Aerolysin, a cytolytic bacterial exotoxin, was radioiodinated by using the Iodogen reagent. Binding of the labeled toxin to rat erythrocytes was inhibited by the native protein and by anti-aerolysin antibody. Toxin, once bound, was not removed by the addition of a large excess of free aerolysin. Binding of the radioactive toxin to erythrocytes of different species paralleled the hemolytic specificity of the unlabeled toxin. Pretreatment of the rat erythrocytes with trypsin, which removed a major membrane glycoprotein, resulted in a dramatic decrease in binding, whereas chymotrypsin treatment had no effect. Binding was inhibited by a glycoprotein fraction isolated from these cells but not by a total rat erythrocyte glycolipid preparation. Aerolysin caused the formation of holes in erythrocytes which were sized by measuring the release of labeled molecular weight markers. Glucagon (molecular weight 3550) and smaller molecules entrapped in human or rat erythrocytes were released by treatment with aerolysin, whereas methoxyinulin (molecular weight 5500) and larger molecules were not. Aerolysin also caused the release of glucose from large unilamellar lipid vesicles. The results indicate that a specific glycoprotein receptor facilitates the interaction of aerolysin with erythrocyte membranes. Binding is followed by the formation of discrete holes or pores, and this results in cell rupture by a colloid-osmotic process.

The six histidines of the channel-forming protein aerolysin have been replaced one at a time with asparagine by site-directed mutagenesis, and each of the modified proteins has been purified. Three proteins had the same hemolytic activity as native toxin, but the others, those changed at His107, His132, or His332, were less able to disrupt both human and rat erythrocytes. The largest reduction in activity, more than 100-fold, was observed with the His132 mutant protein. Studies with radioiodinated samples showed that it had approximately the same affinity as native aerolysin for the rat erythrocyte receptor. However, once bound to either rat or human erythrocytes, it was much less able to carry out the next essential step in hole formation, aggregation to form a stable oligomer. Aggregation was also reduced by replacing His107, but the contrast with native aerolysin and the effect on hemolytic activity were less pronounced. The protein modified at His332 behaved in a different way from those substituted at positions 107 and 132. Its affinity for the rat erythrocyte receptor was considerably lower than the affinity of the wild-type protein, but when bound it appeared to aggregate normally. The results suggest that His132 and perhaps His107 are involved in the aggregation of aerolysin whereas His332 may be at or near the receptor binding site.

No abstract

The substrate specificity of a bacterial analogue of the plasma enzyme lecithin:cholesterol acyltransferase (LCAT) has been examined with small unilamellar liposomes and Triton mixed micelles. In contrast to LCAT, the microbial enzyme is capable of using all of the naturally occurring phospholipids as acyl donors. In general reaction rate depends more on the length or degree of unsaturation of the acyl chains than on the nature of the phospholipid head group. Among a series of disaturated phosphatidylcholines in liposomes, dilauroylphosphatidylcholine is the preferred acyl donor. Like LCAT, the enzyme will catalyze acyl transfer by using other alcohols in addition to cholesterol. Of saturated straight chain primary alcohols 1-decanol is the preferred acyl acceptor. Cholesterol, however, is a far better acceptor than any non-sterol alcohol tested. Other steroids with equatorial hydroxyls at position C-3 and trans-fused A:B rings will also act as acceptors whereas those steroids with axial hydroxyls at C-3 or cis-fused rings are inhibitors of acyl transfer. The ability of steroids to act as acyl acceptors may be due to the nature of their interaction with the phospholipid acyl donor.

The cytolytic toxin aerolysin binds to a receptor on the surface of eukaryotic cells. Murine erythrocytes are among the most sensitive to the toxin. Here we describe the detergent solubilization and partial purification of the receptor from rat erythrocytes. We show that it can be successfully incorporated into planar lipid bilayers, greatly decreasing the concentration of aerolysin required to form channels. Exploiting the ability of the receptor to bind aerolysin after SDS electrophoresis and blotting, we obtain evidence that it is a 47 kDa glycoprotein that is sensitive to proteases and N-glycosidase. It may correspond to CHIP28, the water channel of the human erythrocyte.