Abstract
Human Tamm-Horsfall glycoprotein has been purified from the urine of one male. The Asn-linked carbohydrate chains were enzymically released by peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F, and separated from the remaining protein by gel-permeation chromatography on Bio-Gel P-100. Fractionation of the intact (sulfated) sialylated carbohydrate chains was achieved by a combination of three liquid-chromatographic techniques, namely, anion-exchange FPLC on Q-Sepharose, amine-adsorption HPLC on Lichrospher-NH2, and high-pH anion-exchange chromatography on CarboPac PA1. In total, more than 150 carbohydrate-containing fractions were obtained, some of which still contained mixtures of oligosaccharides. The primary structure of 30 N-glycans, including 10 novel oligosaccharides, were determined by one- and two-dimensional 1H-NMR spectroscopy at 500 MHz or 600 MHz. The types of compounds identified range from non-fucosylated, monosialylated, diantennary to fucosylated, tetrasialylated, tetraantennary carbohydrate chains, possessing the following terminal structural elements: [formula: see text]
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