Glycoinositol Phospholipid Anchor Research Articles

Comparative Analysis of the Prion Protein Open Reading Frame Nucleotide Sequences in Peacock and Parakeet

The open reading frame of peacock and parakeet prion protein (PrP) genes was cloned and sequenced. The peacock and parakeet PrP genes consisted of 833 and 866 nucleotides encoding 266 and 277 amino acids, respectively (GenBank Accession numbers AY365065 and AY365066). Sequence analysis showed that the peacock and parakeet PrP genes had 93.67% homology to each other, 94.04% and 99.64% homology to the chicken PrP gene and 46.0% and 42.1% similarity to the mammalian PrP genes, respectively. The structural features of all known mammalian and avian PrPs, including N-terminal signal peptides, tandem repeats, conserved hydrophobic region, disulfide bridges and glycoinositol phospholipid anchor, were also found in peacock and parakeet PrPs. The parakeet and peacock PrPs, however, differed in the hexarepeat region, with the peacock having six and the parakeet having seven hexarepeats. The phylogenetic analysis showed that the PrP genes in 52 species were clustered into 2 distinct lineages, the avian and the mammalian. The peacock and parakeet PrP genes belonged to the same lineage but the peacock PrP was sub-classed with the pigeon PrP and the parakeet PrP was sub-classed with the duck and chicken PrPs. The present work added two more species data to the collection of the PrP genes and supported the previous findings that the PrP genes are highly conserved across species.

Construction and characterization of secreted and chimeric transmembrane forms of Drosophila acetylcholinesterase: a large truncation of the C-terminal signal peptide does not eliminate glycoinositol phospholipid anchoring.

Despite advances in understanding the cell biology of glycoinositol phospholipid (GPI)-anchored proteins in cultured cells, the in vivo functions of GPI anchors have remained elusive. We have focused on Drosophila acetylcholinesterase (AChE) as a model GPI-anchored protein that can be manipulated in vivo with sophisticated genetic techniques. In Drosophila, AChE is found only as a GPI-anchored G2 form encoded by the Ace locus on the third chromosome. To pursue our goal of replacing wild-type GPI-anchored AChE with forms that have alternative anchor structures in transgenic files, we report the construction of two secreted forms of Drosophila AChE (SEC1 and SEC2) and a chimeric form (TM-AChE) anchored by the transmembrane and cytoplasmic domains of herpes simplex virus type 1 glycoprotein C. To confirm that the biochemical properties of these AChEs were unchanged from GPI-AChE except as predicted, we made stably transfected Drosophila Schneider Line 2(S2) cells expressing each of the four forms. TM-AChE, SEC1, and SEC2 had the same catalytic activity and quaternary structure as wild type. TM-AChE was expressed as an amphiphilic membrane-bound protein resistant to an enzyme that cleaves GPI-AChE (phosphatidylinositol-specific phospholipase C), and the same percentage of TM-AChE and GPI-AChE was on the cell surface according to immunofluorescence and pharmacological data. SEC1 and SEC2 were constructed by truncating the C-terminal signal peptide initially present in GPI-AChE: in SEC1 the last 25 residues of this 34-residue peptide were deleted while in SEC2 the last 29 were deleted. Both SEC1 and SEC2 were efficiently secreted and are very stable in culture medium; with one cloned SEC1-expressing line, AChE accumulated to as high as 100 mg/liter. Surprisingly, 5-10% of SEC1 was attached to a GPI anchor, but SEC2 showed no GPI anchoring. Since no differences in catalytic activity were observed among the four AChEs, and since the same percentage of GPI-AChE and TM-AChE were on the cell surface, we contend that in vivo experiments in which GPI-AChE is replaced can be interpreted solely on the basis of the altered anchoring domain.

Replacement of the glycoinositol phospholipid anchor of Drosophila acetylcholinesterase with a transmembrane domain does not alter sorting in neurons and epithelia but results in behavioral defects.

Drosophila has a single glycoinositol phospholipid (GPI)-anchored form of acetylcholinesterase (AChE) encoded by the Ace locus. To assess the role that GPI plays in the physiology, of AChE, we have replaced the wild-type GPI-AChE with a chimeric transmembrane form (TM-AChE) in the nervous system of the fly. Ace null alleles provided a genetic background completely lacking in endogenous GPI-AChE, and Ace minigene P transposon constructs were used to express both GPI- and TM-AChE forms in the tissues where AChE is normally expressed. Control experiments with the GPI-AChE minigene demonstrated a threshold between 9 and 12% of normal AChE activity for adult viability. Ace mutant flies were rescued by GPI-AChE minigene lines that expressed 12-40% of normal activity and were essentially unchanged from wild-type flies in behavior. TM-AChE minigene lines were able to rescue Ace null alleles, although with a slightly higher threshold than that for GPI-AChE. Although rescued flies expressing GPI-AChE at a level of 12% of normal activity were viable, flies expressing 13-16% of normal activity from the TM-AChE transgene died shortly after eclosion. Flies expressing TM-AChE at about 30% of normal levels were essentially unchanged from wild-type flies in gross behavior but had a reduced lifespan secondary to subtle coordination defects. These flies also showed reduced locomotor activity and performed poorly in a grooming assay. However, light level and electron microscopic immunocytochemistry showed no differences in the localization of GPI- and TM-AChE. Furthermore, endogenous and ectopic-induced expression of both AChEs in epithelial tissues of the adult and embryo, respectively, showed that they were sorted identically. Most epithelial cells sorted GPI- and TM-AChE to the apical surface, but cuticle-secreting epithelia sorted both proteins basolaterally. Our data suggest that rather than having a primary role in protein sorting, the GPI anchor or AChE plays some other more subtle cellular role in neuronal physiology.

Localization of classical and alternative pathway regulatory activity within the decay-accelerating factor.

Decay-accelerating factor (DAF) is a cell-associated C regulatory protein that protects host cells from autologous C attack. It functions intrinsically in host cell surface membranes to rapidly dissociate autologous classical and alternative pathway C3 convertases whenever these amplifying enzymes assemble on host cell surfaces. It is composed of four contiguous approximately 70 amino acid long regions termed short consensus repeats (SCRs) that share homology with similar units in other C3 convertase regulatory proteins. It is attached to the cell surface membrane by a glycoinositol phospholipid (GPI) anchor that is added posttranslationally. In this study, we prepared rGPI-anchored DAF proteins devoid of individual SCRs. We then incorporated the GPI-anchored products into sheep erythrocyte (Esh) hemolytic intermediates and examined their abilities to intrinsically regulate classical or alternative pathway activation. We found that classical pathway C3 convertase regulatory function resides within SCR-2 and SCR-3, while alternative pathway C3 convertase regulatory function resides within SCR-2, -3, and -4. Functional comparisons of the variant DAF proteins in fluid phase C3 activation assays established that the differences reflect domain-specific interactions rather than changes in the spatial arrangement of SCRs above the cell surface. In accordance with these findings, we found that variant DAF molecules containing SCR-1, -2, and -3, but not SCR-4, function to selectively inhibit classical pathway activation.

Glycoinositol phospholipid anchor and protein C-terminus of bovine erythrocyte acetylcholinesterase: analysis by mass spectrometry and by protein and DNA sequencing

Purified bovine erythrocyte acetylcholinesterase (AChE) was radiomethylated on its amine groups and incubated with bacterial phosphatidylinositol-specific phospholipase C to remove the lipid portion of the AChE glycoinositol phospholipid (GPI) anchor, and a C-terminal tryptic fragment that contained the residual GPI glycan was isolated by HPLC. Analysis by electrospray-ionization mass spectrometry revealed a parent ion of m/z 3798. The fragmentation patterns produced by collision-induced dissociation mass spectrometry of the +4 and +5 states of the parent ion indicated a 23-amino acid peptide in amide linkage to ethanolamine-P04-Hex-Hex-Hex(PO4-ethanolamine)(HexNAc)-Hex N(Me)2-inositol phosphate. The glycan structure is completely consistent with that obtained previously for the GPI anchor of human erythrocyte AChE except for the addition of the HexNAc substituent. A nearly complete peptide sequence was deduced from the fragmentation patterns, although four assignments were based only on single fragments of very low abundance. To resolve this uncertainty, a segment of bovine genomic DNA corresponding to the C-terminal AChE sequence was amplified by PCR. DNA sequencing established the 23-amino acid peptide sequence to be FLPKLLSATASEAPCTCSGPAHG, in agreement with the MS data and consistent with results from Edman protein sequencing. Dimerization of AChE polypeptides is mediated by intersubunit disulphide bonding in this C-terminal segment, but the bovine AChE contained two cysteine residues in a ...CTC... motif, in contrast with human AChE which contains only a single cysteine in this segment. Although bovine AChE contained no free thiol groups reactive with iodo[14C]acetamide, partial reduction and alkylation with iodo[14C]acetamide revealed that conversion into monomers occurred with an overall incorporation of only one alkyl group per monomer. An identical level of alkylation was observed when dimeric human AChE was converted into monomers by partial reduction. The question of whether the bovine AChE contains one or two intersubunit disulphide linkages is considered.

Characterization of the Murine PIG-A Promoter Region High Constitutive PIG-A Gene Expression in Brain

PIG-A is an X-linked gene that encodes a synthetic element required to initiate glycoinositol phospholipid (GPI) anchor assembly. In this study we characterized genomic sequence flanking the previously identified 5′ end of the murine PIG-A gene and analyzed constitutive levels of PIG-A gene expression in vivo in different tissues. We found that the 5′-flanking sequence contains 1) alternative transcriptional start sites at −154 and −34/−33 relative to previously known exon 1 sequence, 2) all MRE and multiple GRE, Ap-2 and Sp-1 consensuses, and 3) a 100-bp-long GC-rich segment bounded by NF-IL6 sites. Our survey of PIG-A mRNA levels in vivo unexpectedly revealed that PIG-A expression levels are 2-4 fold higher in the brain than in other tissues

Identification of a PIG-A related processed gene on chromosome 12

PIG-A, the gene encoding the glycoinositol phospholipid anchor synthetic element that is defective in paroxysmal nocturnal hemoglobinuria (PNH), resides on the X chromosome. In the course of analyses of PIG-A genetic alterations in PNH patients, polymerase chain reaction (PCR) amplification of reverse-transcribed RNA from affected leukocytes of a patient with diminished PIG-A mRNA expression yielded a PIG-A related sequence with 126 nucleotide substitutions and five deletions. In the present study, the origin of this product was investigated. Selective reverse transcription (RT)/PCR amplifications with primers specific for PIG-A and this variant sequence, analyses of RNA from unaffected cells, and differential endonuclease digestions showed that mRNA with this sequence is coexpressed with PIG-A mRNA in a wide range of cell types. Mapping of genomic DNA from human/rodent hybrids showed that the sequence derives from a processed gene, designated PIG-A-II, that is encoded on chromosome 12.

Protein Denaturation by Addition and Removal of Acetonitrile: Application to Tryptic Digestion of Acetylcholinesterase

Bovine erythrocyte acetylcholinesterase was prepared for tryptic digestion by radiomethylating with [ 14C]HCHO and NaCNBH 3, cleaving with purified bacterial phosphatidylinositol-specific phospholipase C to remove the lipid portion of the glycoinositol phospholipid anchor, and reducing and alkylating the intersubunit disulfide bonds. Two alternative denaturation procedures were then compared prior to incubation with trypsin. In the conventional procedure, acetylcholinesterase was treated with 6 M guanidine hydrochloride for 40 min at room temperature and dialyzed. In a new procedure, acetonitrile (CH 3CN) was added to 30% v/v for 10-15 min at room temperature and then removed by vacuum evaporation. The CH 3CN concentration during evaporation could be estimated from the apparent pH of the solution (20 mM phosphate buffer), which varied linearly over the range of 0-75% CH 3CN. CH 3CN was removed in a mixture of constant composition (approximately 11% H 2O-89% CH 3CN), so that a final CH 3CN content of 0-5% could be monitored by solution weight alone. The tryptic digests of the two denatured stocks yielded comparable HPLC profiles for A 215 and radioactivity. This new denaturation protocol may be of general utility because of its convenience and gentle conditions.

Glycoinositol phospholipid anchor-defective K562 mutants with biochemical lesions distinct from those in Thy-1- murine lymphoma mutants.

Deficient expression of glycoinositol phospholipid (GPI)-anchored surface proteins has been linked to six different genetic defects in Thy-1- murine lymphoma mutants. In this study, human K562 cell mutants defective in GPI anchoring were derived by anti-decay-accelerating factor (CD55) based negative fluorescent cell sorting of N-methyl-N'-nitro-N-nitrosoguanidine pretreated cells. Homologous cell fusions of six clones that complemented a previously described K562 mutant corresponding to one of the Thy-1- mutant classes (Hirose, S., Mohney, R. P., Mutka, S. C., Ravi, L., Singleton, D. R., Perry, G., Tartakoff, A., and Medof, M. E. (1992) J. Biol. Chem. 267, 5272-5278) showed that they segregated into two complementation groups. In heterologous cell fusions, representative clones of each group complemented Thy-1 expression by all of the previously described GPI anchor pathway-defective Thy-1- murine lymphoma classes (A, B, C, E, F, and H) but not class(es) D (and I) defective in the Thy-1 structural gene. Analyses of putative GPI anchor precursors synthesized by the two lines revealed that one mutant exhibited a complete block in deacetylation of N-acetyl-D-glucosamine-inositol phospholipid to glucosamine (GlcN)-inositol phospholipid, whereas the other mutant assembled GlcN-inositol phospholipid and subsequent mannose (Man)-containing intermediates but showed markedly increased amounts of the terminal ethanolamine (EthN)-phosphate (P)-substituted putative anchor precursors, EthN-P-6ManMan(EthN-P-->)ManGlcN- and EthN-P-6Man(EthN-P-6)Man(EthN- P-->)ManGlcN-acylinositol phospholipid (H7 and H8). We designate these new complementation classes J, harboring a defect in N-acetyl-D-glucosamine-inositol phospholipid deacetylation, and K, deficient in a step preliminary to or associated with protein transfer of assembled anchor precursors. The availability of these new mutant classes should aid in characterization of the GPI anchor pathway enzymes providing for these reactions.

Inositol Glycan Phosphate Derived from Human Erythrocyte Acetylcholinesterase Glycolipid Anchor and Inositol Cyclic 1,2-Phosphate Antagonize Glucagon Activation of Glycogen Phosphorylase

In this study we examine the hypothesis that an inositol glycan phosphate can act similarly to insulin on intact cells. The inositol glycan phosphate used in this study (glycan alpha) was isolated previously from the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase and was shown to have the structure glycine-ethanolamine-PO4-Man-Man-(N,N-dimethylethanolamine-PO4)Man- (N,N-dimethyl)GlcN-inositol-PO4. The cellular response investigated was the glucagon-stimulated activation of glycogen phosphorylase in rat hepatocytes. When hepatocytes were incubated with 20 nM glucagon for 4 min, the ratio of phosphorylase a activity to total phosphorylase increased from a basal value of 0.49 +/- 0.02 to 0.82 +/- 0.03 (mean +/- SE, n = 15). Inclusion of either 100 nM insulin or 3-10 microM glycan alpha during the glucagon incubation significantly decreased the glucagon-stimulated activity ratio to 0.74 +/- 0.03 for either agent. Furthermore, hepatocyte preparations differed in their response to insulin and were divided into insulin-responsive and -resistant groups. Glycan alpha had a significant effect only in the insulin-responsive group for which the observed activity ratio for 10 microM glycan alpha plus glucagon (0.68 +/- 0.05) compared closely with that for insulin plus glucagon (0.70 +/- 0.04). For the insulin-resistant group, the activity ratio in the presence of 10 microM glycan alpha was 0.81 +/- 0.03, unchanged from the control with glucagon alone. Because glycan alpha contains an inositol phosphate group, the effect of inositol cyclic 1,2-phosphate on the glucagon-stimulated activity ratio was determined.(ABSTRACT TRUNCATED AT 250 WORDS)

A candidate mammalian glycoinositol phospholipid precursor containing three phosphoethanolamines

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.

Mannosamine inhibits the synthesis of putative glycoinositol phospholipid anchor precursors in mammalian cells without incorporating into an accumulated intermediate

Mannosamine (ManN) has been reported to inhibit the formation of Man alpha-1,2Man linkages and to prevent the anchoring of membrane proteins by glycoinositol phospholipids (GPIs). In this paper, the effect of ManN on the synthesis of putative GPI anchor precursors in mammalian cells was studied. Three different cell lines were analyzed: HeLa cells and Thy-1 negative lymphoma mutants B and F that accumulate two-mannosyl GPI (Man2GPI) and three-mannosyl GPI (Man3GPI) species, respectively. ManN did not alter the incorporation of [3H]Man into the Man2GPI in mutant B cells. However, when either HeLa cells or mutant F cells were labeled with [3H]Man in the presence of ManN, [3H]Man incorporation into Man3GPi species was blocked and the TLC profile of [3H]Man-labeled glycolipids resembled that obtained with mutant B cells. Further characterization of the predominant labeled glycolipid that accumulated in ManN-treated HeLa cells by enzymatic and chromatographic criteria and permethylation analysis confirmed the structure as a Man2GPI indistinguishable from the Man2GPI that accumulates in mutant B cells. Based on previous reports that ManN inhibits Man alpha-1,2Man linkage formation and that in GPI-anchored proteins the third Man residue is attached by a Man alpha-1,2Man linkage, these results indicate that the third Man residue that distinguishes the putative Man2GPI precursor from the Man3GPI precursor also is attached by a Man alpha-1,2Man linkage. Furthermore, the observation that Man2GPI rather than a new GPI species containing ManN accumulated during ManN treatment revealed that the mechanism of ManN inhibition primarily involved inhibition of an alpha 1,2-mannosyltransferase rather than incorporation of ManN into an aberrant GPI species.

Time-dependent loss of surface complement regulatory activity during storage of donor blood.

The survival of transfused red cells (RBCs) diminishes with time of in vitro storage in blood banks, but the molecular mechanisms underlying the slow but incessant deterioration are incompletely understood. To investigate the possibility that impaired resistance to autologous complement attack could play a role in this phenomenon, packed RBCs stored for variable periods were assayed for decay-accelerating factor (DAF) and CD59, two glycoinositol-phospholipid (GPI)-anchored, membrane-associated complement regulatory proteins that function physiologically to protect blood cells from autologous complement activation on their surfaces. Immunoradiometric and flow cytometric assays employing DAF and CD59 monoclonal antibodies showed that levels of both surface proteins gradually declined over 6 weeks. Digestion analyses with phosphatidylinositol-specific phospholipase C, an enzyme that releases GPI-anchored proteins from cell surfaces, showed that DAF and CD59 molecules with GPI anchors containing unacylated inositol were preferentially lost. These findings suggest: 1) that DAF and CD59 molecules with acylated GPI anchors are more stable in RBC membranes than are molecules with unacylated GPI anchors, and 2) that DAF and CD59 loss may participate with other membrane alterations that occur during in vitro storage in compromising the survival of transfused cells.

Regulation of glycoinositol phospholipid anchor assembly in human lymphocytes. Absent mannolipid synthesis in affected T and natural killer cell lines from paroxysmal nocturnal hemoglobinuria patients.

Glycoinositol phospholipid (GPI) anchor structures derive from sequentially glycosylated inositol phospholipid precursors assembled in the endoplasmic reticulum. To characterize GPI biosynthesis in nontransformed human lymphocytes and to define the GPI synthetic defect underlying deficient expression of GPI-anchored proteins by paroxysmal nocturnal hemoglobinuria (PNH) cells, putative intracellular GPI intermediates were analyzed following [3H]Man labeling of normal and affected lymphocytes. In unstimulated normal peripheral blood lymphocytes, [3H]Man incorporation into GPIs was minimally detectable but after phytohemagglutinin (PHA), allogeneic cell, or anti-CD3 stimulation, assembly of [3H]Man-labeled GPIs markedly increased. Expression of GPIs by prestimulated quiescent PHA blasts could be efficiently induced by phorbol 12-myristate 13-acetate (PMA) and increased by the Ca2+ ionophore A23187 independently of new protein synthesis. Utilizing allogeneically stimulated cells in conjunction with PMA induction, products deriving from [3H]Man labeling of affected CD48- T and natural killer lymphocyte cell lines from five PNH patients were compared to those deriving from unaffected CD48+ cell lines from the same patients or controls. In contrast to unaffected paired control cells, affected cells of all of the patients exhibited a common abnormality in which they assembled dolichol-phosphoryl-Man but failed to express [3H]Man-containing GPIs. These data indicate that 1) significant GPI production in lymphocytes is dependent on prior stimulation of the cells, 2) exposure of lymphocytes to agents which activate protein kinase C induces GPI synthesis, and 3) in five PNH patients affected lymphocytes are uniformly defective in an early GPI biosynthetic step which undermines expression of GPI mannolipids.

Release of the cellular prion protein from cultured cells after loss of its glycoinositol phospholipid anchor.

Secreted forms of the sialoglycoprotein designated cellular prion protein (PrPC) have been identified that cannot be explained by alternative splicing. We report that secreted forms of PrPC derive from precursors that are bound to the plasma membrane by glycoinositol phospholipid (GPI) anchors. Secreted PrPC slowly appeared in the culture medium of metabolically radiolabelled cells after incubations of 8-24 h. Digestion of nascent PrPC with phosphatidylinositol-specific phospholipase C (PIPLC) prevented the appearance of secreted PrPC. Secreted PrPC partitioned into the aqueous phase of Triton X-114 like PrpC-released PrPC. While the M(r) of PIPLC-released PrPC was reduced 2-4 kDa after treatment with aqueous hydroflouric acid, which removes the entire GPI anchor modification, the M(r) of secreted PrPC was unchanged. Both PIPLC-released and secreted PrPC were recognized by antiserum raised against a synthetic C-terminal peptide corresponding to residues 220-233 (amino acid 231 is the site of GPI attachment). We conclude that GPI-anchored PrPC is post-translationally processed to remove most, if not all, of the GPI modification and then shed into culture medium. Whether PrPC is shed after proteolysis near the C-terminus remains to be established. A minority of PrPC in normal Syrian hamster brain partitioned into the aqueous phase of Triton X-114 like shed PrPC, suggesting physiological significance.

Affected paroxysmal nocturnal hemoglobinuria T lymphocytes harbor a common defect in assembly of N-acetyl-D-glucosamine inositol phospholipid corresponding to that in class A Thy-1- murine lymphoma mutants.

Deficient expression of glycoinositol phospholipid (GPI) anchored proteins in affected paroxysmal nocturnal hemoglobinuria (PNH) cells has been traced to a defect in GPI anchor assembly. In a previous study (Schubert, J., Schmidt, R. E., and Medof, M. E. (1993) J. Biol. Chem., in press) we characterized the biosynthesis of putative Man-containing GPI anchor precursors in normal peripheral blood lymphocytes and investigated assembly of these intracellular GPI intermediates in CD48- affected and CD48+ unaffected T and natural killer cell lines of PNH patients. We found that affected T cells from five patients exhibited a uniform defect in which dolichol-phosphoryl-Man was synthesized but no GPI mannolipids were expressed. In this study, membranes of patients' affected T cells were labeled with UDP-[3H]GlcNAc to evaluate earlier steps in GPI synthesis, and intact cells were fused to Thy-1- murine lymphoma mutants harboring different defects in early GPI assembly to test for the presence of corresponding or complementary lesions. In all cases, affected cell membranes failed to assemble GlcNAc-inositol phospholipid, the initial precursor of GPI anchor structures, and the intact cells failed to complement class A mutants while complementing other classes. Affected polymorphonuclear leukocytes from three additional patients of different origin were then labeled with [3H]Man and the labeling patterns found to correspond to those obtained with the T lymphocytes. Taken together the data indicate that the genetic lesion in PNH cells resides in a DNA element which: 1) encodes a product required for the synthesis of GlcNAc-inositol phospholipid, 2) corresponds to that altered in class A Thy-1- murine lymphoma mutants, and 3) is commonly affected in different patients.

Characterization of putative glycoinositol phospholipid anchor precursors in mammalian cells. Localization of phosphoethanolamine.

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.

Glycan components in the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase. Novel fragments produced by trifluoroacetic acid.

Inositol glycans were prepared from reductively radiomethylated human erythrocyte acetylcholinesterase by sequential treatment with Proteinase K, methanolic KOH, and phosphatidylinositol-specific phospholipase C. Four glycans denoted alpha-delta were resolved by anion exchange high performance liquid chromatography (HPLC). Each glycan was subjected to hydrolysis in 4 M trifluoroacetic acid, and their hexose and hexose phosphate compositions were determined by anion exchange HPLC. The predominant glycan alpha showed a relative stoichiometry of 2 mannoses, 1 mannose 6-phosphate, 1 radiomethylated glucosamine, 1 radiomethylated ethanolamine, and 1 inositol. In contrast, the stoichiometry of glycan beta was 1 mannose, 2 mannose 6-phosphates, 1 radiomethylated glucosamine, 2 radiomethylated ethanolamines, and 1 inositol. Glycans alpha and beta were analyzed by electrospray ionization-mass spectrometry, and respective parent ions of m/z 1266 and 1417 were observed. The fragmentation pattern produced by collision-induced dissociation mass spectrometry of these parent ions was consistent with a common linear core glycan sequence prior to radiomethylation of ethanolamine-phosphate-mannose - mannose - mannose - glucosamine - inositol. Glycan alpha contained a single additional radiomethylated phosphoethanolamine branching from the mannose adjacent to glucosamine, whereas glycan beta contained two additional radiomethylated phosphoethanolamines, one branching from each of the mannoses nearest to glucosamine. Trifluoroacetic acid hydrolysis did not cleave within the N,N-dimethylglucosamine-inositol-phosphate moiety in these glycans, and this component was resolved by anion exchange HPLC and structurally confirmed by mass spectrometry. Dephosphorylation of this component by treatment with 50% HF produced N,N-dimethylglucosamine-inositol, and this conjugate was shown to have a characteristic elution time on cation exchange chromatography in an amino acid analyzer. Both of these fragments involving an intact radiomethylated glucosamine-inositol bond are proposed as new diagnostic indicators in the search for minor glycoinositol phospholipids in cells and tissues.

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.

Folate binding protein from kidney brush border membranes contains components characteristic of a glycoinositol phospholipid anchor.

A number of cell surface proteins have been shown to be anchored to the plasma membrane by a covalently attached glycoinositol phospholipid (GPL) in amide linkage to the C-terminus of the mature protein. We applied several criteria to establish that folate binding protein (FBP) in brush border membranes of rat kidney contains a GPL anchor. Brush border membranes were isolated and labeled with [3H]folate, and the complex of FBP and [3H]folate was shown to be released to the supernatant by incubation with purified bacterial phosphatidylinositol-specific phospholipase C (PIPLC) but not by incubation with a purified bacterial phosphatidylcholine-specific phospholipase C. The FBP-[3H]folate complex both in crude extracts and after FBP purification by ligand-directed affinity chromatography interacted with Triton X-114 micelles, and prior incubation with PIPLC prevented this detergent interaction. Individual residues characteristic of GPL anchors were found to be covalently associated with FBP following polyacrylamide gel electrophoresis in sodium dodecyl sulfate. These included glucosamine and ethanolamine, which were radiolabeled by reductive methylation and identified by chromatography on an amino acid analyzer, and inositol phosphate, which was inferred by Western blotting with an anti-CRD antisera. This antisera gave positive immunostaining only after FBP had been cleaved by PIPLC, a reliable diagnostic of a GPL anchor. The relationship between GPL-anchored FBP in biological membranes and soluble FBP in biological fluids also is discussed.