A CANDIDATE MAMMALIAN GLYCOINOSITOL PHOSPHOLIPID PRECURSOR CONTAINING3 PHOSPHOETHANOLAMINES

The glycoinositol phospholipid (GPI) anchors of mammalian proteins con tain linear ethanolamine (EthN)-P-6ManManManGlcN glycan sequences that bear additional EthN-P substituents and in some cases include a fourt h Man and a GalNAc or sialic acid-Gal-GalNAc. Precursors of these anch oring structures are preassembled in the endoplasmic reticulum by sequ ential glycosylation of inositol phospholipid. In previous studies (Hi rose, S., Prince, G. M, Sevlever, D., Ravi, L., Rosenberry, T. L., Ued a, E., and Medof, M. E. (1992) J. Biol. Chem. 267, 16968-16974), a ser ies of putative intermediates of this assembly process were isolated f rom human HeLa cells and murine lymphomas, and several of the more pol ar products were found to contain a second EthN-P attached to the Man residue (Man 1) proximal to GlcN. In this study, the most polar HeLa c ell GPI species was purified by normal phase Iatrobead high performanc e liquid chromatography, and its glycan was characterized. Dionex anio n exchange chromatographic analyses of fragments produced by nitrous a cid deamination, hydrofluoric acid dephosphorylation, and trifluoroace tic acid hydrolysis in conjunction with biosynthetic labeling studies indicated a structure containing a third EthN-P substituent linked to the 6-position of Man 2. The polar GPI product exhibited a ManManManGl cN core lacking additional Man or GalNAc. The implications of the iden tification of this triply phosphoethanolamine-substituted species to m ammalian GPI anchor biosynthesis are discussed.