Plasmodium falciparum GPI mannosyltransferase-III has novel signature sequence and is functional

Abstract

The glycosylphosphatidylinositol (GPI) anchors of Plasmodium falciparum are indispensable for parasite survival since merozoite surface proteins-1, -2, -4, -5 and -10 crucial for erythrocyte invasion are GPI-anchored. Therefore, the GPI biosynthetic pathway can offer potential targets for novel antimalarial drugs. Here, we characterized the putative P. falciparum PIG-B gene (PfPIGB) that encodes mannosyltransferase-III of GPI biosynthesis. PfPIGB mRNA is transcribed in a developmental stage specific manner. A protein corresponding to the expected size of PfPIG-B is expressed by the parasite and is localized in the endoplasmic reticulum. Treatment of parasites with PfPIG-B specific siRNA caused reduced GPI synthesis, affecting the PIG-B specific GPI intermediate. These data demonstrate that PfPIG-B is functional and encodes for mannosyltransferase-III of the parasite GPI biosynthesis. The parasite PfPIG-B is novel in that its signature sequence HKEHKI is unique and is only partially conserved compared to HKEXRF signature motif of mammalian PIG-B enzymes. KeywordsPlasmodium falciparum; glycosylphosphatidylinositol anchors; biosynthesis; mannosyltransferase-III; characterization Malaria caused by Plasmodium falciparum is a major health problem in many countries. About 40% of the global population is at risk to malaria, and in sub-Saharan Africa, millions of deaths, mostly among children less than 5 years old, occur every year [1]. The death toll due to malaria has been rapidly increasing because of drug resistance. Development of novel drugs targeting the essential metabolic pathways of the parasite and therapeutics/vaccine that prevent the infection and/or illness are urgently needed. In this context, glycosylphosphatidylinositol (GPI) biosynthetic pathway can offer important targets [2].The GPIs of all organisms have a conserved glycan core, Manα1-2Manα1-6Manα1-4GlcNα1, covalently linked to myo-inositol of phosphatidylinositol (PI) at O-6 [3]. The mannose-3 of the glycan core is invariably substituted at O-6 with EtN-P, which is used for membrane anchoring of proteins through amide bond formation with the -COOH of the protein C-termini. GPIs from different organisms vary in the PI moiety in having acylated or nonacylated myo-inositol, and diacylglycerols, monoacylglycerols, alkyl/acylglycerols or ceramide [3]. In the case of P. falciparum GPIs, the conserved glycan core is substituted with an additional Man at O-2 of the third Man, and the PI comprised of diacylglycerol and O-2-acylated inositol moieties, both with variable fatty acids [4]. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe biosynthesis of GPIs occurs in the ER by the sequential addition of sugars to the PI by a coordinated action of glycosyltransferases, N-deacetylase, inositol acylase, EtN-P transferase, and several accessory proteins [5]. The preassembled GPIs are transferred enbloc to the Ctermini of proteins that have GPI attachment signal sequence. In animals, hundreds of proteins including cell surface re...

Effect of GPI anchor moiety on the immunogenicity of DNA plasmids encoding the 19‐kDa C‐terminal portion of Plasmodium falciparum MSP‐1

“…Culturing of P. falciparum , and isolation of parasites and parasite total RNA were performed as reported previously (22). The parasite RNA was reverse transcribed, using a oligo (dT) [12][13][14][15][16][17][18] primer and Superscript II reverse transcriptase (Invitrogen), as described (23).…”

Glycosylphosphatidylinositol Mannosyltransferase Ⅰ Protects Chinese Giant Salamander, Andrias davidianus, against Iridovirus

“…GPI-MT-I, a mannosyltransferase required for the addition of the first mannose, is often used as a target for specific inhibitors of GPI-AP biosynthesis [ 24 , 25 ]. In mammals, GPI-MT-I contains a GPI transamidase subunit PIG-U domain and a mannosyltransferase domain (PIG-M), which together form a PMT2 superfamily domain [ 9 , 26 , 27 ]. PIG-U is thought to represent the fifth subunit in this complex and may be involved in the recognition of either the GPI attachment signal or the lipid portion of GPI [ 28 , 29 ].…”

“…The purified GPI-AP MHC class I molecule HLA-A2.1 delivers a hepatitis B virus antigen peptide to the surface of a cytotoxic T cell target, which activates specific T cells [ 8 ]. The biosynthesis of GPI-APs occurs in the ER by the sequential addition of sugars to the phosphatidylinositol through the coordinated activity of several proteins, among which glycosylphosphatidylinositol mannosyltransferase I (GPI-MT-I) is one of the most essential mannosyltransferases [ 9 , 10 ].…”

“…GPI-APs play a crucial role during pathogen invasion, and the essential mannosyltransferase GPI-MT-I during its biosynthetic pathway might be a reasonable target for the development of antipathogen drugs [ 9 , 11 ]. As an essential enzyme for adding mannose on the glycosylphosphatidyl group, protozoan mannosyltransferase (GPI-14) is a key enzyme in the biosynthesis of lipophosphoglycan (LPG) and glycoinositolphospholipids (GIPLs), which play important roles in the parasite infectious cycle [ 12 ].…”

The GPI sidechain ofToxoplasma gondiiprevents parasite pathogenesis

“…The HA tag was visualized by fluorescent microscopy. 8x10 5 HFFs were plated on coverslips in 24-well plates overnight before being infected with 8x10 4 parasites overnight. Cells were fixed with 3% formaldehyde and permeabilized with blocking buffer (1X PBS with 10% normal goat serum, and 0.01% saponin) and stained with rat anti-HA (1:50) (3F10, Sigma).…”