Multifaceted Approaches Including Neoglycolipid Oligosaccharide Microarrays to Ligand Discovery for Malectin

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Multifaceted Approaches Including Neoglycolipid Oligosaccharide Microarrays to Ligand Discovery for Malectin Angelina S. Palma,*,1 Yan Liu,* Claudia Muhle-Goll,†,2 Terry D. Butters,‡ Yibing Zhang,* Robert Childs,* Wengang Chai,* and Ten Feizi* Contents 266 268 268

1. Overview 2. Preparation of Recombinant Soluble Human Malectin 2.1. Materials and equipment 2.2. Generation of plasmids containing the His6-tagged malectin globular domain 2.3. Expression and purification of His6-tagged malectin for microarray analysis 3. Preparation of Glucan Oligosaccharides 3.1. Materials and equipment 3.2. Glucan oligosaccharides di- to heptasaccharides 4. Preparation of Glucosylated High-Mannose N-Glycans 4.1. Triglucosylated high-mannose N-glycans 4.2. Diglucosylated high-mannose N-glycans 5. Preparation of NGL Probes 5.1. Materials and equipment 5.2. Preparation of AO-NGLs of glucan oligosaccharides 5.3. Preparation of AO-NGLs of the glucosylated N-glycans

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* Glycosciences Laboratory, Faculty of Medicine, Imperial College London, Northwick Park Hospital Campus, Harrow, Middlesex, United Kingdom { European Molecular Biology Laboratory, Heidelberg, Germany { Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, Oxford, United Kingdom 1 Present address: REQUIMTE, Departamento de Quı´mica, Centro de Quı´mica Fina e Biotecnologia, Faculdade de Cieˆncias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal 2 Present address: Karlsruhe Institute of Technology (KIT), Institut fu¨r Biologische Grenzfla¨chen (IGB-2), Karlsruhe, Germany Methods in Enzymology, Volume 478 ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)78013-7

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6. Carbohydrate Microarray Analysis of Human Malectin 6.1. Materials and equipment 6.2. Microarray printing 6.3. Probing the microarrays 7. Conclusions Acknowledgments References

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Abstract In this chapter, we describe the key procedures for isolation of the oligosaccharides and the preparation of neoglycolipid probes together with expression of malectin that have enabled the discovery of the highly selective binding of this newly described protein in the endoplasmic reticulum (ER) to a diglucosyl high-mannose N-glycan. This is the first indication of a bioactivity for a diglucosyl high-mannose N-glycan of the type that occurs in the ER of eukaryotic cells and which is an intermediate in the early steps of the N-glycosylation pathway of nascent proteins. The malectin story is an example of a powerful convergence of disciplines in biological sciences: (i) developmental biology, (ii) bioinformatics, (iii) recombinant protein expression, (iv) protein structural studies, (v) glucan biochemistry, and (vi) drug-assisted engineering of oligosaccharide biosynthesis, culminating in (vii) oligosaccharide ‘‘designer’’ microarrays, to clinch the remarkable selectivity of the binding of this newly discovered ER protein. Thus, the way is open to the identification of the role of malectin in the N-glycosylation pathway.

1. Overview The malectin protein gene was originally identified in Xenopus laevis in the search for proteins that are developmentally regulated in the pancreas. However, it was soon found to be broadly expressed in embryonic and adult X. laevis, and moreover detected in all tissues examined (Schallus et al., 2008). Although possibly disappointing initially that this was not a pancreatic developmental marker, bioinformatic studies with the deduced amino acid sequence revealed malectin as a highly conserved protein in the animal kingdom (Fig. 13.1) pointing to an important biological function. A clue for possible ligands for malectin came from its three-dimensional structure resolved by NMR, which showed that the highest hits for fold homologues were microbial carbohydrate-binding modules (CBMs) that recognize glucan polysaccharides (Schallus et al., 2008). Armed with this knowledge, we performed NMR-based ligand-screening studies, using glucose containing oligosaccharides. Maltose was the first disaccharide that was observed to be bound by the xenopus protein, hence its designation ‘‘malectin.’’ The glucan binding property of the xenopus malectin was further

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Figure 13.1 Sequence alignment of malectin protein in animals. The beginning and the end of the expression construct are indicated. The secondary structure elements of the globular domain are shown on top of the amino acid sequence, and the four aromatic residues (Y67, Y89, Y116, and F117) and D186 mediating the carbohydrate interaction are marked by crosses. SP, signal peptide; TM, C-terminal transmembrane helix; Xen, Xenopus laevis; Hum, Homo sapiens; Mou, Mus musculus; Hen, Gallus gallus; Fly, Drosophila melanogaster; Aed, Aedes aegyptii; Cae, Caenorhabditis elegans; Sch, Schistosoma japonicum; Nem, Nematostella vectensis. (The figure was reprinted with kind permission from MBC (Schallus et al., 2008).)

corroborated using microarrays of glucan oligosaccharide probes (Schallus et al., 2008). With the finding soon, thereafter, that malectin is localized in the endoplasmic reticulum (ER) of mammalian cells, we populated our neoglycolipid (NGL)-based oligosaccharide microarrays with the glucosylated high-mannose N-glycans of the type that occur in the ER, having one, two, or three terminal glucosyl residues at the D1 mannosyl branch. These experiments revealed a high selectivity of malectin for a high-mannose N-glycan with two terminal glucose residues (Schallus et al., 2008).

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The NGL technology and the NGL-based microarray system that have come to play in this study have been the subject of chapters in previous volumes of this series (Chai et al., 2003; Feizi and Childs, 1994; Feizi et al., 1994; Liu et al., 2006). Major advantages of the NGL approach are: its inherent flexibility, generation of multivalent probes with increased sensitivity of detection, and the ability to rapidly prepare and populate the arrays with probes containing novel oligosaccharide sequences, especially those of oligosaccharides isolated from biological sources and available only in minute amounts. In this chapter, we dwell in some detail on the multifaceted technical procedures that have been essential to carbohydrate ligand discovery for malectin. These include the procedures for generation of recombinant His-tagged malectin, focusing on how to prepare the human malectin homologue; the preparation of glucan oligosaccharides and glucosylated N-glycans from polysaccharides and drug-treated mammalian cell cultures, respectively; the microscale conjugation of these oligosaccharides to lipid for preparation of NGL probes, followed by NGL-based microarray analysis of recombinant human malectin. Please also read Chapter 19 in Volume 480 of this series, ‘‘Use of glycan microarrays to explore specificity of glycan-binding proteins’’ by David Smith, Xuezheng Song, and Richard Cumming.

2. Preparation of Recombinant Soluble Human Malectin Malectin contains a single highly conserved globular domain (Fig. 13.1; Homo sapiens AAs 42–228; Schallus et al., 2008). Protein domain databases such as SMART (Letunic et al., 2006) and Pfam (Finn et al., 2006) predict an Nterminal signal peptide (H. sapiens: AAs 1–28) and a C-terminal transmembrane helix (H. sapiens: 271–290). Between the globular domain and the C-terminal membrane anchor, a highly charged sequence segment is found, that extends over eight contiguous glutamate residues, for example, in human malectin. The lectin function resides in the globular domain of malectin, which was used for microarray screening of putative ligands. It was cloned into the pET M10 vector of the M-series generated by Gunter Stier, EMBL (Bogomolovas et al., 2009). This vector contains an N-terminal hexahistidine (His6)-sequence that is used as a tag for affinity purification and enables detection of the malectin construct using an anti-His antibody. Further information on the vector can be found at http://www.embl.de/ pepcore/pepcore_services/strains_vectors/index.html.

2.1. Materials and equipment 1. Taq DNA polymerase supplied with appropriate buffer (e.g., AmpliTaq DNA Polymerase, Applied Biosystems)

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2. Sense and reverse primers 3. PCR Nucleotide mix (dATP, dGTP, dCTP, and dTTP; Roche Applied Science) 4. Genomic DNA (e.g., Qiagen) 5. Deionized and sterilized H2O 6. Restriction enzymes (NcoI, Acc65I, supplied with respective buffers; Fermentas) 7. T4 DNA Ligase, supplied with respective buffer (Fermentas) 8. Kits for PCR fragment extraction or plasmid purification (Qiagen) 9. Expression vector (recommended pET system) 10. Escherichia coli BL21[DE3] DH5a (New England Biolabs) for cloning 11. E. coli BL21[DE3] cells (New England Biolabs) for expression 12. LB-medium (10 g/l bactotryptone, 5 g/l yeast extract, 10 g/l NaCl, pH 7.4, supplemented with appropriate antibiotic (e.g., 50 mM kanamycin) and 200 mM isopropylthio-b-galactoside (IPTG) for induction) 13. Lysis buffer (20 mM Tris–HCl, pH 8.0,150 mM NaCl,10 mM Imidazole, 2 mM b-mercaptoethanol, protease inhibitors, for example, Complete Protease Inhibitor Cocktail, Roche) 14. Lysozyme (Sigma) (300 mg/ml) 15. DNase I (Roche Applied Science, 1 mg/ml) þ MgCl2 16. High salt wash buffer (lysis buffer containing 1 M NaCl) 17. Elution buffer (lysis buffer þ 250 mM Imidazole, pH 8.0) 18. Ni NTA resin (Qiagen) 19. Millipore Amicon Ultra-4 centrifugal filter units, 10 kDa cutoff 20. Gel filtration buffer (20 mM Tris–HCl, pH 7.2, 150 mM NaCl, 2 mM 1,4 dithiothreitol (DTT), 1% (w/v) maltose (Sigma) 21. Gel filtration column, for example, Superdex 200 HiLoad 16/60 (GE Healthcare) 22. Dialysis buffer (20 mM Tris–HCl, pH 7.2, 150 mM NaCl, 2 mM DTT) 23. Desalting columns PD10 (Amersham Biosciences) 24. Sonicator (e.g., Sonopuls HD 2070, Bandelin (Berlin)) 25. Ultracentrifuge, for example, Beckmann OptimaTM L-90 26. Spectrophotometer, nanodrop ND-1000, PEQLAB

2.2. Generation of plasmids containing the His6-tagged malectin globular domain The coding sequence for the human malectin globular domain was amplified by PCR from a plasmid carrying the human genomic sequence using the following primers: sense primer MalHsen TTG CCA TGG CCGGG CTG CCCGAGAG and reverse primer MalHrev TTGCGGTACCTT ACTCCAATCCCGGATGAGGCTG.

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The sense primer carries the restriction site for NcoI (CCAGGT). The reverse primer is designed with a stop codon UAA before the Acc65I restriction site (GGTACC). The xenopus malectin domain can, in principle, be amplified using the homologous primers, but an internal NcoI site within the first 40 amino acids of the globular domain should be mutated prior to PCR amplification to avoid internal cleavage. Five PCR cycles were performed at lower annealing temperature of 57  C, which were followed by 20 cycles at 62  C, and the elongation was done at 72  C for 1 min. When the PCR reaction is performed with genomic DNA as a template, 25 cycles at 62  C are recommended to generate enough of the PCR product. The PCR product was cloned using the 30 -Acc65I and 50 -NcoI restriction sites into the pET M10 vector referred above. Plasmids were amplified in E. coli BL21[DE3] DH5a and the kanamycin resistance conferred by the plasmid was used for clone selection. Plasmid purification was achieved using the standard protocols and kits of the Qiagen plamid purification kit. DNA sequencing using T7 sense and reverse primers was used to verify the sequence. The plasmids were transformed into E. coli BL21[DE3] cells for expression.

2.3. Expression and purification of His6-tagged malectin for microarray analysis Five milliliters of an overnight culture of E. coli BL21[DE3] cells with the plasmid containing the gene for the globular segment of human His6malectin gene were diluted in 1 l LB medium containing 50 mg/ml kanamycin. The cells were grown at 37  C to an OD600 of 0.6 (approximately 6–8 h). Expression was induced by adding 0.2 mM isopropyl-b-D-thiogalactopyranoside overnight at 18  C. The cells were harvested by centrifugation (3–5 g/l). At this point, the cell pellet could be stored at 20  C, a step that also facilitated cell rupture. For protein extraction, the cell pellet  was resuspended in lysis buffer (10 ml/g wet cell pellet), chilled to 4 C, complemented with lysozyme, and incubated on ice until cell rupture became visible. To digest genomic DNA, DNase I and 5 mM MgCl2 were added and the cell suspension was incubated at ambient temperature for another 10 min. A sonication step (30–50 short pulses of 5–10 s with pauses 10–20 s) was employed to complete cell rupture. Following ultracentrifugation at 125,000g for 25 min at 4  C (e.g., 40,000 RPM using a Beckmann 45TI rotor), the supernatant was applied onto a Ni-NTA agarose column at ambient temperature (2 ml Ni-NTA resin /1 l cell culture were used). The column was washed with 10–20 column volumes of lysis buffer, followed by 10 column volumes of high salt wash buffer and with 10 volumes of lysis buffer. The His6-tagged proteins were eluted with 5–7 volumes of elution buffer.

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Purity after this step was already high as judged by SDS-PAGE analysis. Nevertheless, a size exclusion purification step on a gel filtration column (Superdex 200) was added to remove spurious copurified proteins that bind nonspecifically to the Ni-NTA column, and might be present at concentrations below the detection limit of SDS-PAGE analysis. On one occasion, we noticed an unidentified glycosidase activity in a protein preparation purified only with Ni-NTA chromatography. This was detected by a change in the color of the protein solution to yellow upon addition of the nitrophenylmaltoside compound, indicating that the nitrophenyl group was cleaved off. The size exclusion matrix material is composed of polymers of dextran. We observed that malectin binds tightly to the column and could not be readily eluted off using standard elution buffers. The inclusion of 1% (w/v) maltose in the gel filtration buffer was sufficient to overcome this problem and the protein eluted as the predicted molecular mass of 18 kDa, which was confirmed by NMR 1H-T2 measurements and dynamic light scattering. Finally, a buffer exchange step was performed by applying the protein to a small size exclusion, desalting column (e.g., PD10) or by dialysis against maltose-free Tris buffer. Up to 20 mg of malectin could be produced from 1 l of bacterial culture following this protocol. Protein concentration was measured by UV measurements at 280 nm using an extinction coefficient of 20,400 M 1 cm 1. The protein was stable under these conditions at 4  C for several weeks at concentrations below 4 mg/ml (20 mM), in the presence of 0.02% sodium azide. The recombinant malectin is soluble up to 20 mg/ml, but on storage tends to partially precipitate at concentrations higher than 10 mg/ml. For long-term storage, the protein can be lyophilized in suitable aliquots with retention of carbohydrate-binding activity and specificity.

3. Preparation of Glucan Oligosaccharides A series of glucan oligosaccharides, in the range of di- to heptasaccharides, with a1,4-, a1,6-, b1,3-, b1,4-, b1,6-linkages and the disaccharides with a1,2- and a1,3-linkages, were either from commercial sources or prepared from polysaccharides after partial depolymerization by chemical means, as described below.

3.1. Materials and equipment 1. Disaccharides nigerose (a1,3-linked) from Wako Chemicals, cellobiose (b1,4-linked) and kojibiose (a1,2-linked) from Sigma 2. Laminarin (b1,3-linked) di-, tri-, and tetrasaccharides from Dextra, penta- and hexasaccharides from Megazyme, and heptasaccharide from Seikagaku America

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3. Malto-di- to heptasaccharides (a1, 4 linked) from Sigma 4. Cello-oligosaccharide (b1,4-linked) mixture obtained by acid hydrolysis of cellulose from Megazyme 5. Glucan polysaccharides pustulan from Umbilicaria papullosa (b1,6linked) (de la et al., 1995) from Calbiochem and dextran (MW 500 kDa, a1,6-linked with 5% a1,3-branches) (de Belder, 1993) from Amersham Biosciences 6. Deionised water 7. HCl and NaOH solutions (0.1 M and 0.2 M) 8. Solvent n-propanol:water (8:3, v/v) 9. Gel filtration columns: Sephadex G10 (1.630 cm, Amersham Biosciences) and Bio-Gel P4 (1.690 cm, Bio-Rad), with an on-line refractive index detector and auto sample collector 10. Silica gel high-performance (HP) TLC plates with aluminium-backing (Merck) 11. Orcinol staining reagent (Chai et al., 2003) 12. MALDI-TOF mass spectrometer, Tof Spec-2E (Waters)

3.2. Glucan oligosaccharides di- to heptasaccharides Partial depolymerization of dextran and pustulan were carried out by acid hydrolysis. Dextran (100 mg) was treated with 0.1 M HCl, at a concentration of 20 mg polysaccharide/ml, at 100  C for 4 h. Pustulan (100 mg) was treated with 0.2 M HCl at a concentration of 10 mg polysaccharide/ml at 100  C for 8 h. The reaction was stopped by neutralization with aqueous NaOH solution. Acid hydrolysis was selected as the depolymerization procedure for pustulan upon our observation that the b-linked oligosaccharide series prepared by acetolysis of the parent polysaccharide contained a percentage of a-anomers. The dextran and pustulan hydrolysates were desalted using a short column (1.630 cm) of Sephadex G10 eluted with deionized water at a flow rate of 20 ml/h monitored on-line by a refractive index detector. The oligosaccharide fraction was collected at the void volume and lyophilized. Oligosaccharide fractions of dextran, pustulan, and cellulose were obtained from gel filtration chromatography on a column (1.690 cm) of Bio-Gel P4. The Bio-Gel P4 column was first equilibrated with deionized water and calibrated with an analytical mixture of dextran hydrolysate by eluting with deionized water at a flow rate of 15 ml/h. The desalted oligosaccharide mixtures (typically 1–2 ml of clear solution, the concentration applied varied according to solubility) were applied to the column and eluted under the same conditions. The eluate was monitored on-line by refractive index and the respective di- to heptasaccharide peaks were pooled according to their predominant glucose units (Fig. 13.2A) and lyophilized.

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Figure 13.2 (A) Gel filtration chromatography of the dextran hydrolysate on a column of Bio-Gel P4; the column was loaded with 4 mg hexose of the hydrolysate mixture in 1 ml deionized water (the dextran polysaccharide, 0.1 mg hexose, was added to the mixture to mark exclusion volume, Vo; the glucose units are indicated for each peak); the inset shows the HPTLC analysis of the isolated dextran oligosaccharide fractions (mono- to heptasaccharide, lanes 1–7, 2 mg hexose per lane) and of the hydrolyzed dextran mixture (total 40 mg hexose) before fractionation (lane 8); (B) MALDI-MS analysis of dextran heptasaccharide fraction. The molecular masses of the sodiated molecules are indicated (major component is the heptasaccharide).

Quantitation of the oligosaccharide fractions, after their reconstitution with deionized water, was carried out by TLC-based orcinol assay using glucose (0.05–1 mg/ml in deionized water) as standard, as described (Chai et al., 1997). Stock solutions were then prepared typically at 5 mg/ml.

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Overall, the solubility in water of fractions up to heptasaccharides is good with the exception of the cello-oligosaccharides with degree of polymerization five or higher, for which the stock solutions were prepared at 1 mg/ml. Aliquots of each fraction (2 mg hexose) were analyzed by HPTLC on silica gel plates, using a solvent system of n-propanol/water, 8:3 (by volume), and stained with orcinol reagent (inset Fig. 13.2A). The molecular masses of the main components of oligosaccharide fractions from gel filtration were corroborated by MALDI-MS (Fig. 13.2B). For MALDI-MS, oligosaccharides solutions were diluted in methanol, at an estimated concentration of 10–20 pmol/ml, and 0.5 ml was deposited on the sample target together with a matrix of 2-(4-hydroxyphenylazo)benzoic acid. The linkage of the oligosaccharide fractions was corroborated by methylation analysis or 1H NMR.

4. Preparation of Glucosylated High-Mannose N-Glycans The monoglucosylated high-mannose N-glycan, Glc1Man9GlcNAc2, was isolated from hen egg yolk IgY using a similar procedure to that previously reported by Ohta et al. (1991). The triglucosylated N-glycan, Glc3Man7(D1) GlcNAc2, was isolated from the recombinant-expressed glycoprotein HIVIIIB gp120, secreted by cells that were treated with an ER-a-glucosidase inhibitor (Petrescu et al., 1997). The diglucosylated N-glycan, Glc2Man7(D1) GlcNAc2, was derived from this Glc3 analogue by digestion with ER-aglucosidase I (Alonzi et al., 2008). The general methodologies for preparation of the tri- and diglucosylated N-glycans are described below and the reader is advised to refer to primary papers cited.

4.1. Triglucosylated high-mannose N-glycans Triglucosylated N-glycans can be obtained from two major sources using tissue cultured cells treated with an ER-a-glucosidase inhibitor: (i) release of the oligosaccharides from secreted glycoproteins; (ii) isolation of free glucosylated oligosaccharides from the cells. 4.1.1. Materials and equipment 1. Chinese hamster ovary (CHO) cell line (Petrescu et al., 1997) stably transfected with highly glycosylated glycoproteins, eg., HIV gp 120 2. a-Glucosidase inhibitor N-butyl-deoxynojirimycin (NB-DNJ) (2–5mM, Toronto Research Chemicals, Inc.) 3. Concanavalin A-Sepharose beads (Sigma) 4. a-Methyl-mannoside (Sigma) 5. Hydrazine reagent (Ludger)

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6. Bacterial peptide N-glycosidase F (PNGase) from NEB or Ludger 7. High-performance anion-exchange chromatography (HPAEC, Dionex) 8. Normal-phase high-performance liquid chromatography NP-HPLC (Anachem) 9. Bio-Gel P4 columns (Bio-Rad) 10. In-line electrochemical detector (Dionex) 4.1.2. Isolation of glycoproteins Tissue cultured cells are grown in the presence of a suitable a-glucosidase inhibitor, such as castanospermine, deoxynojirimycin (DNJ), or NB-DNJ. These are available commercially and DNJ can be conveniently chemically synthesized from inexpensive starting materials such as D-glucuronolactone (Best et al., 2010). N-butylation of DNJ by reductive amination using sodium cyanoborohydride (Mellor et al., 2002) increases the a-glucosidase inhibitory efficacy in cells significantly (Alonzi et al., 2009). CHO cells are grown for 3–4 days in medium containing 2–5 mM sterile filtered NB-DNJ. The choice of CHO is critical for maximizing the synthesis of glucosylated products as it lacks an endomannosidase-mediated salvage pathway that deglucosylates proteins destined for secretion (Spiro, 2004). CHO cells stably transfected with highly glycosylated glycoproteins, for example, HIV gp 120, allow easy isolation of the highly expressed protein by antibody affinity column chromatography, for isolation of glycans. The abundance of glucosylated glycan is increased using lectin-resistant cells that are deficient in complex glycan biosynthesis (Butters et al., 1999). Culture medium that is rich in glucosylated glycoprotein is also used as a convenient source of oligosaccharide. Oligomannosidic glycoproteins can be bound to Concanavalin A-Sepharose beads and subsequently eluted with a-methyl-mannoside (0.5 mM in water). Following dialysis against water (or 0.1% TFA to reduce precipitation) to remove the methyl-mannoside, the glycoprotein-rich extract is lyophilized and subjected to glycan release (Petrescu et al., 1997). 4.1.3. Release and purification of triglucosylated N-glycans from glycoproteins Anhydrous hydrazine is used to cleave the glycan between the terminal nonreducing N-acetylglucosamine residue and the asparagine amino acid. This method is relatively quantitative and preserves the glycan intact. At least 1 mg of protein can be treated with commercially available kit forms of the hydrazine reagent (available from Ludger, UK see http://www.ludger.com/). An alternative method for release is to use bacterial PNGase. For this, the protein is denatured in SDS and disulphide reducing agent to allow enzyme access to all N-glycosylation sites, before dilution and addition of Triton X-100 to preserve PNGase enzymatic activity and incubation at 37  C overnight. For large

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amounts of protein (> 1 mg), the sequential addition of small amounts of enzyme assists hydrolysis. The released oligosaccharides are analyzed by a suitable method to determine structure, such as mass spectrometry and HPAEC for unlabeled glycans, or NP-HPLC using fluorescently labeled glycans (Butters and Neville, 2008). For large-scale purification, Biogel P4 columns are used but smaller amounts are readily isolated using standard HPLC columns where either in-line electrochemical detection or postcolumn labeling and analysis are used to identify and isolate glucosylated glycans (Petrescu et al., 1997). The biosynthesis of glucosylated glycans in CHO cells in the presence of NB-DNJ favors the Glc3Man7(D1)GlcNAc2 oligosaccharide (Petrescu et al., 1997). Smaller amounts of Glc3Man8GlcNAc2 and Glc3Man9GlcNAc2 are also generated. 4.1.4. Triglucosylated N-glycans production and isolation from free glycans As an alternative to using proteins as a source of glucosylated oligosaccharide, treatment of cells with NB-DNJ results in the synthesis of free glycans as a product of glycoprotein degradation following misfolding. The glycan material can be harvested in cell-free extracts from MDBK cells where nonproteasomal hydrolysis in the ER produces Glc3Man7GlcNAc2 in high abundance (Butters and Alonzi, unpublished data). This material does not require chemical or enzyme-mediated release, and can be isolated using HPLC methods described above.

4.2. Diglucosylated high-mannose N-glycans The diglucosylated N-glycan was prepared by enzymatic digestion of Glc3Man7(D1)GlcNAc2 oligosaccharide (10–100 mg), prepared from glucosidase inhibitor treated CHO cells described above, using purified preparations of ER-a1,2-glucosidase I. The enzyme is purified in a single ligand affinity step from detergent extracts of porcine or rat liver, using DNJ linked to chromatography beads. The preparation of the affinity column and general procedures in the affinity chromatography are described below and the reader is advised to refer to primary papers cited. 4.2.1. Materials and equipment 1. N-Carboxypentyl-DNJ (CPDNJ) from Toronto Chemicals, Inc. or chemically synthesized as described below 2. Affi-Gel 102 (Bio-Rad) 3. 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (Sigma) 4. Porcine or rat liver 5. Ultraturrax homogenizer (Janke & Kunkle)

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6. Homogenisation buffer: 50 mM Tris/HCl buffer, pH 7.2, containing 0.25 M sucrose, 5 mM leupeptin, 15 mM pepstatin A, 0.5 mM PMSF, and 1 mM 6-aminohexanoic acid 7. Buffer A (0.1 M sodium phosphate buffer, pH 7.0, containing 0.8% Lubrol PX) described above. 8. Sep-Pak C18 cartridge (1 cc, Waters) 9. NP-HPLC (Anachem) 4.2.2. Preparation of a-glucosidase I affinity ligand Coupling DNJ to chromatography beads provides a suitable ligand affinity support. CPDNJ is available from Toronto Chemicals, Inc., Ontario, Canada or can be synthesized by reacting DNJ with an excess of 6-bromohexanoic acid (Kaushal and Elbein, 1994). An amino-derivatized support such as Affi-Gel 102 allows simple coupling using EDC according to the following protocol. Aqueous solutions of CPDNJ (250 mmol) are adjusted to pH 4.8 with 1 M HCl and added to 5 ml of washed, packed Bio-Rad Affi-Gel 102. The gel is resuspended, adjusted to 9 ml with water, and 1 ml of 100 mM EDC is added. After maintaining the pH at 4.8 for 30 min at room temperature by the addition of 1 M HCl, the coupling reaction is allowed to proceed with gentle mixing for 16 h at 4  C. The gel is filtered using a sintered glass funnel and the filtrate is retained for the estimation of unbound ligand. The derivatized gel is washed sequentially with 100 ml of 50 mM NaOAc buffer, pH 4.5, containing 0.5 M NaCl and with 50 mM Tris/HCl buffer, pH 8.0, containing 0.5 M NaCl. This wash cycle is repeated twice before equilibrating the gel in an appropriate running buffer. The gel can be stored at 4  C in the presence of 0.02% azide and reused several times. To measure the concentration of coupled ligand, a sample of the gel filtrate containing alkylated imino-sugar is taken before and after coupling and subjected to HPAEC using an eluant of 150 mM NaOH/30 mM NaOAc (Dionex BioLC) and a CarboPac column. Alternatively, a Dionex CS10 column eluted with 50 mM sodium sulfate containing 5% (v/v) acetonitrile and in-line micromembrane suppression and electrochemical detection (Mellor et al., 2000) can be used. Typically, values of 20–25 mmol of ligand/ml of gel are obtained. 4.2.3. Isolation of a-glucosidase I The following procedures for extraction and purification of a-glucosidase I are performed at 4  C. Freshly obtained porcine or rat liver (300 g) is homogenized for 2–3 min (Ultraturrax homogenizer) with 0.8 l of 50 mM Tris/HCl buffer, pH 7.2, containing 0.25 M sucrose, 5 mM leupeptin, 15 mM pepstatin A, 0.5 mM PMSF, and 1 mM 6-aminohexanoic acid. The homogenate is filtered through cheesecloth and the filtrate is centrifuged at 15,000g for 30 min. The supernatant is recovered and centrifuged at 150,000g for 60 min

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and the pelleted material is washed with 120 ml of homogenization buffer. The pellet is recovered by centrifugation and resuspended in 75 ml of 10 mM sodium phosphate buffer, pH 6.8, containing 0.5% v/v Triton X-100. The suspension is stirred for 30 min and the membrane fraction is recovered by centrifugation at 150,000g for 30 min. The Triton-extracted pellet is suspended in 120 ml of 0.2 M sodium phosphate buffer, pH 6.8, containing 0.8% Lubrol PX and stirred for 60 min. A glucosidase I-enriched supernatant is recovered by centrifugation at 150,000g for 90 min. 4.2.4. Affinity chromatography of a-glucosidase I For affinity chromatography of a-glucosidase I, the Lubrol PX-extract is mixed with 5 ml of CPDNJ-Affi-Gel (25 mmol ligand/ml gel), prepared as described above, for 18 h and the gel is recovered by low-speed centrifugation. The gel is washed with 250 ml of 0.1 M sodium phosphate buffer, pH 7.0, containing 0.8% Lubrol PX (buffer A) and then eluted with 25 ml of buffer A containing 100 mM NB-DNJ. The column eluate is pooled and the enzyme is stored at 4  C. Before use, an aliquot is dialyzed against 41 l of buffer A to remove NB-DNJ. Enzyme activity and purity is assessed using [14C-Glc]radiolabeled Glc3Man9GlcNAc2 as described (Jacob and Scudder, 1994) or using NP-HPLC with fluorescently labeled substrates (Alonzi et al., 2008). 4.2.5. Hydrolysis of Glc3Man7GlcNAc2 and isolation of Glc2Man7GlcNAc2 Purified Glc3Man7(D1)GlcNAc2 oligosaccharide (10–100 mg) was dried under vacuum and resuspended in 20 ml of a-glucosidase I in buffer A (25 U) and incubated at 37  C for 16 h. An aliquot (1 ml) was taken, labeled with anthranilic acid, 2-AA (Neville et al., 2004) and analyzed by NPHPLC to determine reaction completion. When all the substrate was hydrolyzed to Glc2Man7(D1)GlcNAc2 the enzyme and detergent was removed using a Sep-Pak C18 cartridge and the nonbound eluate containing diglucosylated oligosaccharide was used for further derivatization.

5. Preparation of NGL Probes The glucan oligosaccharides and the glucosylated high-mannose N-glycans prepared above were converted into oxime-linked NGLs (AONGLs) for recognition studies in microarrays. The step by step procedures for the preparation, purification, and quantitation of the AO-NGLs are essentially as described previously (Liu et al., 2006, 2007).

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5.1. Materials and equipment 1. Reducing glucan oligosaccharides and glucosylated high-mannose N-glycans 2. Aminooxy-functionalized 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (AOPE), step by step synthesis procedure described in Liu et al. (2006) 3. Glass microvials with Teflon-lined caps (Chromacol) 4. Aluminium-backed HPTLC plates (Merck) 5. Silica cartridges (Waters or Phenomenex) 6. Primulin and orcinol staining reagents (Chai et al., 2003) 7. Solvents and solutions: methanol and chloroform are of HPLC grade; ammonium acetate solution (0.2 M in deionized water); chloroform/ methanol/water (C/M/W).

5.2. Preparation of AO-NGLs of glucan oligosaccharides In brief, 100 nmol of each glucan oligosaccharide or oligosaccharide fraction (di- to heptasaccharide) was used for conjugation in the presence of 200 nmol of N-aminooxyacetyl-DHPE (AOPE) in 50 ml C/M/W (10:10:1, by volume). After incubation at ambient temperature 16 h, the reaction mixtures were evaporated slowly to dryness at 60  C and reconstituted in 100 ml of C/M/W (25:25:8). The reaction completion was determined by HPTLC analysis (1 ml of the solution applied; developed with C/M/W (60:35:8)) using primulin and then orcinol staining (Chai et al., 2003). The conjugation yields were greater than 80%. AO-NGLs of disaccharides and trisaccharides were purified by semipreparative HPTLC, and those of tetra to heptasaccharides were purified using silica cartridges (stepwise procedures were described in Liu et al. (2006)). Purified AO-NGLs are dissolved in C/M/W (25:25:8) to give an approximate concentration of 100 pmol/ml for HPTLC and MALDI-MS analyses, quantitation (Liu et al., 2006), and for storage (at  20  C). HPTLC analysis of purified AO-NGLs of disaccharides nigerose and kojibiose, and of dextran oligosaccharide fractions is shown in Fig. 13.3.

5.3. Preparation of AO-NGLs of the glucosylated N-glycans The procedures for preparing AO-NGLs of the three glucosylated N-glycans (Fig. 13.4) are similar to those described above, except that a smaller amount, 10 nmol of each N-glycan, was used for conjugation and 200 nmol AOPE was applied. The incubation time was prolonged to 24 h. Aliquots of the reaction mixtures (1/50) were analyzed by HPTLC (developed with C/M/W (55:45:10)) using primulin and orcinol staining.

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Figure 13.3 HPTLC analysis of purified AO-NGLs of kojibiose (lane 1), nigerose (lane 2), and dextran oligosaccharide fractions di- to heptasaccharide (lanes 3–8). The left panel shows primulin staining, and the right panel orcinol staining. For each NGL, 500 pmol was applied.

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Figure 13.4 Sequences and negative-ion MALDI mass spectra of the three glucosylated high-mannose N-glycan AO-NGLs, Glc1Man9GlcNAc2 (A), Glc3Man7(D1) GlcNAc (B), and Glc2Man7(D1)GlcNAc (C). The [MH] ions observed are in accord with their expected values.

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The AO-NGL products were purified by 1 cc silica cartridges (Liu et al., 2006). The purified NGLs can be analyzed by MALDI-MS. For this, the NGLs are dissolved in chloroform/methanol/water (25:25:8) at a concentration of 10 pmol/ml; 1 ml is deposited on the sample target together with a matrix of 2-(4-hydroxyphenylazo)benzoic acid. Negative-ion MALDI spectra of the AO-NGLs of Glc1Man9GlcNAc2, Glc3Man7(D1)GlcNAc, and Glc2Man7(D1)GlcNAc are shown in Fig. 13.4. It should be noted here that the lack of a GlcNAc residue at the chitobiose core in the AO-NGLs of tri- and diglucosylated oligosaccharides (Fig. 13.4B and C) was from the original oligosaccharide materials. The triglucosylated N-glycan material for NGL preparation was recovered from a NMR sample of Glc3Man7(D1) GlcNAc2 which contained filamentous bacteriophages. The loss of the GlcNAc residue was attributed (Schallus et al., 2008) to residual endoglycosidase activity present in the bacteriophages. As the diglucosylated N-glycan Glc2Man7(D1)GlcNAc was prepared from the triglucosylated analogue by enzymatic digestion, it also lacks the core GlcNAc residue.

6. Carbohydrate Microarray Analysis of Human Malectin The microarray used for studies of human malectin encompassed the AO-NGLs of the glucose oligosaccharide sequences and those from the tri-, di-, and monoglucosyl-high-mannose N-glycans (Fig. 13.5). In addition, the microarray included a diverse range of mammalian-type sequences, all lipid-linked: N-glycans of high-mannose and of neutral and sialylated complex-type; O-glycans, blood group-related sequences (A, B, H, Lewisa, Lewisb, Lewisx, and Lewisy) on linear or branched backbones and their sialylated and/or sulfated analogues, gangliosides, and oligosaccharide fragments of glycosaminoglycans and polysialic acid. Also included were homo-oligomers of other monosaccharides.

6.1. Materials and equipment 1. 16 pad nitrocellulose-coated glass slides (Whatman FAST slides, available from Sigma) 2. Noncontact arrayer (Piezorray, Perkin-Elmer LAS) 3. Cy3 mono NHS ester (Amersham Biosciences) 4. Mouse monoclonal anti-polyhistidine (Ab1) (Sigma) 5. Biotinylated antimouse IgG antibody (Ab2) (Sigma) 6. Saline buffer, for example, HBS (5 mM Hepes, pH 7.4, 150 mM NaCl) 7. Blocking solution: HBS containing 3% (w/v) bovine serum albumin (Sigma) and 5 mM CaCl2 8. Alexa Fluor-647-labeled streptavidin (Molecular Probes)

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Figure 13.5 Microarray analyses of the interactions of malectin using Glc3-, Glc2-, and Glc1-high-mannose N-glycans and glucan oligosaccharide probes in the context of a full microarray containing more than 370 mammalian-type sequences and homo-oligomers of other monosaccharides (inset). The asterisk indicates that the binding signal for the Glc2-N-glycan probe was too high to be accurately quantified, using the imaging conditions selected to highlight the binding of malectin to the glucan oligosaccharide sequences. Abbreviations G3N, G2N, and G1N designate Glc3Man7(D1)GlcNAc, Glc2Man7(D1)GlcNAc, and Glc1Man9GlcNAc2 N-glycan probes, respectively; dp, degree of polymerization of the glucan oligosaccharides. The probes and their sequences were described in the Supplementary Table 6 of the publication describing the xenopus malectin (Schallus et al., 2008).

9. Fast frame multislide plate and silicone incubation chambers (16 wells) (Whatman) 10. Fluorescence microarray slide scanner (ProScanArray) and ScanArrayExpress software (Perkin-Elmer LAS)

6.2. Microarray printing The lipid-linked oligosaccharide probes were robotically printed on 16 pad nitrocellulose-coated glass slides using a noncontact arrayer with a spot delivery volume of approximately 330 pl (Palma et al., 2006); further details

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to be described elsewhere. Each probe was printed in duplicate at two levels, 2 and 5 fmol/spot. The Cy3 dye was included in the probe array solution for quality control of sample delivery while arraying and spot visualization while performing the quantitation analysis.

6.3. Probing the microarrays The microarray analysis with his-tagged malectin was performed with the protein precomplexed with mouse monoclonal anti-polyhistidine (Ab1) and biotinylated antimouse IgG antibodies (Ab2) in a ratio of 1:3:3 (by weight). The malectin–antibody complexes were prepared by preincubating Ab1 with Ab2 for 15 min at ambient temperature, followed by addition of malectin and incubation for a further 15 min. The arrayed slides were prewetted with water and blocked for 1 h with blocking solution, rinsed with HBS, and overlaid for 1.5 h with malectin–antibody complexes diluted in the blocking solution, to give a final malectin concentration of 5 mg/ml. Although calcium is not required for binding, we observed an enhancement of the binding signals elicited in the presence of the cation, especially toward the relative low avidity binders, for example, the glucan oligomers. The precomplexation of malectin with the detection antibodies, in order to increase the valency of the interaction, also resulted in enhancement of the binding signals elicited. Binding was detected using Alexa Fluor-647-labeled streptavidin for 45 min at 1 mg/ml in blocking solution. After each overlay step, slides were rinsed with HBS and an additional rinse with water was performed at the end of the binding experiment. The slides were dried and kept in the dark before scanning. The slides were scanned for Alexa Fluor-647 using a fluorescence microarray slide scanner and the spot fluorescence was quantified after background subtraction using the ScanArrayExpress software. Data analysis after quantitation and presentation was performed with a dedicated software developed by Mark S. Stoll of the Glycosciences Laboratory (Stoll and Feizi, 2009). The microarray results presented in Fig. 13.5 are the means of fluorescence intensities of duplicate spots, printed at 5 fmol. The error bars represent half of the difference between the two values. Human malectin, like its xenopus homologue (Schallus et al., 2008), has the property to bind glucan oligosaccharides, predominantly with a1,3-, a1,4-, and a1,6-linked glucose sequences, and shows an intense and highly selective binding to a diglucosylated high-mannose N-glycan (Fig. 13.5), among a diverse range of mammalian-type sequences that were included in the microarray (highlighted in the inset of Fig. 13.5).

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7. Conclusions In conclusion, we illustrate here the flexibility of NGL-based microarrays for generating oligosaccharide probes from desired sources, polysaccharides, and ER N-glycans in this instance. The preparation of native glucosylated glycans from cells offers a number of advantages for the experimentalist. The use of selective metabolic inhibitors for glucosidase and mannosidase enzymes in the ER (e.g., kifunensine) increases the yield of glycoforms that would otherwise be difficult to isolate in amounts sufficient for analyses of bioactivities in recognition systems involved in glycan processing and recognition. Among the oligosaccharide probe sequences analyzed, the high-selective malectin binding is to a diglucosyl high-mannose N-glycan, which is an intermediate oligosaccharide in the N-glycosylation pathway in the ER. This now opens the way to investigations of the possible sites of action of malectin in the ER. In our investigations of the xenopus and human malectin, until now, we have used the truncated diglucosyl N-glycan with seven mannose residues (Man7). This is because the Glc3Man7(D1) GlcNAc2 is the most abundant analogue which accumulates on the glycoproteins of the drug-treated cells (Petrescu et al., 1997). Work is under way to determine which Glc2-high-mannose analogues, Man9, Man8 (two isoforms), Man7, Man4, give the highest binding signals with malectin. These upcoming studies on the size of the determinant may shed light on where the malectin acts in the N-glycosylation pathway.

ACKNOWLEDGMENTS We gratefully acknowledge contributions of our colleagues in the Glycosciences Laboratory: Maria Campanero-Rhodes, Mark Stoll, Alex Lawson, and Colin Hebert; and Thomas Schallus from the EMBL Heidelberg. The Glycosciences Laboratory acknowledges with gratitude the collaborators with whom our microarray probes were studied over the years. For grant support, we acknowledge the U.K. Medical Research Council, the U.K. Research Councils Basic Technology Grant (GR/S79268, ‘‘Glycoarrays’’), Engineering and Physical Research Councils Translational Grant EP/G037604/1, and the NCI Alliance of Glycobiologists for Detection of Cancer and Cancer Risk (U01 CA128416). A. S. P. is a fellow of the Fundac¸a˜o para a Cieˆncia e Tecnologia (SFRH/BPD/26515/2006, Portugal).

REFERENCES Alonzi, D. S., Neville, D. C., Lachmann, R. H., Dwek, R. A., and Butters, T. D. (2008). Glucosylated free oligosaccharides are biomarkers of endoplasmic-reticulum alphaglucosidase inhibition. Biochem. J. 409, 571–580.

Carbohydrate Microarrays and Ligands for Malectin

285

Alonzi, D. S., Dwek, R. A., and Butters, T. D. (2009). Improved cellular inhibitors for glycoprotein processing alpha-glucosidases: Biological characterisation of alkyl- and arylalkyl-N-substituted deoxynojirimycins. Tetrahedron Asymmetry 20, 897–901. Best, D., Wang, C., Weymouth-Wilson, A. C., Clarkson, R. A., Wilson, F. X., Nash, R. J., Miyauchi, S., Kato, A., and Fleet, G. W. J. (2010). Looking glass inhibitors: Scalable syntheses of DNJ, DMDP, and (3R)-3-hydroxy-l-bulgecinine from D-glucuronolactone and of l-DNJ, l-DMDP, and (3S)-3-hydroxy-d-bulgecinine from l-glucuronolactone. DMDP inhibits b-glucosidases and b-galactosidases whereas l-DMDP is a potent and specific inhibitor of a-glucosidases. Tetrahedron Asymmetry 21, 311–319. Bogomolovas, J., Simon, B., Sattler, M., and Stier, G. (2009). Screening of fusion partners for high yield expression and purification of bioactive viscotoxins. Protein Expr. Purif. 64, 16–23. Butters, T. D., and Neville, D. C. A. (2008). In ‘‘Molecular Biomethods Handbook,’’ (J. M. Walker and R. Rapley, eds.), p. 495. Humana Press, Totowa, NJ. Butters, T. D., Sparks, L. M., Harlos, K., Ikemizu, S., Stuart, D. I., Jones, E. Y., and Davis, S. J. (1999). Effects of N-butyldeoxynojirimycin and the Lec3.2.8.1 mutant phenotype on N-glycan processing in Chinese hamster ovary cells: Application to glycoprotein crystallization. Protein Sci. 8, 1696–1701. Chai, W., Feizi, T., Yuen, C.-T., and Lawson, A. M. (1997). Nonreductive release of O-linked oligosaccharides from mucin glycoproteins for structure/function assignments as neoglycolipids: Application in the detection of novel ligands for E-selectin. Glycobiology 7, 861–872. Chai, W., Stoll, M. S., Galustian, C., Lawson, A. M., and Feizi, T. (2003). Neoglycolipid technology—Deciphering information content of glycome. Methods Enzymol. 362, 160–195. de Belder, A. N. (1993). Dextran. In ‘‘Industrial Gums,’’ (R. L. Whistler and J. BeMiller, eds.), pp. 399–426. Academic Press, New York. de la, C. J., Pintor-Toro, J. A., Benitez, T., and Llobell, A. (1995). Purification and characterization of an endo-beta-1,6-glucanase from Trichoderma harzianum that is related to its mycoparasitism. J. Bacteriol. 177, 1864–1871. Feizi, T., and Childs, R. A. (1994). Neoglycolipids: Probes in structure/function assignments to oligosaccharides. Methods Enzymol. 242, 205–217. Feizi, T., Stoll, M. S., Yuen, C.-T., Chai, W., and Lawson, A. M. (1994). Neoglycolipids: Probes of oligosaccharide structure, antigenicity and function. Methods Enzymol. 230, 484–519. Finn, R. D., Mistry, J., Schuster-Bockler, B., Griffiths-Jones, S., Hollich, V., Lassmann, T., Moxon, S., Marshall, M., Khanna, A., Durbin, R., Eddy, S. R., Sonnhammer, E. L., and Bateman, A. (2006). Pfam: Clans, web tools and services. Nucleic Acids Res. 34, D247–D251. Jacob, G. S., and Scudder, P. (1994). In ‘‘Methods Enzymology,’’ (W. J. Lennarz and G. W. Hart, eds.), p. 280. Academic Press, San Diego. Kaushal, G. P., and Elbein, A. D. (1994). In ‘‘Methods Enzymology,’’ (W. J. Lennarz and G. W. Hart, eds.), p. 316. Academic Press, San Diego. Letunic, I., Copley, R. R., Pils, B., Pinkert, S., Schultz, J., and Bork, P. (2006). SMART 5: Domains in the context of genomes and networks. Nucleic Acids Res. 34, D257–D260. Liu, Y., Chai, W., Childs, R. A., and Feizi, T. (2006). Preparation of neoglycolipids with ring-closed cores via chemoselective oxime-ligation for microarray analysis of carbohydrate–protein interactions. Methods Enzymol. 415C, 326–340. Liu, Y., Feizi, T., Campanero-Rhodes, M. A., Childs, R. A., Zhang, Y., Mulloy, B., Evans, P. G., Osborn, H. M., Otto, D., Crocker, P. R., and Chai, W. (2007). Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharide– protein interactions. Chem. Biol. 14, 847–859.

286

Angelina S. Palma et al.

Mellor, H. R., Adam, A., Platt, F. M., Dwek, R. A., and Butters, T. D. (2000). Highperformance cation-exchange chromatography and pulsed amperometric detection for the separation, detection, and quantitation of N-alkylated imino sugars in biological samples. Anal. Biochem. 284, 136–142. Mellor, H. R., Nolan, J., Pickering, L., Wormald, M. R., Platt, F. M., Dwek, R. A., Fleet, G. W., and Butters, T. D. (2002). Preparation, biochemical characterization and biological properties of radiolabelled N-alkylated deoxynojirimycins. Biochem. J. 366, 225–233. Neville, D. C., Coquard, V., Priestman, D. A., te Vruchte, D. J., Sillence, D. J., Dwek, R. A., Platt, F. M., and Butters, T. D. (2004). Analysis of fluorescently labeled glycosphingolipid-derived oligosaccharides following ceramide glycanase digestion and anthranilic acid labeling. Anal. Biochem. 331, 275–282. Ohta, M., Hamako, J., Yamamoto, S., Hatta, H., Kim, M., Yamamoto, T., Oka, S., Mizuochi, T., and Matsuura, F. (1991). Structures of asparagine-linked oligosaccharides from hen egg-yolk antibody (IgY). Occurrence of unusual glucosylated oligo-mannose type oligosaccharides in a mature glycoprotein. Glycoconj. J. 8, 400–413. Palma, A. S., Feizi, T., Zhang, Y., Stoll, M. S., Lawson, A. M., Diaz-Rodrı´guez, E., Campanero-Rhodes, A. S., Costa, J., Brown, G. D., and Chai, W. (2006). Ligands for the beta-glucan receptor, Dectin-1, assigned using ‘designer’ microarrays of oligosaccharide probes (neoglycolipids) generated from glucan polysaccharides. J. Biol. Chem. 281, 5771–5779. Petrescu, A. J., Butters, T. D., Reinkensmeier, G., Petrescu, S., Platt, F. M., Dwek, R. A., and Wormald, M. R. (1997). The solution NMR structure of glucosylated N-glycans involved in the early stages of glycoprotein biosynthesis and folding. EMBO J. 16, 4302–4310. Schallus, T., Jaeckh, C., Feher, K., Palma, A. S., Liu, Y., Simpson, J. C., Mackeen, M., Stier, G., Gibson, T. J., Feizi, T., Pieler, T., and Muhle-Goll, C. (2008). Malectin—A novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein N-glycosylation. Mol. Biol. Cell 19, 3404–3414. Spiro, R. G. (2004). Role of N-linked polymannose oligosaccharides in targeting glycoproteins for endoplasmic reticulum-associated degradation. Cell. Mol. Life Sci. 61, 1025–1041. Stoll, M. S., and Feizi, T. (2009). Software tools for storing, processing and displaying carbohydrate microarray data. (C. Kettner, ed.). Proceeding of the Beilstein Symposium on Glyco-Bioinformatics, 4–8 October, 2009, Potsdam, Germany, Beilstein Institute for the Advancement of Chemical Sciences, Frankfurt, Germany.