Candidate mammalian glycoinositol phospholipid precursor containing three phosphoethanolamines

Etsuko Ueda, Daniel Sevlever, Gregory M. Prince, Terrone L. Rosenberry, Shinichi Hirose, M. Edward Medof

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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-Gal-GalNAc. 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 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.

Original languageEnglish (US)
Pages (from-to)9998-10002
Number of pages5
JournalJournal of Biological Chemistry
Volume268
Issue number14
StatePublished - May 15 1993
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry

Cite this

Ueda, E., Sevlever, D., Prince, G. M., Rosenberry, T. L., Hirose, S., & Medof, M. E. (1993). Candidate mammalian glycoinositol phospholipid precursor containing three phosphoethanolamines. Journal of Biological Chemistry, 268(14), 9998-10002.