The structure of a phytohemagglutinin receptor site from human erythrocytes.

A highly purified glycopeptide with potent phytohemagglutinin (PHA) receptor site activity has been isolated from human erythrocyte membranes. The glycopeptide was released from the membranes by trypsin, treated with alkaline borohydride, and purified by repeated gel filtration, further proteolytic digestion with Pronase, and diethylaminoethyl cellulose chromatography. It has a molecular weight of approximately 2000 and the following composition (in residues): sialic acid, 1; galactose, 2 ; mannose, 2 ; Nacetylglucosamine, 3; aspartic acid, 1.5; serine, 1; and threonine, 1. The sequence of the carbohydrate residues in the oligosaccharide chain was determined by sequential cleavage of the sugars from the nonreducing end with spectic glycosidases. The glycopeptide has a single branched oli.gosaccharide chain contaming two nonreducing termini, one with the composition galactose -%+ N-acetylglucosamine and the other with sialic acid + galactose -% N-acetylglucosamine. Each branch is connected to the inner core which contains 2 mannose and 1 N-acetylglucosamine residues. Since the oligosaccharide chain was not released from the trypsin fragment during the alkaline borohydride treatment, it is probably linked to the peptide backbone by an N-acetylglucosaminylasparagine linkage. Removal of the sialic acid residue does not affect PHAinhibitory activity. When the galactose residues are removed, 90% of the PHA-inhibitory activity is lost. Model compounds having the terminal sequences galactose -4 N-acetylglucosamine have very poor PHA-inhibitory activity unless they also have mannose residues in their interior, showing that the inner core sugars can influence PHAinhibitory activity. The peptide backbone of the glycopeptide also affects the PHA-inhibitory activity of the oligosaccharide chain since higher molecular weight glycopeptides extracted from erythrocyte membranes with chloroformmethanol lose 90% of their PHA-inhibitory activity following proteolytic digestion with trypsin.