Award details

Exploiting a sialic acid binding domain

Reference	BB/E001912/1
Principal Investigator / Supervisor	Professor Garry Taylor
Co-Investigators / Co-Supervisors
Institution	University of St Andrews
Department	Biology
Funding type	Research
Value (£)	273,724
Status	Completed
Type	Research Grant
Start date	01/09/2006
End date	28/02/2010
Duration	42 months

Abstract

We recently discovered that the sialdiase from Vibrio cholerae contains a sialic acid-binding domain that increases the catalytic efficiency of the enzyme by binding to sialic acid rich regions on cells. Using calorimetry and NMR we characterised the binding affinity and specificity of this lectin domain, and found that it recognises the terminal sialic acid (Neu5Ac) with a high binding affinity of 30micromolar. This led us to consider using the lectin in making tools for measuring cell surface sialic acid content and distribution, and as a carrier for delivering other proteins to cells rich in sialic acids. In preliminary experiments, we have linked the lectin to form poly-lectin chains containing 2 or 3 lectin modules to create a molecule with increased cell binding through an avidity effect. We have also introduced a single mutation into the lectin binding site that has increased the affinity 5-fold for Neu5Ac. We will extend these studies to create a series of tools for analysis or treatment of cells with various surface sialic acid profiles. There are over 50 derivatives of Neu5Ac found in nature. In animals, the major variation is acetylation at certain positions. We plan to create mutants of the lectin that will recognise these common variants, and link them together into poly-lectin chains. We will then link green fluorescent protein (GFP) to the poly-lectin chains to create tools for visualising and quantifying cell surface sialylation. We will collaborate with Professor Crocker, an expert on immunoglobulin-like sialic acid binding lectins, who will use our fluorescent poly-lectins in a fluorescent assisted cell sorter (FACS) assay to examine sialylation of a variety of cells. Finally, we will examine the use the poly-lectin to deliver proteins to cell surfaces. Initially we will target a sialidase to remove cell surface sialic acids, which may have a role in removing sialic aids from highly-sialylated cancer cells, reducing their metastatic ability.

Summary

The surfaces of all animal cells are decorated with complex networks of sugar molecules that are attached to proteins sitting in the fatty membrane of the cell. These sugars are there for a variety of reasons, for example to act as attachment sites for proteins from other cells which need to communicate in some way. Many microbes, such as the bacterium that causes cholera and the virus that causes influenza, bind to these sugar molecules as these sugars are the first thing these microbes 'see' as they approach a cell which they are going to infect. The complex networks of sugars are made of strings of molecules such as glucose and galactose, which are quite familiar, but the outermost sugar tends to be a sugar called sialic acid. It is called sialic acid because it was first found in saliva, for which the Greek name is sialon. Unusually for a sugar, sialic acid is negatively charged, and this creates a mask that helps some cells, such as red blood cells, circulate through the body far longer than they would if they didn't have this mask. Sialic acids are also unusual in that over 50 chemical variations of the sugar have been discovered in nature, with the different types of sialic acid appearing on the surface of cells from different animals, or cells that are at different stages in their development. It has also been found that cancerous cells have very large numbers of sialic acids on their surface compared to normal cells. Our research proposal is centred on creating biological tools for detecting different types of sialic acids on cell surfaces. What we have discovered recently is a protein, called a lectin, that specifically binds to sialic acids quite tightly, but not as tight as we would like. We are going to use molecular biology (techniques that enable us to cut and join genes together) to create a string of these lectins to give us a molecule that will bind more tightly to cell surfaces. We are also going to link to our string of lectins a protein derivedfrom a jelly fish that glows green. In this way, we will have a molecule that we can add to cells in a dish, and by looking through a fluoresence microscope we will see exactly where the sialic acids are on cells. We will also use a machine that automatically sorts a mixture of cells depending on how much their fluoresce. This will enable us to measure how much sialic acid there is on the surface of different cells in the body. We will also use molecular biology to change the lectin so that it can recognise other types of sialic acid. In this way we can make tools that will show us the distribution of the different sialic acids on cells. Finally, we are going to attach an enzyme to our string of lectins so that it gets delivered to sialic acid-rich cell surfaces. In this way, we may be able to target cancer cells with enzymes that may destroy them.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Industrial Biotechnology, Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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