Structure of glycopeptides isolated from bovine milk component PP3

The heat-stable acid-soluble phosphoglycoprotein component PP3 was isolated from the bovine milk proteose peptone fraction by concanavalin A affinity chromatography. Glycopeptides were released by pronase digestion of the milk component PP3 and were subsequently separated by high-pH anion-exchange chromatography on CarboPac PA-1. The primary structures of the glycan and peptide moieties of eight N-glycopeptides have been established by combining methylation analysis, mass spectrometry, 400-MHz 1H-NMR spectroscopy, and peptide sequence analysis. All the analyzed fractions contained biantennary N-acetyllactosamine-type carbohydrate chains, some of them with a GalNAc(beta 1-4)GlcNAc or a NeuAc(alpha 2-6)GalNAc(beta 1-4)GlcNAc group. This particular sequence did or did not replace the Gal(beta 1-4)GlcNAc group usually found in most N-linked glycans. Moreover, the sialylated Gal and GalNAc residues were only found on the Man(alpha 1-3) antenna.