Endocrine Abstracts (2008) 15 P202

Attempted detection of recombinant glycosylphosphatidylinositol-anchored proteins using Fluorescently Labelled Aerolysin, FLAER

Yahia Kaabi, Ian Wilkinson, Gareth Richards, Tim Skerry & Richard Ross


University of Sheffield, Sheffield, UK.


Introduction: Glycosylphosphatidylinositol (GPI) anchored proteins are common on the surfaces of eukaryotic cells. An example is alkaline phosphatase. Fluorescently Labelled Aerolysin (FLAER) is a bacterial toxin produced by the gram-negative bacterium Aeromonas hydrophila that binds naturally to GPI anchored proteins and utilizes them as receptors to cause cytotoxicity. FLAER has been used to discriminate the GPI deficient red blood cells in cases of paroxysmal nocturnal haemoglobinuria (PNH). We have been using GPI anchors to express recombinant truncated receptors on the cell surface and this study examined the use of FLAER to detect these proteins.

Method: Growth hormone receptor (GHR) extracellular domain was linked to a GPI signal sequence (GHR-GPI) in the mammalian expression vector, pCR3, and expressed in human kidney embryonic cells, HEK293. FACS analysis was performed to detect the expression using different concentrations of FLAER as well as monoclonal antibodies against GHR.

Results: In untransfected control 293 cells FACS analysis with 5 nM FLAER showed a fluorescence shift presumed to be due to native GPI anchors (mean fluorescence with and without FLAER: 13.89 and 4.5, respectively). This signal was augmented with increasing concentrations of FLAER. In cells transfected with GHR-GPI FACS with a specific mAb to the GHR showed a clear increase in signal (mean fluorescence with and without transfection: 25.2 and 3.67, respectively). However FACS with 5 nM FLAER showed little change in signal compared to background (mean fluorescence with and without transfection: 15.6 and 13.8, respectively) although there was a small percent of cells that showed a higher signal (10%).

Conclusion: FLAER is able to detect the naturally occurring GPI anchored proteins present on the (HEK293) cells; however, it has an insignificant detection of recombinant GPI anchored GHR when compared to the monoclonal antibodies. The failure of FLAER to detect recombinant GPI anchored proteins may relate to the high level of expression or steric hindrance.

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