The spectrum of anionic oligosaccharides released by endo-beta-N-acetylglucosaminidase H from glycoproteins. Structural studies and interactions with the phosphomannosyl receptor.

We have performed a detailed analysis of all the anionic oligosaccharides released by endo-beta-N-acetylglucosaminidase H from the whole cell glycoproteins of P388D1 mouse macrophage-like cells labeled for 14 h with [2-3H]mannose. The major anionic species consisted of phosphorylated high mannose-type oligosaccharides containing one or two phosphomonoesters or phosphodiesters in several different positions. In addition we identified hybrid-type molecules containing one, two, or three sialic acid residues. A subset of the latter molecules also contained phosphodiesters or phosphomonoesters on another branch of the same oligosaccharide. Unlike previously reported hybrid-type molecules, these do not have a "bisecting" N-acetylglucosamine residue on the beta-linked mannose. Some of these oligosaccharides contained an unidentified acid-labile group on the core N-acetylglucosamine or the beta-linked mannose. The glycoproteins secreted by these cells were greatly enriched in hybrid oligosaccharides containing one sialic acid and one phosphomonoester. The interaction of the isolated oligosaccharides with bovine liver phosphomannosyl receptor immobilized on Affigel was analyzed. Oligosaccharides with phosphomonoesters were the only species that interacted with high affinity with the receptor, and molecules with two phosphomonoesters showed the best binding. The location of the phosphomonoester on the oligosaccharide influenced the degree of interaction with the receptor. Removal of accessible nonphosphorylated mannose residues improved the binding in some cases. These findings indicate that the generation of the physiological phosphomannosyl ligand on lysosomal enzymes involves removal of the blocking N-acetylglucosamine residues, trimming of certain mannose residues, and correct positioning of the phosphate esters.