Synthesis of Mannosylglucosaminylinositol

Proc. Nati. Acad. Sci. USA Vol. 89, pp. 6025-6029, July 1992 Medical Sciences Synthesis of mannosylglucosaminylinositol phospholipids in normal but not paroxysmal nocturnal hemoglobinuria cells (complement/decay-accelerating factor/CD59) SHINICHI HIROSE*, LAKSHMESWARI RAVI*, GREGORY M. PRINCE*, MELVIN G. ROSENFELDt, ROBERT SILBERf, STEVEN W. ANDRESEN§, SANDRA V. HAZRA¶, AND M. EDWARD MEDOF*II *Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106; Departments of tCell Biology and *Medicine, New York University Medical Center, New York, NY 10016; §Cleveland Clinic Foundation, Cleveland, OH 44106; and lAkron General Medical Center, Akron, OH 44307 Communicated by Frederick C. Robbins, November 29, 1991 (received for review September 12, 1991) ABSTRACT To identify mannosyl (Man)-containing inter- have been identified, information concerning the biochemical mediates ofthe human glycoinositol phospholipid (GPI) anchor pathway(s) responsible for their intracellular GPI-anchor pathway and examine their expression in paroxysmal nocturnal assembly and protein incorporation is only beginning to hemoglobinuria (PNH), mannolipid products deriving from in emerge. vitro guanosine diphosphate [3HJMan labeling of HeLa cell Among human GPI-anchored proteins are the decay- microsomes were characterized. The defined GPI species were accelerating factor (DAF) and CD59, membrane-associated correlated with products deriving from in vivo [3HJMan label- regulators of the complement cascade which protect host ing of normal and (GPI-anchor defective) affected leukocytes. tissues from autologous complement attack (reviewed in refs. In vitro analyses in HeLa cells showed dolichol-phosphoryl 2-4). Deficient expression of these proteins underlies an (Dol-P)-[3H]Man and a spectrum of [3H]Man lipids exhibiting acquired hemolytic disorder termed paroxysmal nocturnal TLC mobilities approximating those of Trypanosoma brucei hemoglobinuria (PNH), and the lesion responsible for the (Tryp) GPI precursors. Iatrobead HPLC separations and deficit has been traced to defective GPI-anchor pathway partial characterizations ofthe major isolated [3H]Man species function in a bone marrow stem cell(s) (5). The precise site(s) (designated H1-H8) showed that all but Hi (Dol-P-Man) were of interruption in the pathway, however, remains unidenti- sensitive to HNO2 deamination and serum GPI-specific phos- fied. pholipase D digestion but were resistant to phosphatidylinosi- In a previous communication (6), we reported on the use of tol-specific phospholipase C digestion unless previously deac- an in vitro experimental system employing hypotonic cell ylated with mild alkali. [3H]Man label in H3, H4, and H6 but lysates to study mammalian GPI-anchor biosynthesis. The not in H5 or H7 was efficiently released into the aqueous phase experimental system parallels that used for analyses of GPI- by jack bean a-mannosidase digestion. BioGel P-4 and AX-5 anchor structures synthesized in Trypanosoma brucei sizing ofthe dephosphorylated core glycan fragments ofH6 and (Tryps) (7, 8). We previously demonstrated that mammalian H7 gave values that coincided precisely with the corresponding cells transfer GlcNAc from UDP to a PI acceptor and rapidly Tryp anchor donor deacetylate the GlcNAc-PI product to generate GlcN-PI, two glycan fragments from the fully assembled species (designated GPI-A and -B) that correspond to the two A' (P2). Affected leukocytes from four patients with PNH initial intermediates in the Tryp GPI-anchor pathway (7, 8). supported formation of GkcNAc- and GlcN-PI but all failed to We were further able to show that cell lysates ofGPI-anchor- express H6 and H7 as well as H8 and two showed complete defective leukocytes from two PNH patients were able to absence of earlier Man-containing intermediates. These rind- support these biochemical conversions with normal efficien- ings argue that human intracellular GPI mannolipids are built cies. on acylated inositol phospholipids, that H6 and H7 contain In the present communication, we report on the extension differentially phosphoethanolamine-substituted Man3-GIcN- of this experimental system to characterize further steps in inositol cores, and that PNH cells are defective in conversion of the human GPI-anchor pathway. We also report on the partial GlcN-PI into these more mature mannolipid structures. localization within these further steps of the block(s) in GPI-anchor synthesis that undermines expression of DAF, Cell-surface proteins that are membrane anchored by cova- CD59, and other GPI-anchored proteins in affected PNH lently attached glycoinositol phospholipid (GPI) structures cells. are widely distributed on eukaryotic cells. Experimental evidence to date (reviewed in ref. 1) indicates that, despite individual variations, the GPI-anchoring units of these pro- EXPERIMENTAL PROCEDURES teins share a common core structure (Fig. 1) consisting of a Reagents. GDP-[3H]Man (24.3 Ci/mmol; 1 Ci = 37 GBq), phosphoethanolamine (P-Etn), three mannosyl (Man) resi- UDP-[3H]GlcNAc (26.8 Ci/mmol), D-[2-3H]Man (15 Ci/ dues, and a nonacetylated GlcN linked to an inositol phos- mmol), and EN3HANCE spray were obtained from DuPont/ pholipid. This inositol phospholipid is characteristically New England Nuclear. [3H]Glucose oligomer standards were phosphatidylinositol-specific phospholipase C (PI-PLC) sen- provided by J. Baenziger (Washington University, St. Louis, sitive but it may be PI-PLC resistant due to inositol acylation. MO). Tunicamycin, ATP, sphingomyelinase, and all protease In all cases studied, GPI-anchoring moieties appear to be preassembled in the endoplasmic reticulum (ER) and, once Abbreviations: GPI, glycoinositol phospholipid; P-Etn, phosphoeth- synthesized, transferred en bloc to primary translation prod- anolamine; ER, endoplasmic reticulum; DAF, decay-accelerating ucts immediately upon their emergence from ribosomes. factor; PNH, paroxysmal nocturnal hemoglobinuria; Tryps, Try- Although more than 50 proteins containing these structures panosoma brucei; PI-PLC, phosphatidylinositol-specific phospholipase C; GPI-PLD, GPI-specific phospholipase D; PMN, polymorphonuclear leukocyte; Chl, chloroform; Dol-P, dolichol-phosphoryl. The publication costs of this article were defrayed in part by page charge 'To whom correspondence should be addressed at: Institute of payment. This article must therefore be hereby marked "advertisement" Pathology, Case Western Reserve University, 2085 Adelbert Road, in accordance with 18 U.S.C. §1734 solely to indicate this fact. Cleveland, OH 44106. 6025 Downloaded by guest on September 24, 2021 6026 Medical Sciences: Hirose et al. Proc. Natl. Acad. Sci. USA 89 (1992) GPI-PLD - - 'AL E-P EP G PTIp---. E-P-..oK-P--- BUTANOL ALK *atn HN02/NoBH4 P1-PLC *-POO;t E-P-..O E-PmO pO AOUEOUS FIG. 1. A human GPI-anchor core structure characterized by an acylated inositol phospholipid is boxed in the center. Structures and partitioning behaviors of predicted products of various treatments are indicated. *, Effective only in the absence of P-Etn (E-P). inhibitors were purchased from Sigma. Bacillus thuringiensis Mannolipid Purifications and Glycan Analyses. [3H]Man- PI-PLC was provided by T. Rosenberry (Case Western labeled lipid products were separated on a 6RSP-8010 (4.6 x Reserve University, Cleveland, OH). GPI-specific phospho- 250 mm) latrobead HPLC column (latron Laboratories, lipase D (GPI-PLD) was obtained from K.-S. Huang (Hoff- Tokyo) in a Dionex DX-300 apparatus using a linear gradient mann-La Roche). Jack bean a-mannosidase was purchased starting with Chl/methanol (80:20) extending to Chl/ from Oxford. methanol (55:45) followed by water addition to Chl/ Murine anti-human DAF monoclonal antibody (mAb) IA10 methanol/water (41:50:9). One-milliliter fractions were col- (IgG2a) was prepared as described (9) and rat anti-human lected. Reductions of HNO2 deamination fragments and CD59 mAb YTH53.1 (IgG2a) was provided by P. Lachmann dephosphorylations with HF were performed as described (Cambridge University, Cambridge, England). Appropriate (6). P4 (7) and AX-5 (12) chromatography were performed as murine and rat nonrelevant mAbs and fluorescein isothiocy- described. anate-conjugated sheep anti-murine and goat anti-rat immu- noglobulins were purchased from Organon Teknika-Cappel. RESULTS Cell Preparations. Tryps were expanded, human cells were Comparison of Mannoipids Synthesized in Vitro by HeLa cultured, and hypotonic lysates of each were prepared as Cells and Tryps. In initial analyses, HeLa cell lysates were described (6). Subcellular fractionation of HeLa cells was labeled with GDP-[3H]Man. Products were then compared by conducted as described (10). The rough microsome fraction TLC to those generated by Tryps. As shown in Fig. 2B, a was recovered at the 1.55/1.8 M sucrose interface. PNH cells spectrum of HeLa cell-derived [3H]Man-labeled lipids was stained with IA10 and YTH53.1 were analyzed on a Becton identified, the first corresponding to human dolichol- Dickinson FACStarPLUS flow cytometer. In patients (M.W. phosphoryl (Dol-P)-Man, and the others coinciding roughly and S.T.) with DAF/CD59-positive polymorphonuclear leu- in mobility (Fig. 2A) with Tryp GPI intermediates P, y, 6, {', kocytes (PMNs), affected cell subsets were enriched by A', and 0 (see legend for structures). anti-DAF cell-affinity chromatography (5). Next, the HeLa-derived products were tested for proper- Biosynthetic Labeling. Human cell lysates were centrifuged ties of GPI mannolipids. First, the presence in

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