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• W2004402258 abstract "Trypanosoma congolense is an African trypanosome that causes serious disease in cattle in Sub-Saharan Africa. The four major life cycle stages of T. congolense can be grown in vitro, which has led to the identification of several cell-surface molecules expressed on the parasite during its transit through the tsetse vector. One of these, glutamic acid/alanine-rich protein (GARP), is the first expressed on procyclic forms in the tsetse midgut and is of particular interest because it replaces the major surface coat molecule of bloodstream forms, the variant surface glycoprotein (VSG) that protects the parasite membrane, and is involved in antigenic variation. Unlike VSG, however, the function of GARP is not known, which necessarily limits our understanding of parasite survival in the tsetse. Toward establishing the function of GARP, we report its three-dimensional structure solved by iodide phasing to a resolution of 1.65 Å. An extended helical bundle structure displays an unexpected and significant degree of homology to the core structure of VSG, the only other major surface molecule of trypanosomes to be structurally characterized. Immunofluorescence microscopy and immunoaffinity-tandem mass spectrometry were used in conjunction with monoclonal antibodies to map both non-surface-disposed and surface epitopes. Collectively, these studies enabled us to derive a model describing the orientation and assembly of GARP on the surface of trypanosomes. The data presented here suggest the possible structure-function relationships involved in replacement of the bloodstream form VSG by GARP as trypanosomes differentiate in the tsetse vector after a blood meal. Trypanosoma congolense is an African trypanosome that causes serious disease in cattle in Sub-Saharan Africa. The four major life cycle stages of T. congolense can be grown in vitro, which has led to the identification of several cell-surface molecules expressed on the parasite during its transit through the tsetse vector. One of these, glutamic acid/alanine-rich protein (GARP), is the first expressed on procyclic forms in the tsetse midgut and is of particular interest because it replaces the major surface coat molecule of bloodstream forms, the variant surface glycoprotein (VSG) that protects the parasite membrane, and is involved in antigenic variation. Unlike VSG, however, the function of GARP is not known, which necessarily limits our understanding of parasite survival in the tsetse. Toward establishing the function of GARP, we report its three-dimensional structure solved by iodide phasing to a resolution of 1.65 Å. An extended helical bundle structure displays an unexpected and significant degree of homology to the core structure of VSG, the only other major surface molecule of trypanosomes to be structurally characterized. Immunofluorescence microscopy and immunoaffinity-tandem mass spectrometry were used in conjunction with monoclonal antibodies to map both non-surface-disposed and surface epitopes. Collectively, these studies enabled us to derive a model describing the orientation and assembly of GARP on the surface of trypanosomes. The data presented here suggest the possible structure-function relationships involved in replacement of the bloodstream form VSG by GARP as trypanosomes differentiate in the tsetse vector after a blood meal. IntroductionAfrican trypanosomes are protozoan parasites that cause serious disease in humans and their domestic animals in Sub-Saharan Africa. These parasites have influenced the development of Africa and continue to cause socioeconomic devastation, especially by infecting cattle, the mainstay of many smallholder farmers (1Swallow B.M. PAAT Technical and Scientific Series. 2. FAO/WHO/IAEA/OAU-IBAR, Rome2000Google Scholar). Trypanosomes alternate between a mammalian host and an insect vector, the infamous tsetse (Glossina). In mammalian hosts, the trypanosomes live as bloodstream forms (BSFs) 4The abbreviations used are: BSFbloodstream formVSGvariant surface glycoproteinGARPglutamic acid/alanine-rich proteinrGARPrecombinant GARPPCFprocyclic culture formEMFepimastigote form. that are proficient at antigenic variation and host immune system evasion (2Donelson J.E. Acta Trop. 2003; 85: 391-404Crossref PubMed Scopus (136) Google Scholar). Thus, no suitable vaccine candidates for trypanosomiasis have been identified after more than a century of research. In contrast, the trypanosome life cycle stages that exist in the tsetse vector do not undergo antigenic variation. This potentially makes the vector-occupying trypanosomes much better targets for control if strategies can be devised to disrupt their life cycle in the vector or to interfere with their transmission to mammalian hosts. The primary impediment to developing strategies for disruption of trypanosome life cycles in the tsetse is a lack of understanding of the molecular basis of trypanosome-tsetse interactions. In alternating between their mammalian hosts and tsetse vectors, trypanosomes are subject to dramatic changes in environment. Therefore, it is not surprising that their response, in terms of metabolism and surface architecture, is equally dramatic. Several major surface molecules have been identified on insect forms of Trypanosoma brucei ssp. (3Roditi I. Furger A. Ruepp S. Schürch N. Bütikofer P. Mol. Biochem. Parasitol. 1998; 91: 117-130Crossref PubMed Scopus (88) Google Scholar) and on T. congolense (4Beecroft R.P. Roditi I. Pearson T.W. Mol. Biochem. Parasitol. 1993; 61: 285-294Crossref PubMed Scopus (69) Google Scholar, 5Bayne R.A. Kilbride E.A. Lainson F.A. Tetley L. Barry J.D. Mol. Biochem. Parasitol. 1993; 61: 295-310Crossref PubMed Scopus (59) Google Scholar, 6Bütikofer P. Vassella E. Boschung M. Renggli C.K. Brun R. Pearson T.W. Roditi I. Mol. Biochem. Parasitol. 2002; 119: 7-16Crossref PubMed Scopus (34) Google Scholar, 7Utz S. Roditi I. Kunz Renggli C. Almeida I.C. Acosta-Serrano A. Bütikofer P. Eukaryot. Cell. 2006; 5: 1430-1440Crossref PubMed Scopus (27) Google Scholar, 8Sakurai T. Sugimoto C. Inoue N. Mol. Biochem. Parasitol. 2008; 161: 1-11Crossref PubMed Scopus (23) Google Scholar). Although some of these molecules have been extensively studied, no functional role has been assigned to any of them.All of the trypanosome surface proteins so far described are anchored to the surface membrane via glycosylphosphatidylinositol anchors (9Ferguson M.A.J. J. Cell Sci. 1999; 112: 2799-2809Crossref PubMed Google Scholar). During all life cycle stages, the trypanosomes are covered with a continuous dense monolayer of proteins and glycoproteins proposed to protect the parasite from the host immune system or from the proteolytic environment of the tsetse (10Roditi I. Pearson T.W. Parasitol. Today. 1990; 6: 79-82Abstract Full Text PDF PubMed Scopus (45) Google Scholar, 11Stebeck C.E. Pearson T.W. Exp. Parasitol. 1994; 78: 432-436Crossref PubMed Scopus (13) Google Scholar). The parasite surface molecules expressed in the insect stages may play a role in parasite establishment, differentiation, maturation, and tropism (10Roditi I. Pearson T.W. Parasitol. Today. 1990; 6: 79-82Abstract Full Text PDF PubMed Scopus (45) Google Scholar, 11Stebeck C.E. Pearson T.W. Exp. Parasitol. 1994; 78: 432-436Crossref PubMed Scopus (13) Google Scholar). In addition, a role for surface molecules in ligand-associated parasite vector signaling involved in programmed cell death has been proposed (12Pearson T.W. Beecroft R.P. Welburn S.C. Ruepp S. Roditi I. Hwa K.Y. Englund P.T. Wells C.W. Murphy N.B. Mol. Biochem. Parasitol. 2000; 111: 333-349Crossref PubMed Scopus (39) Google Scholar). Both BSFs and metacyclic forms of trypanosomes express a dense surface coat of variant surface glycoprotein (VSG) molecules (13Vickerman K. J. Cell Sci. 1969; 5: 163-193Crossref PubMed Google Scholar) that shields non-variant underlying membrane proteins and protects them from host immune responses. These molecules are involved in antigenic variation, the well known immune evasion strategy evolved by African trypanosomes (2Donelson J.E. Acta Trop. 2003; 85: 391-404Crossref PubMed Scopus (136) Google Scholar). Differentiation of BSFs into procyclic forms in the tsetse vector is characterized by replacement of the VSG coat with a more restricted set of tsetse-specific surface molecules (3Roditi I. Furger A. Ruepp S. Schürch N. Bütikofer P. Mol. Biochem. Parasitol. 1998; 91: 117-130Crossref PubMed Scopus (88) Google Scholar). During this differentiation, at no time are parasites uncoated, as the insect form surface molecules are incorporated into the surface membrane as the VSG coat is replaced (14Roditi I. Schwarz H. Pearson T.W. Beecroft R.P. Liu M.K. Richardson J.P. Bühring H.J. Pleiss J. Bülow R. Williams R.O. Overath P. J. Cell Biol. 1989; 108: 737-746Crossref PubMed Scopus (224) Google Scholar).Trypanosoma species display different subsets of surface proteins. For example, T. brucei ssp. insect forms express the major surface glycoprotein EP and GPEET procyclins (15Roditi I. Clayton C. Mol. Biochem. Parasitol. 1999; 103: 99-100Crossref PubMed Scopus (62) Google Scholar), whereas T. congolense insect forms express four major surface molecules: glutamic acid/alanine-rich protein (GARP) (4Beecroft R.P. Roditi I. Pearson T.W. Mol. Biochem. Parasitol. 1993; 61: 285-294Crossref PubMed Scopus (69) Google Scholar, 5Bayne R.A. Kilbride E.A. Lainson F.A. Tetley L. Barry J.D. Mol. Biochem. Parasitol. 1993; 61: 295-310Crossref PubMed Scopus (59) Google Scholar), a protease-resistant surface molecule (6Bütikofer P. Vassella E. Boschung M. Renggli C.K. Brun R. Pearson T.W. Roditi I. Mol. Biochem. Parasitol. 2002; 119: 7-16Crossref PubMed Scopus (34) Google Scholar), a heptapeptide repeat protein (now considered the T. congolense procyclin) (7Utz S. Roditi I. Kunz Renggli C. Almeida I.C. Acosta-Serrano A. Bütikofer P. Eukaryot. Cell. 2006; 5: 1430-1440Crossref PubMed Scopus (27) Google Scholar), and congolense epimastigote-specific protein (found exclusively on epimastigote forms in the tsetse mouthparts) (8Sakurai T. Sugimoto C. Inoue N. Mol. Biochem. Parasitol. 2008; 161: 1-11Crossref PubMed Scopus (23) Google Scholar). All of these molecules are surface-orientated, immunodominant, and highly charged. GARP is particularly interesting, as its expression coincides with the loss and gain of VSG in the tsetse vector. GARP is expressed by early procyclic forms in the tsetse midgut as VSG is replaced (6Bütikofer P. Vassella E. Boschung M. Renggli C.K. Brun R. Pearson T.W. Roditi I. Mol. Biochem. Parasitol. 2002; 119: 7-16Crossref PubMed Scopus (34) Google Scholar) and is absent in established procyclic midgut forms (6Bütikofer P. Vassella E. Boschung M. Renggli C.K. Brun R. Pearson T.W. Roditi I. Mol. Biochem. Parasitol. 2002; 119: 7-16Crossref PubMed Scopus (34) Google Scholar), where the T. congolense heptapeptide repeat protein procyclin is predominant (7Utz S. Roditi I. Kunz Renggli C. Almeida I.C. Acosta-Serrano A. Bütikofer P. Eukaryot. Cell. 2006; 5: 1430-1440Crossref PubMed Scopus (27) Google Scholar). GARP is also strongly expressed by epimastigotes in tsetse mouthparts (6Bütikofer P. Vassella E. Boschung M. Renggli C.K. Brun R. Pearson T.W. Roditi I. Mol. Biochem. Parasitol. 2002; 119: 7-16Crossref PubMed Scopus (34) Google Scholar) and is lost during replacement by VSG molecules during differentiation to metacyclic forms.Although GARP shows no sequence similarity to VSG molecules, it is tempting to speculate that its coexpression may mitigate the loss of VSG with respect to protecting the parasite membrane during differentiation. VSG molecules are well known to protect bloodstream trypanosomes from host antibody responses; however, the function of GARP is not known, although it has been hypothesized that it serves to protect the parasite membrane molecules from digestion enzymes in the tsetse midgut or to be involved in parasite differentiation and tropism within the tsetse (4Beecroft R.P. Roditi I. Pearson T.W. Mol. Biochem. Parasitol. 1993; 61: 285-294Crossref PubMed Scopus (69) Google Scholar, 10Roditi I. Pearson T.W. Parasitol. Today. 1990; 6: 79-82Abstract Full Text PDF PubMed Scopus (45) Google Scholar, 11Stebeck C.E. Pearson T.W. Exp. Parasitol. 1994; 78: 432-436Crossref PubMed Scopus (13) Google Scholar). A requirement of this prediction is that GARP and VSG share a high degree of structural complementarity and that GARP is appropriately spatially oriented on the parasite cell surface. To address these possibilities, we present a detailed structural, immunofluorescence, and epitope mapping characterization of GARP. Collectively, the data offer a rare insight into the possible function of a trypanosome surface protein. IntroductionAfrican trypanosomes are protozoan parasites that cause serious disease in humans and their domestic animals in Sub-Saharan Africa. These parasites have influenced the development of Africa and continue to cause socioeconomic devastation, especially by infecting cattle, the mainstay of many smallholder farmers (1Swallow B.M. PAAT Technical and Scientific Series. 2. FAO/WHO/IAEA/OAU-IBAR, Rome2000Google Scholar). Trypanosomes alternate between a mammalian host and an insect vector, the infamous tsetse (Glossina). In mammalian hosts, the trypanosomes live as bloodstream forms (BSFs) 4The abbreviations used are: BSFbloodstream formVSGvariant surface glycoproteinGARPglutamic acid/alanine-rich proteinrGARPrecombinant GARPPCFprocyclic culture formEMFepimastigote form. that are proficient at antigenic variation and host immune system evasion (2Donelson J.E. Acta Trop. 2003; 85: 391-404Crossref PubMed Scopus (136) Google Scholar). Thus, no suitable vaccine candidates for trypanosomiasis have been identified after more than a century of research. In contrast, the trypanosome life cycle stages that exist in the tsetse vector do not undergo antigenic variation. This potentially makes the vector-occupying trypanosomes much better targets for control if strategies can be devised to disrupt their life cycle in the vector or to interfere with their transmission to mammalian hosts. The primary impediment to developing strategies for disruption of trypanosome life cycles in the tsetse is a lack of understanding of the molecular basis of trypanosome-tsetse interactions. In alternating between their mammalian hosts and tsetse vectors, trypanosomes are subject to dramatic changes in environment. Therefore, it is not surprising that their response, in terms of metabolism and surface architecture, is equally dramatic. Several major surface molecules have been identified on insect forms of Trypanosoma brucei ssp. (3Roditi I. Furger A. Ruepp S. Schürch N. Bütikofer P. Mol. Biochem. Parasitol. 1998; 91: 117-130Crossref PubMed Scopus (88) Google Scholar) and on T. congolense (4Beecroft R.P. Roditi I. Pearson T.W. Mol. Biochem. Parasitol. 1993; 61: 285-294Crossref PubMed Scopus (69) Google Scholar, 5Bayne R.A. Kilbride E.A. Lainson F.A. Tetley L. Barry J.D. Mol. Biochem. Parasitol. 1993; 61: 295-310Crossref PubMed Scopus (59) Google Scholar, 6Bütikofer P. Vassella E. Boschung M. Renggli C.K. Brun R. Pearson T.W. Roditi I. Mol. Biochem. Parasitol. 2002; 119: 7-16Crossref PubMed Scopus (34) Google Scholar, 7Utz S. Roditi I. Kunz Renggli C. Almeida I.C. Acosta-Serrano A. Bütikofer P. Eukaryot. Cell. 2006; 5: 1430-1440Crossref PubMed Scopus (27) Google Scholar, 8Sakurai T. Sugimoto C. Inoue N. Mol. Biochem. Parasitol. 2008; 161: 1-11Crossref PubMed Scopus (23) Google Scholar). Although some of these molecules have been extensively studied, no functional role has been assigned to any of them.All of the trypanosome surface proteins so far described are anchored to the surface membrane via glycosylphosphatidylinositol anchors (9Ferguson M.A.J. J. Cell Sci. 1999; 112: 2799-2809Crossref PubMed Google Scholar). During all life cycle stages, the trypanosomes are covered with a continuous dense monolayer of proteins and glycoproteins proposed to protect the parasite from the host immune system or from the proteolytic environment of the tsetse (10Roditi I. Pearson T.W. Parasitol. Today. 1990; 6: 79-82Abstract Full Text PDF PubMed Scopus (45) Google Scholar, 11Stebeck C.E. Pearson T.W. Exp. Parasitol. 1994; 78: 432-436Crossref PubMed Scopus (13) Google Scholar). The parasite surface molecules expressed in the insect stages may play a role in parasite establishment, differentiation, maturation, and tropism (10Roditi I. Pearson T.W. Parasitol. Today. 1990; 6: 79-82Abstract Full Text PDF PubMed Scopus (45) Google Scholar, 11Stebeck C.E. Pearson T.W. Exp. Parasitol. 1994; 78: 432-436Crossref PubMed Scopus (13) Google Scholar). In addition, a role for surface molecules in ligand-associated parasite vector signaling involved in programmed cell death has been proposed (12Pearson T.W. Beecroft R.P. Welburn S.C. Ruepp S. Roditi I. Hwa K.Y. Englund P.T. Wells C.W. Murphy N.B. Mol. Biochem. Parasitol. 2000; 111: 333-349Crossref PubMed Scopus (39) Google Scholar). Both BSFs and metacyclic forms of trypanosomes express a dense surface coat of variant surface glycoprotein (VSG) molecules (13Vickerman K. J. Cell Sci. 1969; 5: 163-193Crossref PubMed Google Scholar) that shields non-variant underlying membrane proteins and protects them from host immune responses. These molecules are involved in antigenic variation, the well known immune evasion strategy evolved by African trypanosomes (2Donelson J.E. Acta Trop. 2003; 85: 391-404Crossref PubMed Scopus (136) Google Scholar). Differentiation of BSFs into procyclic forms in the tsetse vector is characterized by replacement of the VSG coat with a more restricted set of tsetse-specific surface molecules (3Roditi I. Furger A. Ruepp S. Schürch N. Bütikofer P. Mol. Biochem. Parasitol. 1998; 91: 117-130Crossref PubMed Scopus (88) Google Scholar). During this differentiation, at no time are parasites uncoated, as the insect form surface molecules are incorporated into the surface membrane as the VSG coat is replaced (14Roditi I. Schwarz H. Pearson T.W. Beecroft R.P. Liu M.K. Richardson J.P. Bühring H.J. Pleiss J. Bülow R. Williams R.O. Overath P. J. Cell Biol. 1989; 108: 737-746Crossref PubMed Scopus (224) Google Scholar).Trypanosoma species display different subsets of surface proteins. For example, T. brucei ssp. insect forms express the major surface glycoprotein EP and GPEET procyclins (15Roditi I. Clayton C. Mol. Biochem. Parasitol. 1999; 103: 99-100Crossref PubMed Scopus (62) Google Scholar), whereas T. congolense insect forms express four major surface molecules: glutamic acid/alanine-rich protein (GARP) (4Beecroft R.P. Roditi I. Pearson T.W. Mol. Biochem. Parasitol. 1993; 61: 285-294Crossref PubMed Scopus (69) Google Scholar, 5Bayne R.A. Kilbride E.A. Lainson F.A. Tetley L. Barry J.D. Mol. Biochem. Parasitol. 1993; 61: 295-310Crossref PubMed Scopus (59) Google Scholar), a protease-resistant surface molecule (6Bütikofer P. Vassella E. Boschung M. Renggli C.K. Brun R. Pearson T.W. Roditi I. Mol. Biochem. Parasitol. 2002; 119: 7-16Crossref PubMed Scopus (34) Google Scholar), a heptapeptide repeat protein (now considered the T. congolense procyclin) (7Utz S. Roditi I. Kunz Renggli C. Almeida I.C. Acosta-Serrano A. Bütikofer P. Eukaryot. Cell. 2006; 5: 1430-1440Crossref PubMed Scopus (27) Google Scholar), and congolense epimastigote-specific protein (found exclusively on epimastigote forms in the tsetse mouthparts) (8Sakurai T. Sugimoto C. Inoue N. Mol. Biochem. Parasitol. 2008; 161: 1-11Crossref PubMed Scopus (23) Google Scholar). All of these molecules are surface-orientated, immunodominant, and highly charged. GARP is particularly interesting, as its expression coincides with the loss and gain of VSG in the tsetse vector. GARP is expressed by early procyclic forms in the tsetse midgut as VSG is replaced (6Bütikofer P. Vassella E. Boschung M. Renggli C.K. Brun R. Pearson T.W. Roditi I. Mol. Biochem. Parasitol. 2002; 119: 7-16Crossref PubMed Scopus (34) Google Scholar) and is absent in established procyclic midgut forms (6Bütikofer P. Vassella E. Boschung M. Renggli C.K. Brun R. Pearson T.W. Roditi I. Mol. Biochem. Parasitol. 2002; 119: 7-16Crossref PubMed Scopus (34) Google Scholar), where the T. congolense heptapeptide repeat protein procyclin is predominant (7Utz S. Roditi I. Kunz Renggli C. Almeida I.C. Acosta-Serrano A. Bütikofer P. Eukaryot. Cell. 2006; 5: 1430-1440Crossref PubMed Scopus (27) Google Scholar). GARP is also strongly expressed by epimastigotes in tsetse mouthparts (6Bütikofer P. Vassella E. Boschung M. Renggli C.K. Brun R. Pearson T.W. Roditi I. Mol. Biochem. Parasitol. 2002; 119: 7-16Crossref PubMed Scopus (34) Google Scholar) and is lost during replacement by VSG molecules during differentiation to metacyclic forms.Although GARP shows no sequence similarity to VSG molecules, it is tempting to speculate that its coexpression may mitigate the loss of VSG with respect to protecting the parasite membrane during differentiation. VSG molecules are well known to protect bloodstream trypanosomes from host antibody responses; however, the function of GARP is not known, although it has been hypothesized that it serves to protect the parasite membrane molecules from digestion enzymes in the tsetse midgut or to be involved in parasite differentiation and tropism within the tsetse (4Beecroft R.P. Roditi I. Pearson T.W. Mol. Biochem. Parasitol. 1993; 61: 285-294Crossref PubMed Scopus (69) Google Scholar, 10Roditi I. Pearson T.W. Parasitol. Today. 1990; 6: 79-82Abstract Full Text PDF PubMed Scopus (45) Google Scholar, 11Stebeck C.E. Pearson T.W. Exp. Parasitol. 1994; 78: 432-436Crossref PubMed Scopus (13) Google Scholar). A requirement of this prediction is that GARP and VSG share a high degree of structural complementarity and that GARP is appropriately spatially oriented on the parasite cell surface. To address these possibilities, we present a detailed structural, immunofluorescence, and epitope mapping characterization of GARP. Collectively, the data offer a rare insight into the possible function of a trypanosome surface protein. African trypanosomes are protozoan parasites that cause serious disease in humans and their domestic animals in Sub-Saharan Africa. These parasites have influenced the development of Africa and continue to cause socioeconomic devastation, especially by infecting cattle, the mainstay of many smallholder farmers (1Swallow B.M. PAAT Technical and Scientific Series. 2. FAO/WHO/IAEA/OAU-IBAR, Rome2000Google Scholar). Trypanosomes alternate between a mammalian host and an insect vector, the infamous tsetse (Glossina). In mammalian hosts, the trypanosomes live as bloodstream forms (BSFs) 4The abbreviations used are: BSFbloodstream formVSGvariant surface glycoproteinGARPglutamic acid/alanine-rich proteinrGARPrecombinant GARPPCFprocyclic culture formEMFepimastigote form. that are proficient at antigenic variation and host immune system evasion (2Donelson J.E. Acta Trop. 2003; 85: 391-404Crossref PubMed Scopus (136) Google Scholar). Thus, no suitable vaccine candidates for trypanosomiasis have been identified after more than a century of research. In contrast, the trypanosome life cycle stages that exist in the tsetse vector do not undergo antigenic variation. This potentially makes the vector-occupying trypanosomes much better targets for control if strategies can be devised to disrupt their life cycle in the vector or to interfere with their transmission to mammalian hosts. The primary impediment to developing strategies for disruption of trypanosome life cycles in the tsetse is a lack of understanding of the molecular basis of trypanosome-tsetse interactions. In alternating between their mammalian hosts and tsetse vectors, trypanosomes are subject to dramatic changes in environment. Therefore, it is not surprising that their response, in terms of metabolism and surface architecture, is equally dramatic. Several major surface molecules have been identified on insect forms of Trypanosoma brucei ssp. (3Roditi I. Furger A. Ruepp S. Schürch N. Bütikofer P. Mol. Biochem. Parasitol. 1998; 91: 117-130Crossref PubMed Scopus (88) Google Scholar) and on T. congolense (4Beecroft R.P. Roditi I. Pearson T.W. Mol. Biochem. Parasitol. 1993; 61: 285-294Crossref PubMed Scopus (69) Google Scholar, 5Bayne R.A. Kilbride E.A. Lainson F.A. Tetley L. Barry J.D. Mol. Biochem. Parasitol. 1993; 61: 295-310Crossref PubMed Scopus (59) Google Scholar, 6Bütikofer P. Vassella E. Boschung M. Renggli C.K. Brun R. Pearson T.W. Roditi I. Mol. Biochem. Parasitol. 2002; 119: 7-16Crossref PubMed Scopus (34) Google Scholar, 7Utz S. Roditi I. Kunz Renggli C. Almeida I.C. Acosta-Serrano A. Bütikofer P. Eukaryot. Cell. 2006; 5: 1430-1440Crossref PubMed Scopus (27) Google Scholar, 8Sakurai T. Sugimoto C. Inoue N. Mol. Biochem. Parasitol. 2008; 161: 1-11Crossref PubMed Scopus (23) Google Scholar). Although some of these molecules have been extensively studied, no functional role has been assigned to any of them. bloodstream form variant surface glycoprotein glutamic acid/alanine-rich protein recombinant GARP procyclic culture form epimastigote form. All of the trypanosome surface proteins so far described are anchored to the surface membrane via glycosylphosphatidylinositol anchors (9Ferguson M.A.J. J. Cell Sci. 1999; 112: 2799-2809Crossref PubMed Google Scholar). During all life cycle stages, the trypanosomes are covered with a continuous dense monolayer of proteins and glycoproteins proposed to protect the parasite from the host immune system or from the proteolytic environment of the tsetse (10Roditi I. Pearson T.W. Parasitol. Today. 1990; 6: 79-82Abstract Full Text PDF PubMed Scopus (45) Google Scholar, 11Stebeck C.E. Pearson T.W. Exp. Parasitol. 1994; 78: 432-436Crossref PubMed Scopus (13) Google Scholar). The parasite surface molecules expressed in the insect stages may play a role in parasite establishment, differentiation, maturation, and tropism (10Roditi I. Pearson T.W. Parasitol. Today. 1990; 6: 79-82Abstract Full Text PDF PubMed Scopus (45) Google Scholar, 11Stebeck C.E. Pearson T.W. Exp. Parasitol. 1994; 78: 432-436Crossref PubMed Scopus (13) Google Scholar). In addition, a role for surface molecules in ligand-associated parasite vector signaling involved in programmed cell death has been proposed (12Pearson T.W. Beecroft R.P. Welburn S.C. Ruepp S. Roditi I. Hwa K.Y. Englund P.T. Wells C.W. Murphy N.B. Mol. Biochem. Parasitol. 2000; 111: 333-349Crossref PubMed Scopus (39) Google Scholar). Both BSFs and metacyclic forms of trypanosomes express a dense surface coat of variant surface glycoprotein (VSG) molecules (13Vickerman K. J. Cell Sci. 1969; 5: 163-193Crossref PubMed Google Scholar) that shields non-variant underlying membrane proteins and protects them from host immune responses. These molecules are involved in antigenic variation, the well known immune evasion strategy evolved by African trypanosomes (2Donelson J.E. Acta Trop. 2003; 85: 391-404Crossref PubMed Scopus (136) Google Scholar). Differentiation of BSFs into procyclic forms in the tsetse vector is characterized by replacement of the VSG coat with a more restricted set of tsetse-specific surface molecules (3Roditi I. Furger A. Ruepp S. Schürch N. Bütikofer P. Mol. Biochem. Parasitol. 1998; 91: 117-130Crossref PubMed Scopus (88) Google Scholar). During this differentiation, at no time are parasites uncoated, as the insect form surface molecules are incorporated into the surface membrane as the VSG coat is replaced (14Roditi I. Schwarz H. Pearson T.W. Beecroft R.P. Liu M.K. Richardson J.P. Bühring H.J. Pleiss J. Bülow R. Williams R.O. Overath P. J. Cell Biol. 1989; 108: 737-746Crossref PubMed Scopus (224) Google Scholar). Trypanosoma species display different subsets of surface proteins. For example, T. brucei ssp. insect forms express the major surface glycoprotein EP and GPEET procyclins (15Roditi I. Clayton C. Mol. Biochem. Parasitol. 1999; 103: 99-100Crossref PubMed Scopus (62) Google Scholar), whereas T. congolense insect forms express four major surface molecules: glutamic acid/alanine-rich protein (GARP) (4Beecroft R.P. Roditi I. Pearson T.W. Mol. Biochem. Parasitol. 1993; 61: 285-294Crossref PubMed Scopus (69) Google Scholar, 5Bayne R.A. Kilbride E.A. Lainson F.A. Tetley L. Barry J.D. Mol. Biochem. Parasitol. 1993; 61: 295-310Crossref PubMed Scopus (59) Google Scholar), a protease-resistant surface molecule (6Bütikofer P. Vassella E. Boschung M. Renggli C.K. Brun R. Pearson T.W. Roditi I. Mol. Biochem. Parasitol. 2002; 119: 7-16Crossref PubMed Scopus (34) Google Scholar), a heptapeptide repeat protein (now considered the T. congolense procyclin) (7Utz S. Roditi I. Kunz Renggli C. Almeida I.C. Acosta-Serrano A. Bütikofer P. Eukaryot. Cell. 2006; 5: 1430-1440Crossref PubMed Scopus (27) Google Scholar), and congolense epimastigote-specific protein (found exclusively on epimastigote forms in the tsetse mouthparts) (8Sakurai T. Sugimoto C. Inoue N. Mol. Biochem. Parasitol. 2008; 161: 1-11Crossref PubMed Scopus (23) Google Scholar). All of these molecules are surface-orientated, immunodominant, and highly charged. GARP is particularly interesting, as its expression coincides with the loss and gain of VSG in the tsetse vector. GARP is expressed by early procyclic forms in the tsetse midgut as VSG is replaced (6Bütikofer P. Vassella E. Boschung M. Renggli C.K. Brun R. Pearson T.W. Roditi I. Mol. Biochem. Parasitol. 2002; 119: 7-16Crossref PubMed Scopus (34) Google Scholar) and is absent in established procyclic midgut forms (6Bütikofer P. Vassella E. Boschung M. Renggli C.K. Brun R. Pearson T.W. Roditi I. Mol. Biochem. Parasitol. 2002; 119: 7-16Crossref PubMed Scopus (34) Google Scholar), where the T. congolense heptapeptide repeat protein procyclin is predominant (7Utz S. Roditi I. Kunz Renggli C. Almeida I.C. Acosta-Serrano A. Bütikofer P. Eukaryot. Cell. 2006; 5: 1430-1440Crossref PubMed Scopus (27) Google Scholar). GARP is also strongly expressed by epimastigotes in tsetse mouthparts (6Bütikofer P. Vassella E. Boschung M. Renggli C.K. Brun R. Pearson T.W. Roditi I. Mol. Biochem. Parasitol. 2002; 119: 7-16Crossref PubMed Scopus (34) Google Scholar) and is lost during replacement by VSG molecules during differentiation to metacyclic forms. Although GARP shows no sequence similarity to VSG molecules, it is tempting to speculate that its coexpression may mitigate the loss of VSG with respect to protecting the parasite membrane during differentiation. VSG molecules are well known to protect bloodstream trypanosomes from host antibody responses; however, the function of GARP is not known, although it has been hypothesized that it serves to protect the parasite membrane molecules from digestion enzymes in the tsetse midgut or to be involved in parasite differentiation and tropism within the tsetse (4Beecroft R.P. Roditi I. Pearson T.W. Mol. Biochem. Parasitol. 1993; 61: 285-294Crossref PubMed Scopus (69) Google Scholar, 10Roditi I. Pearson T.W. Parasitol. Today. 1990; 6: 79-82Abstract Full Text PDF PubMed Scopus (45) Google Scholar, 11Stebeck C.E. Pearson T.W. Exp. Parasitol. 1994; 78: 432-436Crossref PubMed Scopus (13) Google Scholar). A requirement of this prediction is that GARP and VSG share a high degree of structural complementarity and that GARP is appropriately spatially oriented on the parasite cell surface. To address these possibilities, we present a detailed structural, immunofluorescence, and epitope mapping characterization of GARP. Collectively, the data offer a rare insight into the possible function of a trypanosome surface protein. We thank Dr. Lee Haines for generously providing trypanosomes from infected tsetse and for preparing microscope slides." @default.
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• W2004402258 title "Structural Characterization and Epitope Mapping of the Glutamic Acid/Alanine-rich Protein from Trypanosoma congolense" @default.
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