in which C6-OH of GalNAc is free (i.e., Core1, Tn, Core3, and ST), whereas it cannot recognize a GalNAc1-peptide with a substitution at the C6 position (i.e., Core2, Core6, and STn). These findings provide useful information when applying jacalin for functional analysis of mucin-type glycoproteins and glycopeptides. Key words: glycopeptide / Jacalin / mucin / O-glycan
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Introduction |
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Although protein O-glycosylation is a major posttranslational modification, it is poorly understood compared with N-glycosylation. Mucin-type O-glycosyaltion starts with the attachment of alpha N-acetylgalactosamine (
GalNAc) to a Ser/Thr residue and then proceeds through the transfer of various sugars. The addition of GalNAc to a Ser/Thr residue is catalyzed by uridine diphosphate (UDP)–GalNAc : polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-Ts), and recent studies have revealed biochemical characteristics of these glycosyltransferases (White et al., 1995
; Cheng et al., 2004). Although each pp-GalNAc-T may have some preference for the amino acid sequences of the acceptor peptides and glycosylated peptides, there is no reliable method to predict actual GalNAc-attachment sites. In fact, there are more than 15 pp-GalNAc-Ts in humans, and probably they cooperate in various aspects of protein O-glycosylation in a cell (Cheng et al., 2004). Thus, unlike N-glycosylation sites, potential O-glycosylation sites are hardly predictable from the amino acid sequences of the proteins. Poor understanding of protein O-glycosylation, in comparison with N-glycosylation, is largely attributed to technical difficulties in (1) prediction of O-glycan attachment sites in silico-based solely on amino acid sequences, (2) preparation of O-glycosylated proteins in a homogeneous manner, and (3) structural analysis of O-linked glycans and glycopeptides in a sensitive and high-throughput manner, which are due to (a) obvious heterogeneity about carbohydrate structures as well as glycosylation occupancies, (b) substantial inaccessibility to protease digestion because of the presence of O-glycan barriers, and (c) the presence of many O-glycosylation sites often forming a mucin cluster. Because of these basic problems, both structural and functional approaches to O-glycosylated proteins have been considerably retarded.
To solve these problems, development of a reliable method to purify mucin-type O-glycosylated proteins, if possible in a comprehensive manner, is necessary. If one could isolate O-glycosylated proteins and peptides using their O-linked carbohydrates as a tag, studies on structures and functions of O-glycosylated proteins should be much more advanced. However, as described above, mucin-type O-glycans expressed on certain proteins in various cells/tissues are highly heterogeneous. Therefore, as a first step, we aimed to find a method to identify an GalNAc moiety of O-glycans at the reducing end, because such an approach would cover most kinds of mucin-type O-glycosylated proteins. To capture the reducing end GalNAc, we chose lectin, not antibody, because the latter, for example, anti-Tn (i.e., GalNAc) antibody, would fail to capture O-glycans except for this antigen. From this perspective, we screened various plant lectins specific for GalNAc for their ability to bind GalNAc-attached peptides. Among them, however, only a jackfruit-derived lectin, Jacalin, showed sufficient ability to bind GalNAc-attached peptides. Jacalin is known as a specific tool to capture O-glycoproteins, such as IgA1 (Loomes et al., 1991), and its sugar-binding preference is well established for T (Galß1-3GalNAc, i.e., Core1) as well as Tn-antigens (Jeyaprakash et al., 2003; Wu et al., 2003). However, detailed Jacalin’s sugar-specificity, especially for glycopeptides, has not been elucidated. In this article, we describe the binding specificity of Jacalin toward various O-glycosylated peptides for the first time using a quantitative affinity technique, that is, frontal affinity chromatography (FAC). As a result, a novel aspect of Jacalin has been revealed.
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Results and discussion |
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Jacalin recognizes Tn-antigen
Our original motivation was to purify O-glycosylated proteins using lectin affinity chromatography in a comprehensive manner. For this purpose, we screened commercially available agarose-bound lectins, of which binding specificity is defined for GalNAc, for the simplest form of
GalNAc (Tn)-attached peptides. As the first approach, we performed a classical affinity chromatography using the Muc5AC peptide, to which multiple GalNAc residues have been transferred by the action of pp-GalNAc-T. As a negative control, the unglycosylated peptide (with no GalNAc) was also used. Among the lectins tested, however, only one derived from the jackfruit Artocarpus integrifolia, Jacalin, could distinguish GalNAc-attached peptides from the unglycosylated peptide: after separation on the Jacalin–agarose column, 5-carboxyfluorescein succinimidyl ester (FAM)-labeled peptides were detected in the elution fractions (Figure 1A). We further analyzed the peptides in the flow-through and the eluted fractions by high-performance liquid chromatography (HPLC) and found that Jacalin did not bind to the naked Muc5AC peptide (retention time: 20.88 min) but did to GalNAc-attached peptides (retention times: 19.46 and 19.80 min) (Figure 1B).
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We next investigated whether Jacalin could recognize peptides with a single GalNAc residue or not. For this purpose, we used Muc1a peptides, which are the mixture of the unglycosylated peptide and a mono GalNAc-attached peptide (Guo et al., 2002), and evaluated the binding to Jacalin–agarose by the methods described above. FAM-labeled peptides were detected in both pass-through and elution fractions (Figure 1C). However, further analysis by HPLC revealed that Jacalin selectively bound to the mono GalNAc-attached Muc1a peptide (HPLC retention time: 19.03 min), but not to the naked peptide (retention time: 19.73 min) (Figure 1D).
These results clearly show that Jacalin binds to GalNAc-peptides. In contrast, other lectins known as GalNAc-binding lectins, that is, Wisteria floribunda lectin (WFA) (Figure 1E), Vicia villosa agglutinin (VVA), and soybean agglutinin (SBA) (data not shown) did not bind to the GalNAc-peptide.
Binding specificity of Jacalin toward p-nitrophenyl-sugars
It was originally reported that Jacalin primarily bound to the Galß1-3GalNAc- (Core1) structure (Sastry et al., 1986; Ahmed and Chatterjee, 1989). In recent years, however, there have been reports describing its broader binding specificity (Jeyaprakash et al., 2003; Wu et al., 2003). Therefore, we decided to investigate the carbohydrate-binding specificity of Jacalin more in detail by using both pNP-sugars and O-glycosylated peptides. First, we analyzed the binding of pNP-sugars to Jacalin–agarose by column chromatography, followed by HPLC. We first measured the retention time of each pNP-sugar in HPLC and applied a mixture of pNP-sugars with different retention times to the Jacalin–agarose column. After separation by affinity chromatography, flow-through/wash fractions and elution fractions were subjected to HPLC analysis separately. Among the pNP and benzyl (Bz)-sugars tested, Galb-pNP and GalNAcß-pNP were identified only in the pass-through (wash) fractions. This observation indicated that pNP-beta-glycosides did not bind to Jacalin (Figure 2A), consistent with the previous report of thermodynamic study (Gupta et al., 1992) and enzyme-linked lectin-binding assay (Wu et al., 2003). On the other hand, Gal-pNP and GalNAc-Bz were exclusively found in the elution fraction but were also detected in the late wash (retarded) fractions. Notably, GlcNAcß1-6GalNAc-pNP (Core6-pNP) was detected only in the flow-through fraction, whereas its position isomer, GlcNAcß1-3GalNAc-pNP (Core3-pNP) firmly bound to the column and specifically eluted from the column. In another set of experiment, Galß1-3 GalNAc-pNP (Core1-pNP) was identified only in the elution fraction, whereas its branched structure, that is, GlcNAcß1-6(Galß1-3)GalNAc-pNP (Core2-pNP) was completely passed through the column (Figure 2B). These results indicate that Jacalin binds to Core1- and Core3-pNP with high affinity but to Gal-pNP and GalNAc-Bz with significantly lower affinity. More importantly, however, Jacalin was found to lack binding ability to Galß-pNP, GalNAcß-pNP and Core2-pNP. These results suggest that Jacalin binds to -GalNAc, in which C6-OH is free, whereas it cannot bind to those having any substitution at this position.
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As far as we know, this is a novel aspect of Jacalin’s sugar-binding specificity. Therefore, the above results were confirmed by a more quantitative method, FAC. pNP-glycosides dissolved in Tris-buffered saline (TBS) at various concentrations were successively injected into the column, and the retardation of the elution front (V – V0) relative to that of an appropriate standard oligosaccharide (e.g., Fuc-pNP) was determined. Association constants (Ka) determined by Woolf–Hofstee-type plots are summarized in Table I. FAC analysis showed that Jacalin had significant affinity toward Gal-pNP (Ka = 9.3 x 104 M–1) and GalNAc-pNP (Ka = 1.2 x 105 M–1). Although the obtained Ka values were slightly lower than those for coumarin derivatives, that is, Gal–MeUmb (metylumbelliferyl 7-hydroxy-4-methyl-coumarin) and GalNAc–MeUmb, the observed high affinity for these sugars is consistent with a previous result (Gupta et al., 1992). Further stronger binding of Jacalin was observed for Core1-pNP (Ka = 2.9 x 105 M–1). In relative affinity, Core1-pNP has 3.1 and 2.4 times higher affinity than those for Gal-pNP and GalNAc-pNP, respectively. Interestingly, Jacalin showed the greatest affinity for Core3-pNP (Ka = 9.5 x 105 M–1) among the pNP-glycosides tested, although it had previously been reported as Core1 (T-antigen) and GalNAc (Tn)-specific lectin (Sastry et al., 1986; Ahmed and Chatterjee, 1989; Gupta et al., 1992). The affinity for Core3-pNP was 3.3 and 7.9 times that for Core1-pNP and GalNAc-pNP, respectively. On the other hand, Galß-, GalNAcß-pNP, Core2-pNP and Core6-pNP had no ability to bind to Jacalin. Quantitative analysis using the FAC system determined the order of affinity for Jacalin as follows (relative affinity to Gal-pNP); Core3-pNP (10.2) > Core1-pNP (3.1) > GalNAc-pNP (1.3) > Gal-pNP (1.0). The observed binding feature was well consistent with the results obtained in the HPLC analysis described above.
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Binding specificity of Jacalin toward O-glycosylated peptides
Next, we attempted to expand the investigation of Jacalin’s affinity to a variety of O-glycosylated peptides because most of the previous studies on the binding specificity of Jacalin depended on free glycans. Therefore, we labeled the human IgA1 hinge peptide (HP) having GalNAc at the 4th position (GalNAc-HP) with Alexa Fluor 488 (AF) for HPLC analysis. Then, Core1-HP and sialyl-Tn (STn)-HP were generated by a reaction with recombinant Core1GalT or ST6GalNAc1, respectively. Core1-HP was purified by HPLC and further reacted with recombinant Core2Gn-T1 or 2,3-(O)-sialyltransferase to generate Core2-HP and ST–HP. After confirmation of retention time of each peptide in HPLC, mixtures of glycosylated hinge peptides were applied to the Jacalin-affinity column and analyzed by HPLC. As demonstrated in Figure 3A, GalNAc-HP and Core1-HP were identified only in the elution fractions, whereas STn–HP was detected only in the wash fractions. Furthermore, ST–HP was identified only in the elution fractions, whereas Core2-HP was detected only in the wash fractions (Figure 3B).
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The affinity of Jacalin toward AF-labeled HPs was further analyzed by FAC. According to a basic equation of FAC (Eq. 1), retardation of the elution front, that is, V – V0 is proportional to Kd, if the initial concentration of the analyte (here, HPs) is negligibly small compared with the Kd (e.g., >10–6 M) (Hirabayashi et al., 2003; Hirabayashi, 2004). Although it is difficult to determine the accurate concentration of AF-labeled HPs, one can assume that sufficiently diluted AF-labeled HPs are concentration-independent in FAC analysis. This could be simply verified by dilution analysis: since there was no change in V value by further diluting [A]0, the relation Kd > [A]0 was fulfilled (data not shown).
AF-labeled HPs with/without glycans dissolved in TBS were successively injected into the column. Elution profiles obtained for the AF-labeled HPs from the Jacalin–agarose column (0.5 mg/mL) are shown in Figure 4. In this case, however, even the naked AF-labeled HP, showed slight retardation, that is, evidence of nonspecific binding. Therefore, we cannot determine precise Ka values from the observed V – V0. Nevertheless, it is clear from Figure 4 that Jacalin has significant affinity toward AF-labeled-Core1-hinge peptide (Core1-HP), Core3-HP, and ST–HP as well as Tn–HP. On the contrary, Core2-HP and STn–HP were not ligands for Jacalin. These observations were entirely consistent with the results of pNP-sugars (Table I) and of the HPLC analysis for glycopeptides (Figure 3).
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The present findings are summarized in Tables I, II and III. In brief, (1) the GalNAc-, or, Gal-structure at the reducing end is essential for the binding of Jacalin. (2) The presence of an additional carbohydrate, such as ßGlcNAc (in the cases of Core2 and Core6) or sialic acid (STn), at the C6 position of GalNAc abolishes the binding to Jacalin. (3) The presence of an additional carbohydrate, such as ßGalß (Core1), ßGlcNAc (Core3), and Neu5Ac2–3Galß (ST), at the C3 position of GalNAc does not inhibit the binding, but rather increases the affinity for Jacalin. These results were consistent with the recent report of X-ray crystallography of the Jacalin-T antigen complex (Jeyaprakash et al., 2002). In the crystal structure, Jacalin was found to recognize the GalNAc moiety more extensively than the Gal moiety. This suggests that Jacalin can bind to Tn-antigen and possibly to Core3 and to ST-antigen. Also, the C6 position of GalNAc was proposed to be involved in the direct binding between Jacalin and T-antigen. Therefore, any substitution at this site should abolish the binding.
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In this article, we characterized the binding specificity of Jacalin to pNP-sugars and glycopeptides using classical column separation and more quantitative FAC. As a result, a novel aspect of Jacalin specificity has been revealed in a quantitative manner. Emphasis should be given to that by employing a series of glycosyltransferases, various kinds of O-glycopeptides became available, which should be useful for functional analysis of O-glycans/-glycopeptides, that is, O-glycomics. Our analysis has also demonstrated that FAC is a powerful technique for detailed profiling of lectin affinity. Among the Gal/GalNAc reactive lectins, Jacalin still remains as a prime candidate for a tool to select O-glycans. However, since Jacalin does not bind to O-glycans with any C6-substituent at the reducing end GalNAc, careful approach is recommended in the usage of Jacalin for the purification of O-glycosylated proteins, that is, human IgA1. Search by FAC for more versatile lectins showing much broader specificity to O-glycans is in progress in the context of hect-by-hect project (Hirabayashi, 2004).
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Materials and methods |
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Reagents
Agarose-bound lectins, A. integrifolia agglutinin (Jacalin), W. floribunda agglutinin (WFA), and VVA were purchased from Vector Laboratories (Burlingame, CA), and agarose-bound SBA was purchased from Seikagaku (Tokyo, Japan).
Galactose and N-acetyl-D-galactosamine were purchased from Sigma (St. Louis, MO) and Wako (Osaka, Japan), respectively. FAM-labeled peptides of Muc1 (AHGVTSAPDTR) and Muc5AC (GTTPSPVPTTSTTSA) were from Sawady (Tokyo, Japan). A GalNAc was attached to FAM-labeled Muc1 (AHGVT*SAPDTR, * denotes the attachment site) by recombinant pp-GalNAc-T6, and GalNAcs were attached to Muc5AC peptide by pp-GalNAC-T2 as described previously (Cheng et al., 2002; Wang et al., 2003). HPs (VPST*PPTPSPSTPPTPSPSK, * denotes the attachment site), with (GalNAc-HP) and without a GalNAc residue (HP), were obtained from Peptide Institute (Osaka, Japan). Labeling of HP and GalNAc-HP was performed with an AF Protein Labeling kit (Molecular Probes, Eugene, OR) following the manufacturer’s protocol. GalNAc-Bz and GalNAcß-pNP were obtained from Sigma. Gal-pNP and Galß-pNP were obtained from Calbiochem (San Diego, CA). Galß1–3GalNAc-pNP (Core1-pNP), GlcNAcß1–6(Galß1–3)GalNAc-pNP (Core2-pNP), GlcNAcß1–3GalNAc-pNP (Core3-pNP), and GlcNAcß1–6GalNAc-pNP (Core6-pNP) were purchased from Toronto Research Chemicals (North York, Canada).
Column procedures
One milliliter of agarose-bound lectin was packed into a disposable column (7.5 x 20 mm, Seikagaku) and washed with 20 column volumes of wash buffer (100 mM Tris–HCl, pH 7.4). Then, GalNAc-attached peptide or pNP-derivatized sugar solution was added to the column. After washing of the column with 10 column volumes of the wash buffer, bound materials were eluted with elution buffer, 0.8 M galactose in the wash buffer (Figure 1), or, 10 mM GalNAc in the wash buffer (Figures 2 and 3). Both wash and elution fractions were sequentially collected. These column-separated fractions of FAM-labeled peptides with or without GalNAc-residue were determined for fluorescence intensity using a plate reader with the protocol for Fluorescein (ARVO.sx, Perkin Elmer, Wellesley, MA).
HPLC analysis
The column–separated peptides/glycopeptides were evaluated by HPLC as described previously (Iwai et al., 2002; Wang et al., 2003). Briefly, in the case of FAM and AF-labeled peptides, the flow-through and elution fractions were injected onto a C18 reverse-phase column (Waters 5C18-AR, 4.6 x 250 mm), and then, the bound materials were eluted with a linear gradient of acetonitrile (0–50%) in 0.05% trifluoroacetic acid (TFA) at a flow rate of 1.0 mL/min at 40°C. The fluorescence intensity of the eluates were monitored at 520 nm (excitation: 492 nm). In the case of pNP-and Bz-sugars, flow-through and elution fractions were injected onto an ODS-80Ts QA column (4.6 x 250 mm, Tosoh, Tokyo, Japan) and eluted with 9% acetonitrile containing 0.1% TFA at a flow rate of 1.0 mL/min at 50°C. The elution was monitored by absorbance at 210 nm with an ultraviolet spectrophotometer, SPD-10AVP (Shimadzu, Kyoto, Japan)
Biochemical synthesis of glycopeptides
The GalNAc-attached Muc5AC peptide and Muc1 peptide were prepared with FAM-labeled peptides and recombinant pp-GalNAc-Ts as described previously (Guo et al., 2002). Human ST6GalNAc1, Core1Gal-T1 and -T2, ß3Gn-T6 (Core3Gn-T), and Core2GnT1 cDNAs were cloned by real-time polymerase chain reaction (RT–PCR) as reported (Ikehara et al., 1999; Kudo et al., 2002; Iwai et al., 2002; data not shown). The N-terminal truncated forms of these cDNAs were inserted into a modified pFLAG-CMV vector (Sigma). These expression vectors were transfected into HEK293 cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). The N-terminal FLAG-epitope-tagged recombinant glycosyltransferases were obtained from culture supernatants of transfected cells by immunoprecipitation with anti-FLAG mAb conjugated agarose (Sigma). The Galß1-3GalNAc- (Core1) structure was generated in the AF-labeled GalNAc-HP using recombinant human Core1Gal-T and was purified by HPLC. Then, the GlcNAcß1-6 (Galß1-3) GalNAc- (Core2) structure was generated with the recombinant Core2GnT1 or the Neu5AC2-3 Galß1-3 GalNAc- (ST) structure was generated by the recombinant rat 2,3-(O)-sialyltransferase (Calbiochem). The GlcNAcß1-3 GalNAc- (Core3) structure was generated with the recombinant human b3Gn-T6 on the AF-labeled GalNAc-HP. The Neu5AC2-6 GalNAc- (STn) structure was generated on the AF-labeled GalNAc-HP using the recombinant human ST6GalNAc1.
FAC
FAC was performed using an automated system for FAC (FAC-1) as described previously (Hirabayashi et al., 2003; Hirabayashi, 2004, Nakamura et al., 2005). Jacalin–agarose (4.0 and 0.5 mg/mL) was suspended in 10 mM Tris–HCl buffer, pH 7.4, containing 1 mM CaCl2 and 1 mM MnCl2, and the slurry was packed into capsule-type miniature columns (inner diameter, 2 mm; length, 10 mm; bed volume, 31.4 L). The resulting lectin-columns were slotted into a stainless steel holder and were connected to FAC-1. The flow rate and the column temperature were kept at 0.125 mL/min and 25°C, respectively. After equilibrating the miniature columns with 10 mM Tris–HCl buffer, pH 7.4, containing 0.8% NaCl (TBS), 1 mL of pNP glycosides or AF-labeled glycopeptides dissolved in TBS was successively injected into the Jacalin-column by an autosampling system. The elution of pNP-glycosides was monitored by measuring UV absorbance at 280 nm, whereas that of AF-labeled glycopeptides was detected by measuring fluorescence (excitation and emission wavelengths of 495 and 519 nm, respectively). Affinity constants (Ka) of pNP-glycosides were determined by the concentration-dependence analysis as described previously (Hirabayashi et al., 2003). Briefly, various concentrations ([A]0, 10–100 M) of pNP-glycosides dissolved in TBS were applied to the miniature column, and the retardation of the elution front relative to that of an appropriate standard oligosaccharide (V – V0) was determined according to the established procedure (Hirabayashi et al., 2003). Woolf–Hofstee-type plots, that is, (V – V0) versus (V – V0) [A]0, were made to determine a dissociation constant (Kd, in M) and column capacity (Bt in mol) based on the equation of FAC (Eq. 1). In this article, discussion is made in terms of Ka (=1/Kd) for the sake of simplicity.
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Acknowledgements |
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We thank Ms. Fushimi for illustrations. This work was supported by New Energy and Industrial Technology Development Organization (NEDO) and Matching Fund between Mitsubishi Chemical and AIST.
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Abbreviations |
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AF, Alexa Fluor 488; Bz, benzylp; FAC, frontal affinity chromatography; FAM, 5-carboxyfluorescein succinimidyl ester; GalNac, N-acetylgalactosamine; HP, hinge peptide; HP, human IgA1 hinge domain peptide; HPLC, high-performance liquid chromatography; pNP, p-nitrophenyl; pp-GalNAc-T, UDP-GalNAc : polypeptide N-acetylgalactosaminyltransferase; ST, sialyl-T; STn, sialyl-Tn; TBS, Tris-buffered saline; UDP, uridine diphosphate
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References |
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