Characterization of the Cell Surface Receptor for the Agaricus bisporus Hemagglutinin

Abstract

Abstract Two phytohemagglutinins (PHA) with similar specificities for cell surface carbohydrate receptors were partially purified from the commercial mushroom Agaricus bisporus. One of the PHAs, which was greater than 90% pure, was iodinated and used in binding studies to determine the number of receptor sites on different cell types. Human erythrocytes contained 6.8 x 106 receptors per cell, whereas normal human lymphocytes contained 12.7 x 106 receptors and lymphocytes from patients with chronic lymphocytic leukemia had 2.8 x 106 receptors. The ratio of A. bisporus PHA receptors to Phaseolus vulgaris E-PHA receptors was lower on lymphocytes than on erythrocytes, indicating a different surface structure of these two cell types. Although the A. bisporus PHA bound to lymphocytes, it did not act as a mitogen. Trypsin treatment of human erythrocytes resulted in a 50% decrease in the number of mushroom PHA molecules bound to the cells. The glycopeptide material released from the cells by trypsin was a potent haptene inhibitor of mushroom PHA, binding to cells. Removal of the terminal sialic acid residues from the glycopeptide increased its haptene inhibitory activity 8-fold. All of the haptene inhibitory activity of the glycopeptide was due to the O-glycosidically linked oligosaccharides having the structure galactose (β1,3)/→ N-acetylgalactosamine → serine (or threonine). Removal of the galactose by either β-galactosidase or periodate oxidation destroyed all haptene inhibitory activity. The intact glycopeptide was 15,000 times more potent as a haptene inhibitor than the simple sugars galactose and N-acetylgalactosamine, and 65 times more potent than the disaccharide galactose (β1,3)/→ N-acetylgalactosamine. The N-linked oligosaccharide chain of the trypsin-released glycopeptide, which is the receptor for the PHAs of P. vulgaris and Lens culinaris, was devoid of haptene inhibitory activity toward A. bisporus PHA.