Myristate exchange in glycolipid A and VSG of African trypanosomes

The variant surface glycoprotein (VSG) of T. brucei is anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor which is unique in that its fatty acids are exclusively myristate (a fourteen carbon saturated fatty acid). We showed that the myristate is added to the GPI precursor in a remodeling reaction involving deacylation and reacylation. We now demonstrate that trypanosomes have a second pathway of myristoylation for GPI anchors that we call "myristate exchange" which is distinct from the fatty acid remodeling pathway. We propose that this is an exchange of [3H]myristate into both sn-1 and sn-2 positions of glycolipid A, which already contains myristate, and have demonstrated this using inhibitors and a variety of other methods. We have partially characterized myristate exchange with respect to specificity and susceptibility to some inhibitors. The apparent Km for myristoyl CoA is 7 nM. This myristate-specific process may represent a proof-reading system to ensure that the fatty acids on VSG are exclusively myristate. Although myristate exchange was first discovered for glycolipid A, we now believe that VSG is the true substrate of this reaction. VSG is efficiently labeled by exchange in the presence of cycloheximide, which prevents anchoring of newly synthesized protein. Although its location is not yet know, we have evidence that exchange does not localize to either the endoplasmic reticulum or the plasma membrane. We will present data indicating that surface VSG may be internalized and undergo myristate exchange

The role of glycolipid C in the GPI biosynthetic pathway in Trypanosoma brucei bloodstream forms

The glycosylphosphatidylinositol (GPI) biosynthetic pathway in Trypanosoma brucei bloodstream forms includes the formation of glycolipid C. This molecule is the inositol-acylated form of the GPI anchor precursor, glycolipid A. There is no evidence for the transfer of glycolipid C to protein in vivo and the role of glycolipid C is unclear. In this paper we show that glycolipid C is not synthesised in the presence of phenylmethylsulphonyl fluoride (PMSF) and that glycolipid C is not an obligatory intermediate on the pathway to the formation of glycolipid A. Using pulse-chase experiments we show that glycolipid A and glycolipid C are in a dynamic equilibrium and we suggest that only the forward reaction (glycolipid A conversion to glycolipid C) is inhibited by PMSF

Expression cloning of genes for GPI-anchor biosynthesis

Cloning genes for glycosylphosphatydilinositol (GPI)-anchor biosynthesis is important to further understand its mechanisms and regulation. We have been using expression cloning methods in which a cDNA library was transfected into GPI-anchor-deficient mutant cells. The transfectants which restored surface expression of GPI-anchored proteins were isolated and the plasmids were rescued. In this way we previously cloned cDNAs of genes for complementation classes A and F, and named them PIG-A and PIG-F, respectively. In the present study we have cloned the gene for class B, termed PIG-B. In each case we used different methods. For cloning PIG-A cDNA we used a cDNA library made with an Epstein-Barr-virus-based vector and human class A mutant JY5 which expresses EBNA-1 protein. The EBNA-1 protein allows stable replication of oriP-containing plasmids in the episomal form. For cloning PIG-F cDNA we chose a transient expression method and cotransfected a human T-cell cDNA library made with a vector bearing an origin of replication of polyoma virus with a plasmid bearing polyoma virus large T into the class F murine thymona mutant. This cotransfection strategy was unsuccessful for cloning PIG-B due to low transfection efficiency of the class B thymoma mutant SIA-b. Thus, we first established large T-expressing SIA-b cells and then transfected them with cDNA library. PIG-B cDNA restored the surface expression of Thy-1 on SIA-b cells and also synthesis of mature type GPI-anchor precursors in these cells. The cDNA consists of 1929 bp and codes for a putative new protein of 554 amino acid residues

Isolation of virulence genes directing GPI synthesis by functional complementation of Leishmania

Recent advances in molecular genetics of Leishmania parasites prompted us to develop methods of functional genetic complementation in Leishmania and apply them to the isolation of genes involved in the biosynthesis of the virulence determinant LPG, an abundant GPI-anchored polysaccharide. LPG1, the gene product identified by complementation of our R2D2 LPG- mutant, may be a glycosyltransferase responsible for the addition of galactofuranose to the nascent chain. As galactofuranose is not found in mammalian cells, inhibition of the addition of this sugar could be exploited for chemotherapy. Overall, the success of the functional complementation approach opens the way to the identification of a variety of genes involved in pathogenesis and parasitism

The developmental regulation and biosynthesis of GPI-related structures in Leishmania parasites

Most macromolecules on the surface of Leishmania parasites, including the major surface proteins and a complex lipophosphoglycan (LPG) are anchored to the plasma membrane via GPI glycolipids. Free glycoinositol-phospholipids (GIPLs) which are not linked to protein or phosphoglycan are also abundant in the plasma membrane. From structural and metabolic labeling studies it is proposed that most Leishmania species express three distinct pathways of GPI biosynthesis. Some of these pathways (i.e those involved in the protein and LPG anchor biosynthesis) are down-regulated during the differentiation of the insect (promastigote) stage to the mammalian (amastigote) stage. In contrast, the GIPLs are expressed in high copy number in both developmental stages. Based on analysis of the lipid moieties of the different GPI species it is possible that the pathways of GPI anchor and GIPL biosynthesis are located in different subcellular compartments. The relative flux through the GIPL and LPG biosynthetic pathways has been examined in L. Major promastigotes. These studies showed that while the rate of synthesis of the GIPLs and LPG is similar, LPG is shed more rapidly from the plasma membrane and has a higher turnover. The possible metabolic relationship between the GIPL and LPG biosynthetic pathways is discussed

Isolation of temperature-sensitive yeast GPI-anchoring mutants

We are using a genetic approach to explore the synthesis and function of glycosylphosphatidylinositol (GPI). We have developed a novel strategy to isolate Saccharomyces cerevisiae mutants blocked in GPI anchoring by screening colonies of mutagenized yeast cells for those that fail to incorporate [3H]inositol into protein. Among our isolates are strains blocked in mannosylation of the GPI-anchor precursor, and strains defective in the synthesis of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI). We have characterized one mutant, gpil, further. This strain is defective in GlcNAC-PI synthesis and is temperature-sensitive for growth. Completion of the first step in GPI assembly is therefore required for the growth of the unicellular eukaryote S. cerevisiae. We have isolated plasmids that complement the gpil mutation from S. cerevisiae genomic DNA- and fission yeast cDNA libraries

Metabolism of GPIs in mammalian cells

In Trypanosoma brucei, glycosylphosphatidylinositol (GPI) anchors of proteins and free GPIs with identical structures have been characterized. This identity provides strong presumptive evidence that the free GPIs are in fact precursors of the GPI anchors on proteins. In mammalian tissues, however, rather consistent differences in the structures of free GPIs and GPI anchors are observed. The terminal GPIs produced by the mammalian biosynthetic pathway differ from GPI anchors in being almost exclusively fatty acid acylated on the inositol residue, having a greater number of phosphoethanolamine residues, and perhaps in containing a greater percentage of diacylglycerol components. While in principle these differences could be reconciled by remodeling reactions before or after attachment of GPI anchors, it is possible that some of the mammalian free GPIs play cellular roles other than as anchor precursors. We have approached this question by studying the lifetimes of the last three GPIs on the biosynthetic pathway, denoted H6, H7 and H8, in K562 cells and in K562 mutant designated class K that is devoid of GPI-anchored proteins. Pulse-chase metabolic labeling with [3H]-mannose indicated that H6 was a precursor of H7 and H8 and that the H8 lifetime was more than one hour in the parental cells and even longer in the mutant. Preliminary data indicated that the majority of each of the three GPIs was localized in the plasma membrane fraction rather than the endoplasmic reticulum. These observations argue that mammalian GPIs are not utilized exclusively as GPI anchor precursors

GPI biosynthesis in mammalian cells: CoA-dependent acylation of GlcN-PI

We have used microsomes prepared from murine lymphoma cell lines to investigate the individual reactions by glycosylphosphatidylinositol (GPI) is synthesized in mammalian cells. Previously, GTP was found to specifically stimulate the second reaction in the pathway, the deacetylation of GlcNAc-PI to GlcN-PI. An additional GPI precursor was detected in incubations with GTP and was found to be GlcN-PI(acyl), the glycolipid proposed to be the third intermediate in mammalian GPI biosynthesis. Investigation into the factors that affect the formation of GlcN-PI(acyl) revealed that, in the presence of GTP, the addition of either CoA or palmitoyl-CoA to the incubation greatly enhanced the amount of this product made. CoA stimulation of this reaction persisted even when ATP was depleted and no formation of acyl-CoA was possible, indicating that the free CoA rather than an acyl-CoA is the actual effector of GlcN-PI acylation. Therefore, we propose that the third reaction in mammalian GPI biosynthesis is catalyzed by a CoA-dependent transacylase rather than an acyl-CoA acyltransferase

Topology of GPI biosynthesis in the endoplasmic reticulum

Glycosylphosphatidylinositol (GPI) anchors are constructed in the endoplasmic reticulum (ER) through the action of at least seven unique enzymes. Using cell-free systems, mainly derived from African trypanosomes, it has been experimentally possible to re-create many aspects of the GPI biosynthetic pathway in vitro and to obtain a series of glycosylated phosphatidylinositol structures that correspond to biosynthetic intermediates. This approach led to the identification of the biosynthetic donors of individual components of the GPI glycan, and the discovery of unusual fatty acid re-modelling reactions in the GPI pathway in trypanosomes. Despite this progress, questions remain concerning the enzymology of the pathway, particularly the topological distribution of the different assembly steps in the ER membrane. In the work described here we have attempted to define the transbilayer orientation of different GPI biosynthetic intermediates in the ER membrane bilayer. The experiments were performed with a microsomal fraction derived from bloodstream-form Trypanosoma brucei, and standard radiolabeling procedures. The orientation of GPIs was probed with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and the jackbean lectin Concanavalin A. Contrary to expectations based on other ER glycosylation reactions, most notably the reactions involved in the dolichol pathway of N-glycosylation, our results suggest that non-inositol-acylated (PI-PLC-sensitive) GPIs are synthesized in the cytoplasmic leaflet of the ER membrane bilayer and that the final reaction product, a phosphoethanolamine-containing GPI, flips into the luminal leaflet for transfer to protein

GPI phospholipase C from Trypanosoma brucei causes a GPI-negative phenotype in Leishmania major: I. Implications for GPI-negative mammalian cells; II. Compartmentalization of two GPI biosynthetic pathways

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide) are glycosylphosphatidylinositol (GPI)-anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPIPLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPIPLC cell-associated gp63 could not be detected in immunoblots, gp63 was secreted into the culture medium without ever receiving a GPI anchor. Putative protein-GPI intermediates LP-1 and LP-2 decreased about 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. We conclude that reactions specific to the polysaccharide-GPI pathway are compartmentalalized within the endoplasmic reticulum, thereby sequestering those intermediates from GPIPLC cleavage. Protein-GPI synthesis, at least up to production of Man (1Ó6)Man(1Ó4)GlcN(1Ó6)-myo-inositol-1-phospholipid, is cystolic. To our knowledge, this represents the first use of a catabolic enzyme, in vivo, to elucidate the topography of biosynthetic pathways. Intriguingly, the phenotype of GPIPLC-expressing L. major, secretion of proteins with GPI addition signals, and depletion of protein-GPI anchor precursors, is similar to that of some protein-GPI mutants in higher eukaryotes. These findings have implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma

Biochemical analysis of glycophospholipids from wild-type mouse lymphoma cells and from Thy-1 negative mutants which do not add glycolipid-anchored proteins.

Thy-1 is a major glycophospholipid (GPL)-anchored protein found on the surface of neurons, epithelial cells, fibroblasts and murine T-lymphomas. Biochemical studies were undertaken to determine if murine T-lymphomas contain glycolipids which may be on the path of GPL-anchor biosynthesis. Biosynthetic labeling experiments on Thy-1-positive (wild-type) cells followed by battery of chemical and enzymatic diagnostics on the isolated [3H]mannolipids have, for the first time, led to description of a set of glycolipids which have properties consistent with their being GPL-anchor precursors. Using these results as a guide, major differences have been observed upon analysis of the radiolabeled mannolipids of Thy-1-negative mutants from 7 complementation classes, A-C, E, F, H and I. The biosynthetic lesions in anchor synthesis have been identified in some of the mutants.