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James (Jay) Bangs

Picture of James (Jay) BangsProfessor Emeritus, Medical Microbiology and Immunology
No longer on campus
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Email: jdbangs@buffalo.edu
Overview · Publications
  • Silverman JS, Bangs JD (2012) Form and function in the trypanosomal secretory pathway. Curr Opin Microbiol : (PMC3393773) View Abstract · Pubmed Record

    Recent advances in secretory biology of African trypanosomes reveal both similarities and striking differences with other model eukaryotic organisms. Secretion is streamlined for rapid and selective transport of the major cargo, VSG. Selectivity in the early and post-Golgi compartments is dependent on glycosylphosphatidyl inositol anchors. Streamlining includes reduced organellar abundance, and close association of ER exit sites with Golgi and with unique flagellar cytoskeletal elements that govern organellar replication and segregation. These elements include a novel centrin containing bilobe structure. Innate signals for post-Golgi sorting of biosynthetic lysosomal cargo trafficking have been defined, as have pathways for both biosynthetic and endocytic trafficking to the lysosome. Less well-defined secretory organelles such as the multivesicular body and acidocalcisomes are receiving closer scrutiny.

  • Bangs JD (2011) Replication of the ERES:Golgi junction in bloodstream-form African trypanosomes. Mol. Microbiol. 82(6):1433-43 (PMC3237776) View Abstract · Pubmed Record

    The biogenesis of the ER Exit Site/Golgi Junction (EGJ) in bloodstream-form African trypanosomes is investigated using tagged markers for ER Exit Sites, the Golgi and the bilobe structure. The typical pattern is two EGJ in G1 phase (1 kinetoplast/1 nucleus, 1K1N) through S-phase (2K1N), duplication to four EGJ in post-mitotic cells (2K2N) and segregation of two EGJ to each daughter. Lesser cell percentages have elevated EGJ copy numbers in all stages, and blocking cell cycle progression results in even higher copy numbers. EGJs are closely aligned with the flagellar attachment zone (FAZ) indicating nucleation on the FAZ-associated ER (FAZ:ER). Only the most posterior EGJ in each cell is in proximity to the bilobe, which is located at the base of the FAZ filament near the mouth of the flagellar pocket. These results indicate that EGJ replication in bloodstream trypanosomes is not tightly coupled to the cell cycle. Furthermore, segregation of EGJ is not obligately mediated by the bilobe, rather assembly of the EGJ on the FAZ:ER, which is coupled to the flagellar cytoskeleton, apparently ensures segregation with fidelity during cytokinesis. These findings differ markedly from procyclic-form trypanosomes, and models highlighting these stage-specific differences in EGJ biogenesis are proposed.

  • Silverman JS, Schwartz KJ, Hajduk SL, Bangs JD (2011) Late endosomal Rab7 regulates lysosomal trafficking of endocytic but not biosynthetic cargo in Trypanosoma brucei. Mol. Microbiol. 82(3):664-78 View Abstract · Pubmed Record

    We present the first functional analysis of the small GTPase, TbRab7, in Trypanosoma brucei. TbRab7 defines discrete late endosomes closely juxtaposed to the terminal p67(+) lysosome. RNAi indicates that TbRab7 is essential in bloodstream trypanosomes. Initial rates of endocytosis were unaffected, but lysosomal delivery of cargo, including tomato lectin (TL) and trypanolytic factor (TLF) were blocked. These accumulate in a dispersed internal compartment of elevated pH, likely derived from the late endosome. Surface binding of TL but not TLF was reduced, suggesting that cellular distribution of flagellar pocket receptors is differentially regulated by TbRab7. TLF activity was reduced approximately threefold confirming that lysosomal delivery is critical for trypanotoxicity. Unexpectedly, delivery of endogenous proteins, p67 and TbCatL, were unaffected indicating that TbRab7 does not regulate biosynthetic lysosomal trafficking. Thus, unlike mammalian cells and yeast, lysosomal trafficking of endocytosed and endogenous proteins occur via different routes and/or are regulated differentially. TbRab7 silencing had no effect on a cryptic default pathway to the lysosome, suggesting that the default lysosomal reporters p67ΔTM, p67ΔCD and VSGΔGPI do not utilize the endocytic pathway as previously proposed. Surprisingly, conditional knockout indicates that TbRab7 may be non-essential in procyclic insect form trypanosomes.

  • Goren MA, Fox BG, Bangs JD (2011) Amino acid determinants of substrate selectivity in the Trypanosoma brucei sphingolipid synthase family. Biochemistry 50(41):8853-61 (PMC3212988) View Abstract · Pubmed Record

    The substrate selectivity of four Trypanosoma brucei sphingolipid synthases was examined. TbSLS1, an inositol phosphorylceramide (IPC) synthase, and TbSLS4, a bifunctional sphingomyelin (SM)/ethanolamine phosphorylceramide (EPC) synthase, were inactivated by Ala substitutions of a conserved triad of residues His210, His253, and Asp257 thought to form part of the active site. TbSLS4 also catalyzed the reverse reaction, production of ceramide from sphingomyelin, but none of the Ala substitutions of the catalytic triad in TbSLS4 were able to do so. Site-directed mutagenesis identified residues proximal to the conserved triad that were responsible for the discrimination between charge and size of the different head groups. For discrimination between anionic (phosphoinositol) and zwitterionic (phosphocholine, phosphoethanolamine) head groups, doubly mutated V172D/S252F TbSLS1 and D172V/F252S TbSLS3 showed reciprocal conversion between IPC and bifunctional SM/EPC synthases. For differentiation of zwitterionic headgroup size, N170A TbSLS1 and A170N/N187D TbSLS4 showed reciprocal conversion between EPC and bifunctional SM/EPC synthases. These studies provide a mapping of the SLS active site and demonstrate that differences in catalytic specificity of the T. brucei enzyme family are controlled by natural variations in as few as three residue positions.

  • Zhang K, Bangs JD, Beverley SM (2010) Sphingolipids in parasitic protozoa. Adv. Exp. Med. Biol. 688:238-48 (PMC2951629) View Abstract · Pubmed Record

    The surface of most protozoan parasites relies heavily upon lipid-anchored molecules, to form protective barriers and play critical functions required for infectivity. Sphingolipids (SLs) play important roles through their abundance and involvement in membrane microdomain formation, as well as serving as the lipid anchor for many of these molecules and in some but possibly not all species, as important signaling molecules. Interactions of parasite sphingolipid metabolism with that of the host may potentially contribute to parasite survival and/or host defense. In this chapter we summarize current knowledge of SL structure, synthesis and function in several of the major parasitic protozoan groups.

  • Sevova ES, Goren MA, Schwartz KJ, Hsu FF, Turk J, Fox BG, Bangs JD (2010) Cell-free synthesis and functional characterization of sphingolipid synthases from parasitic trypanosomatid protozoa. J. Biol. Chem. 285(27):20580-7 (PMC2898309) View Abstract · Pubmed Record

    The Trypanosoma brucei genome has four highly similar genes encoding sphingolipid synthases (TbSLS1-4). TbSLSs are polytopic membrane proteins that are essential for viability of the pathogenic bloodstream stage of this human protozoan parasite and, consequently, can be considered as potential drug targets. TbSLS4 was shown previously to be a bifunctional sphingomyelin/ethanolamine phosphorylceramide synthase, whereas functions of the others were not characterized. Using a recently described liposome-supplemented cell-free synthesis system, which eliminates complications from background cellular activities, we now unambiguously define the enzymatic specificity of the entire gene family. TbSLS1 produces inositol phosphorylceramide, TbSLS2 produces ethanolamine phosphorylceramide, and TbSLS3 is bifunctional, like TbSLS4. These findings indicate that TbSLS1 is uniquely responsible for synthesis of inositol phosphorylceramide in insect stage parasites, in agreement with published expression array data (17). This approach also revealed that the Trypanosoma cruzi ortholog (TcSLS1) is a dedicated inositol phosphorylceramide synthase. The cell-free synthesis system allowed rapid optimization of the reaction conditions for these enzymes and site-specific mutagenesis to alter end product specificity. A single residue at position 252 (TbSLS1, Ser(252); TbSLS3, Phe(252)) strongly influences enzymatic specificity. We also have used this system to demonstrate that aureobasidin A, a potent inhibitor of fungal inositol phosphorylceramide synthases, does not significantly affect any of the TbSLS activities, consistent with the phylogenetic distance of these two clades of sphingolipid synthases. These results represent the first application of cell-free synthesis for the rapid preparation and functional annotation of integral membrane proteins and thus illustrate its utility in studying otherwise intractable enzyme systems.

  • Sevova ES, Bangs JD (2009) Streamlined architecture and glycosylphosphatidylinositol-dependent trafficking in the early secretory pathway of African trypanosomes. Mol. Biol. Cell 20(22):4739-50 (PMC2777104) View Abstract · Pubmed Record

    The variant surface glycoprotein (VSG) of bloodstream form Trypanosoma brucei (Tb) is a critical virulence factor. The VSG glycosylphosphatidylinositol (GPI)-anchor strongly influences passage through the early secretory pathway. Using a dominant-negative mutation of TbSar1, we show that endoplasmic reticulum (ER) exit of secretory cargo in trypanosomes is dependent on the coat protein complex II (COPII) machinery. Trypanosomes have two orthologues each of the Sec23 and Sec24 COPII subunits, which form specific heterodimeric pairs: TbSec23.1/TbSec24.2 and TbSec23.2/TbSec24.1. RNA interference silencing of each subunit is lethal but has minimal effects on trafficking of soluble and transmembrane proteins. However, silencing of the TbSec23.2/TbSec24.1 pair selectively impairs ER exit of GPI-anchored cargo. All four subunits colocalize to one or two ER exit sites (ERES), in close alignment with the postnuclear flagellar adherence zone (FAZ), and closely juxtaposed to corresponding Golgi clusters. These ERES are nucleated on the FAZ-associated ER. The Golgi matrix protein Tb Golgi reassembly stacking protein defines a region between the ERES and Golgi, suggesting a possible structural role in the ERES:Golgi junction. Our results confirm a selective mechanism for GPI-anchored cargo loading into COPII vesicles and a remarkable degree of streamlining in the early secretory pathway. This unusual architecture probably maximizes efficiency of VSG transport and fidelity in organellar segregation during cytokinesis.

  • Tazeh NN, Silverman JS, Schwartz KJ, Sevova ES, Sutterwala SS, Bangs JD (2009) Role of AP-1 in developmentally regulated lysosomal trafficking in Trypanosoma brucei. Eukaryotic Cell 8(9):1352-61 (PMC2747832) View Abstract · Pubmed Record

    African trypanosomes are the causative agents of human trypanosomiasis (sleeping sickness). The pathogenic stage of the parasite has unique adaptations to life in the bloodstream of the mammalian host, including upregulation of endocytic and lysosomal activities. We investigated stage-specific requirements for cytoplasmic adaptor/clathrin machinery in post-Golgi apparatus biosynthetic sorting to the lysosome using RNA interference silencing of the Tbmu1 subunit of adaptor complex 1 (AP-1), in conjunction with immunolocalization, kinetic analyses of reporter transport, and quantitative endocytosis assays. Tbmu1 silencing was lethal in both stages, indicating a critical function(s) for the AP-1 machinery. Transport of soluble and membrane-bound secretory cargoes was Tbmu1 independent in both stages. In procyclic parasites, trafficking of the lysosomal membrane protein, p67, was disrupted, leading to cell surface mislocalization. The lysosomal protease trypanopain was also secreted, suggesting a transmembrane-sorting receptor for this soluble hydrolase. In bloodstream trypanosomes, both p67 and trypanopain trafficking were unaffected by Tbmu1 silencing, suggesting that AP-1 is not necessary for biosynthetic lysosomal trafficking. Endocytosis in bloodstream cells was also unaffected, indicating that AP-1 does not function at the flagellar pocket. These results indicate that post-Golgi apparatus sorting to the lysosome is critically dependent on the AP-1/clathrin machinery in procyclic trypanosomes but that this machinery is not necessary in bloodstream parasites. We propose a simple model for stage-specific default secretory trafficking in trypanosomes that is consistent with the behavior of other soluble and glycosylphosphatidylinositol-anchored cargos and which is influenced by upregulation of endocytosis in bloodstream parasites as an adaptation to life in the mammalian bloodstream.

  • Dagenais TR, Demick KP, Bangs JD, Forest KT, Paulnock DM, Mansfield JM (2009) T-cell responses to the trypanosome variant surface glycoprotein are not limited to hypervariable subregions. Infect. Immun. 77(1):141-51 (PMC2612290) View Abstract · Pubmed Record

    Variable subregions within the variant surface glycoprotein (VSG) coat displayed by African trypanosomes are predicted sites for T- and B-cell recognition. Hypervariable subregion 1 (HV-1) is localized to an internal amphipathic alpha helix in VSG monomers and may have evolved due to selective pressure by host T-cell responses to epitopes within this subregion. The prediction of T-cell receptor-reactive sites and major histocompatibility complex class II binding motifs within the HV-1 subregion, coupled with the conservation of amino acid residues in other regions of the molecule sufficient to maintain secondary and tertiary VSG structure, prompted us to test the hypothesis that Th cells may preferentially recognize HV-1 subregion peptides. Thus, we examined the fine specificity of VSG-specific T-cell lines, T-cell hybridomas, and Th cells activated during infection. Our results demonstrate that T-cell epitopes are distributed throughout the N-terminal domain of VSG but are not clustered exclusively within HV-1 or other hypervariable subregions. In contrast, T-cell-reactive sites were not detected within the relatively conserved C-terminal domain of VSG. Overall, this study is the first to dissect the fine specificity of T-cell responses to the trypanosome VSG and suggests that evolution of a conserved HV-1 region may be unrelated to selective pressures exerted by host T-cell responses. This study also demonstrates that T cells do not recognize the relatively invariant C-terminal region of the VSG molecule during infection, suggesting that it could serve as a potential subunit vaccine to provide variant cross-specific immunity for African trypanosomiasis.

  • Sutterwala SS, Hsu FF, Sevova ES, Schwartz KJ, Zhang K, Key P, Turk J, Beverley SM, Bangs JD (2008) Developmentally regulated sphingolipid synthesis in African trypanosomes. Mol. Microbiol. 70(2):281-96 (PMC2629665) View Abstract · Pubmed Record

    Sphingolipids are essential components of eukaryotic membranes, and many unicellular eukaryotes, including kinetoplastid protozoa, are thought to synthesize exclusively inositol phosphorylceramide (IPC). Here we characterize sphingolipids from Trypanosoma brucei, and a trypanosome sphingolipid synthase gene family (TbSLS1-4) that is orthologous to Leishmania IPC synthase. Procyclic trypanosomes contain IPC, but also sphingomyelin, while surprisingly bloodstream-stage parasites contain sphingomyelin and ethanolamine phosphorylceramide (EPC), but no detectable IPC. In vivo fluorescent ceramide labelling confirmed stage-specific biosynthesis of both sphingomyelin and IPC. Expression of TbSLS4 in Leishmania resulted in production of sphingomyelin and EPC suggesting that the TbSLS gene family has bi-functional synthase activity. RNAi silencing of TbSLS1-4 in bloodstream trypanosomes led to rapid growth arrest and eventual cell death. Ceramide levels were increased more than threefold by silencing suggesting a toxic downstream effect mediated by this potent intracellular messenger. Topology predictions support a revised six-transmembrane domain model for the kinetoplastid sphingolipid synthases consistent with the proposed mammalian sphingomyelin synthase structure. This work reveals novel diversity and regulation in sphingolipid metabolism in this important group of human parasites.

  • McCann AK, Schwartz KJ, Bangs JD (2008) A determination of the steady state lysosomal pH of bloodstream stage African trypanosomes. Mol. Biochem. Parasitol. 159(2):146-9 (PMC2423349) View Abstract · Pubmed Record

    The lysosomal/endosomal system of African trypanosomes is developmentally regulated and is important in the pathogenesis associated with infection of the mammalian bloodstream. Long considered to be a target for drug development, the internal pH of the lysosome has been variously reported to range from <5.0 to >6.0. We have refined a flow cytometric technique using a pH-sensitive probe that specifically targets the lysosome, tomato lectin:Oregon Green 488 conjugate. The probe is delivered to the lysosome with fidelity, where it is shielded against external pH. Measurement of fluorescent output in the presence and absence of lysomotropic agent (NH(4)Cl) then allows precise titration of steady state lysosomal pH (4.84+/-0.23). Using bafilomycin A1 to inhibit acidification we demonstrate that this method is responsive to pharmacological perturbation of lysosomal physiology. This work should facilitate future studies of the lysosomal function in African trypanosomiasis, as well as other parasitic protozoa.

  • Peck RF, Shiflett AM, Schwartz KJ, McCann A, Hajduk SL, Bangs JD (2008) The LAMP-like protein p67 plays an essential role in the lysosome of African trypanosomes. Mol. Microbiol. 68(4):933-46 View Abstract · Pubmed Record

    RNAi knockdown was employed to study the function of p67, a lysosome-associated membrane protein (LAMP)-like type I transmembrane lysosomal glycoprotein in African trypanosomes. Conditional induction of p67 dsRNA resulted in specific approximately 90% reductions in de novo p67 synthesis in both mammalian bloodstream and procyclic insect-stage parasites. Bloodstream cell growth was severely retarded with extensive death after > 24 h of induction. Biosynthetic trafficking of residual p67, and of the soluble lysosomal protease trypanopain, were unimpaired. Endocytosis of tomato lectin, a surrogate receptor-mediated cargo, was only mildly impaired (approximately 20%), but proper lysosomal targeting was unaffected. p67 ablation had dramatic effects on lysosomal morphology with gross enlargement (four- to fivefold) and internal membrane profiles reminiscent of autophagic vacuoles. Ablation of p67 expression rendered bloodstream trypanosomes refractory to lysis by human trypanolytic factor (TLF), a lysosomally activated host innate immune mediator. Similar effects on lysosomal morphology and TLF sensitivity were also obtained by two pharmacological agents that neutralize lysosomal pH--chloroquine and bafilomycin A1. Surprisingly, however, lysosomal pH was not affected in ablated cells suggesting that other physiological alterations must account for increased resistance to TLF. These results indicate p67 plays an essential role in maintenance of normal lysosomal structure and physiology in bloodstream-stage African trypanosomes.

  • Moraes MC, Jesus TC, Hashimoto NN, Dey M, Schwartz KJ, Alves VS, Avila CC, Bangs JD, Dever TE, Schenkman S, Castilho BA (2007) Novel membrane-bound eIF2alpha kinase in the flagellar pocket of Trypanosoma brucei. Eukaryotic Cell 6(11):1979-91 (PMC2168417) View Abstract · Pubmed Record

    Translational control mediated by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF2alpha) is central to stress-induced programs of gene expression. Trypanosomatids, important human pathogens, display differentiation processes elicited by contact with the distinct physiological milieu found in their insect vectors and mammalian hosts, likely representing stress situations. Trypanosoma brucei, the agent of African trypanosomiasis, encodes three potential eIF2alpha kinases (TbeIF2K1 to -K3). We show here that TbeIF2K2 is a transmembrane glycoprotein expressed both in procyclic and in bloodstream forms. The catalytic domain of TbeIF2K2 phosphorylates yeast and mammalian eIF2alpha at Ser51. It also phosphorylates the highly unusual form of eIF2alpha found in trypanosomatids specifically at residue Thr169 that corresponds to Ser51 in other eukaryotes. T. brucei eIF2alpha, however, is not a substrate for GCN2 or PKR in vitro. The putative regulatory domain of TbeIF2K2 does not share any sequence similarity with known eIF2alpha kinases. In both procyclic and bloodstream forms TbeIF2K2 is mainly localized in the membrane of the flagellar pocket, an organelle that is the exclusive site of exo- and endocytosis in these parasites. It can also be detected in endocytic compartments but not in lysosomes, suggesting that it is recycled between endosomes and the flagellar pocket. TbeIF2K2 location suggests a relevance in sensing protein or nutrient transport in T. brucei, an organism that relies heavily on posttranscriptional regulatory mechanisms to control gene expression in different environmental conditions. This is the first membrane-associated eIF2alpha kinase described in unicellular eukaryotes.

  • Tazeh NN, Bangs JD (2007) Multiple motifs regulate trafficking of the LAMP-like protein p67 in the ancient eukaryote Trypanosoma brucei. Traffic 8(8):1007-17 View Abstract · Pubmed Record

    p67 is a lysosome-associated membrane protein-like lysosomal type I transmembrane glycoprotein in African trypanosomes. The p67 cytoplasmic domain (CD) is both necessary and sufficient for lysosomal targeting in procyclic insect-stage parasites. The p67CD contains two [DE]XXXL[LI]-type dileucine motifs, which function as lysosomal targeting signals in mammalian cells. Using a green fluorescent protein fusion to the p67 transmembrane and cytoplasmic domains as a reporter system, we investigated the role of these motifs in lysosomal targeting in procyclic trypanosomes. Pulse-chase turnover studies, steady-state immunolocalization and quantitative flow cytometry all gave consistent results. Mutagenesis of the membrane-distal dileucine motif impairs lysosomal trafficking leading to partial appearance of the reporter on the cell surface. Mutagenesis of the membrane-proximal motif has little effect on proper targeting. Simultaneous mutagenesis of both motifs results in quantitative delivery to the cell surface. Thus, the distal motif plays a dominant role, but both dileucine motifs are necessary for maximal lysosomal targeting. Additional studies suggest that the upstream acidic residues in each motif influence lysosomal targeting and may also affect forward trafficking in the early secretory pathway. These results strongly suggest an evolutionary conservation in lysosomal trafficking mechanisms in the ancient eukaryote Trypanosoma brucei.

  • Sutterwala SS, Creswell CH, Sanyal S, Menon AK, Bangs JD (2007) De novo sphingolipid synthesis is essential for viability, but not for transport of glycosylphosphatidylinositol-anchored proteins, in African trypanosomes. Eukaryotic Cell 6(3):454-64 (PMC1828920) View Abstract · Pubmed Record

    De novo sphingolipid synthesis is required for the exit of glycosylphosphatidylinositol (GPI)-anchored membrane proteins from the endoplasmic reticulum in yeast. Using a pharmacological approach, we test the generality of this phenomenon by analyzing the transport of GPI-anchored cargo in widely divergent eukaryotic systems represented by African trypanosomes and HeLa cells. Myriocin, which blocks the first step of sphingolipid synthesis (serine + palmitate --> 3-ketodihydrosphingosine), inhibited the growth of cultured bloodstream parasites, and growth was rescued with exogenous 3-ketodihydrosphingosine. Myriocin also blocked metabolic incorporation of [3H]serine into base-resistant sphingolipids. Biochemical analyses indicate that the radiolabeled lipids are not sphingomyelin or inositol phosphorylceramide, suggesting that bloodstream trypanosomes synthesize novel sphingolipids. Inhibition of de novo sphingolipid synthesis with myriocin had no adverse effect on either general secretory trafficking or GPI-dependent trafficking in trypanosomes, and similar results were obtained with HeLa cells. A mild effect on endocytosis was seen for bloodstream trypanosomes after prolonged incubation with myriocin. These results indicate that de novo synthesis of sphingolipids is not a general requirement for secretory trafficking in eukaryotic cells. However, in contrast to the closely related kinetoplastid Leishmania major, de novo sphingolipid synthesis is essential for the viability of bloodstream-stage African trypanosomes.

  • Schwartz KJ, Bangs JD (2007) Regulation of protein trafficking by glycosylphosphatidylinositol valence in African trypanosomes. J. Eukaryot. Microbiol. 54(1):22-4 View Abstract · Pubmed Record

    The structure, biosynthesis, and attachment of glycosylphosphatidylinositol (GPI) anchors were all first determined for the variant surface glycoprotein (VSG) of African trypanosomes, and all of the basic aspects of this work have been shown to be universal in eukaryotic organisms. However, the role of GPI anchors in protein trafficking within trypanosomes has lagged behind the more standard eukaryotic model systems such as yeast and polarized epithelial cells. Trypanosomes are also highly polarized cells in which all endocytosis and exocytosis intersect at a discrete domain of the plasma membrane, the flagellar pocket. Within these convergent pathways trafficking of GPI anchored proteins correlates strongly with valence: homodimeric VSG with two GPIs is stably incorporated into the cell surface coat, heterodimeric transferrin receptor with a single GPI is found in the flagellar pocket and is slowly delivered to the lysosome for degradation, and recombinant GPI minus VSG reporters are rapidly degraded in the lysosome. Here we summarize recent data confirming this correlation using a tool kit of recombinant GPI anchored reporters, including a reporter designed to be conditionally modulated between a GPI valence of one and two.

  • Gruszynski AE, van Deursen FJ, Albareda MC, Best A, Chaudhary K, Cliffe LJ, del Rio L, Dunn JD, Ellis L, Evans KJ, Figueiredo JM, Malmquist NA, Omosun Y, Palenchar JB, Prickett S, Punkosdy GA, van Dooren G, Wang Q, Menon AK, Matthews KR, Bangs JD (2006) Regulation of surface coat exchange by differentiating African trypanosomes. Mol. Biochem. Parasitol. 147(2):211-23 View Abstract · Pubmed Record

    African trypanosomes (Trypanosoma brucei) have a digenetic lifecycle that alternates between the mammalian bloodstream and the tsetse fly vector. In the bloodstream, replicating long slender parasites transform into non-dividing short stumpy forms. Upon transmission into the fly midgut, short stumpy cells differentiate into actively dividing procyclics. A hallmark of this process is the replacement of the bloodstream-stage surface coat composed of variant surface glycoprotein (VSG) with a new coat composed of procyclin. Pre-existing VSG is shed by a zinc metalloprotease activity (MSP-B) and glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC). We now provide a detailed analysis of the coordinate and inverse regulation of these activities during synchronous differentiation. MSP-B mRNA and protein levels are upregulated during differentiation at the same time as proteolysis whereas GPI-PLC levels decrease. When transcription or translation is inhibited, VSG release is incomplete and a substantial amount of protein stays cell-associated. Both modes of release are still evident under these conditions, but GPI hydrolysis plays a quantitatively minor role during normal differentiation. Nevertheless, GPI biosynthesis shifts early in differentiation from a GPI-PLC sensitive structure to a resistant procyclic-type anchor. Translation inhibition also results in a marked increase in the mRNA levels of both MSP-B and GPI-PLC, consistent with negative regulation by labile protein factors. The relegation of short stumpy surface GPI-PLC to a secondary role in differentiation suggests that it may play a more important role as a virulence factor within the mammalian host.

  • Schwartz KJ, Peck RF, Tazeh NN, Bangs JD (2005) GPI valence and the fate of secretory membrane proteins in African trypanosomes. J. Cell. Sci. 118(Pt 23):5499-511 View Abstract · Pubmed Record

    Progression of GPI-anchored proteins in bloodstream African trypanosomes correlates with GPI-valence: homodimeric VSG (2 GPI) is a surface protein; heterodimeric transferrin receptor (1 GPI) localizes in the flagellar pocket; homodimeric GPI-minus VSG (0 GPI) is rapidly degraded in the lysosome. We test this relationship using three native secretory/endocytic proteins as monomeric GPI-plus and -minus reporters. GPI-minus procyclin trafficks to the lysosome and is degraded. GPI-plus procyclin trafficks to the flagellar pocket/cell surface and is released (approximately 50%) with an intact anchor, the remainder (approximately 50%) is degraded in the lysosome. GPI-plus BiPNHP, derived from the ER marker BiP, is released quantitatively (>80%), while GPI-plus p67HP, derived from the lysosomal marker p67, turns over by both release (approximately 15%) and lysosomal degradation (>50%). Turnover of endogenous transferrin receptor occurs primarily by lysosomal degradation (>90%). Thus shedding of monovalent GPI reporters correlates inversely with lysosomal targeting. We propose that mono-GPI reporters cycle through the flagellar pocket and endosome until they are disposed of by either shedding or lysosomal targeting. Partitioning between these fates may be a function of individual physical properties. Release is likely due to the exclusive use of C-14:0 myristate in the bloodstream stage GPI anchor. Up-regulation of transferrin receptor by culture in dog serum resulted in prominent cell surface localization, but not in elevated release. Surface receptor was non-functional for ligand binding suggesting that it may be bivalent homodimers of the GPI-anchored ESAG6 receptor subunit.

  • Gruszynski AE, DeMaster A, Hooper NM, Bangs JD (2003) Surface coat remodeling during differentiation of Trypanosoma brucei. J. Biol. Chem. 278(27):24665-72 View Abstract · Pubmed Record

    African trypanosomes (Trypanosoma brucei) are digenetic parasites whose lifecycle alternates between the mammalian bloodstream and the midgut of the tsetse fly vector. In mammals, proliferating long slender parasites transform into non-diving short stumpy forms, which differentiate into procyclic forms when ingested by the tsetse fly. A hallmark of differentiation is the replacement of the bloodstream stage surface coat composed of variant surface glycoprotein (VSG) with a new coat composed of procylin. An undefined endoprotease and endogenous glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) have been implicated in releasing the old VSG coat. However, GPI hydrolysis has been considered unimportant because (i) GPI-PLC null mutants are fully viable and (ii) cytosolic GPI-PLC is localized away from cell surface VSG. Utilizing an in vitro differentiation assay with pleomorphic strains we have investigated these modes of VSG release. Shedding is initially by GPI hydrolysis, which ultimately accounts for a substantial portion of total release. Surface biotinylation assays indicate that GPI-PLC does gain access to extracellular VSG, suggesting that this mode is primed in the starting short stumpy population. Proteolytic release is up-regulated during differentiation and is stereoselectively inhibited by peptidomimetic collagenase inhibitors, implicating a zinc metalloprotease. This protease may be related to TbMSP-B, a trypanosomal homologue of Leishmania major surface protease (MSP) described in the accompanying paper (LaCount, D. J., Gruszynski, A. E., Grandgenett, P. M., Bangs, J. D., and Donelson, J. E. (2003) J. Biol. Chem. 278, 24658-24664). Overall, our results demonstrate that surface coat remodeling during differentiation has multiple mechanisms and that GPI-PLC plays a more significant role in VSG release than previously thought.

  • LaCount DJ, Gruszynski AE, Grandgenett PM, Bangs JD, Donelson JE (2003) Expression and function of the Trypanosoma brucei major surface protease (GP63) genes. J. Biol. Chem. 278(27):24658-64 View Abstract · Pubmed Record

    The genome of the African trypanosome Trypanosoma brucei (Tb) contains at least three gene families (TbMSP-A, -B, and -C) encoding homologues of the abundant major surface protease (MSP, previously called GP63), which is found in all Leishmania species. TbMSP-B mRNA occurs in both procyclic and bloodstream trypanosomes, whereas TbMSP-A and -C mRNAs are detected only in bloodstream organisms. RNA interference (RNAi)-mediated gene silencing was used to investigate the function of TbMSP-B protein. RNAi directed against TbMSP-B but not TbMSP-A ablated the steady state TbMSP-B mRNA levels in both procyclic and bloodstream cells but had no effect on the kinetics of cultured trypanosome growth in either stage. Procyclic trypanosomes have been shown previously to have an uncharacterized cell surface metalloprotease activity that can release ectopically expressed surface proteins. To determine whether TbMSP-B is responsible for this release, transgenic variant surface glycoprotein 117 (VSG117) was expressed constitutively in T. brucei procyclic TbMSP-RNAi cell lines, and the amount of surface VSG117 was determined using a surface biotinylation assay. Ablation of TbMSP-B but not TbMSP-A mRNA resulted in a marked decrease in VSG release with a concomitant increase in steady state cell-associated VSG117, indicating that TbMSP-B mediates the surface protease activity of procyclic trypanosomes. This finding is consistent with previous pharmacological studies showing that peptidomimetic collagenase inhibitors block release of transgenic VSG from procyclic trypanosomes and are toxic for bloodstream but not procyclic organisms.

  • Triggs VP, Bangs JD (2003) Glycosylphosphatidylinositol-dependent protein trafficking in bloodstream stage Trypanosoma brucei. Eukaryotic Cell 2(1):76-83 (PMC141176) View Abstract · Pubmed Record

    We have previously demonstrated that glycosylphosphatidylinositol (GPI) anchors strongly influence protein trafficking in the procyclic insect stage of Trypanosoma brucei (M. A. McDowell, D. A. Ransom, and J. D. Bangs, Biochem. J. 335:681-689, 1998), where GPI-minus variant surface glycoprotein (VSG) reporters have greatly reduced rates of endoplasmic reticulum (ER) exit but are ultimately secreted. We now demonstrate that GPI-dependent trafficking also occurs in pathogenic bloodstream trypanosomes. However, unlike in procyclic trypanosomes, truncated VSGs lacking C-terminal GPI-addition signals are not secreted but are mistargeted to the lysosome and degraded. Failure to export these reporters is not due to a deficiency in secretion of these cells since the N-terminal ATPase domain of the endogenous ER protein BiP is efficiently secreted from transgenic cell lines. Velocity sedimentation experiments indicate that GPI-minus VSG dimerizes similarly to wild-type VSG, suggesting that degradation is not due to ER quality control mechanisms. However, GPI-minus VSGs are fully protected from degradation by the cysteine protease inhibitor FMK024, a potent inhibitor of the major lysosomal protease trypanopain. Immunofluorescence of cells incubated with FMK024 demonstrates that GPI-minus VSG colocalizes with p67, a lysosomal marker. These data suggest that in the absence of a GPI anchor, VSG is mistargeted to the lysosome and subsequently degraded. Our findings indicate that GPI-dependent transport is a general feature of secretory trafficking in both stages of the life cycle. A working model is proposed in which GPI valence regulates progression in the secretory pathway of bloodstream stage trypanosomes.

  • Alexander DL, Schwartz KJ, Balber AE, Bangs JD (2002) Developmentally regulated trafficking of the lysosomal membrane protein p67 in Trypanosoma brucei. J. Cell. Sci. 115(Pt 16):3253-63 View Abstract · Pubmed Record

    p67 is a lysosomal type I membrane glycoprotein of Trypanosoma brucei. In procyclic stage cells p67 trafficks to the lysosome without modification, but in the bloodstream stage Golgi processing adds poly-N-acetyllactosamine to N-glycans. In both stages proteolytic fragmentation occurs in the lysosome, but turnover is approximately nine times faster in bloodstream cells. Trafficking of wildtype p67 and mutants missing the cytoplasmic (p67DeltaCD) or cytoplasmic/transmembrane domains (p67DeltaTM) was monitored by pulse-chase, surface biotinylation and immunofluorescence. Overexpressed wildtype p67 trafficks normally in procyclics, but some leaks to the cell surface suggesting that the targeting machinery is saturable. p67DeltaCD and p67DeltaTM are delivered to the cell surface and secreted, respectively. The membrane/cytoplasmic domains function correctly in procyclic cells when fused to GFP indicating that these domains are sufficient for stage-specific lysosomal targeting. In contrast, p67 wildtype and deletion reporters are overwhelmingly targeted to the lysosome and degraded in bloodstream cells. These findings suggest that either redundant developmentally regulated targeting signals/machinery are operative in this stage or that the increased endocytic activity of bloodstream cells prevents export of the deletion reporters.

  • Lamb JR, Fu V, Wirtz E, Bangs JD (2001) Functional analysis of the trypanosomal AAA protein TbVCP with trans-dominant ATP hydrolysis mutants. J. Biol. Chem. 276(24):21512-20 View Abstract · Pubmed Record

    TbVCP is a member of the AAA (ATPases Associated with a variety of cellular Activities) family of proteins containing two ATPase domains. Southern analysis indicates TbVCP to have a single-locus, two-copy, genomic organization. One copy, but not both, can be disrupted by targeted gene replacement, suggesting that TbVCP is essential for trypanosome viability. Site-directed mutagenesis of the ATP hydrolysis motifs indicates that the second conserved ATPase domain is essential for TbVCP activity. Constitutive overexpression of TbVCP with a single mutation in the second hydrolysis motif or with mutations in both hydrolysis motifs was not possible. Regulated overexpression of these mutants resulted in cell death as a dominant negative phenotype. In each case cell growth arrested at 24-h post-induction and at all stages of the cell cycle as judged by replication of nuclear and kinetoplast genomes. Onset of growth arrest coincided with the development of severe and characteristic morphological alterations for each mutant. Neither constitutive nor regulated overexpression of wild type TbVCP or the single first hydrolysis domain mutant had any overt effect on cell viability or morphology. However, the distinct phenotype of the double mutant indicates that the first hydrolysis domain, although not essential, does modulate overall TbVCP function. Finally, yeast complementation studies demonstrated that TbVCP can functionally replace the yeast homologue Cdc48p, indicating that protein.protein interactions essential to function have been maintained over great phylogenetic distances.

  • Bangs JD, Ransom DA, Nimick M, Christie G, Hooper NM (2001) In vitro cytocidal effects on Trypanosoma brucei and inhibition of Leishmania major GP63 by peptidomimetic metalloprotease inhibitors. Mol. Biochem. Parasitol. 114(1):111-7 View Abstract · Pubmed Record

    Peptidomimetic inhibitors of mammalian zinc metalloproteases have been tested as potential agents for intervention in disease caused by kinetoplastid protozoa. Certain metalloprotease inhibitors were able to inhibit the release of variant surface glycoprotein from cultured transgenic procyclic Trypanosoma brucei, confirming our previous identification of a cell surface zinc metalloprotease activity in this stage of the trypanosome lifecycle [Bangs, JD et al. Expression of bloodstream variant surface glycoproteins in procyclic stage Trypanosoma brucei: role of GPI anchors in secretion, EMBO J. 1997;16:4285]. Selected peptidomimetics were also found to be toxic for cultured bloodstream trypanosomes with IC50 values in the low micromolar range. The paradigm for zinc metalloproteases in kinetoplastids are the GP63 surface enzymes of Leishmania. Peptidomimetics at low micromolar concentrations were able to inhibit in vitro cleavage of a synthetic peptide substrate by purified GP63 from L. major. Our results suggest that zinc metalloproteases perform essential functions in different stages of the trypanosome lifecycle and we hypothesize that these activities may be affected by the recently discovered trypanosomal homologues of GP63 [El-Sayed, NMA and Donelson, JE. African trypanosomes have differentially expressed genes encoding homologues of Leishmania GP63 surface protease, J. Biol. Chem. 1997;272:26742]. Development of higher affinity metalloprotease inhibitors may provide a novel avenue for treatment of parasitic diseases.

  • Sharma DK, Hilley JD, Bangs JD, Coombs GH, Mottram JC, Menon AK (2000) Soluble GPI8 restores glycosylphosphatidylinositol anchoring in a trypanosome cell-free system depleted of lumenal endoplasmic reticulum proteins. Biochem. J. 351 Pt 3:717-22 (PMC1221412) View Abstract · Pubmed Record

    We previously established an in vitro assay for glycosylphosphatidylinositol (GPI) anchoring of proteins using trypanosome membranes. We now show that GPI anchoring is lost when the membranes are washed at high pH and restored to physiological pH prior to assay. We show that soluble component(s) of the endoplasmic reticulum that are lost in the high-pH wash are required for GPI anchoring. We reconstituted the high-pH extract with high-pH-treated membranes and demonstrated restoration of activity. Size fractionation of the high-pH extract indicated that the active component(s) was 30-50 kDa in size and was inactivated by iodoacetamide. Activity could also be restored by reconstituting the inactivated membranes with Escherichia coli-expressed, polyhistidine-tagged Leishmania mexicana GPI8 (GPI8-His; L. mexicana GPI8 is a soluble homologue of yeast and mammalian Gpi8p). No activity was seen when iodoacetamide-treated GPI8-His was used; however, GPI8-His could restore activity to iodoacetamide-treated membranes. Antibodies raised against L. mexicana GPI8 detected a protein of approx. 38 kDa in an immunoblot of the high-pH extract of trypanosome membranes. Our data indicate (1) that trypanosome GPI8 is a soluble lumenal protein, (2) that the interaction between GPI8 and other putative components of the transamidase may be dynamic, and (3) that GPI anchoring can be biochemically reconstituted using an isolated transamidase component.

  • Sharma DK, Vidugiriene J, Bangs JD, Menon AK (1999) A cell-free assay for glycosylphosphatidylinositol anchoring in African trypanosomes. Demonstration of a transamidation reaction mechanism. J. Biol. Chem. 274(23):16479-86 View Abstract · Pubmed Record

    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.

  • Kelley RJ, Alexander DL, Cowan C, Balber AE, Bangs JD (1999) Molecular cloning of p67, a lysosomal membrane glycoprotein from Trypanosoma brucei. Mol. Biochem. Parasitol. 98(1):17-28 View Abstract · Pubmed Record

    We have previously characterized a highly glycosylated membrane protein (p67) in Trypanosoma brucei spp that is apparently targeted to lysosomes in a developmentally regulated manner. Antibody to native p67 identified a partial cDNA clone from a T. b. rhodesiense expression library and RT-PCR was used to complete the sequence of the cDNA. Equal levels of p67 transcript are detected in both procyclic and bloodstream stages of the life cycle. The 2771 nt cDNA contains a 1980 nt orf encoding a 659 amino acid polypeptide (72,567 Da). Hydropathy analysis predicts a Type I membrane topology (N to C): an N-terminal signal sequence, a large hydrophilic lumenal domain with 14 N-glycosylation sites, a trans-membrane domain (19 aa), and a short (24 aa) cytoplasmic domain. Peptide microsequencing of purified p67 identified nine residues identical to the deduced amino acid sequence, confirming the identity of the cDNA and defining the signal sequence cleavage site. Antibody to p67 protein produced in E. coli recognizes the same spectrum of native p67 glycoforms as the antibody used to clone the cDNA. All features of the deduced amino acid sequence are consistent with the known properties of the native protein and suggest a structure similar to mammalian LAMPS. The cytoplasmic domain contains two putative di-leucine targeting motifs similar to those involved in lysosomal targeting in vertebrate cells. Our results suggest that a single p67 polypeptide, or a group of highly related polypeptides, is synthesized in both bloodstream and procyclic trypanosomes and that subsequent post-translational processing and lysosomal targeting is subject to stage-specific regulation.

  • Roggy JL, Bangs JD (1999) Molecular cloning and biochemical characterization of a VCP homolog in African trypanosomes. Mol. Biochem. Parasitol. 98(1):1-15 View Abstract · Pubmed Record

    Through reverse transcription-polymerase chain reaction using degenerate oligonucleotide primers, a VCP homolog was identified in African trypanosomes. Sequence analysis shows a 72 and 64% deduced amino acid identity, respectively, with mouse VCP and yeast Cdc48p. Southern analysis indicates tbVCP to have a single locus with two alleles. Antibodies generated against recombinant protein recognize a 95 kDa protein in whole cell lysates of both procyclic and bloodstream trypanosomes. There is an approximately four-fold greater expression of TbVCP protein in the procyclic stage of the trypanosome life cycle. Subcellular fractionation and immunofluorescence with anti-TbVCP antibodies indicate the majority of TbVCP to be cytoplasmically localized with a small subset associated with membranes. Sucrose velocity sedimentation and gel filtration size analysis studies suggest that TbVCP is a homohexameric particle as has been demonstrated with other VCP homologs. Also like other VCP homologs, TbVCP contains an NEM-inhibitable ATPase activity. This is the first characterization of an AAA (ATPases Associated with a variety of cellular Activities) family member in African trypanosomes.

  • McDowell MA, Ransom DM, Bangs JD (1998) Glycosylphosphatidylinositol-dependent secretory transport in Trypanosoma brucei. Biochem. J. 335 ( Pt 3:681-9 (PMC1219832) View Abstract · Pubmed Record

    We have investigated the role of glycosylphosphatidylinositol (GPI) anchors in forward secretory trafficking using African trypanosomes as a model system. Soluble GPI-minus forms of variant surface glycoprotein (VSG), in which the C-terminal GPI-addition peptide signal is deleted, are secreted from transformed procyclic trypanosomes with 5-fold reduced kinetics, relative to matched GPI-anchored constructs. Cell fractionation and immunofluorescence localization studies indicate that the GPI-minus VSG reporters accumulate in the endoplasmic reticulum (ER). This transport defect is specific, since overexpression of GPI-minus VSG has no effect on the rate of transport of a second soluble secretory reporter (BiPN) when co-expressed in the same cells. Two results suggest that delayed forward transport cannot be accounted for by failure to fold/assemble in the absence of a GPI anchor, thereby leading to prolonged association with ER quality-control machinery. First, no evidence was found for elevated association of GPI-minus VSG with the ER molecular chaperone, BiP. Secondly, newly synthesized GPI-minus VSG is dimerized efficiently, as judged by velocity-sedimentation analysis. GPI-dependent transport is not confined to the VSG reporters, because a similar dependence is found with another trypanosomal GPI-anchored protein, trans-sialidase. These findings suggest that GPI structures act in a positive manner to mediate efficient forward transport of some, and perhaps all, GPI-anchored proteins in the early secretory pathway of trypanosomes. Possible mechanisms for GPI-dependent transport are discussed with respect to current models of vesicular trafficking.

  • Bangs JD (1998) Surface coats and secretory trafficking in African trypanosomes. Curr. Opin. Microbiol. 1(4):448-54 View Abstract · Pubmed Record

    Recent advances in transfection technology have been exploited to address fundamental questions relating to secretory trafficking in African trypanosomes. Targeted gene disruptions and ectopic expression of the major stage-specific surface proteins have provided unexpected insights into both the function and assembly of the essential parasite surface coats. A growing list of novel secretory cargo molecules, as well as advances in the characterization of trypanosomal secretory machinery, provide a unique model system for the study of eukaryotic secretory processes.

  • Bangs JD, Ransom DM, McDowell MA, Brouch EM (1997) Expression of bloodstream variant surface glycoproteins in procyclic stage Trypanosoma brucei: role of GPI anchors in secretion. EMBO J. 16(14):4285-94 (PMC1170054) View Abstract · Pubmed Record

    Using transformed procyclic trypanosomes, the synthesis, intracellular transport and secretion of wild-type and mutant variant surface glycoprotein (VSG) is characterized. We find no impediment to the expression of this bloodstream stage protein in insect stage cells. VSG receives a procyclic-type phosphatidylinositol-specific phospholipase C-resistant glycosyl phosphatidylinositol (GPI) anchor, dimerizes and is N-glycosylated. It is transported to the plasma membrane with rapid kinetics (t(1/2) approximately 1 h) and then released by a cell surface zinc-dependent metalloendoprotease activity, a possible homolog of leishmanial gp63. Deletion of the C-terminal GPI addition signal generates a soluble form of VSG that is exported with greatly reduced kinetics (t(1/2) approximately 5 h). Fusion of the procyclic acidic repetitive protein (PARP) GPI anchor signal to the C-terminus of the truncated VSG reporter restores both GPI addition and transport competence, suggesting that GPI anchors play a critical role in the folding and/or forward transport of newly synthesized VSG. The VSG-PARP fusion is also processed near the C-terminus by events that do not involve N-linked oligosaccharides and which are consistent with GPI side chain modification. This unexpected result suggests that GPI processing may be influenced by adjacent peptide sequence or conformation.

  • Bangs JD, Brouch EM, Ransom DM, Roggy JL (1996) A soluble secretory reporter system in Trypanosoma brucei. Studies on endoplasmic reticulum targeting. J. Biol. Chem. 271(31):18387-93 View Abstract · Pubmed Record

    A homolog of the endoplasmic reticulum (ER) hsp70 protein, binding protein (BiP), from the parasitic protozoan Trypanosoma brucei (Bangs, J. D., Uyetake, L., Brickman, M. J., Balber, A. E., and Boothroyd, J. C.(1993) J. Cell Sci. 105, 1101-1113) is further characterized. In co-precipitation experiments, BiP transiently associates with newly synthesized secretory proteins, including variant surface glycoprotein (VSG), confirming its role as a molecular chaperone. To study the molecular signals targeting BiP to the ER, we have developed soluble secretory reporters for expression in transformed procyclic trypanosomes. Deletion of the BiP C-terminal tetrapeptide (MDDL) and the glycosylphosphatidylinositol-anchor addition sequence of VSG converts these proteins to secreted forms. Attachment of MDDL to VSG results in intracellular retention confirming that MDDL is a trypanosomal ER localization signal. Secretion of both reporters is inefficient, but further truncation of the BiP C-terminal peptide-binding domain allows quantitative export ( t1/2 approximately 1 h) of the N-terminal ATPase domain (BiPN), consistent with the conserved domain structure of hsp70 proteins. This is the first demonstration of soluble protein secretion in African trypanosomes. Using the BiPN reporter, the sequence specificity of C-terminal tetrapeptide retention signals in trypanosomes is analyzed and found to be similar to higher eukaryotes. These results indicate that the basic signals mediating protein targeting to the ER lumen are conserved throughout the wide range of eukaryotic evolution.

  • Bangs JD, Uyetake L, Brickman MJ, Balber AE, Boothroyd JC (1993) Molecular cloning and cellular localization of a BiP homologue in Trypanosoma brucei. Divergent ER retention signals in a lower eukaryote. J. Cell. Sci. 105 ( Pt 4:1101-13 View Abstract · Pubmed Record

    Using the polymerase chain reaction with degenerate primers, three new members of the hsp70 gene family of Trypanosoma brucei have been identified. A genomic clone of one of these, gA, has been fully sequenced and the corresponding gene product has been characterized using antibody to recombinant gA fusion protein. gA is the trypanosomal homologue of BiP, an endoplasmic reticulum resident hsp70 gene family member, based on four lines of evidence: (1) gA protein has 64% deduced amino acid identity with rat BiP; (2) the deduced amino acid sequence has a putative secretory signal peptide; (3) the gA gene product is a soluble luminal resident of a trypanosomal microsome fraction; (4) the gA polypeptide does not cofractionate with mitochondrial markers. Trypanosomes are the most primitive eukaryote yet in which BiP has been identified. The gA polypeptide has been used as a specific marker for the direct visualization of endoplasmic reticulum in trypanosomes by both indirect immunofluorescence and cryoimmuno electron microscopy. The endoplasmic reticulum is seen as a tubular network that extends throughout the cell excluding the flagellum. The C-terminal tetrapeptide of gA is MDDL, which, together with the C-terminal tetrapeptide (KQDL) of a trypanosome protein disulfide isomerase homologue (Hsu et al. (1989) Biochemistry 28, 6440-6446), indicates that endoplasmic reticulum retrieval signals in trypanosomes may be as divergent and heterogeneous as any seen in the other eukaryotes yet studied.

  • Elmendorf HG, Bangs JD, Haldar K (1992) Synthesis and secretion of proteins by released malarial parasites. Mol. Biochem. Parasitol. 52(2):215-30 View Abstract · Pubmed Record

    Controlled mechanical homogenization of Plasmodium falciparum-infected erythrocytes releases parasites of a quality sufficient for studying the export of newly synthesized plasmodial proteins. Protein synthesis occurs within intact released parasites as defined by resistance of acid-insoluble incorporation of radiolabel to high levels of exogenously added EDTA, hexokinase, and RNaseA. While exogenously added ATP and erythrocyte cytosol were not essential for biosynthetic activity at levels comparable to that seen in infected erythrocytes, the addition of an extracellular ATP regenerating system (ARS) stimulated the synthesis of parasite proteins. Conversely, parasite viability and biosynthetic activity are decreased by the addition of a non-hydrolyzable ATP analogue (ATP gamma S), ADP, or ATP in the absence of a regenerating system. These data suggest a metabolic interdependence between extracellular energy metabolism and biosynthetic functions within the parasite. The export of a predominant subset of proteins was retarded in the presence of Brefeldin A, indicating the existence of a classical secretory pathway characteristic of that seen in higher eukaryotic cells. Interestingly, a Brefeldin A-insensitive component of export was also consistently observed; this may suggest the existence of an additional alternative secretory mechanism in malaria.

  • Bangs JD, Crain PF, Hashizume T, McCloskey JA, Boothroyd JC (1992) Mass spectrometry of mRNA cap 4 from trypanosomatids reveals two novel nucleosides. J. Biol. Chem. 267(14):9805-15 View Abstract · Pubmed Record

    Synthesis of mRNA in kinetoplastid protozoa involves the process of trans-splicing, in which an identical 39-41-nucleotide (depending on the species) mini-exon is placed at the 5' end of mature mRNAs. The mini-exon sequence is highly conserved among all members of the Kinetoplastida, nucleotides 1-6 being identical in the four genera so far examined. Prior to trans-splicing, the mini-exon donor RNA is capped by the addition of a (5'-5') triphosphate-linked 7-methylguanosine, followed by modification of the first four transcribed nucleotides. Partial structures have been previously deduced for this cap 4 moiety from Trypanosoma brucei and Leptomonas collosoma. We have purified enough cap 4 from T. brucei and Crithidia fasciculata to allow definitive structural analysis by combined liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry. The results, together with the known mini-exon sequence, show that cap 4 in both species has the structure m7G(5')ppp(5')m6(2)AmpAmpCmpm3Ump. The presence of N6,N6,2'-O-trimethyladenosine and 3,2'-O-dimethyluridine, nucleosides previously unknown in nature, were confirmed by rigorous comparison with synthetic standards. The conservation of cap 4 between these divergent genera suggests that this structure may be common to most if not all Kinetoplastida.

  • Bean MF, Bangs JD, Doering TL, Englund PT, Hart GW, Fenselau C, Cotter RJ (1989) Assessing heterogeneity of the high-mannose glycopeptide gp432 on the variant surface glycoprotein of trypanosomes: a comparison of plasma desorption mass spectrometry and radiolabeling techniques. Anal. Chem. 61(23):2686-8 View Abstract · Pubmed Record
  • Bangs JD, Doering TL, Englund PT, Hart GW (1988) Biosynthesis of a variant surface glycoprotein of Trypanosoma brucei. Processing of the glycolipid membrane anchor and N-linked oligosaccharides. J. Biol. Chem. 263(33):17697-705 View Abstract · Pubmed Record

    The variant surface glycoprotein (VSG) of the ILTat 1.3 variant of Trypanosoma brucei has two asparagine-linked glycan moieties, as well as a phosphatidylinositol glycan membrane anchor. We have investigated the structure and processing of each of these oligosaccharides through analysis of the intact protein and of glycopeptides. Processing has been examined by comparing glycan structures purified from an immature intracellular form (58 kDa) of VSG with those of the mature form (59 kDa) found on the parasite surface. We find exclusively high mannose oligosaccharides (Man4-7-GlcNAc2) at Asn-432 in both the immature 58-kDa and mature 59-kDa forms. In contrast, the "core" oligosaccharide of Asn-419 (Man3-GlcNAc2) appears to be nearly quantitatively processed to a complex biantennary structure [Gal-GlcNAc-Man)2-Man-GlcNAc2) during VSG maturation. The asparagine-linked structures at Asn-419, but not those at Asn-432, are resistant to endo-beta-N-acetylglucosaminidase H within 30 s of biosynthesis. This suggests possible novel and selective mechanisms for glycosylation in African trypanosomes. Finally, we show that the carboxyl-terminal glycolipid is galactosylated (3-4 residues) relatively late in VSG biosynthesis. Phosphatidylinositol glycans have been identified on a growing number of eukaryotic membrane proteins. This report provides a direct demonstration of the processing of such a glycolipid anchor following its attachment to protein.

  • Hereld D, Krakow JL, Bangs JD, Hart GW, Englund PT (1986) A phospholipase C from Trypanosoma brucei which selectively cleaves the glycolipid on the variant surface glycoprotein. J. Biol. Chem. 261(29):13813-9 View Abstract · Pubmed Record

    The surface coat of Trypanosoma brucei is composed of 10(7) molecules of the variant surface glycoprotein (VSG). Each VSG molecule is tethered to the cell membrane by a glycolipid moiety which contains 1,2-dimyristoyl-sn-phosphatidylinositol (Ferguson, M. A. J., Low, M. G., and Cross, G. A. M. (1985) J. Biol. Chem. 260, 14547-14555). Following cell lysis, an endogenous phospholipase C cleaves dimyristoyl glycerol from the glycolipid, releasing soluble VSG. We have purified this enzyme, which we designate VSG lipase, by detergent extraction, (NH4)2SO4 fractionation, hydrophobic chromatography, and cation exchange chromatography. It is purified 2600-fold and is virtually homogeneous. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular mass is 37 kDa. In solutions containing the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), the Stokes radius (2.6 nm), S20,w (3.7 S), and v (0.77 cm3/g) of VSG lipase suggest a molecular mass for the native enzyme of about 47 kDa, part of which may be due to bound CHAPS. Therefore, it is probably monomeric. VSG lipase does not require Ca2+; it is stimulated by chelating agents or dithiothreitol, and it is inhibited by some sulfhydryl reagents. The purified enzyme appears to be highly specific. Under the conditions of our assay, it cleaves the VSG glycolipid, a biosynthetic precursor of the VSG glycolipid, and, to a much lesser extent, 1,2-dimyristoyl-sn-phosphatidylinositol. There was no apparent cleavage of other myristate-containing lipids of trypanosomes or 1-stearoyl-2-arachidonoyl-sn-phosphatidylinositol.

  • Krakow JL, Hereld D, Bangs JD, Hart GW, Englund PT (1986) Identification of a glycolipid precursor of the Trypanosoma brucei variant surface glycoprotein. J. Biol. Chem. 261(26):12147-53 View Abstract · Pubmed Record

    The variant surface glycoprotein (VSG) of Trypanosoma brucei has a glycolipid covalently attached to its C terminus. This glycolipid, which anchors the protein to the cell membrane, is attached to the VSG polypeptide within 1 min after translation (Bangs, J. D. Hereld, D., Krakow, J.L., Hart, G. W., and Englund, P. T. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3207-3211). This rapid processing suggests that, prior to incorporation, the glycolipid may exist in the cell as a preformed precursor which is transferred to the VSG polypeptide en bloc. We have isolated a molecule which has properties consistent with it being a VSG glycolipid precursor. It is highly polar and can be labeled by [3H] myristate but not by [3H]palmitate. It reaches steady state during continuous labeling with [3H]myristate and shows rapid turnover in pulse-chase experiments, suggesting that it is a metabolic intermediate rather than an end product. When treated with HNO2 it liberates phosphatidylinositol, as does VSG (Ferguson, M. A. J., Low, M. G., and Cross, G. A. M. (1985) J. Biol. Chem. 260, 14547-14555). Also, like VSG, it releases a compound which co-migrates on thin layer chromatography with dimyristylglycerol when treated with the purified endogenous phospholipase C from trypanosomes. After treatment with this lipase, the putative precursor can be immunoprecipitated by antibodies directed against the C-terminal cross-reactive antigenic determinant of the VSG. These data provide strong evidence that this glycolipid is a VSG precursor.

  • Bangs JD, Andrews NW, Hart GW, Englund PT (1986) Posttranslational modification and intracellular transport of a trypanosome variant surface glycoprotein. J. Cell Biol. 103(1):255-63 (PMC2113794) View Abstract · Pubmed Record

    After synthesis on membrane-bound ribosomes, the variant surface glycoprotein (VSG) of Trypanosoma brucei is modified by: (a) removal of an N-terminal signal sequence, (b) addition of N-linked oligosaccharides, and (c) replacement of a C-terminal hydrophobic peptide with a complex glycolipid that serves as a membrane anchor. Based on pulse-chase experiments with the variant ILTat-1.3, we now report the kinetics of three subsequent processing reactions. These are: (a) conversion of newly synthesized 56/58-kD polypeptides to mature 59-kD VSG, (b) transport to the cell surface, and (c) transport to a site where VSG is susceptible to endogenous membrane-bound phospholipase C. We found that the t 1/2 of all three of these processes is approximately 15 min. The comparable kinetics of these processes is compatible with the hypotheses that transport of VSG from the site of maturation to the cell surface is rapid and that VSG may not reach a phospholipase C-containing membrane until it arrives on the cell surface. Neither tunicamycin nor monensin blocks transport of VSG, but monensin completely inhibits conversion of 58-kD VSG to the mature 59-kD form. In the presence of tunicamycin, VSG is synthesized as a 54-kD polypeptide that is subsequently processed to a form with a slightly higher Mr. This tunicamycin-resistant processing suggests that modifications unrelated to N-linked oligosaccharides occur. Surprisingly, the rate of VSG transport is reduced, but not abolished, by dropping the chase temperature to as low as 10 degrees C.

  • Bangs JD, Hereld D, Krakow JL, Hart GW, Englund PT (1985) Rapid processing of the carboxyl terminus of a trypanosome variant surface glycoprotein. Proc. Natl. Acad. Sci. U.S.A. 82(10):3207-11 (PMC397744) View Abstract · Pubmed Record

    The variant surface glycoprotein of the parasite Trypanosoma brucei contains a glycolipid of unknown structure covalently attached to its COOH terminus. We have shown, by using metabolic labeling with [35S]methionine or [3H]myristic acid, precipitation with specific antibodies, and NaDodSO4/polyacrylamide gel electrophoresis, that this glycolipid is attached to the variant surface glycoprotein polypeptide within 1 min after its translation.