Abstract
A number of mammalian cell surface proteins are anchored by glycoinositol phospholipid (GPI) structures that are preassembled and transferred to them in the endoplasmic reticulum. The GPIs in these proteins contain linear ethanolamine (EthN)-phosphate (P)-6ManManManGlcN core glycan sequences bearing an additional EthN-P attached to the Man residue (Man 1) proximal to GlcN. The biochemical precursors of mammalian GPI anchor structures are incompletely characterized. In this study, putative [3H]Man-labeled GPI precursors were obtained by in vitro GDP-[3H] Man labeling of HeLa cell microsomes and by in vivo [3H]Man labeling of class B and F Thy-1 negative murine lymphoma mutants known to accumulate incomplete GPIs. The high performance liquid chromatography-purified in vitro and accumulated in vivo GPI products were structurally analyzed by nitrous acid deamination, hydrofluoric acid, trifluoroacetic acid hydrolysis, biosynthetic labeling, and exoglycosidase treatment. The data were consistent with a biosynthetic scheme in which Man and EthN-P are added stepwise to the developing glycan. Several additional points were demonstrated: 1) putative mammalian GPI precursors contain incomplete core glycans corresponding to those in previously characterized trypanosome GPI precursors. 2) The proximal EthN-P found in mature mammalian GPI anchor structures is added to Man 1 prior to incorporation of Man 2 and Man 3. 3) Glycans in the incomplete GPIs that accumulate in classes B and F lymphoma mutants consist of Man2- and Man3GlcN in which EthN-P is linked to Man 1. 4) Distal EthN-P linked to the 6-position of Man, characteristic of the complete GPI core, is found both in a subsequent GPI species with the glycan sequence EthN-P-6ManMan(EthN-P----)ManGlcN and in a more polar GPI product.
Highlights
E anchored by glycoinositol phospholipid (GPI) structures that aprereassembled and transferred thoem in the endoplasmic reticulum
Correspondence of inVitroand in Vivo Generated GPI Products-To determine how previously defined GPI species H2-H8 synthesized in vitro [13] relateto GPI species detectable following in vivo labeling of intact cells [14, 15], GPI products deriving from GDP-[3H]Man labeling of HeLa cell lysates and from [3H]Man labeling of culturedHeLa cells were compared by TLC
To establish if immature GPImannolipid intermediates that accumulate in GPI anchor defective class F and B Thy-1 negative lymphoma mutants [15] correlate with any of the less polar in vitro GPI species, comparative studies were repeated with thesemutant cells
Summary
One consistently reported feature of mannitol; GPI-PLD, GPI-specific phospholipase D; PI-PLC, phosphatidylinositol-specific phospholipase C; PE, phosphatidylethanolamine; TLC, thin layer chromatography; HPLC, high performance the glycans in the anchors of mammalian proteins [4, 5, 7, 8] is that theycontain an additional EthN-P (see Fig. 1).In the liquid chromatography; HEPES, 4-(2-hydroxyethyl)-l-piperazineeth- anchors of rat brain Thy-1,human erythrocyte acetylcholinanesulfonic acid.
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