#sse2013

[sugar-complexity]

How Cell Surface Sugars Can Help Treat Disease and Injury

Heparan sulphate is a complex long chain sugar (a polysaccharide) strategically positioned at the cell surface (Pic 1 – top left).  Heparan sulphate is critical for regulation of the growth and differentiation of stem cells by protein growth factors. It binds selectively to proteins such as growth factors and enzymes and controls their activities via a code of specific structures (Pic 2 – top right). It often acts as a “molecular matchmaker” that influences the assembly of multiple protein complexes. These mechanisms of action are central to a wide range of normal biological processes, and are relevant to many diseases and medical conditions.  Recent studies have provided new insights into this area of biology, and have real potential for translation into novel treatments for diseases including neurodegeneration, cancer, inflammation, infections, and also for tissue repair.  Chemical methods for synthesis of these complex sugars have recently been developed, including compounds found to be potent inhibitors of an enzyme central to causing Alzheimers disease (AD)1.  These compounds could be developed to treat the underlying cause of AD for the first time.  In spinal cord injury, such compounds have the potential to reduce scarring reactions which prevent nerve regrowth (Pic 3 – middle left), and improve the efficacy of cell transplantation therapies designed to aid nerve repair2.   Mechanisms for natural repair of muscle injury depend on stem cells present in muscle tissue (Pic 4), and proper activation of these cells depends on heparan sulphate3.  This has the exciting potential for development of drug-based therapies to enhance muscle repair, and for treatment of muscular dystrophies. 

Pic 1 – top left - Mouse embryonic stem cells stained for cell surface heparan sulphate (green), with additional staining for a marker of stem cells, (red) and nuclei (blue).

Pic 2: top right - Heparan sulphate – a long chain sugar with specific protein binding sites that controls protein functions and regulates biological processes relevant to many disease processes.

Pic 3: middle left - Schwann cells, a supporting nerve cell type, can be transplanted at sites of spinal cord injury to enhance repair, but this can cause scarring reactions which prevent nerve regrowth. However, as this image shows, Schwann cells (green) and astrocytes (red), which normally form a distinct boundary between each other in the scarring response, can be induced to mingle freely after treatment with a low sulphated modified heparin. Such compounds have exciting potential for enhancing repair of spinal cord injuries.

Pic 4: middle right - Muscle stem cells are tiny cells normally resident in a dormant state attached to muscle fibres. In response to injury, these stem cells become activated, divide and then repair damaged muscle fibres. In this fluorescent picture the red staining identifies Syndecan-4, a protein with heparin sulphate side chains,expressed on the surface of muscle stem cells (indicated here by an arrow). The two muscle stem cells arrowed are the result of a recent cell division and are activated (as shown by green staining indicating high levels of phospho-tyrosine (note: the sum of red and green appears orange in fluorescence microscopy).

Pic 5: bottom - Muscle stem cells can be isolated from muscle tissue and grown in a petri dish. In this picture individual muscle stem cells are identified by the blue staining in the nucleus; they have proliferated and express on their surface Syndecan-4(stained in green), a protein with heparan sulphate side chains. Cultured muscle stem cells can also differentiate in a petri dish and fuse one to another to form large cells called myotubes, which eventually become mature muscle fibres. The nuclei in myotubes appear only blue, since they do not express Pax7 (stained in red, so that these nuclei become purple).  Pax7 is only expressed by the non-differentiated progenitor cells, but still express Syndecan-4, which is now localized to special adhesion structures on the surface of myotube membranes called focal adhesions. In non-differentiated muscle stem cells Syndecan-4 controls and modulates growth factor signals through its heparin sulphate sugar chains, as well as mediating cell adhesion to the extracellular matrix.

Authors: Professor Jerry Turnbull and Dr Dada Pisconti, Liverpool University

References:

1.    Schwörer, R; et al., Synthesis of a Targeted Library of Heparan Sulfate Saccharides as Inhibitors of b-Secretase: Potential Therapeutics for Alzheimer’s disease. Chemistry European Journal. 2013, DOI: 10.1002/chem.201204519

2.    Higginson, J R; et al., Differential Sulfation Remodelling of Heparan Sulfate by Extracellular 6-O-sulfatases Regulates Fibroblast Growth Factor-induced Boundary Formation By Glial Cells: Implications for Glial Cell Transplantation, Journal of Neuroscience, 2012, 32, 15902-15912. DOI:10.1523/JNEUROSCI.6340-11.2012

3.  Pisconti A., et al., Syndecan-3 and Notch cooperate in regulating adult myogenesis.  Journal of Cell Biology, 2010, 190(3): 427-41. DOI:10.1083/jcb.201003081

[sugar-complexity]

Glycotherapeutics - How Sugars Can Help Us

Sugars are central to biology, controlling many vital processes relevant to health and a broad range of diseases. This provides many targets for therapeutic interventions. Existing sugar-based therapeutics – “glycotherapeutics” - cover a wide range of applications. On cell surfaces, they are attachment points for many disease pathogens, which has led to the development of drugs like Relenza (a neuraminidase enzyme inhibitor to treat influenza1) and anti-bacterial vaccines such as Haemophilus influenzae type B (Hib vaccine).  Sugars interact with and control many proteins such as enzymes: the understanding of these mechanisms has resulted in the development of effective drugs such as heparin and low molecular weight heparins  (for reducing blood clotting after surgery and for deep vein thrombosis - DVT); acarbose (for diabetes); and hyaluranon polysaccharide (for eye surgery, arthritis and wound healing). Sugars linked to the surface of proteins are also critical for their activity and stability, and this is a vital aspect for quality control of proteins made synthetically by recombinant technologies; these include therapeutic antibodies for cancer and autoimmune diseases, and protein drugs such as erythropoietin (EPO) for anaemia (see picture of EPO showing sites of sugar attachment on the surface). New insights into the biology of sugars are constantly providing new targets, so there is huge potential for new sugar-based drugs for diverse diseases.  For example, advanced synthetic chemistry can now produce specific heparin sugars with potential to treat Alzheimers disease2, spinal cord injury3 and cancer, and might be used in the future to enhance muscle repair4, and control stem cells for tissue repair.  

Author: Professor Jerry Turnbull, University of Liverpool

References:

1  von Itzstein M; et al. Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature, 1993, 363 (6428): 418–23.

2 Schwörer, Ralf; et al., Synthesis of a Targeted Library of Heparan Sulfate Saccharides as Inhibitors of b-Secretase: Potential Therapeutics for Alzheimer’s Disease. Chemistry European Journal, 2013,DOI: 10.1002/chem.201204519

3 Higginson, Jennifer R; et al., Differential Sulfation Remodelling of Heparan Sulfate by Extracellular 6-O-sulfatases Regulates Fibroblast Growth Factor-induced Boundary Formation By Glial Cells: Implications for Glial Cell Transplantation.  Journal of Neuroscience,2012,32, 15902-15912. DOI:10.1523/JNEUROSCI.6340-11.2012

4  Pisconti Addolorata; et al., Syndecan-3 and Notch cooperate in regulating adult myogenesis.  J Cell Biol, 2010, 190(3): 427-41. DOI:10.1083/jcb.201003081

[sugar-complexity]

Tools for studying cell surface sugars

Cells are coated with a sugar-rich layer called the glycocalyx.  Understanding its interaction with other molecules will provide a wide range of opportunities for the development of new foods, medicines and healthcare treatments.  As the glycocalyx is very dynamic and changes between individuals or during some diseases, it provides a method for producing advanced diagnostic tools for personalised medicines.

It is very difficult to study the sugars of cell surfaces because cells are very small and fragile. We therefore have built new tools that allow us to measure communication events on cell surfaces, and one of these tools are glycoarrays.  We use a gold plate because it is conducting and we can connect it up to many analytical instruments used in diagnostics in the clinic. We have developed methods to stick the sugars of the glycocalyx onto these gold surfaces so that is looks like a cell surface and then measure how diverse biomolecules react with these sugars.  In the future, this high-throughput technology could be used for the discovery of sugar biomarkers in diseases such as cancer.  It will also help us prevent and treat microbial infections, if we know how different microbes such as viruses and bacteria bind to the cell surface sugars.  This strategy has recently been shown to be successful in new anti-flu therapies.

Author: Professor Sabine Flitsch, The University of Manchester

References

1.       Šardzík, Robert; et al., Chemoenzymatic synthesis of O-mannosylpeptides in solution and on solid phase. J Am Chem Soc, 2012, 134, 4521-4524. DOI:10.1021/ja211861m

2.       Noble, Gavin; et al. Accelerated Enzymatic Galactosylation of N-Acetylglucosaminolipids in Lipid Microdomains, J. Am. Chem. Soc., 2012, 134, 31, 13010–13017. DOI:10.1021/ja302506t

3.       Rannes, Julie; et al. Glycoprotein labelling using engineered variants of galactose oxidase obtained by directed evolution, J. Am. Chem. Soc., 2011, 133(22), 8436-9. DOI:10.1021/ja2018477

4.       Liu Y; et al.,Receptor specificity and cell tropism of D222G hemagglutinin mutants isolated from fatal cases of pandemic A(H1N1) 2009 influenza virus. J Virol. 2010, 84, 12069-12074. DOI:10.1128/JVI.01639-10.

[sugar-complexity]

Plant sugars - the next generation of renewable energy

Plant cell walls are important for a whole range of industrially important processes, from food processing to paper production. Recently they have received considerable attention as a potential sustainable source of CO2 neutral biofuels, however, it also possesses particular problems when trying to utilise the constituent components efficiently. Consequently, we are interested both in the fundamental research, but also how we might apply this research to generate an applied output.

Our focus has been on the woody, secondary cell wall since this constitutes the majority of plant Biomass. The secondary cell wall is composed predominantly of three polymers: cellulose, xylan and lignin. While cellulose and xylan may are potential sources of sugars for biofuels production lignin must be removed and utilized separately. While it is possible to identify a number of targets that could improve cell wall quality, such as less cross-linking of carbohydrates to phenolics and greater cellulose content our lack of basic understanding of how cell wall polymers are made, what controls cell wall quantity and composition and what physical properties different polymers confer upon the wall limit our ability to make these beneficial changes.

We identified a series of mutants that were deficient in deposition of cellulose, xylan and lignin. These have allowed us to identify several of the proteins required for cellulose and xylan biosynthesis that had previously not been possible to do using biochemical approaches. One of these xylan mutant appears to greatly increase the rates at which sugars can be released from the cell wall and should enhance our ability to breakdown plant biomass to use for sugar and biofuel production (Brown et al. 2011).

The mutants also allowed us to identify the CESA proteins that are presumed to be the catalytic subunit from the complex that makes cellulose. We are currently combining genetics with biochemistry to understand the composition and organization of the large complex that makes up cellulose with the aim of understanding what regulates the rates of cellulose synthesis and the crystalinity and organization of the cellulose microfibril. While we have used epitope tagging to generate highly pure preparation of oligomers of the catalytic CESA proteins (Atanassov et al. 2009) isolating an intact and functional complex remains one of the most important aims in this field. We are currently exploiting the information gained from epitope tagging to modify our approach and address this question.

Finally, generating large quantities of biofuels or other products from plant cell walls will require huge quantities of biomass. We have been looking at how to increase the number of cells that make these thick woody cell walls and have recently identified a receptor kinase and its ligand that regulate this process. Over expressing these genes gives a huge increase in biomass in the model plant Arabidopsis and we are currently testing how far we can extend these results into more useful plants and particularly trees (Etchells and Turner 2012).

Author: Professor Simon Turner, The University of Manchester

References:

1.       Etchells, J.P., et al., Plant Vascular Cell Division Is Maintained by an Interaction between Pxy and Ethylene Signalling. PLoS Genet. 2012, 8, e1002997-e1002997. DOI: 10.1371/journal.pgen.1002997.

2.       Brown, D; et al., Arabidopsis Genes Irregular Xylem (Irx15) and Irx15l Encode Duf579-Containing Proteins That Are Essential for Normal Xylan Deposition in the Secondary Cell Wall. Plant J, 2011, 66, 401-413. DOI: 10.1111/j.1365-313X.2011.04501.x.

3.       Atanassov, I.I; et al., Elucidating the Mechanisms of Assembly and Subunit Interaction of the Cellulose Synthase Complex of Arabidopsis Secondary Cell Walls. J. Biol. Chem., 2009, 284, 3833-3841. DOI: 10.1074/jbc.M807456200.

[sugar-complexity]

How pandemic, seasonal and bird flu differ in their cell surface sugar recognition

Influenza (flu) viruses infect cells by attaching to bead-like sugar chains at the surface of the cells of the respiratory tract, referred to as sugar receptors. Different viruses attach to different sugar receptors, and if a virus cannot find its specific sugar receptor, it cannot get into the cell. Once inside the cell, the virus uses the cell’s machinery to make thousands more viruses, which then burst out of the cell and infect neighbouring ones, establishing an infection.

Seasonal influenza viruses attach to sugar receptors found on cells in the nose, throat and upper airway, enabling them to infect a person’s respiratory tract. The ability of human influenza viruses to stick to the sugars on the cells of the upper airway may explain why the virus spreads so fast from sneezing and coughing symptoms.

We have shown that 2009 pandemic H1N1 swine flu can attach also to cell surface sugars found on cells deep inside the lungs, to which bird flu virus can bind, and cause severe lung infection. We found that 2009 pandemic H1N1 influenza bound only weakly to the types of sugars that occur in the lungs. This is why most people infected with the virus experienced mild infection.

We compared the way seasonal and pandemic H1N1 flu viruses infect cells by identifying which sugar receptors each virus binds to. To do this, we used a glass surface with different sugars attached to it, called a carbohydrate microarray. When viruses were added to the glass surface, they stuck to specific sugars and the corresponding areas on the array ‘lit up’. This is how we could see which receptors the different viruses attached to.

Pandemic H1N1 influenza could bind strongly to sugars called ‘α2-6 receptors’, which are found in the nose, throat and upper airway, and it could also attach more weakly to ‘α2-3 receptors’, which are found on cells deeper inside the lungs. In contrast, the seasonal H1N1 influenza could only attach to α2-6 receptors.

Sugar-binding determines which cells the viruses can infect. Our follow-on study of the viruses found in the lungs of cases of severe or fatal infections, has shown that the virus can mutate during infection and bind more strongly to the α2-3 receptors. Our findings uncover potential mechanisms linking sugar-binding to severity of disease.

If the mutant virus were to acquire the ability to spread more widely, the potential consequences would be of great significance, hence the need to closely monitor the evolution of influenza virus!

The carbohydrate microarray technique is being used to study how other viruses, bacteria and parasites bind to cells that they infect.

  Author: Professor Ten Feizi – Imperial College London

References:

1 Liu Y; et al.,Receptor specificity and cell tropism of D222G hemagglutinin mutants isolated from fatal cases of pandemic A(H1N1) 2009 influenza virus. J Virol. 2010, 84, 12069-12074. DOI:10.1128/JVI.01639-10.

2 Childs, Robert A; et al., Receptor-binding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray. Nature Biotechnology, 2009, 27, 797-799. DOI:10.1038/nbt0909-797.

3 Feizi Ten; et al., Oligosaccharide microarrays to decipher the glyco code. Nat Rev Mol Cell Biol., 2004, 5, 582-588.

[sugar-complexity]

Using Sugars to Combat Global Food Shortages

Plants make extensive use of sugars, for energy storage, to maintain their upright structure, to control their complex metabolism and to counteract attack by pathogens. Research at the John Innes Centre is directed towards understanding how plants make and use complex sugars, with a view to developing a range of new crops that are optimal for specific applications.  This research is of global importance as the world population and demand for crops and crop-based products escalate.

Key food crops, such as wheat, rice and barley, are rich sources of sugars in the form of starch, a polymer of glucose. Starch makes up nearly half of the entire calories in the human diet; it is also a billion tonnes a year bulk commodity that is used in many different applications – a thickener in ketchup; a hyper-absorbant material in nappies; a plasticiser in concrete; a finishing agent in paper; a feed-stock for beer, whisky and bioethanol production. Despite its global importance, there are still many aspects of starch metabolism that we do not understand. The JIC team are using a combination of chemical and genetic approaches to define the biochemical pathways that plants use to make and digest granular starch. There are parallels between how plants metabolise starch and how it is digested by humans. Restricting the digestion of dietary starch, which generates free glucose, is a drug target for the control of type-II diabetes. Extrapolating from medicine to agriculture, we are therefore exploiting anti-diabetic drugs as tools to dissect the detail and timing of plant starch metabolism. This so-called chemical genetic approach is both fast and direct, over-coming the issues associated with genetically modifying plants. On one hand our studies have demonstrated that the accepted text book pathway for cereal starch metabolism is incorrect. On the other hand, the tools that we have developed are being applied to the control root growth during barley malting, a key process for the brewing industry. 

References:

1  Rejzek, Martin; et al., Chemical genetics and cereal starch metabolism: structural basis of the non-covalent and covalent inhibition of barley β-amylase. Mol. Biosyst., 2011, 7, 718-730. DOI: 10.1039/c0mb00204f.

2  Stanley, Duncan; et al., The role of α-glucosidase in germinating barley grains. Plant Physiol., 2011, 155, 932-943. DOI:10.1104/pp.110.168328.

3  Rejzek, Martin; et al., Versatile oligosaccharide microarrays for plant glycobiology and cell wall research. J. Biol. Chem., 2012, 287, 39429-39438.

Author: Professor Rob Field, John Innes Centre

[sugar-complexity]

The Role of Sugars in Toxin Invasion

Cholera, travellers’ diarrhoea and E. coli O157 food poisoning are all caused by protein toxins that are released by bacteria in the intestine. The toxins stick to glycolipids that consist of specific sugars with greasy lipid tails that are embedded in the cell membrane. After sticking to the cell surface, the toxins can enter the cells where they cause diarrhoea.

In some cases, the severity of the disease is influenced by other cell surface sugars. For example, your blood group is defined by which sugars you have on the surface of your cells, and some of the blood group sugars can also stick to the protein toxins. The consequence is that if you are blood group O and you get cholera, you will probably be more severely affected than if you are blood group A or B. In our research, we aim to get a better understanding of these diseases by measuring how tightly different sugars can stick to the protein toxins.

As binding to the glycolipids is a prerequisite for entering cells and causing diarrhoea, anything that can prevent the protein-sugar interaction could potentially become an anti-diarrhoeal drug. We make synthetic mimics of the cell surface sugars and study their ability to block the toxins from sticking to and entering the cells. As the toxins typically have five sugar binding sites, a good strategy for inhibitor design is to make compounds that have multiple copies of the sugars so that all of binding sites can be blocked by a single molecule of the inhibitor.

  In a different project we are starting to investigate if we can convert the toxins into safe tools for delivering drugs into cells. Here we re-engineer the protein to remove its toxic section and then we exploit its natural ability to stick to sugars and enter cells as a strategy to transport medicinally useful molecules into the target cells, e.g. motor neurones.

References:

1 W. B. Turnbull, Thermodynamic dissection of the cholera toxin-ganglioside GM1 interaction, Int. Drug Discovery 2011, 6, 56-57. ISSN:2150-4792.

2 P. K. Mandal, T. R. Branson, E. D. Hayes, J. F. Ross, J. A. Gavín, A. H. Daranas and W. B. Turnbull, Towards a structural basis for the relationship between blood group and the severity of El Tor cholera, Angew. Chem.Int. Ed. 2012, 51, 5143-5146. DOI:10.1002/anie.201109068.

3 T. R. Branson and W. B. Turnbull, Bacterial toxin inhibitors based on multivalent scaffolds. Chem. Soc. Rev. 2013, DOI: 10.1039/C2CS35430F

  Author: Dr Bruce Turnbull, Leeds University.

[sugar-complexity]

Introduction to the science behind The Complex Life of Sugars

Sugars are not just the white granules you stir into your tea, they are all around us.  In fact, you wouldn't be here today without them!

Sugars provide the building blocks of life, and most of the Earth’s biomass. They are generated through photosynthesis from carbon dioxide, water and light. Sugars are used as food to provide energy and biomaterials such as cellulose in wood. We are only just starting to uncover the role of sugars in complex biological processes in cells, tissues and organisms. Our fundamental understanding of the science of sugars will help us to address the important challenges society will face in the future as petroleum and gas resources diminish. Sugars can provide renewable substitutes for energy and material production. They will also play a part in emerging and future medicines, such as biopharmaceuticals, vaccines or antimicrobials.

This exhibit will introduce you to the science of sugars and recent discoveries made in our laboratories. We will show how we can apply our discoveries in the areas of bioenergy and biomaterial production, as well as in food and health. We will show you how sugars are produced in plants and can be converted to biofuels. Plant sugars such as starch and cellulose can also be used to make novel high-tech smart materials that are biorenewable and biodegradable, and which will replace petroleum derived plastics in the future. Sugars are also important components of a new generation of medicines called biopharmaceuticals, which include many new effective anti-cancer treatments. We have recently discovered that sugars are important in many cell surface events, including the most fundamental of life processes: fertilization! Complex sugar molecules coat the outer layer of eggs making them ‘sticky’ which helps the sperm and egg bind together.

The following series of blog posts will reveal more about The Complex Life of Sugars!

Author: Professor Sabine Flitsch at The University of Manchester.