Development of new methods to study cell surface glycosylation
O'Connell, Tríona M. (2016) Development of new methods to study cell surface glycosylation. PhD thesis, Dublin City University.
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This thesis seeks to develop new methods to study the glycosylation of cell surfaces, with a particular interest in apoptosis. Existing methods in glyco- biology include mass spectrometry and glycan identification by lectin bind- ing. The use of new carbohydrate binding proteins (CBP) and microfluidic techniques will improve the field of glycobiology and its relevance in the area of apoptosis.
A method using the Lab in a Trench (LiaT) platform was developed to allow sequential labelling of cell surface glycans using lectins. Cells are captured in the trench, fluorescently labelled lectins are then added and allowed time to bind. The lectins are then released by their corresponding free sugar, allowing probing with subsequent lectins without steric hindrance due to adjacent sugars of interest. This study represents the first sequential labelling of the same cell surface by lectins.
It has been established, that exposure of terminal N-acetylglucosamine (GlcNAc) occurs in late apoptosis and has a role in immune recognition and clearance of dead cells. AAL-2 is a recombinantly produced lectin or CBP with an affinity for the terminal sugar N-acetyl glucosamine (GlcNAc). It has a binding profile matching that of the commercial GSL II in flow cy- tometry and western blot, but has the advantage of not requiring additional ions in its buffer. Using flow cytometry, this study has shown that AAL- 2 binds exclusively to late apoptotic cells. GafD is another recombinantly produced CBP with an affinity for terminal O-linked GlcNAc (O-GlcNAc). It was determined by flow cytometry that GafD binds to a subset very late apoptotic cells. The proteins to which GafD binds were identified through isolation of the cell membrane followed by mass spectrometric analysis. The proteins were found to be predominantly cytosolic, indicating a migration of the intracellular membrane to the cell surface during late apoptosis.
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