Glycosyltransferase Assay System Utilizing an Acylated Glycopeptide Acceptor

Abstract

A glycosyltransferase assay system was devised utilizing as acceptor a purified glycopeptide which was acylated at its N-terminus using caprylic (C 8) anhydride. The glycopeptide contained five amino acids and an N-linked biantennary oligosaccharide, and it was purified from a pronase digest of bovine fibrinogen. Desialylation and β-galactosidase digestion conditions were developed to produce asialo- and asialo-agalacto glycopeptides. Using fatty acid anhydrides, N-acylation conditions for these glycopeptides were then optimized. The products formed when the appropriate acylated glycopeptide was incubated with either of two N-acetylglucosaminyltransferases and UDP-[ 3H] N-acetylglucosamine were easily separated from unused sugar nucleotide and breakdown products by exploiting the affinity of the radiolabeled acylated glycopeptide products for pellicular C 18 cartridges. The products of the enzymatic reactions bound quantitatively to the cartridges and could be eluted in small amounts of methanol. The K m values for the unacylated and acylated glycopeptide accepters were similar when measured using either N-acetylglucosaminyltransferase V or the N-acetylglycosaminyltransferase which transfers N-acetylglucosamine in β(1,3) linkage to N-acetyllactosamine (or lactose). This assay system can be used to measure many glycosyltransferases and other enzymes which transfer to N-linked biantennary oligosaccharides and is applicable to additional glycosyltransferases that transfer to other oligosaccharides which can be prepared as glycopeptides.