The promastigote surface protease (gp63) of Leishmania is expressed but differentially processed and localized in the amastigote stage

Molecular and Biochemical Parasitology, 37 (1989) 263-274 Elsevier

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The promastigote surface protease (gp63) of Leishmania is expressed but differentially processed and localized in the amastigote stage E n r i q u e M e d i n a - A c o s t a 1, R o g e r E. K a r e s s 2, H e i n z S c h w a r t z 3, a n d D a v i d G. Russell 1 Departments of tPathology and 2Biochemistry, NYU Medical Center, New York, NY, U.S.A., and 3Max-Planck-lnstitut fi~r Biologie, Tiibingen, F.R. G. (Received 6 July 1989; accepted 8 August 1989)

The expression, processing and localization of the promastigote surface glycoprotein, gp63, in the amastigote form of Leishmania mexicana was examined. Metabolically labeled protein was immunoprecipitated from promastigotes and amastigotes. The isolated proteins were subjected to deglycosylation and partial peptide mapping. The cleavage products generated migrated similarly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the proteins were closely related. The majority of gp63 in amastigotes was inaccessible to surface-labeling procedures, and lacked the phosphatidylinositol membrane anchor. Immunolocalization of this subpopulation of gp63 revealed it to be present within the parasite's flagellar pocket. Despite the relative paucity of "membrane-form' gp63, isolation and analysis of surface proteins from lesion amastigotes indicated that gp63 was the most abundant protein on the amastigote surface. Key words: Leishmania; Amastigote; Glycoprotein; Flagellar pocket

Introduction Leishmania are parasitic protozoa that alternate between a uniflagellate promastigote form in the insect vector, and an aflagellate amastigote form in the vertebrate host. Once the promastigote form is introduced into the tissue of the vertebrate host by a feeding sandfly, it must gain entry into a macrophage in order to survive and develop into the amastigote stage [1-3]. The major surface glycoprotein (gp63) of Leishmania promastigotes has been implicated in the survival of the parasite in its vector, as well as initiation of infection within the vertebrate host. gp63 is known to express protease activity [4,5], and has been shown to mediate attachment of promastiCorrespondence address: David G. Russell, Dept. of Pathology, NYU Medical Center, 550 First Avenue, New York, NY 10016, U.S.A. Abbreviations: gp63, 63-kDa surface glycoprotein of promastigotes; NCS; N-chlorosuccinimide; PIPLC, phosphatidylinositol specific phospholipase C, isolated from Bacillus thuringiensis.

gotes to macrophages [6-8]. Binding of gp63 to the macrophage surface is via complement receptor type 3 (CR3), which recognises an Arg-GlyAsp-containing region of the parasite glycoprotein [8,9]. The genes encoding gp63 constitute a multigene family of tandemly linked copies [10,11], and the gp63s from different Leishmania species show extensive structural, molecular and immunological homology [11-14[. Although it is known that the gp63 gene locus is constitutively transcribed throughout the life cycle of the parasite [11], attempts to demonstrate the existence of the protein within the amastigote stage of Leishmania have yielded contradictory results [12,15-17]. In this present study we demonstrate that amastigotes express gp63, but the protein is processed differently from gp63 in promastigotes, gp63 in amastigotes is present on the surface membrane, where it appears to be the most abundant surface protein, and within the flagellar pocket. The gp63 in the flagellar pocket appears to lack the phosphatidylinositol membrane anchor.

0166-6851/89/$03.50 t~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)

264 Materials and Methods

Parasites. Promastigotes of Leishmania mexicana mexicana (strain MNYC/BZ/62/M379) were cultured in semi-defined medium SDM79 [18] supplemented with 10% colostrum-free bovine serum (Irvine Scientific) at 26°C and used within 8 weeks of transformation from freshly isolated amastigotes or from frozen stabilates of freshly transformed parasites. Amastigotes were isolated as previously described [19], omitting the column fractionation step, from cutaneous lesions in BALB/c or CBA/ca mice and used immediately after isolation.

Biosynthetic labeling and detergent extracts of parasites. Parasites were washed 3 x in ice-cold PBS and resuspended (4 × 108 cells ml -l) in methionine-free RPMI-1640 medium (Gibco Laboratories) supplemented with 35 txCi m1-1 [ssS]methionine (Amersham) or in SDM containing 50 I~Ci m1-1 of [3H]myristic acid (Amersham) in a preformed complex with defatted BSA [20]. Incubation conditions were: (a) promastigotes at 26°C for 4 h; (b) amastigotes at 35°C for 6 h under a 5% CO 2 atmosphere. Labeled cells were washed 3 times and extracted with lysis buffer LB (N-Octyl 13-D-glucopyranoside at 20 mg ml-~ in PBS containing 1 IxM TLCK, Leupeptin and 1,10orthophenanthroline; all from Sigma). For in vitro transformation experiments, 1.6 × 10 9 freshly isolated amastigotes were resuspended in SDM and transferred to 26°C for 48 h. 4 x 108 ceils were withdrawn at 0 h, 4 h, 8 h and 24 h following temperature shift, washed 3 times and labeled with 35 ~Ci ml-I of [35S]methionine for 6 h. Labeled cells were washed and detergent extracted as described above. SDS-PAGE was performed with samples normalized for amounts of radioactivity.

Surface iodination. Late log-phase promastigotes and lesion-derived amastigotes (4 x 108 cells m1-1) were surface iodinated by either Iodogen (Pierce Chemicals Co.) [21], or using iactoperoxidase/glucose oxidase [22] with 100 ixCi of carrier-free Na125I (Amersham) for 10 min on ice. Parasites were washed several times with ice-cold PBS and detergent-extracted.

Surface biotinylation. [35S]methionine-labeled living cells and surface-iodinated living cells were washed 3 times and incubated with 1 mg ml-I of either sulfo-NHS-biotin or the reduction-sensitive analogue, NHS-SS-biotin (Pierce) in PBS for 45 min at room temperature. Cells were then extensively washed and detergent-extracted. Cells derivatized with sulfo-NHS-biotin were immediately prepared for 2-D gel electrophoresis, followed by electroblotting onto nitrocellulose and probing with [125I]streptavidin (Amersham). On cells derivatized with NHS-SS-biotin, exposed peptides and proteins were recovered from lysates by incubation with streptavidin-agarose (Sigma). The complexes were washed 3 times in LB and solubilized in sample buffer containing 2ME for 2-D PAGE.

lmmunoprecipitation and lectin affinity isolation. Parasite lysates were clarified by centrifugation at 10000 rev./min for 10 min at 4°C. 60 ~1 samples of the supernatants were diluted 10 times in LB and incubated for 1 h at 4°C, with 60 I~1 of a 30% suspension of purified Igs, coupled directly to activated CH-Sepharose (Pharmacia). Antigen-antibody complexes were washed 5 times with LB, and antigens were eluted in 60 i~1 of boiling electrophoresis sample buffer. A similar protocol was used for lectin isolation of proteins. Treatment of immunoisolated gp63 with phosphatidyl inositolspecific phospholipase C (PIPLC) from Bacillus thuringiensis (a gift from Dr. Martin Low, Columbia University) was conducted on material bound to antibody-Sepharose, immediately prior to the addition of PAGE sample buffer. Quantitation of the relative levels of protein synthesis was assessed by the incorporation of [3SS]methionine into TCA-precipitable material before and after immunoprecipitation and expressed as percentage of counts recovered. 5 Ixl samples were withdrawn in duplicate and processed for TCA precipitation and liquid scintillation counting.

Deglycosylation and chemical peptide mapping. Affinity purified glycoproteins were digested with the endoglycosidase peptide: N-glycosidase F (Genzyme) as specified by the manufacturers. All reaction mixtures were supplemented with TLCK,

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leupeptin and 1,10-orthophenantroline (each at 1 I~g ml-l). Samples were analyzed by SDS-PAGE. For partial peptide mapping by chemical cleavage, gel slices containing labeled proteins were treated with N-chlorosuccinimide (Sigma) as previously described [23]. Gel pieces were loaded onto 17.5% acrylamide slab gels.

Electrophoresis and immunoblotting. Protein samples were analysed by SDS-PAGE on 1-mmthick minigels of 10.5% acrylamide with 3% acrylamide stacking gels, using the buffer system of Laemmli [24]. 2-D PAGE was performed as described by Russell et ai. [25] in a pH gradient formed by mixing LKB ampholines, pH 3.5-10 (1.2%) and pH 5-7 (0.8%), and developed for 9000 V h. All samples were solubilized before 2-D PAGE by incubation in sample buffer containing 6 M urea and 1% Nonidet P-40. Gels were processed for fluorography with AutoFluor (National Diagnostics) and exposed with Kodak XAR.5 film at -70°C. Electroblotting of SDSPAGE fractionated material to nitrocellulose was carried out using the transfer buffer system previously described [26].

hyde in PBS. Material was then embedded in agarose, dehydrated and infiltrated with Lowicryl K4M resin as described [27]. Ultrathin sections were cut, blocked with 0.5% gelatine, and 0.2% human placental albumin, prior to incubation with primary antiserum. After incubation with TiIL3.8 in blocking buffer, grids were washed in PBS on a rocking table, and labeled with either protein G-gold (5 nm) or goat anti-mouse-gold (10 nm) (Janssen Pharmaceuticals) according to the manufacturers' instructions. The grids were finally washed, fixed with gluteraldehyde, and stained with uranyl acetate and lead citrate. Results

Expression of gp63-related proteins by the amastigote. The expression of gp63, and gp63-related proteins, was examined throughout the Leishmania life cycle, by immunoprecipitation of extracts from amastigotes, metabolically labeled immediately following isolation, and at stages during transformation into promastigotes. The

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Antibodies. Monoclonal antibodies against promastigote gp63 were formed by fusing BALB/c mouse spleen cells from an immunized mouse with the myeloma line NS1. One cell line, TOL3.8, secreted an IgG1 antibody that recognized amastigote gp63. TiiL3.8 antibody was isolated from ascites fluid by affinity chromatography on protein A-Sepharose.

lmmunofluorescence and immunoelectron microscopy. Immunofluorescence was performed on cells fixed in 2.5% formaldehyde in serum-free DMEM for 15 min at room temperature. The cells were then detergent extracted for 30 s in 1.5 mg ml-1 Zwittergent 3.12 (Calbiochem) and blocked for 15 min in DMEM with 10% FCS. All subsequent incubations were conducted in serum containing medium. The second antibody used was FITC-conjugated goat anti-mouse IgG/IgM (Jackson Immunoresearch Labs.) and the cells were counter-stained with Hoechst dye. Immunoelectron microscopy was performed on cells fixed in 4% formaldehyde and 0.1% gluteralde-

Fig. 1. Expression of gp63 during amastigote to promastigote transformation. An autoradiograph from a 9% SDSPAGE of TiiL3.8-immunoprecipitated material from amastigotes isolated fresh from a lesion (1) and at different time points after temperature shift to 26°C. In each case the cells were labeled with [3-~S]methionine for 2 h prior to processing for immunoprecipitation. The time points taken were at 0 h (1) (before altering the temperature), and at 4 h (2), 8 h (3), and 24 h (4). The arrows on the right-hand side indicate molecular weight standards of 90.5, 69, and 46 kDa.

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monoclonal antibody used, TiaL3.8 (IgG1), had been raised against promastigote gp63. SDSPAGE analysis of immunoprecipitated material from [35S]methionine-labeled cells, during transformation from amastigotes into promastigotes, shows expression of different forms of gp63-related proteins (Fig. 1). gp63-related proteins from amastigotes migrate as a hetero-disperse complex from 60-72 kDa. Expression of the high-molecular-weight forms ceases between 4 to 8 h of incubation at 26°C. In promastigotes, in agreement with previously published results [4,28], gp63 accounted for 0.8-1.2% of total TCA-precipitable radioactivity, whilst in amastigotes the 60-72 kDa complex represented only 0.09-0.11% of the TCA-precipitable material. No metabolically labeled proteins could be recovered from the medium, following removal of the parasites, indicating that gp63-related proteins were not being secreted.

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In contrast to the above results, immunoprecipitation of lysates of surface-iodinated amastigotes with mAb TiiL3.8 identified a cross-reactive protein migrating as a single band of 60 kDa (Fig. 2a). This protein was found to incorporate [3H]myristic acid (Fig. 2b), which could be liberated by incubation in PIPLC from B. thuringiensis, suggesting that it possessed a phosphatidylinositol anchor, as previously reported for gp63 in promastigotes [29,30]. The loss of [3H]myristic acid was accompanied by a decrease in electrophoretic mobility, observed in protein labeled by surface iodination (Fig. 2).

Deglycosylation and peptide mapping of gp63-related protein(s) from amastigotes. The nature of the relationship between promastigote gp63 and the gp63-related molecule(s) found in amastigotes was investigated by enzymatic deglycosylation and chemical peptide mapping. Incubation of radiolabeled, mAb-affinity purified protein

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Fig. 2. Analysis of gp63-related proteins from amastigotes. Autoradiographs from SDS-PAGE (9%) gels of surface-iodinated (a) or [3H]myristic acid metabolically labeled (b) amastigotes demonstrate that the gp63-related protein(s) immunoprecipitated by anti-gp63 mAb TiiL3.8 possess a PI anchor. (a) Radiolabeled amastigotes (1) were detergent-extracted, and surface glycoproteins were isolated by precipitation with either Concanavalin A-Sepharose (2) or Sepharose coupled with TiiL3.8 IgG (3 and 4). The protein in lane 4 was subjected to treatment with PIPLC prior to electrophoresis. (b) [3H]Myristic acid-labeled protein immunoprecipitated with TiiL.3.8 IgG coupled Sepharose (1). The protein run in lane 2 represents a comparable loading of the same sample of protein following treatment with PIPLC. The arrows indicate molecular weight standards of 90.5, 69 and 46 kDa.

Fig. 3. SDS-PAGE analysis of deglycosylated gp63. An autoradiograph of a 10.5% SDS-PAGE of TiiL3.8-immunoisolated promastigote (1) and amastigote (2 and 3) proteins following radiolabeling reveals that both proteins contain a peptide backbone of comparable size. The amastigote gp63related proteins were divided into high- (3) and low- (2) molecular-weight forms. All samples were excised from a gel, treated in situ, and equilibrated in sample buffer prior to reelectrophoresis. The preparations shown in lanes 4, 5, and 6 represent the same samples as 1, 2, and 3, respectively, after treatment with N-glycanase. Following this procedure all the proteins co-migrate. The arrows on the right-hand side indicate molecular weight standards of 90.5, 69 and 46 kDa.

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Fig. 4. Peptidemapping of amastigote gp63by partial digest. An autoradiograph from a 17.5% SDS-PAGE gel of N-chlorosuccinimide promastigote (1 and 3) and amastigote (2 and 4) gp63 both prior (1 and 2) to and following(3 and 4) deglycosylation with N-glycanase, indicates that both molecules contain a similar peptide backbone. In both instances the digestion pattern produced by N-chiorosuccinimide, which cleaves at tryptophan residues, are very similar, showingthat the proteins are closelyrelated.

from both promastigotes and amastigotes with Nglycanase resulted in the formation of comigrating 58-kDa protein species (Fig. 3). Partial proteolysis of the deglycosylated proteins with Nchlorosuccinimide, generated similar digestion products, indicating a high degree of structural homology between the peptide backbones of the proteins (Fig. 4). From these data we conclude that the surface glycoprotein, gp63, of promastigotes and amastigotes are structurally related, beating at least one common epitope and having similar primary structures. It is likely that differences in SDS-PAGE migration patterns are, for the most part, attributable to differential glycosylation of amastigote and promastigote gp63. To simplify exposition of the two forms, these polypeptides will be referred to as gp63.a (amastigotes) and gp63.p (promastigotes).

Two-dimensional PAGE analysis of gp63 from promastigotes and lesion amastigotes. We had

267 noted that metabolically labeled gp63.a generated a more complex pattern on SDS-PAGE than gp63.a recovered from surface-iodinated amastigotes (Figs. 1 and 2). To compare further the different forms of gp63 in both amastigotes and promastigotes, we carried out 2-D PAGE analysis (Fig. 5). gp63.p, immunoprecipitated from promastigotes metabolically labeled with [35S]methionine (Fig. 5a), and gp63.p, immunoprecipitated from surface-iodinated promastigotes (Fig. 5b), both migrated as a heterogeneous complex of glycoproteins. The qualitative similarity of the patterns suggested that the majority of forms of gp63.p were represented on the promastigote surface. In contrast, there was a profound difference between the 2D-PAGE profiles generated by gp63.a, isolated from amastigotes metabolically labeled with [35S]-methionine (Fig. 5d), and gp63.a, from surface-iodinated amastigotes (Fig. 5e). gp63.a, radio-iodinated following isolation, produced a profile (not illustrated) comparable to metabolically labeled protein (Fig. 5d), indicating that the different pattern was not due to an inability to radiolabel the other gp63.a forms. The different 2D-PAGE patterns from surface and metabolically labeled gp63.a strongly suggest that only a subpopulation of gp63.a is present on the amastigote surface.

Distribution of the phosphatidylinositol anchor in amastigote gp63. 2D-PAGE analysis was also conducted on promastigote (Fig. 5c) and amastigote (Fig. 50 gp63 following metabolic labeling with [3H]myristic acid, which is known to be incorporated into the PI anchor structure of the glycoprotein. Promastigote gp63 labeled with [3H]myristic acid generated a 2D-PAGE profile comparable to [35S]methionine-labeled and surface-iodinated gp63.p (Fig. 5a and b), indicating that all or most of the forms of the protein possessed a PI anchor. [3H]Myristic acid-labeled amastigote gp63 migrated in 2D-PAGE in a manner similar to that shown for surface-iodinated gp63.a (Fig. 5e). This pattern was again simpler than the profile generated by [35S]methionine-labeled gp63.a, and suggests that, in amastigotes, the PI anchor is restricted to only those forms of gp63.a that can be surface iodinated. Taken together, the results of the 2D-PAGE analysis

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Fig. 5. Two-dimensional PAGE analysis of differentially labeled gp63 isolated Irom both promastigotes and amastigotes. Autoradiographs of immunoprecipitated gp63 from both promastigotes (a,b,c) and amastigotes (d,e,f) reveals that, in contrast to promastigotes, not all the forms of gp63 are exposed on the surface of the amastigote or label with [3H]myristic acid. The promastigotes were labeled by (a) surface iodination, and by metabolic labeling with either (b) [35S]methionine or (c) [3H]myristic acid. Similarly, amastigotes were labeled by (d) surface-iodination, or metabolic labeling with (b) [35S]methionine or (c) [~H]myristic acid. Interestingly, although in promastigotes all the forms of gp63 can be labeled by all three methods, in amastigotes not all forms of gp63 are accessible to surface iodination, or possess, or retain, a glycolipid anchor that can be labeled with myristic acid.

shown in Fig. 5, indicate that most gp63 in amastigotes is situated where it is inaccessible to surface labeling protocols.

lmmunolocalization of amastigote gp63. Immunofluorescence studies conducted on amastigotes in in vitro infected macrophages (infected 5 days previously with amastigotes isolated from lesions) showed uneven distribution of fluorescent label on the parasite (Fig. 6). Immunoelectron microscopy of lesion amastigotes, using TiiL3.8 and either goat anti-mouse gold or protein G gold, shows that, although the surface membrane is labeled, the majority of gp63 in amastigotes is situated in the flagellar pocket

of the parasite (Fig. 7). The label appears to be associated with electron-dense amorphous material lying within the lumen of the pocket structure. In view of our surface labeling results, this cellular compartment must be inaccessible to the compounds used in the labeling procedures.

Amastigote surface exposed proteins. Our results demonstrate that one form of gp63.a is expressed on the amastigote surface; we are, however, also interested in determining the identity and relative abundance of other amastigote surface proteins. Previous studies involving surface labeling of freshly isolated lesion amastigotes have yielded profiles difficult to interpret because of possible

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Fig. 6. lmmunofluorescence of infected macrophages with anti-gp63 monoclonal antibody. An immunofluoreseence micrograph of mouse bone marrow macrophages infected with L. m. mexicana amastigotesisolated directly from a lesion. The culture was maintained for a week before processing for immunofluorescence. The preparation is labeled with Hoechst dye (a) for visualization of the host and parasite nuclei, and with TiiL3.8 and FITC anti-mouse IgG/IgM (b) for visualization of gp63. The TiiL3.8 IgG staining pattern observed suggests that although label can be seen over the entire parasite, the gp63 is not evenly distributed on, or in, the amastigote. contamination with host protein [31,32]. We have developed a procedure for visualising those surface proteins that can be metabolically labeled, thereby excluding host-derived proteins. Fig. 8a shows an autoradiograph from a 2-D gel of freshly isolated lesion amastigotes following biotin derivatization, electrotransfer to nitrocellulose and probing with [~25I]streptavidin. The polypeptides visualized include host proteins as well as externally orientated parasite proteins. The most abundant polypeptide is host actin. In contrast, Fig. 8b illustrates an autoradiograph from a 2-D gel of [35S]methionine-labeled surface proteins from lesion amastigotes that were metabolically labeled for 1 h, then derivatized with NHS-SSbiotin. Following derivatization, the cells were detergent-extracted, and biotinylated proteins were recovered with streptavidin-agarose. A major parasite protein of 60 k D a is visible in both the preparations. More detailed analysis of this molecule (not shown) confirmed it to be a form of gp63.a. Discussion The existence of gp63 in the amastigote stage has been the subject of some debate in the literature [11,12,15-17]. In this present report we be-

Fig. 7. Immunolocalization of amastigote gp63 by electron microscopy. High-resolution localization of amastigote gp63 was conducted on Lowicryl embedded material isolated directly from an L. m. m e x i c a n a lesion. These sections were labeled with Ti)L.3.8 IgG and goat anti-mouse antibody conjugated with 10-nm gold particles. The amastigotes (a) were labeled sparsely around the periphery of the cell (arrows) and more intensely within the region of the flagellar pocket (arrowheads). Higher magnification of the flagellar pocket region (b) shows that the label is primarily associated with dense material in the lumen of the pocket, and not with the membrane.

iieve, for the following reasons, that we provide formal proof that amastigotes express gp63. First, both promastigote and amastigote synthesize proteins recognized by the monoclonal antibody TiiL3.8, which was raised against promastigote gp63. Second, immunoprecipitated protein from both promastigotes and amastigotes was shown to comigrate in S D S - P A G E , following deglycosylation with N-glycanase. Finally, partial digestion of deglycosylated protein with N-chlorosuccinimide, which cleaves at tryptophan residues, yielded similar digestion products. It is likely that the reported stage specificity [15,16] is due to differential glycosylation of gp63. The m A b used in this study, TiiL3.8, came from series of m A b s raised

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OHFig. 8. Two-dimensional PAGE of surface-labeled amastigotes. Autoradiographs from 2D-PAGE gels run with surfacc-derivatized amastigotes isolated from infected mice reveal that the major protein, of parasite origin, on the surface of lesion amastigotes is a gp63.a. (a) The amastigotes in this gel were derivatized with NHS-sulfobiotin, washed, fractionated by 2D-PAGE, and transferred to nitrocellulose. The membrane was then probed with [~25I]streptavidin and autoradiographed. The most abundant polypeptide is host actin (open arrow). The other arrowed protein is amastigote gp63, in the form present on the surface. (b) Amastigotes metabolically labeled for 3 h with [35S]methionine, derivatized with NHS-SS-biotin and then detergent-extracted. The metabolically labeled, biotin-derivatized polypeptides were recovered with streptavidin agarose, removed with 2-ME and separated by 2D-PAGE. By this procedure parasite surface proteins can be discriminated from those of host origin. The most abundant amastigote surface protein is similar in molecular weight and PI to promastigote gp63.

against promastigote gp63. From twelve such mAbs, only two recognized gp63.a, and both of these were directed against peptide determinants (DGR unpublished results). We have generated a profile of the overall complexity of the amastigote surface by derivatization of exposed, metabolically labeled polypeptides with NHS-biotin. This approach circumvents potential problems of contamination with host proteins. Barring differential turnover of membrane proteins, and possible disproportion in either [35S]methionine-incorporation or interaction with the hydroxysuccinimide ester; the 2DPAGE profile shown in Fig. 8 strongly suggests that gp63 is the most abundant surface-exposed polypeptide on the amastigote stage of Leishmania. gp63 has been shown to have several properties important to the survival of the promastigote stage of Leishmania, many of these properties could also be envisaged as playing key roles in

amastigote survival, gp63 is a highly active protease [4,5], and is also capable of mediating attachment of the parasite to macrophages [6-9]. First, with respect to protease activity, it has been suggested the enzyme plays a role protecting the parasite from attack by lysosomal hydrolases [17]. Our demonstration that gp63 is present on the surface of amastigotes indicates that such a scenario is physically possible. It is, however, interesting that the bulk of gp63 in the amastigote is not on the cell's plasmalemma but is confined to the flagellar pocket region (Fig. 7). The flagellar pocket of trypanosomatids has been the subject of several studies, all of which recognise the pocket as the primary point of membrane activity in the cell [33,34]. Experiments on Trypanosoma brucei have shown that the flagellar pocket is involved in distribution and internalization of variant surface protein [34]. It is also involved in receptor-mediated endocytosis of low-density lipoproteins [35,36}. Although

271 similar studies have not been conducted on Leishmania spp., the membranous structures subtending this part of the cell suggest comparable activity. Cytochemical studies have shown that Leishmania d o n o v a n i have several enzymatic activities, such as acid phosphatase [37], and 3' and 5' nucleotidase [38], associated with this region of the cell. In light of our present results, one can speculate that the primary role of the proteolytic activity of gp63 in amastigotes is participation in the breakdown of host macromolecules, either for protection, or to provide nutrition for the parasite. The other possible role for gp63 in amastigote biology is related to its ability to interact with the macrophage surface receptor CR3 [7,8]. Promastigote gp63 from L. m. mexicana has been shown to bind directly to the complement receptor CR3. This is achieved by virtue of an Arg-Gly-Aspcontaining region of the gp63 amino acid sequence [8,9]. Given this binding activity, it is feasible that gp63.a could contribute to the attachment of amastigotes to macrophages during the amplification of an infection. Existing studies on the interaction of amastigotes with macrophages have implicated fibronectin in the adhesion process [39,40]; possible interpretations of these observations have been discussed elsewhere [8,9,41-43]. Further experimentation is required to assess the relative contribution of host protein, present in considerable quantities on isolated lesion amastigotes, and other as yet unidentified amastigote surface molecules, to the attachment of amastigotes to their host cells. These findings also relate to the use of gp63 as a vaccine candidate. We previously demonstrated that CBA mice vaccinated with gp63 reconstituted into liposomes generated a protective immune response to subsequent homologous chal-

lenge with promastigotes [44,45]. Immunity could be transferred with T cells, and results from other studies [46,47], indicate that this is probably achieved by secretion of r-interferon and activation of infected macrophages. The demonstration of gp63 in amastigotes indicates that gp63-induced immunity is probably not promastigotespecific and raises the possibility of its use as an immunotherapeutic agent for the treatment of established Leishmania infections by a procedure analogous to that of Convit and colleagues [48,49]. Finally, in this study we have shown that gp63 in the amastigote is heterogenously processed, both in terms of glycosylation and the restriction of the phosphatidylinositol anchor to a subpopulation of gp63. We also known that, in L. m. mexicana, the genes encoding gp63 are arranged in three different tandemly-linked clusters on the same chromosome [11,50]. The individual genes in each cluster produce identical restriction enzyme digests, however, the digests from genes in different clusters show restriction site polymorphism (ref. 50, and Medina-Acosta et al., unpublished results). The possible inter-relationship between the heterogeneity of gp63 genes and the subsequent generation of differentially processed polypeptides is currently under study.

Acknowledgements This study was supported by grants from the MacArthur Foundation, from the World Health Organisation, and a Whitehead Presidential fellowship awarded to DGR. The authors wish to thank Dr. Rocilda Schenkman for her assistance throughout the project. We also wish to thank Dr. Victor Nussenzweig for his encouragement and guidance.

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