Abstract
The human brain protein, secreted amyloid precursor protein alpha (sAPPα), derived by proteolytic processing of a parent protein amyloid precursor protein (APP), contributes to healthy functioning of the brain as a neuroprotective protein. sAPPα is a counter to the neurotoxic effects of amyloid beta. Elucidating the molecular mechanisms by which sAPPα delivers these benefits to a healthy brain may be critical to developing meaningful therapies for Alzheimer’s disease.
This project characterises the potential of a tri-functional crosslinking reagent Sulfo-N-hydroxysuccinimidyl-2-(6-[biotinamido]-2-(p-azido benzamido)-hexanoamido) ethyl-1,3'-dithioproprionate (Sulfo-SBED) to identify the elusive receptor(s) for sAPPα. This reagent has three functional groups on three arms: (i) an NHS-Ester which forms covalent bonds with primary amines and can be used to attach the reagent to a ‘bait’ protein, in this project, sAPP; (ii) a phenyl azide which forms a covalent bond under UV exposure to proximal molecules. This is used to UV-link interacting proteins such as receptor(s) of the bait protein. In this project the interaction would bring the putative receptor into proximity of the phenyl azide on the reagent that is attached to the sAPPmolecule; (iii) a biotin molecule which can be used either for purification of the captured interacting protein(s) on an avidin affinity column and/or for Western blot analysis.
For attachment of the reagent to sAPPα, 200 mM Hepes buffer with 1 mM of reagent gave the best results to achieve a combination of maximum reagent attached with minimal loss of protein from precipitation. At higher concentrations of SBED significant loss of protein occurred. Mass spectrometry showed that of the lysine residues detected the reagent was attached specifically within sAPPα, and not randomly.
ii
Of a number of methods tested, the most effective way to remove excess SBED reagent was with a Zeba Spin column to achieve maximum removal of reagent and highest recovery of SBED labelled sAPPα. Dialysis caps, dialysis tubing, Viva Spin columns or G50 columns by comparison gave poor results.
Sensitivity and specificity were modelled with known interacting proteins of sAPP (specific antibodies and amyloid beta). A number of potential negative controls were evaluated and none were adequate. Results from UV-linking the sAPPα:reagent conjugate to neuroblastoma and primary hippocampal cells did not show specificity when comparing positive and negative controls. As non-specific UV-linking had also been observed when UV-linking to specific versus non-specific antibodies, it is possible that specificity could be significantly improved by reducing the residual reagent even further with multiple consecutive Zeba Spin columns.
The SBED reagent may be a good tool for identifying interactions between proteins of relatively high abundance but not so useful to identify receptors that may at any one time be present on cells in low numbers.