Sequence analysis of the Rhop-3 gene of Plasmodium yoelii.

The 110 kDa/Rhop-3 rhoptry protein of Plasmodium falciparum is non-covalently associated with two other proteins, the 140 kDa Rhop-1 and the 130 kDa Rhop-2. cDNAs encoding Rhop-3 from Plasmodium yoelii were isolated using rhoptry-specific antisera from Plasmodium falciparum, P. yoelii, and Plasmodium chabaudi. The cDNAs encoded peptides with partial homology to the C-terminal region (residues 541-861) of P. falciparum Rhop-3. Core regions of homology to the P. falciparum gene will be useful in determining the biological role of Rhop-3 and its potential as a vaccine candidate for malaria.

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.

Rapid analysis of glycolipid anchors in amphiphilic dimers of acetylcholinesterases.

1. We describe two simple procedures for the rapid identification of certain structural features of glycolipid anchors in acetylcholinesterases (AChEs). 2. Treatment with alkaline hydroxylamine (that cleaves ester-linked acyl chains but not ether-linked alkyl chains) converts molecules possessing a diacylglycerol, but not those with an alkylacylglycerol, into hydrophilic derivatives. AChEs in human and bovine erythrocytes possess an alkylacylglycerol (Roberts et al., J. Biol. Chem. 263:18766-18775, 1988; Biochem. Biophys. Res. Commun. 150:271-277, 1988) and are not converted to hydrophilic dimers by alkaline hydroxylamine. Amphiphilic dimers of AChE from Drosophila, from mouse erythrocytes, and from the human erythroleukaemia cell line K562 also resist the treatment with hydroxylamine and likely possess a terminal alkylacylglycerol. This indicates that the cellular pool of free glycolipids used as precursors of protein anchors is distinct from the pool of membrane phosphatidylinositols (which contain diacylglycerols). 3. Pretreatment with alkaline hydroxylamine is required to render the amphiphilic AChE from human erythrocytes susceptible to digestion by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC) (Toutant et al., Eur. J. Biochem. 180:503-508, 1989). We show here that this is also the case for the AChE from mouse erythrocytes, which therefore likely possesses an additional acyl chain in the anchor that prevents the action of PI-PLC. 4. In two sublines of K562 cells (48 and 243), we observed that AChE either was directly susceptible to PI-PLC (243) or required a prior deacylation by alkaline hydroxylamine (48). This suggests that glycolipid anchors in AChE of K562-48 cells, but not those in AChE of K562-243 cells, contain the additional acylation demonstrated in AChE from human erythrocytes. These observations illustrate the cell specificity (and the lack of species-specificity) of the structure of glycolipid anchors.

Molecular forms of acetylcholinesterase in two sublines of human erythroleukemia K562 cells. Sensitivity or resistance to phosphatidylinositol-specific phospholipase C and biosynthesis.

Acetylcholinesterase (AChE) in K562 cells exists in two molecular forms. The major form, an amphiphilic dimer (G2a) which sediments at 5.3 S, and the minor form, an amphiphilic monomer (G1a) which sediments at 3.5 S. Extraction in the presence of the sulfhydryl alkylating agent N-ethylmaleimide was required to preserve the G2a form. In Triton X-100 extracts of the subline K562-243, phosphatidylinositol-specific phospholipase C (PtdIns-PLC) from Bacillus thuringiensis converted most of the G2a AChE into a hydrophilic dimer (G2h), indicating that the G2a form possessed a hydrophobic glycoinositol phospholipid that mediated its attachment to the membrane. Treatment of intact K562-243 cells with PtdIns-PLC released approximately 60% of the total AChE activity and provided an estimate of the externally exposed AChE. The direct conversion from an amphiphilic to a hydrophilic dimeric form by PtdIns-PLC was not obtained in extracts or intact cells of the subline K562-48. Instead, pretreatment with alkaline hydroxylamine was necessary to render the amphiphilic G2 form of this subline susceptible to digestion by the phospholipase. In this respect, the amphiphilic dimer of K562-48 AChE resembles the G2a form of human erythrocyte AChE, which is resistant to PtdIns-PLC because of the direct palmitoylation of an inositol hydroxyl group in the anchor [Roberts et al. (1988) J. Biol. Chem. 263, 18766-18775]. Release of this acyl chain by hydroxylamine renders the enzyme susceptible to PtdIns-PLC [Toutant et al. (1989) Eur. J. Biochem. 180, 503-508]. In both K562 sublines, sialidase decreased the migration of the G2a form but not of the G1a form of AChE. G1a forms thus appear to represent an intracellular pool of newly synthesized molecules residing in a compartment proximal to the trans-Golgi apparatus. The sialidase-resistant G1a molecules were also resistant to PtdIns-PLC digestion; possible explanations for this resistance are presented.

Biosynthetic incorporation of [3H]ethanolamine into protein synthesis elongation factor 1 alpha reveals a new post-translational protein modification.

Biosynthetic incorporation of [3H]ethanolamine into proteins was assessed in the human erythroleukemia cell line K562. A single predominant labeled protein of about 50 kDa was observed following electrophoresis of cell extracts on polyacrylamide gels in the presence of sodium dodecyl sulfate. Subcellular fractionation showed this protein to distribute similarly to a 46-kDa [3H]ethanolamine-labeled protein reported previously (Tisdale, E. J., and Tartakoff, A. M. (1988) J. Biol. Chem. 263, 8244-8252). In particular, the protein was enriched in cytosolic and microsomal fractions relative to plasma membrane and thus did not appear to correspond to the class of proteins with glycoinositol phospholipid anchors, the only post-translational protein modification involving ethanolamine that had been described previously. Two-dimensional polyacrylamide gel analysis involving isoelectric focusing followed by electrophoresis in sodium dodecyl sulfate indicated that the protein was very basic, and nitrocellulose blots of one- and two-dimensional gels subjected to 3H autoradiography and immunostaining with antisera to purified rabbit elongation factor (EF) 1 alpha revealed that the protein was EF-1 alpha. Copurification of rabbit EF-1 alpha and the [3H]ethanolamine-labeled protein from K562 cells further supported this identification. Analysis of tryptic fragments produced from the copurified proteins by reverse-phase high pressure liquid chromatography showed two radiolabeled peptides. Amino acid analysis demonstrated 1 residue of ethanolamine in each peptide, and peptide sequencing revealed that the ethanolamine-containing component(s) was attached to Glu301 and Glu374 in the EF-1 alpha protein sequence deduced from a human EF-1 alpha cDNA. These data confirm a new class of post-translational protein modifications involving ethanolamine.