Differential glycosylation of epitope-tagged glycoprotein Gp72 during the Trypanosoma cruzi life cycle

MOLECULAR

ii%HEMIcAL PARAsIToLoGy

ELSEVIER

Molecular and Biochemical Parasitology 83 (1996) 253-256

Short communication

Differential glycosylation of epitope-tagged glycoprotein during the Trypanosoma cruzi life cycle Paul A. Haynes’,

George

Gp72

A.M. Cross*

Lahorarory of’ Molecular Parasitology, The Rockefeller Unicersity, 1230 York Avenue, New York, 10021. Neiv York. USA

Received 19 August 1996: revised 27 September 1996; accepted 30 September 1996

Keywrds:

Trvpznosorna

cruzi; Glycoprotein

72; Epitope

Trypanosoma crud undergoes distinct biochemical and morphological changes during a complex life-cycle. In epimastigote cells, a 72 kDa glycoprotein, Gp72, was first identified on the cell surface using a carbohydrate-specific monoclonal antibody, WIC29.26 [l]. Further studies showed that WIC29.26 also reacted with the surface of metacyclic trypomastigotes [2], but the WIC29.26 epitope was not found in the amastigote or tissueculture trypomastigote life-cycle stages [l]. Although it has often been regarded as specific for or synonymous with Gp72, WIC29.26 recognizes a glycan epitope that is present on several glycoproteins, including Gp72 [3]. The deletion of gp72 caused an unexpected phenotype, in which the flagellum was detached

* Corresponding author. Tel: + 1 212 3277571; fax: + 1 212 3277845; e-mail: [email protected] ’ Present address: Department of Molecular Biotechnology, School of Medicine, University of Washington. Seattle, WA 98195-7730. USA.

tag; Developmental

regulation;

Glycosylation

from the cell body after emerging from the flagellar pocket, and the overal shape of the parasite was dramatically altered [3]. Complementation of null-mutant cells suggested that glycosylation of the mature protein was the limiting step in expressing functional Gp72 in epimastigotes [4]. The glycan structures that react with WIC29.26 are very unusual: they contain galactofuranose, rhamnose, fucose and xylose and appear to be phosphodiester-linked to threonine residues in the polypeptide [5]. The1 precise function of Gp72 remains uncertain, although it clearly plays an important role in the maintenance of normal parasite morphology. It has been suggested to play an important role in differentiation since WlC29.26 can inhibit metacyclogenesis in a model system [6]. As WIC29.26 only reacts with epimastigotes and metacyclic trypomastigotes, it is not possible to use this antibody to study Gp72 in other life-cycle stages. Attempts to use antisera raised against the peptide backbone of Gp72 were not successful in mi-

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254

P.A. Ha.vnes. G.A.M. Cross ! Molecular and Biochemical Parasitology 83 (1996) 253-256

croscopy [3], suggesting that little of the polypeptide chain is exposed on the cell surface. We overcame this problem by expressing an epitope-tagged version of Gp72. Three tandem copies of an influenza hemagglutinin nonapeptide sequence (HA tag; Tyr-Pro-Tyr-Asp-Val-Pro-AspTyr-Ala), inserted into the amino terminal region of the mature gp72 coding sequence, produced a functional epitope-tagged Gp72, designated Gp72HAN3 [7]. In epimastigotes, Gp72HAN3 was distributed over the cell body, but confined to the proximal region of the flagellum. In amastigotes, it was distributed uniformly [7]. In this report, we describe the expression of Gp72HAN3 in four life-cycle stages of T. crud in vitro. T.cru-_i strain Dm28c epimastigote cells were transfected with linearized pUBG72HAN3 (Fig. I), and a representative clone, designated lOC12,

0 7 ‘kh

Fig. I. The gp72han3 expression plasmid. The construction of this plasmid has been described in detail elsewhere [7]. Briefly. an oligonucleotide encoding three copies of an HA nonapeptide (Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) was inserted between residues Asp-38 and Gln-41 of the native Gp72 sequence. To construct pUBG72HAN3, a 1.6 kb fragment containing the ble gene flanked by gp72 upstream region (5’) and glyceraldehyde S-phosphate dehydrogenase intergenic region (3’) was excised from p72BGL [ 131 and ligated into pCR-script (Stratagene). The 1.7 kb gp72han3 coding region was amplified by the polymerase chain reaction, with the addition of HindI and X/r01 sites at the 5’ and 3’ termini. respectively, and ligated immediately downstream of the glyceraldehyde 3-phosphate dehydrogenase intergenic region to produce pUBG72HAN3. Parasites at mid-log phase (0.5 ml) were mixed with 50 pg of linearized pUBG72HAN3 (previously digested with Not1 and KpnI to linearize the insert and PunI to minimize remaining plasmid) in Zimmerman fusion medium [3] and electroporated using a BTX electro cell manipulator set at I.5 kV and 24 ohms resistance. Parasites were diluted into IO ml of liver infusion tryptose medium and incubated for 48 h before adding 200 pg/ml of phleomycin. Surviving transfected parasites reached mid log phase after 28-35 days, and were cloned by limited dilution in 96-well plates, in the absence of drug selection, for a further 28-35 days.

was analyzed further. Southern blotting showed that the lOCl2 epimastigote cells contained several copies of the pUBG72HAN3 plasmid replicating as a stable episome (data not shown). The difficulty in obtaining direct gene replacements in T. cruzi, as was originally intended, rather than episome formation or plasmid integration, has been noted previously [8]. The lOC12 cells maintained this episome through many generations of growth in the absence of drug selection, with no significant decrease in copy number. This is in contrast to results observed with Gp72HAN3 expressed using the pTEX shuttle vector [9], where the initial copy number of Gp72HAN3 in transformants was very high, but decreased in the absence of drug selection [7]. The lOC12 epimastigote cells transformed into metacyclic trypomastigotes when cultures were allowed to grow to stationary phase. These metacyclic trypomastigotes were used to infect monolayers of mammalian cells to produce tissueculture trypomastigotes, which were then harvested or transformed into amastigotes. Lysates were prepared from cells at each of the four life-cycle stages and analyzed by Western blotting. WIC29.26 showed a strong reaction with epimastigotes and metacyclic trypomastigotes, as expected, and no reaction with the other two stages (Fig. 2A). An amastigote-specific antibody, 2C2 [lo], reacted only with the amastigote lysate (Fig. 2B), as expected, and tissue-culture trypomastigote-specific antibody 2Al [lo] reacted only with the trypomastigote lysate (Fig. 2C). Anti-HA tag antibody reacted with a single band, of approximately equal intensity, in all four life-cycle stages (Fig. 2D). Thus the Gp72HAN3 polypeptide was expressed at similar levels in all life-cycle stages examined, but amastigotes and tissue-culture trypomastigotes did not react with WIC29.26, indicating developmentally regulated differences in glycosylation. In a previous report, we showed that Gp72HAN3 is a functional, normally glycosylated version of Gp72 that is localized to the cell surface and proximal region of the flagellum in epimastigote cells and uniformally distributed over amastigotes [7]. In this study, we used a different strain of T. cruri (Dm28c) to that used previously

P.A. Ha\nrs. G.A.M. Cross

! Molecular and Biochemical Parasitolog? 83 (1996) 253 -156

kDa 97 ‘d?

69

4%

-

46EMAT

WIC29.26

EMAT

2C2

EMAT

2A1

EMAT

anti-HA

Fig. 2. Analysis of Gp72HAN3 expression and glycosylation in the lOC12 cell line. Antibodies used in each panel are as indicated. Lane assignments: E, epimastigote; M. metacyclic trypomastigote; A, amastigote; T, tissue-culture trypomastigote. Western blots were performed with 1:5000 dilutions of the anti-HA and WIC29.26 primary antibodies, and I:1000 dilution of the 2C2 and 2Al antibody in phosphate-buffered saline. 5% nonfat milk, followed by 15000 dilutions of appropriate secondary antibody, and detected by either chemiluminescence or 5-bromo-4-chloro-3-indolylphosphate p-toluidineinitro-blue tetrazolium chloride. Epimastigote stocks of clone lOC12 were grown in liver infusion tryptose medium at 26°C without agitation. Metacyclic trypomastigotes were purified from stationary-phase epimastigote cultures by lysing remaining epimastigotes with guinea pig complement. Tissue-culture trypomastigotes were produced by incubating metacyclic trypomastigotes in the presence of gamma-irradiated L6E9 rat skeletal muscle cell monolayers maintained in Dulbecco’s Modifed Eagle Medium supplemented with 10% heat-inactivated fetal bovine serum [14]. Extracellular amastigotes were obtained by incubating released trypomastigotes in liver infusion tryptose medium at 37°C HO].

(Y-NIH), as it is more amenable to passage through the life cycle in vitro. The plasmid pUBG72HAN3 was designed to function as an integration vector, which would provide an intact, authentic gp 72 3’-untranslated region. This did not occur, however, due to the formation of a very stable episome, presumably involving the remaining undigested plasmid used in transfection. It appears from our work, and that of others (B. Burleigh and N. Andrews, personal communication), that the Dm28c strain of T. cm5 displays a marked preference for the formation of stable episomes in transformants rather than homologous integration. Because the lOC12 cells are not expressing Gp72HAN3 from a locus with authentic gp72 flanking sequences, we must be cautious in extrapolating our data to imply that the wild-type gp7-3 is expressed in all stages of the life cycle, because flanking sequences, and 3’-untranslated regions in particular, can influence the developmental regulation of gene expression in T. crlCi [ 111.

Despite these caveats, the results presented here clearly indicate that the WIC29.26 glycosylation, as carried by Gp72HAN3 epimastigotes, is developmentally regulated. The fact that the bands on anti-HA Western blots of all life-cycle stages were of a very similar intensity and size suggested that the protein was being expressed at similar levels and that the lack of recognition by WIC29.26 was not caused by a simple absence of glycosylation. If that were the case, a noticeable decrease in apparent molecular weight would have been expected as the predicted size of the unglycosylated protein is 62 kDa [12]. Thus, the Gp72HAN3 glycoprotein expressed in amastigotes and tissueculture trypomastigotes must contain most of the structural features of the epimastigote glycoprotein, but must also contain a modification that is not recognized by or prevents recognition by the WIC29.26 antibody. This is most likely caused by addition or removal of a small number of carbohydrate or phosphate residues. In further experiments, we observed that Gp72HAN3 in

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other life-cycle stages was modified by mild acid labile glycosylation similar to that found in epimastigotes [5], indicated by a change in electrophoretic mobility after acid hydrolysis (data not shown). Detailed investigations of developmental differences in glyan structures were beyond the resources of the current investigations. We have recently shown that Flal, a homolog of Gp72 found in T. brucei, is essential for viability and is expressed in both bloodstream and procyclic forms. Flal is also heavily glycosylated, but the glycosylation is different in each life-cycle stage [13]. If glycosylation of Gp72 in T. cruz-i is similarly developmentally regulated, the glycosyltransferases or glycosidases involved in this process must act on unusual sugars, both as donor and substrate, due to the novel nature of the structures involved [.5]. The isolation and characterization of enzymes involved in such pathways would be of fundamental biological interest, as well as having possible implications for control and treatment of the parasite and disease.

Acknowledgements

The authors would like to thank Barbara Burleigh and Norma Andrews for providing antibodies and sharing unpublished results, Tomoyoshi Nozaki for invaluable assistance, and Simone Lea1 for thoughtful discussions. This work was supported by the National Institutes of Health (grant AI 26197).

References [l] Snary, D., Ferguson, M.A.J., Scott, M.T. and Allen, A.K. (1981) Cell surface antigens of Trypuflosoma 0x5: use of monoclonal antibodies to identify and isolate an epimastigote specific glycoprotein. Mol. Biochem. Parasitol. 3. 343-356.

PI

Kirchhoff, L.V. and Sher, A. (1985) Modifications of a 72k surface glycoprotein (GP-72) of Trypanosoma cruzi during development. Fed. Proc. 44, 1332. [31 Cooper, R.. Ribeiro de Jesus, A. and Cross, G.A.M. (1993) Deletion of an immunodominant Trypanosoma cruzi surface glycoprotein disrupts flagellum-cell adhesion J. Cell Biol. 122, 149-156. [41 Nozaki. T. and Cross, G.A.M. (1994) Functional complementation of glycoprotein 72 in a Trypanosoma cru-_i glycoprotein 72 null mutant. Mol. Biochem. Parasitol. 67. 91llO2. M.A.J.F. and Cross, G.A.M. PI Haynes, P.A., Ferguson, (1996) Structural characterization of novel oligosaccharides of cell surface glycoproteins of Trypanosoma cru:i. Glycobiology, in press. WI Sher, A. and Snary, D. (1982) Specific inhibition of the morphogenesis of Trypanosoma cruzi by a monoclonal antibody. Nature 300, 6399640. [71 Haynes, P.A.. Russell, D.G. and Cross, G.A.M. (1996) Subcellular localization of Trypanosoma cruri glycoprotein Gp72. J. Cell Sci. In press. S., Ajioka, J. and Swindle, J. (1993) Stable PI Hariharan, transformation of Trvpanosoma cruzi: Inactivation of the PUB12.5 polyubiquitin gene by targeted gene disruption. Mol. Biochem. Parasitol. 57. 15-30. PI Kelly. J.M.. Ward, H.M., Miles, M.A. and Kendall, G. (I 992) A shuttle vector which facilitates the expression of transfected genes in Trypanosoma cru5 and Leishmania. Nucleic Acids Res. 20, 3963-3969. UOI Andrews. N.W., Hong, K.S.. Robbins, E.S. and Nussenzweig. V. (1987) Stage-specific surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruri. Exp. Parasitol. 64, 474-484. [Ill Nozaki, T. and Cross, G.A.M. (1995) Effects of 3’ untranslated and intergenic regions on gene expression in Trypunosoma cruri. Mol. Biochem. Parasitol. 75, 55-67. LIZI Cooper, R., Inverso, J.A.. Espinosa, M.. Nogueird, N. and Cross, G.A.M. (1991) Characterization of a candidate gene for Gp72, an insect stage-specific antigen of Trypanosoma cruzi. Mol. Biochem. Parasitol. 49, 45560. iI31 Nozaki, T., Haynes, P.A. and Cross, G.A.M. (1996) Structure and developmentally regulated glycosylation of an essential Trypanosoma hrucei homologue of a Tr>>panosoma cru;i flagellar adhesion glycoprotein. Mol. Biochem. Parasitol, in press. u41 Ribeiro de Jesus. A., Cooper, R., Espinosa, M., Gomes, J.E.P.L., Garcia, ES., Paul, S. and Cross, G.A.M. (1993) Gene deletion suggests a role for Trypanosoma cruzi surface glycoprotein Gp72 in the insect and mammalian stages of the life cycle. J. Cell Sci. 106. 1023-1033.