Ligand identification of carbohydrate-binding proteins employing a biotinylated glycan binding assay and tandem mass spectrometry

Analytical Biochemistry 406 (2010) 132–140

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Ligand identification of carbohydrate-binding proteins employing a biotinylated glycan binding assay and tandem mass spectrometry Manfred Wuhrer *, Alexandra van Remoortere, Crina I.A. Balog, André M. Deelder, Cornelis H. Hokke Department of Parasitology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands

a r t i c l e

i n f o

Article history: Received 21 April 2010 Received in revised form 22 June 2010 Accepted 12 July 2010 Available online 16 July 2010 Keywords: Biotinylated carbohydrates Protein–carbohydrate interaction Mass spectrometry

a b s t r a c t Characterization of protein–carbohydrate interactions at the molecular level is important for understanding many glycan-mediated processes. Here we present a method for the identification of glycan ligands of carbohydrate-binding proteins. The glycans released from natural sources are labeled with biotinamidocaproyl hydrazide (BACH) and subsequently fractionated by high-performance liquid chromatography. Glycan fractions are screened for binding to carbohydrate-binding proteins (CBPs) using a microtitration plate binding assay; CBPs are immobilized, BACH–glycan fractions are added, and bound BACH–glycans are detected using alkaline phosphatase-conjugated streptavidin. The glycan structures in binding fractions are studied by (tandem) mass spectrometry, exoglycosidase treatment, and rechromatography, thereby revealing the glycan motifs recognized by the CBPs. Subsequent surface plasmon resonance experiments using a reverse setup with immobilization of the BACH–glycan ligands on streptavidincoated surfaces provide more information on glycan–CBP interactions via association and dissociation curves. The presented method is easy and fast, and the required instrumentation is available in many laboratories. The assay is very sensitive given that both the mass spectrometric analysis and the microtitration plate binding assay can be performed on femtomole amounts of BACH–glycans. This approach should be generally applicable to study and structurally identify carbohydrate ligands of anti-glycan antibodies and lectins. Ó 2010 Elsevier Inc. All rights reserved.

Multicellular organisms display a variety of glycans at the surface of their cells. These glycans are either protein linked as O-glycans and N-glycans or ceramide linked as glycan parts of glycosphingolipids. Cell surface glycans are involved in cellular communication, signaling, and cellular adhesion, and they can serve as attachment sites for invading pathogens [1–5]. Moreover, many proteins secreted into biological fluids are glycosylated, and glycans on plasma glycoproteins mediate and modulate interactions with receptors and determine protein half-life [6,7]. Glycans and glycoconjugates of viruses, bacteria, and parasites often mediate or provoke immune responses in infected hosts, including the induction of glycan-specific antibodies [8–12]. Glycans often act through interaction with a protein counterpart such as a lectin or an immunoglobulin, and knowledge of the molecular details of these interactions is of utmost importance for understanding glycan-mediated processes. Depending on the addressed research questions, various techniques that can be applied to the analysis of protein–carbohydrate interactions are

* Corresponding author. Fax: +31 71 526 6907. E-mail address: [email protected] (M. Wuhrer). 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.07.008

available, including calorimetry [13], surface plasmon resonance (SPR)1 spectroscopy [14], affinity purification of glycan ligands using immobilized carbohydrate-binding proteins (CBPs) [15,16], and frontal affinity chromatography [17]. Many protein–carbohydrate interactions are characterized by high off-rates [18,19], and stable interactions are often achieved by concerted polyvalent interactions of multiple oligosaccharide ligands with CBPs exhibiting multiple binding sites. To be able to address this issue, synthesized or purified carbohydrates are commonly attached to carrier proteins, synthetic polymers, or various surfaces, resulting in polyvalent presentation and allowing their use in enzyme-linked immunosorbent assays (ELISAs), glycan microarrays, and SPR analysis [14,20–22]. Both microtitration plate binding assays and glycan microarrays generally 1 Abbreviations used: SPR, surface plasmon resonance; CBP, carbohydrate-binding protein; ELISA, enzyme-linked immunosorbent assay; BACH, biotinamidocaproyl hydrazide; HPLC, high-performance liquid chromatography; MS/MS, tandem mass spectrometry; KLH, keyhole limpet hemocyanin; SDS, sodium dodecyl sulfate; PBS, phosphate-buffered saline; PNGase F, peptide-N-glycosidase F; MALDI–MS, matrixassisted laser desorption/ionization mass spectrometry; HILIC, hydrophilic interaction liquid chromatography; HCT, high-capacity trap; ESI, electrospray ionization; IT, ion trap; TOF, time-of-flight; BSA, bovine serum albumin; AP, alkaline phosphatase; p-NPP, para-nitrophenylphosphate; Con A, concanavalin A; RU, response units; mAb, monoclonal antibody; Hex, hexose; HexNAc, N-acetylhexosamine; dHex, deoxyhexose; SEA, soluble egg antigen; IgM, immunoglobulin M.

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rely on detection of CBPs by direct fluorescent labeling or via secondary antibodies [22–25]. Notably, candidate ligands of CBPs are often not readily available in pure synthesized form. As an alternative, glycans from biological sources can be tested in CBP binding assays. For this purpose, potential CBP ligands may be purified from biological sources such as target glycoproteins, cells, and organisms. This approach has been applied in glycan microarrays using fluorescence labeling [26] or SPR analysis [21] for the detection of bound CBPs. In the current study, we present a complementary method to the above-mentioned CBP binding assays that is also based on glycans isolated from natural sources, but in this case avoiding the need for glycan immobilization and CPB detection. Instead, the presented glycan–CPB binding assay employs a microtitration plate format that relies on immobilized (adsorbed) CBPs that are screened for binding of glycans in solution. Glycans are isolated directly from a natural source and labeled with biotinamidocaproyl hydrazide (BACH) that specifically labels the reducing end of the glycan in a 1:1 stoichiometry. Next, BACH-labeled glycans are fractionated by high-performance liquid chromatography (HPLC). CBPs are adsorbed to the microtitration plate, and aliquots of the biotinylated glycan fractions are added. Bound glycans are chromogenically detected using enzyme-labeled streptavidin. Fractions containing glycan ligands are then characterized by tandem mass spectrometry (MS/MS) in conjunction with exoglycosidase treatment and rechromatography. The method is exemplified by analyzing keyhole limpet hemocyanin (KLH) N-glycans for their reactivity with anti-KLH antibodies as well as cross-reactive antibodies directed against the human parasite Schistosoma mansoni. This approach allows the unambiguous identification and structural characterization of glycan ligands from complex biological sources without the need for synthetic approaches, labeling of CPBs, or the technically more challenging construction of a microarray. Materials and methods Monoclonal antibodies The production of the monoclonal antibodies (mAbs) used in this study (Table 1) has been described previously [27]. The selected panel came from the extensive collection of hybridomas produced in the Department of Parasitology at the Leiden University Medical Center and was known to show reactivity with fucosylated carbohydrate epitopes [14,28,29]. Release and purification of N-glycans from KLH Here 90 ll of 10% sodium dodecyl sulfate (SDS) and 4.5 ll of 2mercaptoethanol were added to 900 ll of a 5.6-mg/ml solution of KLH in phosphate-buffered saline (PBS, pH 7.8, Sigma–Aldrich, Zwijndrecht, Netherlands). The sample was incubated for 10 min at 100 °C and allowed to cool down to room temperature. CHAPS

(9 mg) was added, and the sample was thoroughly mixed. After that, 5 mU of peptide-N-glycosidase F (PNGase F, Roche Diagnostics, Mannheim, Germany) was added. After overnight incubation at 37 °C, the sample was fractionated by gel filtration on a Superdex 75 HiLoad column (16  600 mm, GE Healthcare, Piscataway, NJ, USA) at a 1-ml/min flow of 25 mM NH4HCO3 (pH 8.0). Fractions of 5 ml were collected and glycans were detected by carbohydrate constituent analysis [30]. Fractions containing glycans were pooled and applied to a self-packed porous graphitized carbon column (8  50 mm, Carbograph, Alltech, Deerfield, IL, USA). After washing the carbon column with water, glycans were eluted with 25% aqueous acetonitrile. Fractions of 1 ml were collected and assayed for the presence of glycans by matrix-assisted laser desorption/ionization mass spectrometry (MALDI–MS). Glycan-containing fractions were pooled and lyophilized. BACH labeling and fractionation of N-glycans BACH labeling through glycosylhydrazide formation of the oligosaccharides’ reducing ends was performed according to the method of Leteux and coworkers [23]. The sample of dried KLH N-glycans obtained by N-glycan release was mixed with BACH (500 nmol in 100 ll of 30% aqueous acetonitrile, Sigma–Aldrich), evaporated to dryness, and then dissolved in 25 ll of methanol/ water/acetic acid (95:4:1, v/v). The reaction was performed overnight at 60 °C, and the sample was subsequently dried. BACH–glycans were dissolved in 80% acetonitrile and fractionated by hydrophilic interaction liquid chromatography (HILIC)– HPLC on a TSK-Amide 80 column (5 lm beads, 4  250 mm, Tosoh Biosciences, Stuttgart, Germany) at 0.4 ml/min [31]. Solvent A was 50 mM formic acid adjusted to pH 4.4 with ammonia. Solvent B was 20% solvent A in acetonitrile. The following gradient conditions were used: t = 0 min, 100% solvent B; t = 152 min, 52.5% solvent B; t = 155 min, 0% solvent B; t = 162 min, 0% solvent B; t = 163 min, 100% solvent B. The total run time was 180 min. Fractions (1 min) were collected, dried in a vacuum centrifuge, and dissolved in 200 ll of water. nano-LC ESI–IT–MS BACH–glycan fractions were separated on a Pep-Map column (75 lm  100 mm, Dionex/LC Packings, Amsterdam, Netherlands) using an Ultimate nano-LC system (Dionex). The system was directly coupled with an Esquire high-capacity trap (HCT), electrospray ionization ion trap (ESI–IT) mass spectrometer (Bruker Daltonics, Bremen, Germany). The column was equilibrated with eluent A (0.1% formic acid and 0.8 mM sodium hydroxide) at a flow rate of 200 nl/min. After injecting the sample, the column was run isocratically for 5 min, followed by a linear gradient to 50% eluent B (H2O/acetonitrile [95:5,v/v] containing 0.1% formic acid) in 30 min and a final wash with 100% B for 5 min. For positive mode nano-ESI (1200–2500 V), capillaries (360 lm o.d. and 20 lm i.d. with 10-lm

Table 1 Monoclonal antibodies used in this study. mAb

(Sub)-class

Binds to:

Reference(s)

128-1E7-C 291-5D5-A 258-3E3 290-4A8-A 114-5B1-A 114-4D12-A 290-2E6-A 273-3F2 291-4D10-A

IgM IgM IgM IgM IgG1 IgG1 IgM IgM IgM

F-LDN-F (Fuca1-3GalNAcb1-4(Fuca1-3)GlcNAcb1-); Fuca1-3GlcNAcb1F-LDN (Fuca1-3GalNAcb1-4GlcNAcb1-) F-LDN (Fuca1-3GalNAcb1-4GlcNAcb1-); Fuca1-2Fuca1-3GlcNAcb1-; Fuca1-2Fuca1-2Fuca1-3GlcNAcb1LDN-DF (GalNAcb1-4(Fuca1-2Fuca1-3)GlcNAcb1-) LDN-DF (GalNAcb1-4(Fuca1-2Fuca1-3)GlcNAcb1-) DF-LDN-DF (Fuca1-2Fuca1-3GalNAcb1-4(Fuca1-2Fuca1-3)GlcNAcb1-) LDN-F (GalNAcb1-4(Fuca1-3)GlcNAcb1-) LDN (GalNAcb1-4GlcNAcb1-) LewisX (Galb1-4(Fuca1-3)GlcNAcb1-)

[28,29] and unpublished data [28] and unpublished data [29] and unpublished data [14,28] [14,28] [15] [14] [14] [14,28]

Note. IgM, immunoglobulin M; IgG, immunoglobulin G.

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opening) from New Objective (Cambridge, MA, USA) were used. The solvent was evaporated at 150 °C with a nitrogen stream of 6 L/min. Ions from m/z 50 to 3000 were registered. MS/MS was performed in the automated mode, and the three most intense precursors from each mass spectrum were selected for fragmentation. Exoglycosidase treatment BACH–oligosaccharides were dissolved in 200 ll of 50 mM sodium acetate buffer (pH 5.0) and incubated overnight at 37 °C with a mix of 5 mU each of a-mannosidase (Sigma–Aldrich), b-galactosidase (Seikagaku, Falmouth, MA, USA), and b-N-acetylhexosaminidase (Sigma–Aldrich) from jack beans. After exoglycosidase treatment, BACH–glycans were fractionated by HILIC–HPLC. MALDI–MS MALDI time-of-flight (TOF) MS data were obtained using an Ultraflex TOF mass spectrometer (Bruker Daltonics) equipped with a ‘‘LIFT” MS/MS facility. 6-Aza-2-thiothymine (5 mg/ml, Sigma–Aldrich) was used as matrix, and spectra were acquired in the positive ion reflectron mode. ELISA KLH (50 ll of 10 lg/ml in PBS) was adsorbed to microtitration plates (Maxisorp, Nunc, Roskilde, Denmark). Plates were washed three times with PBS (1:20 diluted containing 0.005% Tween 20), followed by treatment with 100 ll of 20 mM sodium periodate (Merck, Darmstadt, Germany) in 100 mM sodium acetate (pH 5.5). Control wells were treated with 100 ll of 100 mM sodium acetate (pH 5.5). Plates were washed three times, followed by the addition of mouse hybridoma cell culture supernatants (diluted 1:20 in PBS containing 0.5% bovine serum albumin [BSA] and 0.1% Tween 20). For detection of bound antibodies, peroxidase-conjugated rabbit anti-mouse immunoglobulin was added (diluted 1:2000). 3,3,5,50 -Tetramethylbenzidine and H2O2 were used as substrates for peroxidase in 0.1 M acetate (pH 5.5). The reaction was terminated by the addition of H2SO4, and absorbance was measured at 450 nm. Background was subtracted and signals were normalized (highest signal set to 1.0).

As a variant of this approach, KLH (60 ll of 1 mg/ml KLH in PBS) was adsorbed to microtitration plates. Plates were washed three times, followed by the addition of mouse hybridoma cell culture supernatants (diluted 1:20). Alternatively, concanavalin A (Con A, Sigma–Aldrich) was adsorbed to the microtitration wells (1 mg/ ml in PBS). The rest of the procedure, including the incubation with BACH–glycans, was performed as described above. SPR All SPR binding studies were conducted at 25 °C in HBS–EP running buffer (GE Healthcare) using a Biacore 3000 SPR system (GE Healthcare). Fractions 20–29, 49–58, and 55 were bound to an SA sensor chip (research grade, GE Healthcare); levels of 200 response units (RU) were obtained. As a reference channel, a blank surface was used. Subsequently, several monoclonal antibodies (mAbs) reactive with LDN-F, F-LDN, F-LDN-F, LDN-DF, and DF-LDN-DF and Lewis X [14,28,29] (see Table 1) were applied to the surfaces at concentrations of approximately 20 lg/ml and at a flow rate of 20 ll/min for 5 min, followed by a dissociation time of 10 min. The surfaces were regenerated after each cycle with one 60-s pulse of 10 mM HCl, followed by a stabilization time of 5 min. SPR data were analyzed with BiaEvaluation (version 4.1, GE Healthcare). Reference surface and blanks were subtracted from all binding curves. Results KLH carries a variety of carbohydrate structures, and the Fuca13GalNAcb1-4(Fuca1-3)GlcNAcb1-[F-LDN-(F)] element has been described as the structural unit responsible for cross-reactivity of antibodies against schistosome glycoconjugates carrying the same unit [14,28,29]. Here six mAbs directed against schistosome carbohydrate structures (Table 1) were tested for their cross-reactivity with KLH by ELISA (Fig. 1). Three of them, which were known from previous studies to recognize enzymatically or chemically synthesized glycan structures containing a terminal fucose [14,28,29], were found to bind to KLH (Fig. 1). Reduced binding was observed when KLH glycans were destroyed by periodate treatment after

1.2

BACH–glycan microtitration plate binding assays

0.8 0.6 0.4

1:20

1:20

1:20

291-4D10-A (Lewis X)

114-5B1 (LDN-DF)

290-4A8 (LDN-DF)

1:200

1:20

258-3E3 (F-LDN)

1:200

1:20

291-5D5-A (F-LDN)

1:200

0.0

1:20

0.2

128-1E7-C (F-LDN-F)

Rabbit anti-mouse immunoglobulin (1:2000 in PBS, Dako, Glostrup, Denmark), rabbit anti-hamster immunoglobulin (1:2000, Nordic Immunological Laboratories, Tilburg, The Netherlands), and swine anti-rabbit immunoglobulin (1:2000 in PBS, Dako) were adsorbed to 96-well microtitration plates (Maxisorp) by 15 min of incubation at 37 °C. Plates were washed three times with PBS (1:20 diluted containing 0.005% Tween 20), followed by the addition of murine S. mansoni infection serum (diluted 1:500 in PBS containing 0.5% BSA and 0.1% Tween 20), mouse hybridoma cell culture supernatants (diluted 1:20), hamster anti-S. mansoni egg antigen hyperimmune serum (diluted 1:2000), and rabbit anti-KLH hyperimmune serum (diluted 1:2000). After 15 min at 37 °C, plates were washed three times, followed by the addition of BACH–glycans in 50 ll of PBS. After 15 min of incubation at 37 °C, no wash was performed, but alkaline phosphatase (AP)-labeled streptavidin was added (50 ll, diluted 1:2000, in PBS containing 0.1% Tween 20, Dako). After another 15 min of incubation at 37 °C, plates were washed three times, followed by staining for 1 h at room temperature in the dark using 0.1% para-nitrophenylphosphate (p-NPP) in 100 mM diethylamine (pH 9.6) containing 0.5 mM MgCl2. Absorption was measured at 405 nm.

Absorption (450 nm)

1.0

Fig. 1. ELISA recognition of KLH by anti-F-LDN-(F) monoclonal antibodies. KLH (500 ng/well) was adsorbed, followed by incubation with or without periodate (black or white bars, respectively). Subsequently, mAbs directed against schistosome glycoconjugates were assayed for their binding to KLH at dilutions 1:20 and 1:200.

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antigen coating, suggesting that the mAbs recognized carbohydrate epitopes on KLH. To characterize the recognized KLH glycans, enzymatically released N-glycans were labeled with biotin, fractionated by HPLC, and analyzed using a microtitration plate binding assay, MS, and exoglycosidase treatment in conjunction with rechromatography. The released oligosaccharides were studied by MALDI–TOF–MS, which revealed a typical KLH N-glycan profile (not shown) in accordance with previous reports [32–35]. Based on the observed masses and peak patterns, compositions were assigned in terms of hexose (Hex), N-acetylhexosamine (HexNAc), pentose (pent), and deoxyhexose (dHex). KLH N-glycans were labeled with BACH and fractionated by HILIC–HPLC. Microtitration plate assays were set up to screen the HPLC fractions using CBPs. Wells were coated with Con A, and aliquots of fractionated KLH BACH–glycans were added. KLH N-glycans captured by Con A were detected using AP-labeled streptavidin (Fig. 2A). Con A-bound glycans were detected in the pools of fractions 20–29, 30–39, and 40–49 with strong signal intensity. A similar binding assay was performed with anti-carbohydrate antibodies (Fig. 2B,C). In this assay, wells were coated with anti-rabbit and anti-mouse immunoglobulins. Subsequently, anti-KLH rabbit hyperimmune serum (Fig. 2B) and mAb 128-1E7-C directed against a schistosome carbohydrate antigen (Fig. 2C) were applied, followed by the addition of KLH BACH–glycans and AP-labeled streptavidin. Chromogenic detection highlighted fractions 50–59 as the major BACH–glycan fractions bound by both the mAb and polyclonal serum antibodies (Fig. 2B,C). To get a more detailed picture of the KLH N-glycans recognized by anti-schistosome antibodies, pairs of fractions in the elution range from fractions 29–67 were screened for binding to antibodies of murine S. mansoni infection serum (Fig. 3A), hamster antisoluble egg antigen (SEA) hyperimmune serum (Fig. 3B), and the three mAbs 291-5D5-A, 258-3E3, and 128-1E7-C that bound to KLH (Figs. 1 and 3C–E). All antibodies resulted in very similar glycan fraction-binding patterns (Fig. 3A–E), which pointed to a shared set of glycan epitopes recognized by the different polyclonal and monoclonal antibodies. Next to the immobilization of monoclonal and hyperimmune antibodies using secondary antibodies,

an alternative sandwich method was applied for antibody immobilization as follows. Wells were coated with KLH to capture mAb 128-1E7-C (Fig. 3G). Subsequently, Fab portions of the mAb that were not occupied by KLH antigens were used for capturing KLH BACH–glycans, followed by detection with AP-conjugated streptavidin. It must be noted that immobilization of the mAb 128-1E7-C with anti-mouse immunoglobulin (Fig. 3E) and with KLH antigen (Fig. 3G) gave virtually identical results. The glycan fractions that gave strong signals with the tested schistosome-directed antibodies (Fig. 3) were analyzed by MALDI–TOF–MS (Fig. 4). Fractions 49 and 50 (Fig. 4A) and fractions 51 and 52 (Fig. 4B) contained complex mixtures of glycan species. Fractions 53 and 54 (Fig. 4C), fraction 55 (Fig. 4D), and fractions 56 and 57 (Fig. 4E) (cf. Fig. 3A–F), which in ELISA showed strongest reactivity with schistosome-directed antibodies, were dominated by species of composition H5,6N4F2,3P0,1. Next, the heterogeneity of the glycan fractions recognized by schistosome-directed antibodies was reduced by exoglycosidase treatment. We chose to apply a combination of the exoglycosidases b-N-acetylhexosaminidase, a-mannosidase, and b-galactosidase because we expected that these enzymes would not affect the fucosylated motifs recognized by schistosome-directed antibodies (Table 1). Therefore, aliquots of fractions 53–57, which contained the major cross-reactive glycans (Fig. 3A–F), were pooled and incubated with the three above-mentioned exoglycosidases. Cleavage products were rechromatographed by HPLC, and fractions were assayed using mAb 128-1E7-C as described above. Fractions 49–51 of the exoglycosidase-treated KLH N-glycans showed strong reactivity with this mAb (Fig. 3G). Analysis of these fractions by MALDI–TOF–MS revealed BACH–glycans of composition Hex4HexNAc4dHex2 (fraction 49), Hex4HexNAc4dHex2Pent1 (fraction 50), and Hex5HexNAc4dHex2 (fraction 50) (Fig. 5). Taken together, MALDI–TOF–MS of the binding fractions of the first- and second-dimension HPLC separation (Figs. 4 and 5) indicated Hex4–6HexNAc4dHex2,3Pent1 as a common composition of the glycans recognized by schistosome-directed antibodies. To get more structural information, BACH–glycans were further analyzed by MS/MS to allow identification of the binding motif of the mAb. To this end, glycans were separated by reverse-phase na-

1

A

Con A

D

0.5 AP

AP

mAb 128-1E7-C

C

-BACH

B

-BACH

anti-KLH 0.5

-BACH

1 -BACH

Relative intensity

0

0 1

0.5

antibody

80-89

70-79

60-69

50-59

40-49

30-39

20-29

10-19

0

secondary antibody

Fractions Fig. 2. ELISA detection of fractionated KLH glycans. KLH BACH–glycans were tested for binding to CBPs using a glycan capture assay. Con A (A), antibodies from an anti-KLH hyperimmune serum (B), and the anti-F-LDN-(F) mAb 128-1E7-C (C) were trapped on microtitration plates. Pools of 10 fractions of KLH-derived biotinylated N-glycans were added, and binding of biotinylated glycans to lectin or antibodies was detected using AP-conjugated streptavidin. For each experiment, the most intense signal is normalized to 1.0. (D) Schematic representation of the binding assay as performed in panels B and C.

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1

1

A

S. mansoni infection serum

0.5

0

0

1

B

Relative intensity

anti-SEA 0.5 0

Relative intensity

0.5

mAb 128-1E7-C

E

mAb 128-1E7-C, KLH adsorbed

F

mAb 128-1E7-C, after cleavage

G

1

0.5 0 1

1

C

mAb 291-5D5-A 0.5

0.5

0

0 29 31 33 35 37 39 41 43 45 47 49 51 53 55 56 58 60 62 64 66

1

D

mAb 258-3E3

Fraction

0.5 0 29 31 33 35 37 39 41 43 45 47 49 51 53 55 56 58 60 62 64 66

Fraction

2146.8 H4N4F2 2162.7 H5N4F1

H6N3 H5N3F1P1 H6N3P1 H6N3F1 H7N3 1975.7 2091.7 2107.7 2121.7 2137.7

1934.7 H7N2

Fig. 3. ELISA detection of KLH glycans cross-reactive with schistosome-directed antibodies. HPLC fractions of KLH BACH–glycans were analyzed for antibody binding using a glycan capture assay. Antibodies from murine S. mansoni infection serum (A), different schistosome-reactive mAbs (C–E,G), and immunoglobulins from a hamster anti-SEA hyperimmune serum (B) were trapped on microtitration plates using anti-murine immunoglobulin or anti-hamster immunoglobulin. Alternatively, KLH was adsorbed and a mAb was trapped on the basis of its anti-KLH reactivity (F). KLH BACH–glycans were added to the trapped antibodies, and binding of biotinylated glycans to antibodies was detected using AP-conjugated streptavidin. Whereas intact BACH–glycans from KLH were applied in panels A–F, in panel G BACH–glycans of fractions 48–59 after enzymatic treatment with a-mannosidase, b-galactosidase, and b-N-acetylhexosaminidase and HPLC fractionation were applied. All fractions except fraction 55 were applied as a pool of two fractions (fractions 29 and 30 were combined, etc.). For each experiment, the most intense signal was normalized to 1.0.

Fractions 49-50

A

2308.6 H5N4F2

Fractions 53-54

2324.6 H6N4F1

2283.6 H7N3F1

2440.6 H5N4F2P1

Fraction 55

2440.6 H5N4F2P1

B

2308.8 H5N4F2 2324.8 H6N3F1

2278.8 H4N4F2P1 2294.7 H5N4F1P1

2253.7 H6N3F1P1

2162.7 H5N4F1

1934.7 H7N2

2121.7 H6N3F1 2137.7 H7N3

Fractions 51-52

1893.7 *

C

2324.6 H6N4F1

2456.6 H6N4F1P1

2308.6 H5N4F2

2470.5 H6N4F2

Fractions 56-57

D

E

2470.5 H6N4F2 2486.6 H7N4F1 2586.5 H5N4F3P1

2000

2200

2400 m/z

2600

2000

2200

2400

2600

m/z

Fig. 4. MALDI–TOF–MS analysis of BACH–glycan fractions from KLH that are recognized by schistosome-directed antibodies. Biotinylated N-glycan fractions from KLH exhibiting strong reactivity with schistosome-directed antibodies (cf. Fig. 3A–F) were analyzed by MALDI–TOF–MS. Sodium adducts are labeled with registered mass and deduced composition. H, hexose; N, N-acetylhexosamine; F, fucose; P, pentose; D, potassium adduct; *, contaminant. Compositions of H4–6N4F2,3P0,1 are highlighted in bold.

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Fuc(a1-3)GalNAc(b1-4)[Fuc(a1-3)]GlcNAcb1-(F-LDN-F) antenna structure. Fragmentation spectra of the other binding candidates (fractions 55–57 [Fig. 4D,E]) likewise gave indications for F-LDNF antenna structures. The BACH–glycan fractions that were consistently identified as the major targets of both monoclonal and polyclonal antibodies in the microtitration plate binding assay (Fig. 3) were used to study antibody binding by SPR. For this purpose, both a pool of the BACH–glycan fractions recognized by anti-schistosome antibodies (fractions 49–58 [Fig. 3]) and the high-binder fractions (fraction 55 [Fig. 3]) that showed rather low heterogeneity in mass spectrometric analysis (fraction 55 [Fig. 4]) were immobilized on an SPR chip via covalently attached streptavidin. Fractions 20–29, which were not bound by schistosome-directed antibodies (Fig. 2), were immobilized as negative controls. The SPR chip exhibiting BACH– glycans was used to screen a set of mAbs known to be directed against various schistosome glycoconjugates (Table 1). One SPR chip was used for all of the analyses, and regeneration of the chip surface was performed after each analysis to remove binding antibodies from the chip surface. These experiments showed that only the three F-LDN(-F)-directed mAbs 128-1E7-C, 258-3E3, and 2915D5-A were binding to the fraction pool 49–58 and to fraction 55 (Fig. 7). None of the antibodies showed binding to control fractions (pool 20–29 [data not shown]). Other mAbs directed against schistosome glycoconjugates, such as Lewis X (291-4D10-A [Fig. 7D]), LDN, LDN-F, and DF-LDN-DF, showed no binding to the KLH BACH–glycan fractions (data not shown), in accordance with their described binding properties (Table 1). Notably, the three KLH-binding mAbs differed in their association and dissociation properties. mAbs 128-1E7-C and 291-5D5-A showed a slow dissociation, whereas mAb 258-3E3 exhibited a fast association and fast dissociation.

Fraction 49 2146.5 H4N4F2 Δ

2278.5 H4N4F2P1

2308.6 H5N4F2 Δ

2200

2300

2400

m/z Fig. 5. MALDI–TOF–MS analysis of exoglycosidase-treated KLH BACH–glycans by schistosome-directed antibodies. KLH BACH–glycans after exoglycosidase treatment and HPLC fractionation, which exhibited strong reactivity with schistosomedirected antibodies (cf. Fig. 3G) were analyzed by MALDI–TOF–MS. H, hexose; N, Nacetylhexosamine; F, fucose; P, pentose; D, potassium adduct.

7

Y5α Y6

H3

B3 Y4α

N

N

F

F Y5β

Discussion We have presented a microtitration plate-based method that offers a simple solution for identifying CBP-binding glycans released from a natural source. As an example, the method was successfully used to identify and characterize F-LDN-F units on KLH Nglycans as binding motifs for anti-schistosome mAbs. Carbohydrate determinants shared between S. mansoni antigens and KLH

2,4A 7 0,2A 7

H

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B6 Y1 0,2A

597.1

B3

7Y5α 0,2A

7Y5αY5β

N

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7Y 6

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F

1092.8

N

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F 2,4A

1019.3

F

991.3

[M+2Na]2+ 918.3

575.1

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N2 F 2

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7Y4α

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379.1

noLC coupled online to ESI–IT MS/MS. Because collision-induced fragmentation of proton adducts is often associated with monosaccharide rearrangement reactions leading to ‘‘internal residue loss” resulting in misleading fragment ions that complicate structural elucidation, 0.6 mM sodium hydroxide was added to the running solvent to create sodium adducts that provide more informative fragment spectra [36–38]. BACH–glycan Hex4HexNAc4dHex2 from fraction 54 (see Fig. 4C) was registered as a double-sodiated species. The fragmentation spectrum showed the presence of a HexNAc2dHex2 antenna structure (Fig. 6). Notably, fragmentation clearly shows that one fucose is easily lost from the precursor (signals at m/z 1092.8 and 1707.5), followed by an N-acetylhexosamine (m/z 991.3 and 1504.6). A second N-acetylhexosamine is lost only after the loss of the second fucose (signals at m/z 1358.6 and 1155.7), which indicates a dHex-HexNAc-(dHex-)HexNAc terminal motif. These findings are in accordance with a

1853.4

2100

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1358.6

A

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m/z Fig. 6. nanoLC–MS/MS of KLH BACH–glycan species H5N4F2. Biotinylated KLH N-glycans of fraction 54 were analyzed by nanoLC–ESI–MS/MS. The fragment spectrum of the double-sodiated, biotinylated species H5N4F2 (precursor at m/z 1165.8) is shown. Fragments are assigned according to Ref. [45]. H, hexose; N, N-acetylhexosamine; F, fucose; P, pentose.

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B mAb 258-3E3

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Fig. 7. SPR sensorgrams illustrating the binding of mAbs with KLH BACH–glycans. (A–C) Interaction of mAbs 291-5D5-A (A), 258-3E3 (B), and 128-1E7-C (C) with captured BACH–glycan fractions 49–58 (dotted lines) and fraction 55 (solid lines). mAb 291-4D10-A (D), which is known to bind to LewisX, did not recognize these fractions. All sensorgrams were double referenced (corrected for the negative control and buffer injections). As negative control, a blank surface loaded with biotin was used.

are the molecular basis for the cross-reactivity of schistosomiasis infection sera with KLH [39–41]. KLH has been explored to detect antibodies for diagnosis of schistosomiasis. Moreover, KLH has been tested as a vaccine against schistosomiasis in animal models [42,43]. In accordance with the literature [16,33], our method highlighted a specific set of KLH N-glycans carrying the Fuca13GalNAcb1-4(Fuca1-3)GlcNAcb1-[F-LDN(-F)] structural unit as the major targets for anti-schistosome polyclonal and monoclonal antibodies. Compared with other techniques for studying protein–carbohydrate interactions such as glycan microarrays, the microtitration plate binding assay is easier and faster and can be performed in any laboratory equipped with basic MS instrumentation. CBPs are immobilized by adsorption to microtitration plates either directly or via a secondary antibody. Oligosaccharides are labeled with BACH, the labeled glycans are fractionated by HILIC–HPLC, and aliquots of the fractions are added to each well, where binding between glycan ligands and CBPs occurs. The addition of AP-conjugated streptavidin may add to the stability of the interaction thanks to its four biotin-binding sites that may cross-link the biotin-tagged glycans. After a short washing step, bound BACH–glycans are detected via AP staining. The sensitivity of both mass spectrometric analysis and microtitration plate binding assay is in the femtomole range. Whereas earlier microtitration plate binding assays with BACH–glycans relied on glycan immobilization [23,24], our reverse setup is based on immobilized CBPs. Because the captured BACH–glycans are detected via AP-labeled streptavidin, the method works with unlabeled CBPs such as antibodies and lectins. This is in contrast to most glycan microarray approaches

that work with immobilized or adsorbed glycans and glycoconjugates and require labeling of CBPs or the use of labeled secondary antibodies. BACH labeling was performed following a procedure described in the literature [23]. For the analysis of samples containing labile monosaccharides such as sialic acids, milder labeling conditions may need to be employed by, for example, reducing acid content and shortening incubation times to avoid degradation. In our current experiments, which establish a proof of principle by characterization of nonsialylated Schistosoma crossreactive glycans from KLH, we found no indications of glycan degradation. BACH–glycan fractions identified as binders can be easily analyzed by MALDI–TOF–MS and nanoLC–ESI–MS/MS, providing structural information of the analyzed glycans [44]. However, many of the fractions were still heterogeneous, and this hindered the unambiguous identification of glycan ligands. An approach for reducing the heterogeneity of the fractions would be seconddimension chromatography on, for example, reverse-phase or graphitized carbon or exoglycosidase treatment in combination with rechromatography, as performed in this study. This approach allowed us to efficiently separate the glycans carrying the target epitope from nonrelevant BACH–glycans, and performance of the BACH–glycan binding assay on the fractions of this second separation readily highlighted several fractions that were homogeneous in terms of glycan molecular composition (Fig. 5). Interestingly, treatment of BACH–glycans with a mixture of exoglycosidases still left four or five hexoses on the N-glycan. Although most of these hexoses will represent mannoses of the trimannosyl core, in KLH

Ligand identification of carbohydrate-binding proteins / M. Wuhrer et al. / Anal. Biochem. 406 (2010) 132–140

mannoses may be capped by galactoses in various linkages [33]. These galactoses have been shown to be in part resistant to treatment with a- and b-galactosidase [33]. MS/MS of BACH–glycans allowed the identification of F-LDN-F as the mAb target structure (Fig. 6), in accordance with earlier findings for the mAb M2D3H, which likewise recognizes the schistosome–KLH cross-reactive determinant [16,32,33]. Whereas anti-schistosome antibodies bound predominantly large, hybrid-type glycans eluting in between fractions 49 and 57 (Fig. 3), Con A was found to bind predominantly to the elution range of fractions 20–49. These fractions are presumably dominated by the major KLH N-glycan species, which are paucimannosidic N-glycans that may carry a core fucose optionally decorated with galactose residues [32–35]. It has been described previously, using glycan microarray technology, that these paucimannosidic KLH N-glycans are recognized by Con A [26]. In addition to the microtitration plate binding assay, the BACH– glycans were used for SPR analysis of antibody binding. To this end, BACH–glycans were immobilized on the SPR chip via streptavidin and antibody binding was detected by SPR. The chip could be regenerated by an acid wash, and the same surface was used to characterize the binding of multiple mAbs in consecutive experiments. The SPR experiments showed marked differences in the binding kinetics of the mAbs; two mAbs were characterized by rather slow binding and showed a slow dissociation, whereas one mAb was characterized by fast association and dissociation behavior. Notably, all of the mAbs tested in SPR are of the immunoglobulin M (IgM) class, and one may assume that the interaction of the mAbs with the BACH–glycan-coated chip surface is happening in a multivalent manner. Therefore, the observation of rather stable interactions for two of the mAbs (Fig. 7A,C) may be explained, at least in part, by multivalency. It is noteworthy that the ligands for all three mAbs were readily bound in the microtitration plate assay, further indicating that the assay performs with both slow and fast dissociating antibodies. Whereas the microtitration plate binding assay described here works with immobilization of CBPs, in the SPR assays the glycans were immobilized and CBPs were applied in solution. The SPR assay shares this configuration with glycan microarrays. For polyvalent CBPs, binding will be influenced by the density and presentation of the immobilized glycans. Likewise, for the microtitration plate binding assay described here, the CBP coating or immobilization efficacy together with CBP presentation is expected to influence binding. For example, direct coating of the CBP versus immobilization of the CBP via a secondary antibody may result in very different presentations of glycan binding sites, and this will influence the formation of complexes of CBPs, BACH–glycans and streptavidin conjugates at the plate surface. Enrichment or purification of glycans by affinity chromatography followed by, for example, mass spectrometric analysis of bound and unbound fractions represents another useful method for oligosaccharide ligand identification [15]. However, in general, rather high affinities and low off-rates are needed for purification of glycan ligands by affinity chromatography using immobilized CBPs. Hence, the choice of the assay (affinity purification, glycan array, SPR, or the microtitration plate binding assay described here) will determine, at least in part, which glycans will be detected as binders or not, and results obtained with the various approaches may vary considerably. In conclusion, we have established a method comprising the biotinylation of natural glycans, oligosaccharide HPLC fractionation, screening of the fractions in microtitration plate binding assays, and (tandem) mass spectrometric characterization of glycan ligands. The method is suitable for identification and structural characterization of glycan ligands of carbohydrate-binding proteins. Moreover, in combination with SPR, information on association and dissociation properties can be obtained.

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[35]

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