Identification and characterization of the aerolysin receptor from rat erythrocyte
Date
1995
Authors
Cowell, Simon Piers
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Abstract
A protocol was devised for the isolation of a 47 kDa glycoprotein from the rat erythrocyte membrane which was able to bind the Gram negative bacterial protein toxin aerolysin with high affinity. The protein was extracted in non-ionic detergent and purified using ion-exchange chromatography. This was followed by affinity chromatography using immobilized wheat germ agglutinin. A partial N-terminal sequence was obtained for the purified protein. It revealed homology with a family of
glycosylphosphatidylinositol-anchored proteins including muscle cell ADP-ribosyltransferases and rat and mouse T-cell antigens. The 47 kDa aerolysin binding protein (ABP) was released into the supernatant during treatment of rat erythrocyte
membranes with phosphatidylinositol specific phospholipase C (PI-PLC; which cleaves GPI-anchors). This further supported the classification of the rat erythrocyte ABP as a novel member of this protein family. The treated rat erythrocytes were less sensitive to aerolysin than control cells not treated with the enzyme. Supernatants containing the ABP were able to inhibit toxin-mediated haemolysis after preincubation with aerolysin. Analysis of a number of other cell lines revealed that rat T-cell line EL-4, and mouse erythrocytes also possess a single high-affinity aerolysin binding species.
The effects of enzymatic treatments upon the protein's aerolysin binding activity were examined. Treatment of the ABP with peptide N-glycosidase F removed N-linked oligosaccharides, and revealed that the protein carries two or more such moieties, but that these are not required for aerolysin binding. The protein was found to be sensitive to trypsin but not to chymotrypsin, and trypsin treatment abolished aerolysin binding activity.