Introduction Human CD36 is a class B scavenger receptor expressed in a variety of cell types including macrophages and endothelial cells. This heavily glycosylated membrane protein has a large ectodomain (ED) responsible for binding a variety of ligands. These include oxidised LDL and long chain fatty acids, which link CD36 to the development of atherosclerosis and insulin resistance, respectively. CD36 has also been implicated in the phagocyte-uptake of apoptotic cells, anti-angiogenic effects in endothelial cells and development of a variety of fibrotic diseases, through interaction with thrombospondin-1. The main objective of this study was to express, purify and characterise the ligand binding ectodomain of CD36 with a view to better understanding how CD36 binds multiple ligands. Methods A baculovirus expression system was used to express and secrete CD36 ED with a 12-His tag from insect cells using an N-terminal secretion sequence. Subsequent purification was performed using nickel affinity chromatography with functionality of the ED assessed through the ability to bind modified LDL in a solid phase binding assay. The N-glycosylation status of the purified ED was explored through use of the N-glycosidase PNGase F and mass spectrometric analysis. Initial studies to measure binding kinetics involved use of surface plasmon resonance (SPR) and binding of commercially available antibodies, mAb1955, mAb1258 and mAbFA6-152, to purified CD36 ED immobilised on an NTA SPR sensor chip. Results The N-glycosylation status of CD36 ED produced in Sf21 and Hi5 insect cells was different with heterogeneous N-glycosylation being identified at individual glycosylation sites. Solid-phase ligand binding revealed that both glycosylated and deglycosylated ED retain affinity for modified LDL. The purified CD36 ED was found to homo-oligomerise particularly at higher concentrations. MAb1955 and mAb1258 were found to bind to the nickel on the sensor chip precluding microkinetic analysis of binding to CD36. However, mAbFA6-152 was found to bind to the immobilised CD36 ED with high avidity and two-step binding kinetics. Conclusions and future direction This study shows for the first time that N-glycosylation is not important for the binding of modified LDL to CD36. Furthermore it was demonstrated that native CD36 ED produced in Sf21 insect cells can be deglycosylated without denaturation, which may be critical for the success of future crystallisation trials. The characterisation of mAbFA6-152 binding by SPR is proof-of-principle that CD36 ED can be studied using this technique paving the way for future biophysical analysis of ligand binding.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:667358 |
| Date | January 2014 |
| Creators | Sanders, David John |
| Publisher | Queen Mary, University of London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://qmro.qmul.ac.uk/xmlui/handle/123456789/9003 |
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