Imaging the Glycome - Report Example

Imaging the glycome A lot of information about bio-molecules such as RNAs, sub-cellular localization of proteins, protein expression patterns, ion concentration and protein-protein interactions can be revealed through molecular imaging while in their original environment and with minimal disturbance to the organism being studied. Proteins can be easily manipulated with genetics to generate auto-fluorescent protein chimeras enabling them most accessible to in vivo visualization. Moreover, imaging can be done on small molecules and ions such as copper, calcium, lead, zinc, hydrogen peroxide, cAMP and mercury by using reengineered fluorescent proteins or designed small molecule fluorophores (Laughlin and Bertozzi 12). In contrast, in vivo imaging of varieties of bio-molecules such as glycans is challenging despite the power of molecular imaging. Inaccessibility of glycans is attributed to their incompatibility with genetically encoded reporters (Laughlin, Baskin and Amacher 664). Glycans play a key role in many biological processes including metastasis in cancer cell, leukocyte homing, and cell-cell interactions involved in embryonic development. The wide range of functions indicates the variety in structure of the glycans. Vertebrate glycans may be secreted, intracellular, or membrane associated. They make up the proteoglycans, glycoplipids and glycoproteins. They are more structurally diversified than linear biopolymers because the building blocks are monossacharides that are connected in both linear and branched geometries. In addition, the cell’s genome, transcriptome, proteome, nutrients and the environment influence the cell glycome, which is the totality of glycans produced in a cell. Generally the physiological state of the cell can be depicted by the glycome whereas changes in the glycome may be associated with disease. Therefore, the understanding of biological systems will be enhanced by the ability to notice changes in the glycome that will lead to provision of new clinical tools for diagnosing disease. Glycans can be imaged using techniques that depend on genetic reporters due to their indirect encoding in the genome. Molecular recognition with probe-bearing lectins or antibodies can be used to monitor antibodies. Lectins are glycan bindin proteins that are used for enrichment and detection of glyconjgates. They are able to recognize varied structures such as fucose, monossacharide sialic acid and higher order structures. The use of lectins in vivo is limited because they require multivalency for high avidity binding since they have a low affinity for their glycan epitope. The other reason is that lectins are tissue permeable and often toxic. Previously, lectins have been used to visualize glycans on tissue sections in mouse, chick and fly embryogenesis. They have also been used in the visualization of rat endothelial vasculature mature mouse thymus, and human kidney. Antibodies also enable limited in vivo visualization of very specific glycan structure. They are also permeable into tissue and most antibodies are generated against low affinity IgM subtype glycan epitopes. Antibodies have been used in glycan visualization as well as chick cornea sections, rabbit appendix and human thymus. Other than limited in vivo applicability, both antibodies and lectins require removal of cells or tissues from their original environment before analysis. In this regard, methods that permit in vivo analysis of continuous changes in the glycome have been developed. In imaging glycans with biorthogonal chemical receptors, a two step approach is used. First, metabolic labeling with an unnatural monossacharide substrate is used to chemically incorporate reactive moiety into target glycans. The second step the reporter group is visualized by covalent reaction with imaging probe. The reporter must be minute to avoid being digested by the metabolic enzymes of the cells. It should also be inert to the endogenous chemical functionality of the cell. This chemical reporter is of ketone group and can be detected by oxime or hydrazone with formation with aminooxy or hydrazine fuctionalised probes. They limit in vivo since these covalents reactions perform best at reduced pH. This can be alleviated by use of azides and alkynes as biorthorgonal functional groups at physiological pH. Azides and alkynes are minute, inert and capable of reacting with biorthogonal functional groups at physiological pH. Metabolic labeling of glycans with azido sugars enables visualization of glycans and enriches specific glycoproteins types for proteomic analysis. Metabolic labeling with azido or alkynyl monosaccharide as chemical reporters has been used to image glycan subtypes except glycosainoglycans and glycosylphosphatidylinositol. The first glycan subtypes, were glycoconjugates bearing the terminal monossacharide N-acetylneuraminic acid. N-acetylneuraminic acid is a member of sialic acid family. It is a determinant of leukocyte endothelial cell adhesion, determinant of viral infection, immune cell activation, neuronal development and cancer metastasis. Metabolic labeling with analogs of its biosynthetic precursor N-acetylmannosamine enables visualization sialic acid containing glycans. Chemical reporters could be added to the N-acyl group with AC4ManNAz and alkynyl ManNAc which have been used to visualize sialic acids in living ice and human cancer cell lines respectively. Chemical reporter strategy has been used to study mucin type O structures that share a common core N-acetylgalactosamine residue that links the glycan to serine or treonine residues within the underlying protein. Fluorophore-conjugated phosphines and alkynes enable imaging of glyans that have been labeled by azide and alkyne on cultured cells. Staudinger litigation with fluorescein-, rhodamine and Cy5.5-conjugated phosphine reagents was used to visualize cell surface SiaNAz residues on live cells. Cy5.5-conjugated phosphine reagent was most sensitive due to its low background fluorescence and non-specific cell binding. The fluorescence background causes poor clearance of unreacted fluorophore. This can be minimized using smart probes which only fluorescent only after reaction with the target. Smart probes have been made based on coumarin scaffold and ManNAc labeled glycans fixed on cells using CuAAC reaction. Figure 1: Staudinger ligation activates fluorogenic phosphine dyes with azides In living organisms, the constituents of glycome must be visualized in living organisms to capture glycans as they perform their various functions. The reagents involved are both bioorthogonal and do not cause toxicity therefore strain promoted reaction and Staudinger litigation of azides and cyclooctynes are applied to in vivo imaging. Staudinger litigation has been used in imaging live mice. However, the method has slow kinetic reactions making it difficult to study biological processes in real time. The reaction of azides with cyclooctynes has improves kinetics over physiological conditions. DIFO, a cyclooctyne reagent that has a gem difluoro group adjacent to alkyne reacts faster with azide than Staudinger reactions or nonfluorinated cyclooctynes. The cycloaddition of azides with DIFO reagents has enabled visualization of glycans in developing zebrafish. Li, Mock and Wu (2012) describes the introduction of glycans bearing LacNAc disaccharides andbioorthogonal chemical reporters onto the cell surface fucosylated glycans. The tagged glycans can be conjugated to imaging probes. These approcahes can also be used to image gycans in living systems. In vivo molecular imaging techniques target glycans, due to bioorthogonal chemical reporter approach that has been successfully implemented to image glycans in living organisms such as zebra fish. This has enhanced probing the dynamic glycome with applications in other organisms such as Drosophila and to mammalian disease models and human clinical settings. Reagents that metabolically stable and pharmacokinetic properties as well as selectivity and kinetics need to designed. The fraction of the glycome that is revealed by a chemical reporter is labels only a fraction of the glycome therefore multiple sugars are required to make complete coverage. Additional chemical reporters are required to distinguish multiple sugars without use of azides and alkynes. In summary, metabolic labeling with chemical reporters and litigation to fluorescent probes has enabled in vivo visualization of glycans, a possibility that seemed challenging (Laughlin and Bertozzi 17). Works Cited Laughlin, S T and C R Bertozzi. "Metabolic labeling of glycans with azido sugars and subsequent glycan-profiling and visualization via Staudinger litigation." Nature Protocols 2.11 (2007): 2930-44. Laughlin, S T, et al. "In vivo imaging of membrane associated glycans in developing zebrafish." Science 320.5876 (2008): 664-667. Laughlin, Scott T and R Carolyn Bertozzi. "Imaging the glycome." PNAS 106.1 (2009): 12-17. Li, B, F Mock and P Wu. "Imaging the glycome in living systems." Methods Enzymology 505 (2012): 401-419. Read More