Synthesis of mannosylglucosaminylinositol phospholipids in normal but not paroxysmal nocturnal hemoglobinuria cells.
To identify mannosyl (Man)-containing intermediates of the human glycoinositol phospholipid (GPI) anchor pathway and examine their expression in paroxysmal nocturnal hemoglobinuria (PNH), mannolipid products deriving from in vitro guanosine diphosphate [3H]Man labeling of HeLa cell microsomes were characterized. The defined GPI species were correlated with products deriving from in vivo [3H]Man labeling of normal and (GPI-anchor defective) affected leukocytes. In vitro analyses in HeLa cells showed dolichol-phosphoryl (Dol-P)-[3H]Man and a spectrum of [3H]Man lipids exhibiting TLC mobilities approximating those of Trypanosoma brucei (Tryp) GPI precursors. Iatrobead HPLC separations and partial characterizations of the major isolated [3H]Man species (designated H1-H8) showed that all but H1 (Dol-P-Man) were sensitive to HNO2 deamination and serum GPI-specific phospholipase D digestion but were resistant to phosphatidylinositol-specific phospholipase C digestion unless previously deacylated with mild alkali. [3H]Man label in H3, H4, and H6 but not in H5 or H7 was efficiently released into the aqueous phase by jack bean alpha-mannosidase digestion. BioGel P-4 and AX-5 sizing of the dephosphorylated core glycan fragments of H6 and H7 gave values that coincided precisely with the corresponding glycan fragments from the fully assembled Tryp anchor donor A' (P2). Affected leukocytes from four patients with PNH supported formation of GlcNAc- and GlcN-PI but all failed to express H6 and H7 as well as H8 and two showed complete absence of earlier Man-containing intermediates. These findings argue that human intracellular GPI mannolipids are built on acylated inositol phospholipids, that H6 and H7 contain differentially phosphoethanolamine-substituted Man3-GlcN-inositol cores, and that PNH cells are defective in conversion of GlcN-PI into these more mature mannolipid structures.
Hirose
S
S
Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106.
Ravi
L
L
Prince
G M
GM
Rosenfeld
M G
MG
Silber
R
R
Andresen
S W
SW
Hazra
S V
SV
Medof
M E
ME
D015703:Antigens, CD / Q000096:biosynthesis; D018958:CD55 Antigens; D018957:CD59 Antigens; D002478:Cells, Cultured; D006017:Glycolipids / Q000096:biosynthesis*; D017261:Glycosylphosphatidylinositols; D006367:HeLa Cells; D006457:Hemoglobinuria, Paroxysmal / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008362:Mannosides / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D010716:Phosphatidylinositols / Q000096:biosynthesis*; D011499:Protein Processing, Post-Translational
D015703:Antigens, CD; D018958:CD55 Antigens; D018957:CD59 Antigens; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D008362:Mannosides; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010716:Phosphatidylinositols