Abstract
The glycoinositol phospholipid (GPI) anchors of mammalian proteins contain linear ethanolamine (EthN)-P-6ManManManGlcN glycan sequences that bear additional EthN-P substituents and in some cases include a fourth Man and a GalNAc or sialic acid-GalGalNAc. Precursors of these anchoring structures are preassembled in the endoplasmic reticulum by sequential glycosylation of inositol phospholipid. In previous studies (Hirose, S., Prince, G. M, Sevlever, D., Ravi, L., Rosenberry, T. L., Ueda, E., and Medof, M. E. (1992) J. Biol. Chem. 267, 16968-16974), a series of putative intermediates of this assembly process were isolated from human HeLa cells and murine lymphomas, and several of the more polar 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 cell GPI species was purified by normal phase HeLa cell GPI species was purified by normal phase Iatrobead high performance liquid chromatography, and its glycan was characterized. Dionex anion exchange chromatographic analyses of fragments produced by nitrous acid deamination, hydrofluoric acid dephosphorylation, and trifluoroacetic 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 ManManManGlcN core lacking additional Man or GalNAc. The implications of the identification of this triply phosphoethanolamine-substituted species to mammalian GPI anchor biosynthesis are discussed.
Highlights
The glycoinositopl hospholipid (GPI)anchors of are variably decoratedindifferentorganisms.InGPI-anmammaliapnroteincsontailnineaerthanolamine chored proteins that have been examined from mammalian (EthN)-P-6ManManManGlcNglycansequences that cells [3,4,5], a second EthN-P substituent is characteristically bear additional EthN-P substituents andsoinme cases present on the Manresidue (Man 1)proximal toGlcN, and a include a fourth Man and a GalNAc or sialic acid-Gal- third EthN-Pmay be present on the adjacent Man2 residue
Chem. 267, 16968-16974), a series of putative intermediates of this assembly process were isolated from human HeLa cells and murine lympho
HeLa cell GPI species was purified by normal phase tured human HeLa and murine lymphomceall lines, putative
Summary
The glycoinositopl hospholipid (GPI)anchors of are variably decoratedindifferentorganisms.InGPI-anmammaliapnroteincsontailnineaerthanolamine chored proteins that have been examined from mammalian (EthN)-P-6ManManManGlcNglycansequences that cells [3,4,5], a second EthN-P substituent is characteristically bear additional EthN-P substituents andsoinme cases present on the Manresidue (Man 1)proximal toGlcN, and a include a fourth Man and a GalNAc or sialic acid-Gal- third EthN-Pmay be present on the adjacent Man residue. 267, 16968-16974), a series of putative intermediates of this assembly process were isolated from human HeLa cells and murine lympho-. Studies in these cells [7, 8] showed that the first two intermediates of the pathway, GlcNAc- and GlcN-PI, are formed by transfer of GlcNAc from UDP to an inositpohlospholipid and deacetylationof the product. HeLa cell GPI species was purified by normal phase tured human HeLa and murine lymphomceall lines, putative. The mammalianGPI change chromatographic analyses of fragments pro- mannolipid species (designatedH2-H8in Refs. 9 and 11)
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