Expression of bloodstream variant surface glycoproteins in procyclic stage Trypanosoma brucei: role of GPI anchors in secretion
1997; Springer Nature; Volume: 16; Issue: 14 Linguagem: Inglês
10.1093/emboj/16.14.4285
ISSN1460-2075
Autores Tópico(s)Toxin Mechanisms and Immunotoxins
ResumoArticle15 July 1997free access Expression of bloodstream variant surface glycoproteins in procyclic stage Trypanosoma brucei: role of GPI anchors in secretion James D. Bangs Corresponding Author James D. Bangs Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, 1300 University Avenue, Madison, WI, 53706 USA Search for more papers by this author Dawn M. Ransom Dawn M. Ransom Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, 1300 University Avenue, Madison, WI, 53706 USA Search for more papers by this author Mary Ann McDowell Mary Ann McDowell Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, 1300 University Avenue, Madison, WI, 53706 USA Search for more papers by this author Eric M. Brouch Eric M. Brouch Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, 1300 University Avenue, Madison, WI, 53706 USA Search for more papers by this author James D. Bangs Corresponding Author James D. Bangs Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, 1300 University Avenue, Madison, WI, 53706 USA Search for more papers by this author Dawn M. Ransom Dawn M. Ransom Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, 1300 University Avenue, Madison, WI, 53706 USA Search for more papers by this author Mary Ann McDowell Mary Ann McDowell Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, 1300 University Avenue, Madison, WI, 53706 USA Search for more papers by this author Eric M. Brouch Eric M. Brouch Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, 1300 University Avenue, Madison, WI, 53706 USA Search for more papers by this author Author Information James D. Bangs 1, Dawn M. Ransom1, Mary Ann McDowell1 and Eric M. Brouch1 1Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, 1300 University Avenue, Madison, WI, 53706 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:4285-4294https://doi.org/10.1093/emboj/16.14.4285 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 (t1/2 ∼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 (t1/2 ∼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. Introduction African trypanosomes are pathogenic kinetoplastid protozoa responsible for human and veterinary trypanosomiasis in sub-Saharan Africa. They are digenetic parasites with a life cycle that alternates between the insect vector (tsetse flies) and the bloodstream of mammalian hosts. Both forms are commonly studied in the laboratory and each elaborates a distinct stage-specific glycosyl phosphatidylinositol (GPI)-anchored protein: variant surface glycoprotein (VSG) in bloodstream trypanosomes (Cross, 1975) and procyclic acidic repetitive protein (PARP, also called procyclin) in the procyclic insect stage (Mowatt and Clayton, 1987; Roditi et al., 1987; Richardson et al., 1988). These molecules comprise the major cell surface component, and consequently are the major secretory cargo, of their respective stages of the life cycle. Not surprisingly then, VSG and PARP have provided many insights into the secretory pathway of these ancient eukaryotes. The adaptation of VSG as a recombinant secretory reporter (Bangs et al., 1996) promises even more insights to come. VSG (10–20% of total bloodstream protein) constitutes a dense monolayer surface coat that completely envelopes the cell (Vickerman, 1969; Cross, 1975). It is by the sequential expression of distinct VSG genes, a process called antigenic variation, that the parasite avoids the host immune response (reviewed in Vickerman et al., 1993). A homodimer (Auffret and Turner, 1981), VSG is synthesized in the endoplasmic reticulum (ER) where it is modified rapidly by core N-linked glycosylation and GPI addition (Bangs et al., 1985, 1986; Ferguson et al., 1986; Duszenko et al., 1988). Newly synthesized VSG has been shown to interact physically with BiP (Bangs et al., 1996), an ER molecular chaperone. It is presumably at this early stage that dimerization takes place, as is the case with secretory macromolecules in other eukaryotic cells (Hurtley and Helenius, 1989). Thereafter, VSG is transported through the cells (t1/2 15 min), with concomitant oligosaccharide processing, to the cell surface where it is incorporated into the surface coat. In contrast to VSG, PARP (1–3% of total procyclic protein) has an essentially non-variant sequence (Mowatt and Clayton, 1987) consisting of an ∼40 amino acid N-terminal domain containing a single N-linked glycan followed by a C-terminal domain composed of a variable number (∼30) of Glu–Pro dipeptide repeats. The repeat domain is believed to have a rigid and extended conformation (Roditi et al., 1989). The precise function of PARP is not known, but its relative abundance suggests that it forms a surface coat for protection in the hydrolytic environment of the tsetse fly midgut (Stebeck and Pearson, 1994); the dipeptide repeat domain has been demonstrated to be essentially protease resistant (Ferguson et al., 1993). Nothing is known concerning the post-translational processing and intracellular transport of PARP. The VSG GPI anchor and PARP GPI precursor (and presumably the mature PARP anchor) have identical Manα1–2Manα1–6Manα1–4GlcN glycan core structures linked α1–6 to phosphatidylinositol (reviewed in Englund, 1993). The VSG anchor contains exclusively dimyristoyl glycerol (Ferguson et al., 1985) while the PARP anchor contains 1-stearoyl-2-lyso glycerol and is palmitylated on the inositol (Field et al., 1991). A consequence of this lipid arrangement is that, unlike the diacylglycerol (DAG) VSG anchor, the PARP structure is resistant to phosphatidylinositol-specific phospholipase C (PI-PLC; Field et al., 1991). Both structures bear distinct side chain modifications to the core glycan. VSG has a variable number of α-linked galactose residues (Ferguson et al., 1988) and, although its precise structure is not known, the PARP GPI anchor has a poly-N-acetyllactosamine side chain with terminal sialic acids (Ferguson et al., 1993). Both of these side chains are synthesized after attachment of the core GPI anchor to protein (Bangs et al., 1988; Ferguson et al., 1993). Presumably, the N-acetyllactosamine structures of the PARP anchor are attached during intracellular transport, but sialylation occurs after export and is mediated by a cell surface trans-sialidase activity (Pontes de Carvalho et al., 1993). Expression of VSG and PARP are coordinately regulated; bloodstream trypanosomes that are induced in vitro to differentiate to procyclic forms simultaneously repress expression of VSG and induce the expression of PARP (Roditi et al., 1989). During the differentiation process, the turnover of VSG increases dramatically (from t1/2 ∼32 h to ∼13 h) as VSG is shed from the surface of differentiating cells (Bulow et al., 1989). Release occurs by proteolytic cleavage of VSG near the C-terminus, generating a soluble truncated form (Ziegelbauer et al., 1993). The protease(s) mediating release, which is apparently activated or expressed during differentiation, is not susceptible to many commonly used protease inhibitors. Using a stable transformation system, we have shown previously that a recombinant form of VSG, truncated at the site of GPI addition, is exported in a soluble manner from procyclic cells (Bangs et al., 1996). In this study, we extend these results to include both full-length wild-type and truncated forms of two distinct VSGs. We find that wild-type VSG is synthesized, dimerized and GPI anchored in procyclic trypanosomes and that it is exported to the cell surface efficiently (t1/2 ∼1 h). It is then shed into the medium by a proteolytic event that mimics the release of VSG from differentiating bloodstream cells. Proteolysis is sensitive to chelating agents, including 1,10-phenanthroline, suggesting involvement of a cell surface zinc metalloprotease. Export of the truncated form of VSG is greatly reduced (t1/2 ∼5 h) relative to the rate of GPI-anchored VSG transport, suggesting a role for GPI anchors in protein folding and/or forward transport from the ER. Fusion of the PARP GPI addition sequence to truncated VSG not only restores rapid transport, but also results in apparent modification of the GPI anchor during intracellular transport. This finding suggests that adjacent protein sequence may influence modification of the core GPI structure. Results Expression of VSG in procyclic trypanosomes In the process of developing soluble secretory reporters for expression in transformed trypanosomes, we truncated a VSG gene (VSG 117) such that the C-terminal peptide sequence specifying GPI addition was deleted (Bangs et al., 1996). This reporter, 117Δgpi, is secreted from procyclic trypanosomes, albeit inefficiently. To investigate this phenomenon further and to determine if full-length wild-type VSG can be synthesized, GPI anchored and transported with fidelity in procyclic insect stage trypanosomes, we generated a procyclic cell line expressing unmodified VSG 117 (117wt) (see Figure 1). In addition, an analogous set of reporters was generated using the distinct VSG 221 gene (221wt and 221Δgpi). All cell lines were analyzed in parallel by metabolic radiolabeling and specific immunoprecipitation from cell and media fractions (Figure 2). Figure 1.VSG secretory reporters. (A) Diagrams (not to scale) of the various VSG 117-derived secretory reporters used. Domain regions are: N-terminal signal sequence (); mature VSG coding region (□); VSG GPI anchor sequence (); HA9 epitope (■); PARP GPI anchor sequence (). The approximate positions of the single N-linked glycosylation site at Asn453 (●) and the C-terminal proteolytic cleavage site (▲) are indicated. Asterisks; analogous VSG 221wt and 221Δgpi reporters were also prepared. (B) C-terminal amino acid sequences of the VSG 117 reporters. Sequences are aligned from the C-terminus of mature VSG 117. Sequences derived from the PARP C-terminus (underlined) and the HA9 epitope (bold) are indicated. Asterisks denote sites of GPI addition in the native VSG 117 and PARP proteins. Sequences are from Boothroyd et al. (1981) and Clayton and Mowatt (1989), and amino acid numbering, here and in the text, is relative to the initiation methionine residue of VSG 117. Download figure Download PowerPoint Figure 2.Expression of recombinant VSG reporters. Transformed procyclic cell lines expressing different VSG 117 and VSG 221 reporters, as denoted, were radiolabeled with [35S]methionine for 4 h. Cell (c) and media (m) fractions were prepared, and labeled polypeptides were immunoprecipitated with anti-VSG 117 (lanes 1–4) or anti-VSG 221 (lanes 5–8) antibody. Immunoprecipitates were analyzed by SDS–PAGE and fluorography. Lanes 5 and 6, 107 cell equivalents; all other lanes, 5×106 cell equivalents. A scan of a 36 h exposure is presented. Scale refers to relative molecular mass in kDa. Download figure Download PowerPoint As previously reported, a polypeptide of the expected Mr (∼59 kDa) is synthesized in, and secreted from, the 117Δgpi cell line (lanes 3 and 4). However, the ratio of exported to cell-associated protein is low, suggesting that this reporter is poorly secreted. Likewise, deletion of the GPI anchor addition sequence results in low level secretion of VSG 221 (lanes 7 and 8). In the 117wt cell line, an abundant cell-associated polypeptide of the predicted Mr (∼59 kDa) is also detected (lane 1), but two unexpected results were obtained. First, a smaller minor form (∼53 kDa) of cell-associated VSG is also apparent (lane 1) and, second, this 53 kDa form is abundant in culture medium derived from these cells (lane 2). A similar pattern of expression is seen in the 221wt cell line (lanes 5 and 6), although the soluble fragment is smaller, indicating that release of truncated VSG (tVSG) is not restricted to a single variant antigen. The magnitude of the truncations, especially that of 221 tVSG, suggests that release occurs by proteolytic cleavage upstream of the C-terminal membrane anchor. Recombinant 117wt protein is GPI anchored Before proceeding with an analysis of intracellular transport of recombinant VSGs, we wished to prove that the full-length reporters are indeed properly GPI anchored in procyclic cells. Therefore, both 117wt and 117Δgpi cells were metabolically radiolabeled with [3H]palmitate (Figure 3). Palmitate is a component of the PARP GPI anchor (Field et al., 1991) and would be expected to specifically label GPI structures on recombinant VSG synthesized in procyclic cells. Labeled full-length VSG was detected in the 117wt cell line (lane 1), suggesting that VSG is properly GPI anchored. The failure to detect labeled VSG in 117Δgpi cells (lane 2) indicates that labeling is dependent on the presence of the C-terminal GPI addition peptide. To confirm this, we restored palmitate labeling (lane 3) by placing the authentic PARP GPI addition peptide at the C-terminus of the 117Δgpi reporter (117HP, see Figure 1). Collectively, these data indicate that the label has been incorporated into a bona fide GPI structure. No labeled polypeptides corresponding to tVSG were observed in any cell line, but palmitate was incorporated into small polypeptides derived from both the 117wt (lane 1) and 117HP (lane 3) cell lines. In keeping with our conclusion that tVSG release results from cleavage proximal to the C-terminus, these species must be the residual C-terminal GPI-anchored fragment. The larger size of the 117HP fragment is due, in part, to the inclusion of extra amino acids during the construction of this reporter (see Figure 1). Figure 3.[3H]Palmitate labeling of 117 VSGs. 117wt (lane 1), 117Δgpi (lane 2) and 117HP (lane 3) cell lines were metabolically radiolabeled with [3H]palmitate as described in Materials and methods. Radiolabeled polypeptides were immunoprecipitated with anti–VSG 117, fractionated on a 15% SDS–polyacrylamide gel and analyzed by fluorography. A scan of a 6 week exposure is shown. All lanes contain 4×107 cell equivalents. Full-length polypeptides (F) and C-terminal fragments (C) are denoted. Scale refers to relative molecular mass in kDa. (Note: discontinuities in the lower molecular mass region of the gel are cracks caused by drying.) Download figure Download PowerPoint To characterize the nature of the VSG GPI anchor further, we tested for sensitivity to GPI-specific phopholipase C (GPI-PLC). If a PARP-type inositol-acylated anchor is attached to VSG in procyclic cells, it should be resistant to the action of this enzyme (Field et al., 1991). Radiolabeled 117wt cells were extracted by Triton X-114 phase separation and the hydrophobic proteins were subjected to digestion with trypanosomal GPI-PLC. Purified bloodstream membrane-form 221 VSG was included in the digests as a positive control. Reaction products were fractionated by phase separation and analyzed (Figure 4) by immunoblotting (221 VSG) or immunoprecipitation (117 VSG). Untreated 221 VSG partitions exclusively in the detergent phase (Figure 4A, lane 1 versus 2) and GPI-PLC treatment substantially reverses this pattern (Figure 4A, lane 3 versus 4), consistent with the removal of DAG from the GPI anchor. Untreated procyclic 117wt VSG is also found in the detergent phase (Figure 4B, lane 1 versus 2), but GPI-PLC treatment has no effect on this distribution. This result indicates that a PARP-type anchor is added to VSG when expressed in procyclic cells. Figure 4.GPI-PLC treatment of procyclic VSG. 117wt procylic cells were radiolabeled with [35S]methionine for 2 h and extracted in 1% Triton X-114. Purified bloodstream membrane-form 221 VSG was added to the extract and samples were incubated for 2 h with (+) or without (−) GPI-PLC. Aqueous (A) and detergent (D) fractions were generated by phase separation and analyzed for 221 (A) and 117 (B) VSG by immunoblotting and immunoprecipitation, respectively. Scale refers to relative molecular mass in kDa. A scan of an overnight exposure is presented in (B). (Note: 117 tVSG is not detected in this exposure because the labeling period is shorter than in Figure 2.) Download figure Download PowerPoint Kinetics of VSG transport We next determined the precise kinetics for intracellular transport of VSG in procyclic cells. Both the 117wt and 117Δgpi cell lines were analyzed by pulse–chase radiolabeling followed by immunoprecipitation of VSG polypeptides from cell and media fractions. Transport and release of 117wt VSG was monitored over a 4 h chase (Figure 5A). Initially, all of this reporter is cell associated and is detected exclusively as full-length VSG (lane 1). During the chase, this form disappears from the cell (lanes 2–5), concomitant with the appearance of 117 tVSG in the medium (lanes 6–10). The kinetic half-time of 117wt transport and release, as measured by the disappearance of cell-associated full-length VSG, is ∼2.5 h (Figure 5B). However, there is a distinct lag in export, best visualized in the curve for appearance of tVSG in the medium. Identical results were obtained for the 221wt cell line (data not shown). Figure 5.Kinetics of 117wt VSG secretion. Transformed procyclic cells expressing recombinant 117wt VSG were pulse-labeled with [35S]methionine for 15 min and then chased for 4 h. (A) At the indicated chase times, aliquots were separated into cell and medium fractions and radiolabeled VSG polypeptides were analyzed as in Figure 2. All lanes contain 5×106 cell equivalents. A scan of a 3 day exposure is presented. Scale refers to relative molecular mass in kDa. (B) Repetitions of this experiment (n = 3) were analyzed by densitometry, and the kinetics of transport were determined as described previously (Bangs et al., 1996). Data are presented as percentage of total reporter at time zero (ordinate) versus chase time (abscissa). ▲, cell-associated reporter; ●, secreted reporter. Download figure Download PowerPoint These results are consistent with a two-step process in which newly synthesized VSG is first transported to the cell surface where it transiently resides as a GPI-anchored homodimer. Subsequent proteolytic cleavage, proximal to the C-terminus of each monomer, results in quantitative release as dimeric tVSG. The smaller size of 221 tVSG (see Figure 2) can be accounted for, in part, by cleavage further from the C-terminus and in part by an N-linked oligosaccharide, immediately upstream of the GPI anchor (Holder, 1985; Carrington et al., 1991), that remains with the C-terminal fragment. Inherent in the model is the presence at the cell surface of a heterodimeric VSG intermediate, composed of one full-length GPI-anchored monomer and one tVSG monomer. Consistent with this prediction is the presence of a small level of cell-associated 117 tVSG later in the chase period (Figure 5A, lanes 2–5). Another prediction is that the true rate of intracellular transport should be faster than the measured rate of release. Additional data to support this model are presented below. Export of 117Δgpi was monitored over a 24 h chase period (Figure 6A) and a t1/2 for export of ∼5 h, with no apparent lag, was determined (Figure 6B). While this is slow relative to export of both 117wt VSG and another soluble recombinant reporter, BiPN (t1/2 ∼1 h) (Bangs et al., 1996), it is nevertheless efficient since essentially all of the labeled 117Δgpi VSG is exported to the medium by the end of the chase period (compare lanes 1 and 12). Identical results were obtained for the 221Δgpi cell line (data not shown). Figure 6.Kinetics of 117Δgpi VSG secretion. Transformed procyclic cells expressing recombinant 117Δgpi VSG were pulse-labeled for 15 min and then chased for 24 h. At the indicated chase times, aliquots were separated into cell and medium fractions and analyzed as in Figure 5. A representative fluorogram is presented in (A). Densitometric analyses of repetitions (n = 3) are presented in (B). ▲, cell-associated reporter; ●, secreted reporter. Download figure Download PowerPoint The PARP GPI sequence restores transport competence The data presented above suggest that the absence of a GPI anchor reduces the efficiency of VSG transport. If so, the 117HP reporter, with the PARP GPI anchor sequence fused at the C-terminus, should display wild-type kinetics. To control for the presence of the HA9 epitope in 117HP, a matched GPI-minus reporter (117H) was also prepared (see Figure 1). Pulse–chase analyses (Figure 7) indicate that neither the manner nor rate of export of 117HP (Figure 7A) or 117H (Figure 7B) VSG differ significantly from that of the original 117 constructs. Thus, addition of a GPI signal is sufficient to restore transport competence, confirming that the transport defects in 117Δgpi and 117H are related to the absence of a GPI anchor. However, in contrast to 117wt (see Figure 5A), the size of 117HP VSG increases during intracellular transport. Initially detected as an immature form (∼65 kDa; Figure 7A, lane 1), during the chase period it is converted to a mature form (∼68 kDa) and subsequently is released from cells (Figure 7A, lanes 2–5). These findings suggest that 117HP, but not 117wt, VSG is modified during intracellular transport. Figure 7.Restoration of the GPI-plus phenotype. Transformed procyclic cell lines expressing recombinant VSG 117 reporters were pulse-labeled for 15 min and then chased for 4 h (A, 117HP) or 24 h (B, 117H). At the indicated chase times, aliquots were separated into cell and medium fractions and analyzed as in Figure 5, except that (A) is an 8–15% gradient gel. All lanes contain 5×106 cell equivalents. Scans of 2–3 day exposures are presented. Scale refers to relative molecular mass in kDa. Download figure Download PowerPoint Endoglycosidase treatment of procyclic VSGs One possibility for post-translational processing of 117HP is the N-linked oligosaccharide at Asn453 of the 117 VSG sequence (see Figure 1). Although processing of N-linked oligosaccharides does occur in bloodstream forms, it has not been reported in procyclic trypanosomes. To address this question, we treated radiolabeled procyclic VSG with endo-β-N-acetylglucosaminidase H (Endo H), an enzyme that removes unprocessed N-linked oligosaccharides (Figure 8). Endo H treatment coordinately decreased the sizes of both full-length and truncated forms of 117wt (lanes 1 and 2) and 117HP (lanes 3 and 4) VSG by an amount consistent with the removal of a single high-mannose (i.e. unprocessed) oligosaccharide. It is also apparent that tVSG fragments derived from both reporters are essentially the same size (compare lanes 1 versus 3, and 2 versus 4). Since the 117HP and 117wt proteins (i) both have C-terminal GPI anchors, (ii) differ only in their C-terminal amino acid sequences and (iii) generate identical glycosylated tVSG fragments, a common cleavage site must exist downstream of Asn453 in the shared VSG 117 amino acid sequence, consistent with the generation of different sized C-terminal fragments (Figure 3). These results also demonstrate that the N-linked oligosaccharide is not involved in maturation of 117HP and that the site of processing is proximal to the C-terminus. Figure 8.Endoglycosidase treatment of 117wt and 117HP VSG. Cell lines were radiolabeled for 4 h, and 117wt (lanes 1 and 2) and 117HP (lanes 3 and 4) VSG polypeptides were immunoprecipitated from cell lysates. Immunoprecipitates were mock treated (−, lanes 1 and 3) or digested with Endo H (+, lanes 2 and 4). Reactions were fractionated on 8–15% SDS–polyacrylamide gels and visualized by fluorography. All lanes contain 107 cell equivalents. Scans of 36 h exposures are presented. Scales refer to relative molecular mass in kDa. Download figure Download PowerPoint We have also found that the two N-linked oligosaccharides attached to 221wt VSG in procyclic trypanosomes are fully Endo H sensitive (unpublished observations). In bloodstream cells, this VSG bears one high-mannose (sensitive) and one complex (resistant) oligosaccharide (Zamze et al., 1991). This result, together with similar findings for endogenous p67, a highly glycosylated lysosomal membrane protein (Brickman and Balber, 1994; Kelley et al., 1996), suggests that procyclic cells are unable to process protein-linked high-mannose oligosaccharides to complex structures. Release of VSG by a metalloprotease The release of tVSG from procyclic cells provides a convenient assay that in reality measures a two-step process, transport and release. To study the release process alone, we surface-biotinylated the 117wt cell line and then followed the appearance of biotinylated 117 tVSG as a function of time (Figure 9A). Initially, all biotinylated VSG was present on the cell surface as both full-length and truncated VSG in approximately equal proportions. During the subsequent incubation, this doublet of surface VSG coordinately decreases concomitant with the exclusive appearance of tVSG in the medium. The presence of a large steady-state pool of cell surface tVSG, that has no membrane anchor of its own, is consistent with our interpretation that a heterodimer of full-length VSG and tVSG is an intermediate of the release process. Unlike the transport and release of biosynthetically labeled VSG, there is no lag when only the surface pool of VSG is assayed. These results indicate that the estimated t1/2 for release of biosynthetically labeled VSG is an underestimate of the actual rate for intracellular transport. Figure 9.Release of cell surface 117wt VSG from procyclic trypanosomes. 117wt cells were surface biotinylated as described in Materials and methods. (A) Kinetics of release of cell surface 117wt VSG. Biotinylated cells were cultured at 27°C in TM-P medium and, at the indicated times, aliquots were separated into cell and media fractions. (B) Inhibition of release by chelators of divalent cations. Biotinylated cells were cultured for 4 h at 27°C in supplemented PBS containing 3 mM EDTA, EGTA or 1,10-phenanthroline and then separated into cell and media fractions. Control biotinylated cells were analyzed with (Mock) or without (Cont) incubation. (A and B) 117 VSG polypeptides were specifically immunoprecipitated from all samples and fractionated by SDS–PAGE. Following electrophoretic transfer to nitrocellulose, biotinylated VSG was detected with streptavidin–HRP conjugate. All lanes contain 5×106 cell equivalents. Full-length (F) and tVSG (T) forms of 117wt VSG are indicated. Scale refers to relative molecular mass in kDa. Download figure Download PowerPoint The data presented thus far are consistent with the release of tVSG by a proteolytic event, but attempts to block release with a mixture of protease inhibitors (antipain, chymostatin, pepstatin and leupeptin) were unsuccessful. As none of these inhibitors would be expected to effect metalloproteases, we tested the ability of chelators of divalent cations to block release of tVSG (Figure 9B). Biotinylated cells were incubated in physiological saline in the presence of EDTA (lanes 5 and 6), EGTA (lanes 7 and 8) or 1,10-phenanthroline (lanes 9 and 10). Millimolar concentrations of each of these chelators substantially reversed the pattern of release seen in the untreated cells (lanes 1–4), indicating a divalent cation requirement. In an identical assay performed in complete TM-P medium, which is replete with millimolar levels of Ca2+ and Mg2+, only phenanthroline inhibited release (unpublished observations). The ability of phenanthroline to inhibit in the presence of these cations strongly implicates some other metal ion, presumably Zn2+, as the active cofactor. Discussion VSG is the major surface protein of the bloodstream stage of the trypanosome life cycle; its expression is tightly repressed in the insect stage. Using cell lines stably transformed with genes for two distinct VSGs (117 and 221), we find that there is no impediment to the ‘inappropriate’ expression of these proteins in procyclic cells. Collectively, our data
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