Niels-Christian Reichardt

Construction of N‐Glycan Microarrays by Using Modular Synthesis and On‐Chip Nanoscale Enzymatic Glycosylation

An effective chemoenzymatic strategy is reported that has allowed the construction, for the first time, of a focused microarray of synthetic N-glycans. Based on modular approaches, a variety of N-glycan core structures have been chemically synthesized and covalently immobilized on a glass surface. The printed structures were then enzymatically diversified by the action of three different glycosyltransferases in nanodroplets placed on top of individual spots of the microarray by a printing robot. Conversion was followed by lectin binding specific for the terminal sugars. This enzymatic extension of surface-bound ligands in nanodroplets reduces the amount of precious glycosyltransferases needed by seven orders of magnitude relative to reactions carried out in the solution phase. Moreover, only those ligands that have been shown to be substrates to a specific glycosyltransferase can be individually chosen for elongation on the array. The methodology described here, combining focused modular synthesis and nanoscale on-chip enzymatic elongation, could open the way for the much needed rapid construction of large synthetic glycan arrays.

Fucosyltransferases as synthetic tools: glycan array based substrate selection and core fucosylation of synthetic N-glycans.

Two recombinant fucosyltransferases were employed as synthetic tools in the chemoenzymatic synthesis of core fucosylated N-glycan structures. Enzyme substrates were rapidly identified by incubating a microarray of synthetic N-glycans with the transferases and detecting the presence of core fucose with four lectins and one antibody. Selected substrates were then enzymatically fucosylated in solution on a preparative scale and characterized by NMR and MS. With this approach the chemoenzymatic synthesis of a series of α1,3-, α1,6-, and difucosylated structures was accomplished in very short time and with high yields, which otherwise would have required extensive additional synthetic effort and a complete redesign of existing synthetic routes. In addition, valuable information was gathered regarding the specificities of the lectins employed in this study.

MALDI-TOF Mass Spectrometric Analysis of Enzyme Activity and Lectin Trapping on an Array of N-Glycans†

Glycan microarrays are an established platform for the highthroughput screening of substrate specificities of carbohydrate-binding proteins and processing enzymes. The most common detection method, which is based on fluorescently tagged lectins, can only give a measure of binding specificity, as quantification is often compromised by the specific lectin affinity. Therefore, a label-free technique that could overcome these analytical problems is needed for the analysis of array spot compositions. MALDI-TOF-based assays on surface-bound carbohydrates have been reported. Mrksich and co-workers have shown that this technique can be used to study enzyme activity or to trap affinity ligands on biofunctionalized selfassembled monolayers (SAMs) of oligoethylene glycols on gold surfaces. Unfortunately, the covalent attachment of glycans to the monolayer requires high ligand concentrations that are not suitable when working with complex oligosaccharides. More recently, Wong, Siuzdak, and co-workers studied 2,3-sialyltransferase and b-galactosidase activity on fluorous-tagged lactose immobilized on a perfluorinated surface. The difficult preparation of the nanostructured surface and the multistep tagging procedure, however, render this approach less appealing for routine and high-throughput use in on-chip mass spectrometric analysis of enzyme activity. Inspired by the flexible and mobile organization of glycolipids in lipid bilayers, we present herein a novel strategy for the surface-based MALDI-TOF analysis of glycan arrays that is conceptually a fusion of the approaches of Wong, Siuzdak, Mrksich, and their respective co-workers. Glycans are functionalized with a lipid tag (Scheme 1) and noncovalently immobilized on the MALDI plate by insertion into a self-assembled alkylthiolate monolayer. This setup was tested in a series of glycomics applications. The facile preparation of both a hydrophobic MALDI plate and tagged ligands, as well as the general applicability for large oligosaccharides make this method stand out from other surface-based MALDI-TOF approaches. Hydrophobic surfaces have been applied to MALDI-TOF-based proteomics for sample desalting, but, to the best of our knowledge, these surfaces have not been applied to the oriented immobilization of lipid-tagged biomolecules for surface MALDI-TOF analysis. We chose a commercially available gold-coated MALDI sample plate as surface to prepare a hydrophobic selfassembling monolayer of 1-undecanethiol by following a published procedure. Analysis of the monolayer under standard MALDI-TOF conditions showed only peaks corresponding to known matrix ions, while no disulfide or thiolate ions were detected (Figure 1). The hydrophobically tagged carbohydrate ligands 1–10 used in this study were prepared from synthetic N-glycan structures (2–8) by conjugation with stearic acid or from commercial reducing sugars by reductive amination (1, 9, and 10). Glycan arrays (see the Supporting Information) were made by spotting the conjugates 2–10 onto individual wells of the hydrophobic sample plate, drying, and rinsing the plates with water to remove unbound material. MALDI-TOF analysis using 2,4,6trihydroxy-acetyophenone (THAP) as matrix showed strong ion signal intensities with signal-to-noise values of typically 100 or higher for all compounds; these values are comparable to those reported by Siuzdak and co-workers. Ions were detected as sodium adducts with a detection limit of around 5 picomol. The maximum surface capacity for glycan immobilization was determined by deposition of increasing amounts of conjugate 2, washing, and measurement of the signal for 2 normalized to an internal standard. Surface saturation was reached after deposition of around 2 nmol of conjugate 2 per well, which translates to a surface concentration of 0.2 mmolmm . To avoid the unnecessary waste of valuable analytes, glycans were spotted at half the saturation concentration without compromising signal intensity. The stability of the immobilized glycans to repeated washing with aqueous buffers, water, or organic solvents was then determined. Tagged glycans typically resisted 3–5 wash cycles of 1 minute duration without significant reduction of signal intensity, and the intensity of the model glycan 2 was essentially unchanged after 60 seconds of continuous sonication (Figure 1d). However, the glycans were completely removed from the surface [*] Dr. A. Sanchez-Ruiz, Dr. S. Serna, Dr. N. Ruiz, Prof. Dr. M. Martin-Lomas, Dr. N.-C. Reichardt Biofunctional Nanomaterials Unit, CICbiomaGUNE Paseo Miramon 182, 20009 San Sebastian (Spain) Fax: (+ 34)943-005-314 E-mail: nreichardt@cicbiomagune.es

Microarray-based identification of lectins for the purification of human urinary extracellular vesicles directly from urine samples.

As cellular‐derived vesicles largely maintain the biomolecule composition of their original tissue, exosomes, which are found in nearly all body fluids, have enormous potential as clinical disease markers. A major bottleneck in the development of exosome‐based diagnostic assays is the challenging purification of these vesicles; this requires time‐consuming and instrument‐based procedures. We employed lectin arrays to identify potential lectins as probes for affinity‐based isolation of exosomes from the urinary matrix. We found three lectins that showed specific interactions to vesicles and no (or only residual) interaction with matrix proteins. Based on these findings a bead‐based method for lectin‐based isolation of exosomes from urine was developed as a sample preparation step for exosome‐based biomarker research.

Lectin-array blotting: profiling protein glycosylation in complex mixtures.

By combining electrophoretic protein separation with lectin-array-based glycan profiling into a single experiment, we have developed a high-throughput method for the rapid analysis of protein glycosylation in biofluids. Fluorescently tagged proteins are separated by SDS-PAGE and transferred by diffusion to a microscope slide covered with multiple copies of 20 different lectins, where they are trapped by specific carbohydrate protein interactions while retaining their relative locations on the gel. A fluorescence scan of the slide then provides an affinity profile with each of the 20 lectins containing a wealth of structural information regarding the present glycans. The affinity of the employed lectins toward N-glycans was verified on a glycan array of 76 structures. While current lectin-based methods for glycan analysis provide only a picture of the bulk glycosylation in complex protein mixtures or are focused on a few specific known biomarkers, our array-based glycoproteomics method can be used as a biomarker discovery tool for the qualitative exploration of protein glycosylation in an unbiased fashion.

Nanostructured indium tin oxide slides for small-molecule profiling and imaging mass spectrometry of metabolites by surface-assisted laser desorption ionization MS.

Due to their electrical conductivity and optical transparency, slides coated with a thin layer of indium tin oxide (ITO) are the standard substrate for protein imaging mass spectrometry on tissue samples by MALDI-TOF MS. We have now studied the rf magnetron sputtering deposition parameters to prepare ITO thin films on glass substrates with the required nanometric surface structure for their use in the matrix-free imaging of metabolites and small-molecule drugs, without affecting the transparency required for classical histology. The custom-made surfaces were characterized by atomic force microscopy, scanning electron microscopy, ellipsometry, UV, and laser desorption ionization MS (LDI-MS) and employed for the LDI-MS-based analysis of glycans and druglike molecules, the quantification of lactose in milk by isotopic dilution, and metabolite imaging on mouse brain tissue samples.

Synthesis and microarray-assisted binding studies of core xylose and fucose containing N-glycans.

The synthesis of a collection of 33 xylosylated and core-fucosylated N-glycans found only in nonmammalian organisms such as plants and parasitic helminths has been achieved by employing a highly convergent chemo-enzymatic approach. The influence of these core modifications on the interaction with plant lectins, with the human lectin DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Nonintegrin), and with serum antibodies from schistosome-infected individuals was studied. Core xylosylation markedly reduced or completely abolished binding to several mannose-binding plant lectins and to DC-SIGN, a C-type lectin receptor present on antigen presenting cells. Employing the synthetic collection of core-fucosylated and core-xylosylated N-glycans in the context of a larger glycan array including structures lacking these core modifications, we were able to dissect core xylose and core fucose specific antiglycan antibody responses in S. mansoni infection sera, and we observed clear and immunologically relevant differences between children and adult groups infected with this parasite. The work presented here suggests that, quite similar to bisecting N-acetylglucosamine, core xylose distorts the conformation of the unsubstituted glycan, with important implications for the immunogenicity and protein binding properties of complex N-glycans.

Toward the solid-phase synthesis of heparan sulfate oligosaccharides: evaluation of iduronic acid and idose building blocks.

Glycan arrays have been established as the premier technical platform for assessing the specificity of carbohydrate binding proteins, an important step in functional glycomics research. Access to large libraries of well-characterized oligosaccharides remains a major bottleneck of glycan array research, and this is particularly true for glycosaminoglycans (GAGs), a class of linear sulfated polysaccharides which are present on most animal cells. Solid-supported synthesis is a potentially powerful tool for the accelerated synthesis of relevant GAG libraries with variations in glycan sequence and sulfation pattern. We have evaluated a series of iduronic acid and idose donors, including a couple of novel n-pentenyl orthoester donors in the sequential assembly of heparan sulfate precursors from monosaccharide building blocks in solution and on a polystyrene resin. The systematic study of donor and acceptor performance up to the trisaccharide stage in solution and on the solid support have resulted in a general strategy for the solid-phase assembly of this important class of glycans.

Chemo-Enzymatic Synthesis of (13)C Labeled Complex N-Glycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry.

Methods for the absolute quantification of glycans are needed in glycoproteomics, during development and production of biopharmaceuticals and for the clinical analysis of glycan disease markers. Here we present a strategy for the chemo-enzymatic synthesis of (13)C labeled N-glycan libraries and provide an example for their use as internal standards in the profiling and absolute quantification of mAb glycans by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. A synthetic biantennary glycan precursor was (13)C-labeled on all four amino sugar residues and enzymatically derivatized to produce a library of 15 glycan isotopologues with a mass increment of 8 Da over the natural products. Asymmetrically elongated glycans were accessible by performing enzymatic reactions on partially protected UV-absorbing intermediates, subsequent fractionation by preparative HPLC, and final hydrogenation. Using a preformulated mixture of eight internal standards, we quantified the glycans in a monoclonal therapeutic antibody with excellent precision and speed.

Array-assisted Characterization of a Fucosyltransferase Required for the Biosynthesis of Complex Core Modifications of Nematode N-Glycans

Background: The chitobiose region of nematode N-glycans can be modified with three fucose residues. Results: Glycan arrays and other analytical techniques facilitated the definition of the biologically relevant activity of Caenorhabditis FUT-6. Conclusion: The concerted action of Caenorhabditis FUT-1, FUT-6, and FUT-8 is required for trifucosylation of worm N-glycan cores. Significance: New approaches for studying glycans from parasitic nematodes are now possible. Fucose is a common monosaccharide component of cell surfaces and is involved in many biological recognition events. Therefore, definition and exploitation of the specificity of the enzymes (fucosyltransferases) involved in fucosylation is a recurrent theme in modern glycosciences. Despite various studies, the specificities of many fucosyltransferases are still unknown, so new approaches are required to study these. The model nematode Caenorhabditis elegans expresses a wide range of fucosylated glycans, including N-linked oligosaccharides with unusual complex core modifications. Up to three fucose residues can be present on the standard N,N′-diacetylchitobiose unit of these N-glycans, but only the fucosyltransferases responsible for transfer of two of these (the core α1,3-fucosyltransferase FUT-1 and the core α1,6-fucosyltransferase FUT-8) were previously characterized. By use of a glycan library in both array and solution formats, we were able to reveal that FUT-6, another C. elegans α1,3-fucosyltransferase, modifies nematode glycan cores, specifically the distal N-acetylglucosamine residue; this result is in accordance with glycomic analysis of fut-6 mutant worms. This core-modifying activity of FUT-6 in vitro and in vivo is in addition to its previously determined ability to synthesize Lewis X epitopes in vitro. A larger scale synthesis of a nematode N-glycan core in vitro using all three fucosyltransferases was performed, and the nature of the glycosidic linkages was determined by NMR. FUT-6 is probably the first eukaryotic glycosyltransferase whose specificity has been redefined with the aid of glycan microarrays and so is a paradigm for the study of other unusual glycosidic linkages in model and parasitic organisms.