To date, several carbohydrate microarray platforms have been developed that: 1) use alternative chemical strategies to overcome the limitation of direct immobilization of oligosaccharides onto solid matrices; 2) differ on the type of carbohydrates and how they are displayed on the array surface; and 3) are based on covalent or non-covalent immobilization to different surfaces. These are reviewed in detail in recent references73,75,76,78–81. Here, some high-throughput platforms that
contain a high diversity of oligosaccharide probes and that use different strategies for their immobilization and presentation will be highlighted.
Feizi and colleagues have developed a microarray system based on the neoglycolipid (NGL) technology82, in which the oligosaccharides are linked to a lipid67,71,72,78,83,84. A summary of the
key steps involved in the construction of the NGL-based microarrays and their analysis to reveal carbohydrate-binding patters is depicted in Figure 1.5. Central to this microarray system is the microscale lipid conjugation of oligosaccharides (natural or chemically synthesized sequence-defined or as mixtures) to an aminophospholipid to produce NGLs. The generated NGL
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Figure 1.5. Schematic overview of the main steps comprising the analysis using neoglycolipid (NGL)-based carbohydrate microarrays. The key steps of this methodology are depicted
in three stages: 1) Construction of the NGL-based carbohydrate microarrays: the probes are all lipid-linked and comprise both NGLs prepared from natural or chemically synthesized oligosaccharides and glycolipids, natural or synthetic; the interface with mass spectrometry (MS) and HPTLC or HPLC, enables purification and characterization of oligosaccharides or NGL mixtures; the NGLs can also be prepared from oligosaccharide mixtures derived from ligand-bearing glycomes, to reveal and characterise the oligosaccharide ligands they harbour (‘designer’ microarray methodology); the NGL and glycolipid probes are robotically dispensed onto nitrocellulose-coated glass slides using a liposome formulation in the presence of carrier lipids. 2) Probing and Fluorescence imaging: Cyanine 3 fluorophore is included in the NGLs liposome formulation, so that the immobilized probes can be visualised by fluorescence imaging at 532 nm; the microarrays can then be probed for carbohydrate-binding by monoclonal antibodies, CBMs, lectins and viruses or other pathogens; the binding signals are revealed by scanning for Alexa-fluor647 emission at 647 nm. 3) Microarray data analysis and display: the fluorescence intensities are quantified and analysed to reveal the carbohydrate binding patterns using a dedicated software, which includes a database that holds all of the microarray data and metadata on experimental conditions and information on probes and proteins; interactive tools are then used for semi-automatic presentation of microarray data by filtering, sorting and deep mining every data point. Figure adapted from Palma et al. 201478 and Palma et al. 201532.
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probes have amphipathic properties, which enables efficient display onto nitrocellulose-coated glass slides using a liposome formulation in the presence of carrier lipids85 (Figures 1.5 and 1.6A).
The reducing oligosaccharides can be conjugated through reductive amination to the aminolipid 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE, DH-NGLs) (Figure 1.6C). This procedure yields the ring-opening of the monosaccharide at the reducing end74. To overcome this
limitation, NGLs with ring-closed monosaccharide cores have been introduced by Liu and
colleagues86. These are prepared by conjugating reducing oligosaccharides to an
aminooxy-functionalized DHPE by oxime ligation (without reduction) (AOPE, AO-NGLs) (Figure 1.6C). This procedure enables the efficient presentation of short oligosaccharides for direct binding assays86. The non-covalent immobilization of NGLs in a lipid environment onto a
nitrocellulose surface introduces an element of mobility. This mode of presentation simulates to some extent the cell surface display of glycans and may be advantageous for detection of binding for particular recognition systems78. The NGL-based microarray system currently contains a
repertoire of around 900 sequence-defined probes, with a high content of natural oligosaccharide sequences, including NGLs derived from various oligosaccharides of mammalian sources, from polysaccharides of bacterial, fungal, and plant origins, and natural and synthetic glycolipids78
(accessed through the link https://glycosciences.med.ic.ac.uk/glycanLibraryIndex.html).
The microarray platform of the Consortium for Functional Glycomics (CFG) developed by the early work of Blixt and colleagues70,87 is also based upon amine chemistry, whereby
oligosaccharides linked at the reducing end with an amine-terminating linker are covalently immobilized onto N-hydroxysuccinimide (NHS) ester-derivatized glass slides (Figure 1.6B). Recent microarray versions are composed of around 600 mammalian-type probes (mammalian printed array version 5.2). Other strategies that also use an amino-linker involve immobilization of the amine-terminated oligosaccharides onto epoxide-derivatized slides (Figure 1.6B). Examples are by Cummings and colleagues that used this method for immobilization of naturally-derived oligosaccharide libraries88 and by Varki and colleagues who developed a
structurally diverse microarray of sialylated oligosaccharides89. Gildersleeve and colleagues
demonstrated that oligosaccharides conjugated to bovine serum albumin (BSA) or human serum albumin (HSA) (displayed as neoglycoproteins) may also be efficiently immobilized using amine chemistry onto epoxide functionalized glass slides for binding studies90. Other groups, have
developed covalent microarrays based on different chemistries, such as the early work by Shin and colleagues using the thiol chemistry, whereby maleimide-functionalized oligosaccharides are immobilized onto thiol-derivatized slides91. In these covalent oligosaccharide microarray
platforms, the nature and length of the linkers between the oligosaccharide and the array surface are important for accessibility of the oligosaccharide to the protein and detection of specific binding.
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Figure 1.6. Graphic representation of examples of immobilization strategies used to generate carbohydrate microarrays. (A) Non-covalent microarrays: immobilization onto nitrocellulose-coated glass
slides of reducing oligosaccharides derivatized by reductive amination to an aminophosholipid, to prepare neoglycolipids (NGLs)78, or to BSA, to prepare neoglycoproteins92. (B) Covalent microarrays: immobilization of synthetic oligosaccharides derivatized at the reducing end to an amino-terminating linker onto
N-hydroxysuccinimide (NHS)-functionalized glass slides70 or onto epoxide-functionalized glass slides89.
(C) NGL probes prepared from reducing oligosaccharides by reductive amination (DHPE, DH-NGLs)93 and by oxime ligation (AOPE, AO-NGLs)86; the derivatization of the oligosaccharide by oxime ligation produces an equilibrium between the open- and closed-ring form of the reducing monosaccharide86. Examples of carbohydrate structures in the different libraries are shown using the symbol nomenclature for glycans (SNFG) according to Varki et al., 201594.
The NGL-based microarray facility and that of the Consortium of Functional Glycomics (accessed through links https://www.imperial.ac.uk/glycosciences/ and http://www.functionalglycomics.org/, respectively) are the two largest platforms assembled to date that are open to the broad scientific community for microarray screening analyses of carbohydrate-binding proteins in different biological contexts.
Although the number of sequence-defined probes in carbohydrate microarrays has been expanding, the increase in diversity to date has been mainly on mammalian-type sequences. Some groups, however, have focused on development of microarrays from microbial32,95,96 or
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natural polysaccharides and the development of methods for fine-tuned depolymerisation, purification, high-sensitive sequencing and structural characterisation of the oligosaccharide fragments are crucial32,92. Methods for chemical103–105 or chemo-enzymatic synthesis106 of
structural elements from complex microbial or plant cell wall polysaccharides offer powerful complementary approaches to develop sequence-defined microarrays. Recently, Seeberger et al. combining different carbohydrate synthesis approaches including automated glycan assembly, solution-phase synthesis and chemoenzymatic methods, successfully obtained a library of over 300 structures of different microbial oligosaccharides, which were used to develop the most diverse microbe-focused carbohydrate microarray platform to date95. The genetic
engineering of bacterial strains with specific CAZymes gene deletions to produce oligosaccharides in the presence of a target substrate107, offer an alternative approach to achieve
the much needed structural diversity.
In the context of the work developed in this thesis, some selected examples of microarray approaches to study plant-carbohydrate recognition by CBMs will be highlighted in the sections below.