Functional influence of VASE insertion on neural cell adhesion molecule homophilic binding

Neural cell adhesion molecule (NCAM) is a transmembrane, homophilic binding protein which has important roles in cellular migration, nervous system development, learning and memory. NCAM is estimated to have at least 27 different isoforms and the functional significance of one alternatively spliced isoform, called the variable alternatively spliced exon (NCAM-VASE) is the subject of this research. NCAM-VASE differs from NCAM by the insertion of an extra ten amino acids into the fourth immunoglobulin domain. This converts NCAM function from a promoter of axonal outgrowth to an inhibitor. NCAM-VASE is expressed in nervous system areas of low synaptic plasticity in contrast to NCAM, which is generally found in areas of high plasticity. it has been postulated that NCAM-VASE functions differently by altering the homophilic binding strength of NCAM. To test this hypothesis, single molecule spectroscopy (SMFS) was carried out using an atomic force microscope (AFM) to measure the adhesion between single molecule NCAM±VASE proteins. A novel method for AFM surface functionalisation was developed to affinity capture Fc-tagged NCAM±VASE proteins via an adsorbed α Fc antibody. Adhesion measurements revealed that the homophilic binding strength of NCAM and NCAM-VASE proteins was similar but this data was only preliminary. Due to the potential problems associated with the use of a dimerising Fc tag and non-specific tip-sample interactions, another model was developed for measuring the adhesion between covalently bound PEGylated surfaces coordinating His-tagged NCAM±VASE proteins. Due to time constraints AFM measurements were not taken using this system. A novel form of AFM, termed single cell force spectroscopy (SCFS), was used and an experiment was developed to measure the adhesion between parental cells and those expressing the NCAM or NCAM-VASE transgene. At long contact times (10 min), not short (5 s) NCAM-VASE cell adhesion was increased three-fold over two NCAM cells.EThOS - Electronic Theses Online ServiceChemical Biology Centre and EPSRCGBUnited Kingdo