Glycosylphosphatidylinositol Signal Sequence Research Articles

Nanoparticle formulation enhanced protective immunity provoked by PYGPI8p-transamidase related protein (PyTAM) DNA vaccine in Plasmodium yoelii malaria model

We have previously reported the new formulation of polyethylimine (PEI) with gamma polyglutamic acid (γ-PGA) nanoparticle (NP) to have provided Plasmodium yoelii merozoite surface protein-1 (PyMSP-1) plasmid DNA vaccine with enhanced protective cellular and humoral immunity in the lethal mouse malaria model. PyGPI8p-transamidase-related protein (PyTAM) was selected as a possible candidate vaccine antigen by using DNA vaccination screening from 29 GPI anchor and signal sequence motif positive genes picked up using web-based bioinformatics tools; though the observed protection was not complete. Here, we observed augmented protective effect of PyTAM DNA vaccine by using PEI and γ-PGA complex as delivery system. NP-coated PyTAM plasmid DNA immunized mice showed a significant survival rate from lethal P. yoelii challenge infection compared with naked PyTAM plasmid or with NP-coated empty plasmid DNA group. Antigen-specific IgG1 and IgG2b subclass antibody levels, proportion of CD4 and CD8T cells producing IFN-γ in the splenocytes and IL-4, IFN-γ, IL-12 and TNF-α levels in the sera and in the supernatants from ex vivo splenocytes culture were all enhanced by the NP-coated PyTAM DNA vaccine. These data indicates that NP augments PyTAM protective immune response, and this enhancement was associated with increased DC activation and concomitant IL-12 production.

Retrotranslocation of Prion Proteins from the Endoplasmic Reticulum by Preventing GPI Signal Transamidation

Neurodegeneration in diseases caused by altered metabolism of mammalian prion protein (PrP) can be averted by reducing PrP expression. To identify novel pathways for PrP down-regulation, we analyzed cells that had adapted to the negative selection pressure of stable overexpression of a disease-causing PrP mutant. A mutant cell line was isolated that selectively and quantitatively routes wild-type and various mutant PrPs for ER retrotranslocation and proteasomal degradation. Biochemical analyses of the mutant cells revealed that a defect in glycosylphosphatidylinositol (GPI) anchor synthesis leads to an unprocessed GPI-anchoring signal sequence that directs both ER retention and efficient retrotranslocation of PrP. An unprocessed GPI signal was sufficient to impart ER retention, but not retrotranslocation, to a heterologous protein, revealing an unexpected role for the mature domain in the metabolism of misprocessed GPI-anchored proteins. Our results provide new insights into the quality control pathways for unprocessed GPI-anchored proteins and identify transamidation of the GPI signal sequence as a step in PrP biosynthesis that is absolutely required for its surface expression. As each GPI signal sequence is unique, these results also identify signal recognition by the GPI-transamidase as a potential step for selective small molecule perturbation of PrP expression.

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

The glycosylphosphatidylinositol (GPI)-anchored Plasmodium falciparum merozoite surface protein 1 (MSP-1) is a widely studied malaria vaccine candidate. The C-terminal 19-kDa portion of MSP-1 (MSP-1(19)) is of particular interest because this polypeptide moiety remains bound to the parasite even after erythrocyte invasion, while the remainder of MSP-1 is shed during invasion. Studies have shown that antibodies against MSP-1(19) inhibit merozoite invasion of erythrocytes efficiently, and that MSP-1(19) produces protective immunity in mice and monkeys. To investigate the efficacy of MSP-1(19 )DNA vaccine and role of GPI anchor moiety in the immunogenicity of MSP-1(19), we constructed expression vectors that produce MSP-1(19) as either secretory or GPI-anchored polypeptide. Both constructs efficiently expressed MSP-1(19) in transfected HEK-293 cells. While the recombinant plasmid lacking GPI anchor signal sequence expressed MSP-1(19) mainly as secreted polypeptide, that containing GPI anchor signal sequence produced GPI-anchored MSP-1(19 )on cell surface. In immunized mice, both constructs produced substantial levels of MSP-1(19)-specific IgG1, IgG2a, IgG2b, IgG3, IgA and IgM antibodies. In both cases, the IgG1 level was significantly higher than other isotypes. Interestingly, the plasmid containing GPI anchor signal sequence produced significantly higher levels of IgG2a and IgG2b than the plasmid that lacks GPI signal sequence.

Expression and immunogenicity of the Plasmodium falciparum circumsporozoite protein: The role of GPI signal sequence

Previous studies have shown that the immunogenicity of rodent malaria parasite-derived circumsporozoite protein (CS) can be improved by deleting the glycosyl-phosphatidyl-inositol (GPI) signal sequence. To study whether GPI signal sequence deletion would also improve immunogenicity of CS derived from the major plasmodium species causing mortality in humans ( P. falciparum), we tested different variants of the P. falciparum CS protein in the context of a live vector-based vaccine carrier (rAd35). We demonstrate that deletion of the GPI signal sequence from CS did not result in altered expression or secretion. In contrast, cellular localization was clearly altered, which perhaps helps to explain the significant improvement of anti-CS antibody and T-cell responses observed in mice using deletion variants in the context of the rAd35 carrier. Our results show that rational design of antigens is warranted for further development of malaria vaccines.

Ethanolamine Phosphate Linked to the First Mannose Residue of Glycosylphosphatidylinositol (GPI) Lipids Is a Major Feature of the GPI Structure That Is Recognized by Human GPI Transamidase

Glycosylphosphatidylinositol (GPI) anchoring of proteins is catalyzed by GPI transamidase (GPIT), a multisubunit, endoplasmic reticulum (ER)-localized enzyme. GPIT recognizes ER-translocated proteins that have a GPI-directing C-terminal signal sequence and replaces this sequence with a preassembled GPI anchor. Although the GPI signal sequence has been extensively characterized, little is known about the structural features of the GPI lipid substrate that enable its recognition by GPIT. In a previous study we showed that mature GPIs could be co-immunoprecipitated with GPIT complexes containing functional subunits (Vainauskas, S., and Menon, A. K. (2004) J. Biol. Chem. 279, 6540-6545). We now use this approach, as well as a method that reconstitutes the interaction between GPIs and GPIT, to define the basis of the interaction between GPI and human GPIT. We report that (i) human GPIT can interact with GPI biosynthetic intermediates, not just mature GPIs competent for transfer to protein, (ii) the ethanolamine phosphate group on the third mannose residue of the GPI glycan is not critical for GPI recognition by GPIT, (iii) the ethanolamine phosphate residue linked to the first mannose of the GPI structure is a major feature of GPIs that is recognized by human GPIT, and (iv) the simplest GPI recognized by human GPIT is EtN-P-2Manalpha1-4GlcN-(acyl)-phosphatidyl-inositol. These studies define the molecular characteristics of GPI that are recognized by GPIT and open the way to identifying GPIT subunits that are involved in this process.

Generation of GPI-linked CCL5 based chemokine receptor antagonists for the suppression of acute vascular damage during allograft transplantation

Limiting the acute vascular damage associated with leukocyte infiltration is a central issue in solid organ transplantation. The family of chemotactic cytokines (chemokines) helps to regulate leukocyte recruitment. Systemic treatment with the chemokine ligand-5 (CCL5) based antagonist Met-RANTES has previously shown to suppress acute damage to transplanted kidneys by blocking effector cell recruitment. To address problems associated with systemic long-term administration of chemokine receptor antagonists, a chemokine based reagent was designed to be integrated into endothelial surfaces of the organ just before transplantation. Proteins anchored by glycosylphosphatidylinositol (GPI), when purified and added to cells, are efficiently incorporated into their cell surface membranes. A series of modifications were introduced into the CCL5 protein to generate a functional antagonist. These included the addition of an N-terminal methionine group, a mutation to render the protein a dimer and a GPI signal sequence for surface expression. The resultant protein was stably expressed in CHO cells, GPI anchorage was confirmed and the protein purified by FPLC. Exogenously administered Met-CCL5(dimer)-GPI was efficiently inserted into the membrane of microvascular endothelial cells. The reagent is being tested in murine models of renal transplantation. The effect on subsequent immune induced damage will be assessed.

Differential insertion of GPI‐anchored GFPs into lipid rafts of live cells

Partitioning of proteins in cholesterol and sphingolipid enriched plasma membrane microdomains, called lipid rafts, is critical for many signal transduction and protein sorting events. Although raft partitioning of many signaling molecules remains to be determined, glycosylphosphatidyl-inositol (GPI)-anchored proteins possess high affinity for lipid rafts and are currently exploited as markers to investigate fundamental mechanisms in protein sorting and signal transduction events. In this study, we demonstrate that two recombinant GPI-anchored green fluorescent proteins (GFP-GPIs) that differ in their GPI signal sequence confer distinct localization in plasma membrane microdomains. GFP fused to the GPI signal of the decay accelerating factor GFP-GPI(DAF) partitioned exclusively in lipid rafts, whereas GFP fused to the GPI signal of TRAIL-R3, GFP-GPI(TRAIL-R3), associated only minimally with microdomains. In addition, we investigated the unique ability of purified GFP-GPIs to insert into membrane microdomains of primary lymphocytes. This cell surface painting allows rapid, stable, and functional association of the GPI-anchored proteins with the target cell plasma membrane. The distinct membrane localization of the two GFP-GPIs was observed irrespective of whether the GPI-anchored molecules were painted or transfected. Furthermore, we show that painted GFP-GPI(DAF) was totally dependent on the GPI anchor and that the membrane insertion was increased by the addition of raft-associated lipids such as cholesterol, sphingomyelin, and dipalmitoyl-phosphatidylethanolamine. Thus, this study provides evidence that different GPI signal sequences lead to distinct membrane microdomain localization and that painted GFP-GPI(DAF) serves as an excellent fluorescent marker for lipid rafts in live cells.

Characterization of two novel proteins, NgRH1 and NgRH2, structurally and biochemically homologous to the Nogo-66 receptor.

Nogo-66 receptor (NgR) has recently been identified as the neuronal receptor of the myelin-associated proteins Nogo-A, oligodendrocyte protein (OMgp) and myelin-associated glycoprotein (MAG), and mediates inhibition of axonal regeneration both in vitro and in vivo. Through database searches, we have identified two novel proteins (NgRH1 and NgRH2) that turned out to be homologous in their primary structures, biochemical properties and expression patterns to NgR. Like NgR, the homologues contain eight leucine-rich repeats (LRR) flanked by a leucine-rich repeat C-terminus (LRRCT) and a leucine-rich repeat N-terminus (LRRNT), and also have a C-terminal GPI signal sequence. Northern blot analysis showed predominant expression of NgRH1 and NgRH2 mRNA in the brain. In situ hybridization and immunohistochemistry on rat brain slices revealed neuronal expression of the genes. NgRH1 and NgRH2 were detected on the cell surface of recombinant cell lines as N-glycosylated GPI anchored proteins and, consistent with other GPI anchored proteins, were localized within the lipid rafts of cellular membranes. In addition, an N-terminal proteolytic fragment of NgR comprising the majority of the ectodomain was found to be constitutively secreted from cells. Our data indicate that NgR, NgRH1 and NgRH2 constitute a novel receptor protein family, which may play related roles within the CNS.

Structural features affecting variant surface glycoprotein expression in Trypanosoma brucei.

The glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) of Trypanosoma brucei is the most abundant GPI-anchored protein expressed on any cell, and is an essential virulence factor. To determine what structural features affect efficient expression of VSG, we made a series of mutations in two VSGs. Inserting 18 amino acids, between the amino- and carboxy-terminal domains, reduced the expression of VSG 221 to about 3% of the wild-type level. When this insertion was combined with deletion of the single carboxy-terminal subdomain, expression was reduced a further three-fold. In VSG 117, which contains two carboxy-terminal subdomains, point mutation of the intervening N-glycosylation site reduced expression about 15-fold. Deleting the most carboxy-terminal subdomain and intervening region, including the N-glycosylation site, reduced expression to 15-20% of wild type VSG, and deletion of both subdomains reduced expression to <1%. Despite their low abundance, all VSG mutants were GPI anchored on the cell surface. Our results suggest that, for a protein to be efficiently displayed on the surface of bloodstream-form T. brucei, it is essential that it contains the conserved structural motifs of a T. brucei VSG. Serum resistance-associated protein (SRA), which confers human infectivity on T. brucei, strongly resembles a VSG deletion mutant. Expression of three epitope-tagged versions of SRA in T. brucei conferred total resistance to human serum. SRA possesses a canonical GPI signal sequence, but we were unable to obtain unequivocal evidence for the presence of a GPI anchor. SRA was not released during osmotic lysis, indicating that it is not GPI anchored on the cell surface.

Expression and purification of functional, recombinant Trypanosoma cruzi complement regulatory protein

The complement regulatory protein (CRP) of Trypanosoma cruzi is a developmentally regulated glycosylphosphatidylinositol (GPI)-anchored membrane protein that protects the parasite from complement-mediated killing, and is an important virulence determinant of the microorganism. CRP binds human complement components C3b and C4b to restrict activation of the complement cascade. Here, we report production of functional, recombinant T. cruzi CRP in mammalian cells and a one-step purification of the recombinant protein. Exchange of the crp DNA sequence encoding the carboxy-terminal GPI signal sequence with the corresponding sequence of decay accelerating factor (DAF) was necessary for recognition, cleavage, and addition of GPI in mammalian cells. CRP production was assessed in two mammalian cell lines with crp-daf gene expression driven by three different transcription control regions: Rous sarcoma virus long terminal repeat, cytomegalovirus (CMV) immediate early gene, and chicken β-actin promoter/CMV enhancer. We present evidence that CRP produced in transfected Chinese hamster Ovary (CHO) cells was functional and protected the cells from complement-mediated lysis. To facilitate purification of the recombinant protein, a hexahistidyl tag was incorporated at 3 ′ end of the cDNA upstream of the GPI anchor addition sequence. An additional histidine fusion construct was made that allowed for secretion and recovery of recombinant protein from culture supernatant fluid. Both membrane and secreted forms of the protein were purified in one step by nickel nitrilotriacetic acid. The production and purification of functionally active CRP in a non-infectious expression system will allow for structure and function studies aimed at identifying the active site(s) of this protein.

The glycosylphosphatidylinositol (GPI) signal sequence of human placental alkaline phosphatase is not recognized by human Gpi8p in the context of the yeast GPI anchoring machinery.

Biosynthesis of glycosylphosphatidylinositol (GPI)-anchored proteins involves the action of a GPI trans-amidase, which replaces the C-terminal GPI signal sequence (GPI-SS) of the primary translation product with a preformed GPI lipid. The transamidation depends on a complex of four proteins, Gaa1p, Gpi8p, Gpi16p and Gpi17p. Although the GPI anchoring pathway is conserved throughout the eukaryotic kingdom, it has been reported recently that the GPI-SS of human placental alkaline phosphatase (hPLAP) is not recognized by the yeast transamidase, but is recognized in yeast that contain the human Gpi8p homologue. This finding suggests that Gpi8p is intimately involved in the recognition of GPI precursor proteins and may also be responsible for the subtle taxon-specific differences in transamidase specificity that sometimes prevent the efficient GPI anchoring of heterologously expressed GPI proteins. Here, we confirm that the GPI signal sequence of hPLAP is indeed not recognized by the yeast GPI-anchoring machinery. However, in our hands, GPI attachment cannot be restored by the co-expression of human Gpi8p in yeast cells under any circumstances.

Mutational analysis of the variant surface glycoprotein GPI-anchor signal sequence in Trypanosoma brucei.

The variant surface glycoproteins (VSG) of Trypanosoma brucei are anchored to the cell surface via a glycosylphosphatidylinositol (GPI) anchor. All GPI-anchored proteins are synthesized with a C-terminal signal sequence, which is replaced by a GPI-anchor in a rapid post-translational transamidation reaction. VSG GPI signal sequences are extraordinarily conserved. They contain either 23 or 17 amino acids, a difference that distinguishes the two major VSG classes, and consist of a spacer sequence followed by a more hydrophobic region. The omega amino acid, to which GPI is transferred, is either Ser, Asp or Asn, the omega+2 amino acid is always Ser, and the omega+7 amino acid is almost always Lys. In order to determine whether this high conservation is necessary for GPI anchoring, we introduced several mutations into the signal peptide. Surprisingly, changing the most conserved amino acids, at positions omega+1, omega+2 and omega+7, had no detectable effect on the efficiency of GPI-anchoring or on protein abundance. Several more extensive changes also had no discernable impact on GPI-anchoring. Deleting the entire 23 amino-acid signal sequence or the 15 amino-acid hydrophobic region generated proteins that were not anchored. Instead of being secreted, these truncated proteins accumulated in the endoplasmic reticulum prior to lysosomal degradation. Replacing the GPI signal sequence with a proven cell-surface membrane-spanning domain reduced expression by about 99% and resulted not in cell surface expression but in accumulation close to the flagellar pocket and in non-lysosomal compartments. These results indicate that the high conservation of the VSG GPI signal sequence is not necessary for efficient expression and GPI attachment. Instead, the GPI anchor is essential for surface expression of VSG. However, because the VSG is a major virulence factor, it is possible that small changes in the efficiency of GPI anchoring, undetectable in our experiments, might have influenced the evolution of VSG GPI signal sequences.

The carboxyl terminus of Dictyostelium discoideum protein 1I encodes a functional glycosyl-phosphatidylinositol signal sequence

The 1I gene is expressed in the prespore cells of culminating Dictyostelium discoideum. The open reading frame of 1I cDNA encodes a protein of 155 amino acids with hydrophobic segments at both its NH 2- and COOH-termini that are indicative of a glycosyl-phosphatidylinositol (GPI)-anchored protein. A hexaHis-tagged form of 1I expressed in D. discoideum cells appeared on Western blot analysis as a doublet of 27 and 24 kDa, with a minor polypeptide of 22 kDa. None of the polypeptides were released from the cell surface with bacterial phosphatidylinositol-specific phospholipase C, although all three were released upon nitrous acid treatment, indicating the presence of a phospholipase-resistant GPI anchor. Further evidence for the C-terminal sequence of 1I acting as a GPI attachment signal was obtained by replacing the GPI anchor signal sequence of porcine membrane dipeptidase with that from 1I. Two constructs of dipeptidase with the 1I GPI signal sequence were constructed, one of which included an additional six amino acids in the hydrophilic spacer. Both of the resultant constructs were targeted to the surface of COS cells and were GPI-anchored as shown by digestion with phospholipase C, indicating that the Dictyostelium GPI signal sequence is functional in mammalian cells. Site-specific antibodies recognising epitopes either side of the expected GPI anchor attachment site were used to determine the site of GPI anchor attachment in the constructs. These parallel approaches show that the C-terminal signal sequence of 1I can direct the addition of a GPI anchor.

The Glycosyl Phosphatidylinositol Anchor of Human T-Cadherin Binds Lipoproteins

T-cadherin (T-cad) is a Ca2+-dependent cell adhesion glycoprotein bound to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. T-cad expressed on vascular smooth muscle cells (SMC) binds lipoproteins on blot. To analyze the molecular basis for the interaction of T-cad with lipoproteins we expressed recombinant human T-cad in HEK293 cells. Whereas membrane-bound T-cad from SMC and T-cad transfected HEK293 cells bind lipoproteins, T-cadherin proteins cleaved from the cell surface by phosphatidylinositol-specific phospholipase C (PI-PLC) do not. The lipoprotein-binding function is also lacking both for a recombinant human T-cad expressed in HEK293 cells without the GPI signal sequence, and for a human T-cad form expressed in Escherichia coli that contains the signal sequence for GPI attachment but is not modified with a GPI. We conclude that the GPI moiety of T-cadherin is necessary and sufficient to mediate lipoprotein binding.

A Cell-free Assay for Glycosylphosphatidylinositol Anchoring in African Trypanosomes: DEMONSTRATION OF A TRANSAMIDATION REACTION MECHANISM

We established an in vitro assay for the addition of glycosyl-phosphatidylinositol (GPI) anchors to proteins using procyclic trypanosomes engineered to express GPI-anchored variant surface glycoprotein (VSG). The assay is based on the premise that small nucleophiles, such as hydrazine, can substitute for the GPI moiety and effect displacement of the membrane anchor of a GPI-anchored protein or pro-protein causing release of the protein into the aqueous medium. Cell membranes containing pulse-radiolabeled VSG were incubated with hydrazine, and the VSG released from the membranes was measured by carbonate extraction, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis/fluorography. Release of VSG was time- and temperature-dependent, was stimulated by hydrazine, and occurred only for VSG molecules situated in early compartments of the secretory pathway. No nucleophile-induced VSG release was seen in membranes prepared from cells expressing a VSG variant with a conventional transmembrane anchor (i.e. a nonfunctional GPI signal sequence). Pro-VSG was shown to be a substrate in the reaction by assaying membranes prepared from cells treated with mannosamine, a GPI biosynthesis inhibitor. When a biotinylated derivative of hydrazine was used instead of hydrazine, the released VSG could be precipitated with streptavidin-agarose, indicating that the biotin moiety was covalently incorporated into the protein. Hydrazine was shown to block the C terminus of the released VSG hydrazide because the released material, unlike a truncated form of VSG lacking a GPI signal sequence, was not susceptible to proteolysis by carboxypeptidases. These results firmly establish that the released material in our assay is VSG hydrazide and strengthen the proof that GPI anchoring proceeds via a transamidation reaction mechanism. The reaction could be inhibited with sulfhydryl alkylating reagents, suggesting that the transamidase enzyme contains a functionally important sulfhydryl residue.

The Carboxyl Terminus of Pneumocystis cariniiGlycoprotein A Encodes a Functional Glycosylphosphatidylinositol Signal Sequence

Pneumocystis carinii pneumonia is a hallmark disease associated with AIDS. An abundant glycoprotein, termed gpA, on the surface of P. carinii is considered an important factor in host-parasite interactions. The primary structure of ferret P. carinii gpA contains a carboxyl-terminal sequence characteristic of a signal for glycosylphosphatidylinositol (GPI) anchors. Here we report the capacity for this gpA carboxyl sequence to direct attachment of a secreted protein, human growth hormone (hGH), to the membranes of COS cells. A control fusion protein (hGHDAF37) was obtained which, under the direction of the GPI signal from decay accelerating factor, directs hGH cell surface expression. A construct (phGH2-1A30) was created similar to hGHDAF37 by fusing hGH to the putative GPI signal sequence encoded in the terminal 30 residues from a ferret P. carinii gpA cDNA clone. By indirect immunofluorescent staining, hGH was detected on the surface of COS cells transfected with phGH2-1A30; this surface location was confirmed by confocal laser cytometry. Metabolic labeling with [3H]ethanolamine and subsequent immunopurification of hGH from cells transfected with phGH2-1A30 confirmed that a lipid moiety characteristic of a conventional GPI anchor was linked covalently to hGH, and cell surface hGH2-1A30 fusion protein was sensitive to enzymatic cleavage by phosphatidylinositol-phospholipase C. Furthermore, hGH2-1A30 recombinant protein cofractionated with 5'-nucleotidase, a classical GPI-anchored membrane marker. Together, these results indicate that the carboxyl-terminal residues of ferret P. carinii gpA constitute a biologically functional GPI consensus domain, thus providing a potential mechanism for antigenic variation of P. carinii gpA during P. carinii pneumonia.

GPI-anchor synthesis in mammalian cells: genes, their products, and a deficiency.

Protein GPI anchors are ubiquitous in eukaryotic cells. More than 50 mammalian proteins are anchored to the membrane via GPI. GPI anchoring is a posttranslational modification occurring in the endoplasmic reticulum where preassembled GPI anchor precursors are transferred to proteins bearing a C-terminal GPI signal sequence. The GPI anchor precursors are synthesized in the endoplasmic reticulum by sequential addition of sugar and other components to phosphatidylinositol. More than ten genes participate in this biosynthetic pathway, eleven of the mammalian genes having been cloned by means of complementation of mutant cells that are defective in this pathway or based on sequence homology to previously cloned yeast counterparts. A somatic mutation in one of those genes, PIG-A, involved in the first reaction step, is responsible for the hemolytic disease, paroxysmal nocturnal hemoglobinuria.

Proteins with Glycosylphosphatidylinositol (GPI) Signal Sequences Have Divergent Fates during a GPI Deficiency: GPIs ARE ESSENTIAL FOR NUCLEAR DIVISION IN TRYPANOSOMA CRUZI

Glycosylphosphatidylinositols (GPIs) are membrane anchors for cell surface proteins of several major protozoan parasites of humans, including Trypanosoma cruzi, the causative agent of Chagas' disease. To investigate the general role of GPIs in T. cruzi, we generated GPI-deficient parasites by heterologous expression of T. brucei GPI-phospholipase C. Putative protein-GPI intermediates were depleted, causing the biochemical equivalent of a dominant-negative loss of function mutation in the GPI pathway. Cell surface expression of major GPI-anchored proteins was diminished in GPI-deficient T. cruzi. Four proteins that are normally GPI-anchored in T. cruzi exhibited different fates during the GPI shortage; Ssp-4 and p75 were secreted prematurely, while protease gp50/55 and p60 were degraded intracellularly. These observations demonstrate that secretion and intracellular degradation of GPI-anchored proteins may occur in the same genetic background during a GPI deficiency. We postulate that the interaction between a protein-GPI transamidase and the COOH-terminal GPI signal sequence plays a pivotal role in determining the fate of these proteins. At a nonpermissive GPI deficiency, T. cruzi amastigotes inside mammalian cells replicated their single kinetoplast but failed at mitosis. Hence, in these protozoans, GPIs appear to be essential for nuclear division, but not for mitochondrial duplication.

Functional dissociation of CD8 alpha's Ig homologue and connecting peptide domains.

The contribution of the CD8 alpha.IgV homologue domain to class I MHC binding was evaluated using a series of chimeric human CD8 alpha:Fc polypeptides incorporating alternative CD8 alpha extracellular domain components. Using a nonisotopic cellfree physical binding assay, those Fc chimeras encompassing the CD8 alpha.IgV homologue domain only (dissociated from the 48-amino acid CD8 alpha connecting peptide) were shown to retain the capacity of the complete CD8 alpha extracellular domain to bind to a recombinant soluble class I MHC alpha 3 domain unit or to intact class I MHC. The specificity of the CD8 alpha:class I MHC alpha 3 domain interaction was verified by mAb and soluble polypeptide blocking experiments. Furthermore, co-precipitation of an Fc chimera incorporating only the CD8 alpha.IgV homologue domain and a recombinant soluble class I MHC alpha 3 domain unit was accomplished. In addition, a glycosylphosphatidylinositol (GPI)-modified variant of the CD8 alpha.IgV homologue domain was generated via chimerization with the GPI signal sequence from decay-accelerating factor. GPI anchorage for this truncated CD8 alpha polypeptide was verified, and its capacity to promote intercellular adhesion through class I MHC binding was shown in a cell:cell binding assay. The findings indicate that the CD8 alpha.IgV homologue domain acts as an independent structural unit when dissociated from the CD8 alpha connecting peptide, and in so doing retains class I MHC binding capacity. This further establishes the principle that Ig superfamily domains from receptor:counter-receptor pairs can interact with each other as isolated units, providing an experimental path for tailoring therapeutically useful IgSF protein derivatives.

Identification, Isolation, and Cloning of a Bacillus thuringiensis CryIAc Toxin-binding Protein from the Midgut of the Lepidopteran Insect Heliothis virescens

Bacillus thuringiensis toxins are insecticidal to a variety of insect species. The selectivity of the toxins produced by these bacteria is dependent on both the toxin structure and the receptor sites that are present in different insect species. One of these toxins, CryIAc, is highly insecticidal to the noctuid pest Heliothis virescens. Using toxin overlay assay, a 120-kDa glycoprotein was identified as a toxin-binding protein. This protein was partially purified, its N-terminal sequence was determined, and the full-length cDNA encoding this protein was isolated from a H. virescens midgut library. The B. thuringiensis toxin-binding protein, BTBP1, has high homology to aminopeptidase N from eukaryotes and prokaryotes.