Monoclonal antibodies to Leishmania mexicana promastigote antigens

I. Secreted acid phosphatase and other proteins share epitopes with Iipophosphogiycan

THOMAS ELG\ BEATRICE MENZ1-*, GERHARD WINTER1, DAVID G. RUSSELL2,

ROBERT ETGES1^, DIETMAR SCHELL1 and PETER OVERATH1'*1Max-Planck-Institut fUr Biologic, Corrensstrasse 38, D74O0 Tubingen, Federal Republic of Germany2Department of Pathology, NYU Medical Center, 560 First Avenue, New York, NY 10016, USA

•Present address: London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, Englandt Present address: Institut de Biochimie, University de Lausanne, Chemin des Boveresses, CH-1066 Epalinges, Switzerlandt Author for correspondence

Summary

The abundant surface glycolipid, lipophosphoglycan(LPG), of Leishmania promastigotes is composed ofphosphosaccharide repeating units linked via aphosphosaccharide core to a conserved lyso alkyl-phosphatddylinositol membrane anchor. It is shownin this paper that monoclonal antibodies (mAbs)directed against LPG also react with an acid phos-phatase secreted by L. mexicana promastigotes. Acidphosphatase purified by column chromatography(apparent Mr=100000) reacts in immunoblots withthe anti-LPG mAb AP3 and another mAb, L3.13,which does not recognize LPG. mAb L3.13 was used

to purify the enzyme by affinity chromatography.The resulting glycoprotein has the same molecularweight and binds AP3 on immunoblots. The secretedphosphatase is non-covalently associated with a highmolecular weight, galactose-containing glycan orproteoglycan that is recognized by both AP3 andL3.13. In addition to acid phosphatase, other parasiteproteins appear to be modified by LPG epitopes.

Key words: lipophosphoglycan, acid phosphatase, monoclonalantibodies, Leishmania mexicana, post-translationalmodification, immunoblote.

Introduction

Leishmania is a dimorphic protozoan parasite living as aflagellated promastigote in the gut of the sandfly vector,and as a non-motile amastigote in the phagolysosomes ofmammalian macrophages. The promastigote expresses onits surface an abundant lipopolysaccharide, termed lipo-phosphoglycan or LPG (cf. reviews by Turco, 1988, 1990).The structure of LPG has been elucidated for three speciesof Leishmania: L. donovani (Turco etal. 1989; Turco, 1990),L. mexicana (Ilg et al. unpublished experiments) and L.major (McConville et al. 1991) revealing both conservedand species-specific features. LPG from all species con-tains [PO4—>6Gal(/31-4)Man(al)] phosphosaccharide re-peats linked to a conserved phosphosaccharide core and aconserved lyso alkylphosphatidylinositol residue. In con-trast to L. donovani LPG, which exclusively contains thephosphodisaccharide repeats, LPG in L. mexicana iscomposed of repeats modified in part by glucosyl residues,forming trisaccharides and by galactosyl, glucosyl andarabinosyl residues in L. major LPG forming tri-, tetra-and pentasaccharides. In L. major, the non-reducingterminus of phosphoglycan is capped with the neutraldisaccharide Manp(Q-l,2)Man(o'l) (McConville et al. 1991).

For a number of years, Dwyer and co-workers havestudied a tartrate-sensitive phosphatase of L. donovanithat is secreted into the medium of cultured promastigotesin vitro (Gottlieb and Dwyer, 1982; Lovelace et al. 1986).This highly glycosylated enzyme contains N-linked gly-

Journal of Cell Science 99, 175-180 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

cans that can be removed by treatment with Af-glycosidaseF (Bates and Dwyer, 1987; Bates et al. 1990). As expectedfor N-glycosylation in the endoplasmic reticulum, theaddition of these glycans is inhibited by tunicamycin(Bates and Dwyer, 1987). A second type of modificationoccurs in the Golgi complex because incubation ofpromastigotes with monensin reduces both the apparentmolecular weight and the degree of heterogeneity of thesecreted form of the enzyme (Bates and Dwyer, 1987).Recently, Bates et al. (1990) demonstrated that this latterchange can also be achieved in vitro by treatment of theglycoprotein at pH2.0 and 60°C, conditions that areknown to cleave the phosphodiester bonds between therepeat units of LPG (Turco et al. 1984). Since mild acidhydrolysis concomitantly removed most of the phosphateresidues previously shown to be linked to the enzyme(Lovelace and Gottlieb, 1987), it was proposed that thesecretory acid phosphatase is post-translationally modi-fied in the Golgi by addition of an acid-labile phosphogly-can related to LPG.

In a search for monoclonal antibodies (mAbs) againstacid phosphatases of L. mexicana promastigotes, one of usisolated an antibody, designated AP3, which reacted withpart of the cell-associated and all of the secreted acidphosphatase (Menz, 1990). Surprisingly, this mAb alsoreacted strongly with the surface of promastigotes, aproperty characteristic of anti-LPG antibodies (De Ibarraet al. 1982; Handman and Hocking, 1982; Greenblatt et al.1983; Tolson et al. 1989) but unexpected for mAbs against

175

the secreted polypeptide chain (Bates et al. 1989). It will beshown in this paper that the secreted acid phosphatase andother promastigote proteins of L. mexicana share immuno-logical epitopes with LPG.

Materials and methods

Cell culturePromastigotes of L. mexicana MNYC/BZ/62/M379 were grownas described (Menz et al. 1991).

Hybridoma cell linesSpleen cells of Balb/c mice immunized with concanavalin A(ConA)-binding membrane components from L. mexicana werefused with the plasmacytoma line P3-NSl-Ag4-l (Menz et al.1991). The hybridoma supernatants were screened with anenzyme-linked immunoadsorbent assay (ELISA) against L.mexicana promastigotes fixed in 2 % formaldehyde/0.05 % glutar-aldehyde. This assay led to the isolation of the line secreting mAbL7.25 (subclass IgM). mAb L3.13 (subclass IgGi) was raised frommice inoculated with ConA-binding proteins and selected byELISA against detergent extracts from a crude promastigotemembrane preparation. Finally, the culture supernatants weredirectly screened for their ability to precipitate acid phosphataseactivity from a detergent extract of promastigote membranes(Menz et al. 1991). This screening assay led to the isolation of aline secreting mAb AP3, a protein A-binding IgM antibody.

Isolation and immobilization of monoclonal antibodiesThe IgM antibodies AP3, L7.25 and WIC108.3 were isolated fromascites fluid by PEG-6000 precipitation (Neoh et al. 1986). mAbsL3.13 and AP3 were also isolated from ascites fluid using eitherprotein A-Sepharose CL4B or protein G-Sepharose CL4B FastFlow (Pharmacia, Freiburg, FRG). The antibodies were immobi-lized at a ratio of 5mgml~ antibody to activated CH-Sepharose4B according to the manufacturer's instructions (Pharmacia).

SDS—polyacrylamide gel electrophoresis (PAGE) andimmunoblottingSDS-PAGE was performed on 10 % or linear gradient gels (8 % to16%) according to the method of Laemmli (1970). Gels werestained with Coomassie Blue, permanganate/silver (Ansorge,1986), periodate/silver (Tsai and Frasch, 1982).

Blotting was conducted as described by Towbin et al. (1979),omitting 20% methanol in the blotting buffer. The preferredmembrane for blotting of LPG and acid phosphatase wascationized nylon (Zeta Probe, Bio-Rad, Munchen, FRG), and forblotting of other proteins, polyvinylidenedifluonde (PVDF, Immo-bilon, Millipore, Eschborn, FRG). Transferred proteins on PVDFwere reversibly stained with Ponceau S. Subsequently, the blotswere treated with blocking buffer (5% non-fat milk powder inPBS) for 1 h at 37 °C. The blots were incubated overnight at 4°C or1 h at 37 °C with purified monoclonal antibody in blocking buffer(0.5-8 /<g ml"1), washed three times in PBS and then furtherincubated with a 1:5000 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG/IgM in blocking buffer for 1 h at37 °C. After three washings in PBS and one washing inphosphatase buffer (50mM NaHCO3> 2mM MgCl2, pH9.6), theblots were developed in phosphatase buffer containing 50 jig ml"1

nitrobluetetrazolium chloride and 25/igml"1 disodium-5-bromo-4-chloro-3-indolylphosphate. The presence of antigen was indi-cated by a blue (PVDF) or brown (Zeta Probe) color development.

Metabolic labeling of promastigotes with [3SS]methionine'and [3H]galactose and immunoprecipitationA washed cell pellet of 2xlO8 promastigotes was suspended in5 ml methionine-free RPMI medium containing 7/*g heminml"1

and lmCi of [^SJmethionine or in 5 ml glucose-free RPMImedium containing 7^g heminml"1 and 300 fjCi of [6-3H]galac-tose. After incubation for 14 h at 27 °C, cells were washed in PBS

and extracted in 5 ml PBS containing 50/IM tosyl-L-lysinechloromethyl ketone, 15 ^M leupeptin and Smgml"1 Zwittergent3.12. The lysate was centrifuged at 100000g for l h at 4°C. Theculture supernatant was retained and passed through a 0.45 fxmnitrocellulose filter. Detergent extract (250 /d) or supernatant(250 /d) was added to the various antibodies coupled to activatedCH-Sepharose. After a 90 min incubation on a shaker in the cold,the immunoadsorbent was washed four times in extraction bufferand then denatured with 50/d SDS sample buffer at 100 °C for2 min. Samples were subjected to SDS-PAGE and autoradiogra-phy.

Determination of acid phosphatase activitySamples were incubated for 60 min at 37 °C in 0 . 1 M sodiumacetate, pH5.0, containing 5mM p-nitrophenylphosphate (finalvolume 1 ml). This substrate concentration was near saturation ofthe enzyme. After stopping the reaction with 100 /d 1 M NaOH theabsorbance of nitrophenol was measured at 405 nm (e= 18 500 M" 1

cm"1). One unit of enzyme activity is defined as the amount ofenzyme hydrolyzing 1 fjiaol substrate per minute.

Purification of secreted acid phosphatasePromastigotes grown in SDM 79 medium plus 10% iFCS(inactivated fetal calf serum) to a density of 4xlO7 cells ml"1

(Menz et al. 1991) were harvested by centrifugation andresuspended in an equal volume of SDM 79 lacking iFCS. Aftershaking for 24h at 27°C, the culture contained 8xlO7 cells ml"1.Parasites were recovered by centrifugation and the supernatantsubjected to ultracentrifugation at 100 000 g for 1 h at 5°C.

A 500 /J sample of mAb L3.13-containing ascites fluid wasdiluted with 1.5 ml of 100 HIM sodium phosphate buffer, pH8.8,and gently shaken overnight with 2 ml protein G-Sepharose. Thebeads were collected by centrifugation, washed with 20 mMTris-HCl, pH8.0, and packed into a column. The culturesupernatant containing 80mi.u. enzyme activity ml"1 was ap-plied to the column at a rate of lm lh" 1 until the column wassaturated. After washing the column with 10 volumes 20 mMTris-HCl, pH8.0, bound acid phosphatase was eluted with100 mM sodium acetate, 0.5 M NaCl, pH4.0. Fractions of 0.5 mlwere collected and assayed for activity. The column bound about1.4 units of enzyme activity, from which 0.75 unit (53%) wasrecovered in the eluate.

Alternatively, secreted acid phosphatase was purified bycolumn chromatography (Ilg et al. unpublished results).

Protein determinationProtein in cell lysates was estimated by the Peterson modificationof Lowry's method; all other protein determinations wereperformed according to the Peterson modification of Bradford'sdye binding method (Peterson, 1983).

Results

Reactivity of monoclonal antibodies againstlipophosphoglycan

mAb 7.25 was isolated as an antibody recognizing fixed L.mexicana promastigotes, while mAb AP3 was detected in ascreen for antibodies precipitating acid phosphataseactivity from a detergent extract of the total membranefraction of L. mexicana. The antibodies were tested inimmunoblots against cellular or culture supernatant LPGfrom L. mexicana promastigotes and compared in theirreactivity with an established anti-LPG antibody,WIC108.3 (Greenblatt etal. 1983; Handman etal. 1984).All three antibodies detect LPG as a broad smearextending from the middle of the gels to the front. Thedistributions of the cellular and the culture supernatantLPG are similar. The bulk of the LPG as revealed by thecarbohydrate stain runs in a more restricted area at thefront of the gel (Fig. 1).

176 T. Ilg et al.

Mr

x10 '116.97"

67-

43-

29-

14.3-

AP3 L7.25WIC108.3 L3.13

HIO4/Silver

IIcs c cs c cs c cs c cs c

Fig. 1. Reactivity of monoclonal antibodies with LPG. LPG(5 /ig/lane) isolated from cells (C) or the culture supernatant(CS) of Leishmania mexicana waa separated on 8 % to 16 %SDS-PAGE gels and either processed for immunoblottingusing the indicated monoclonal antibodies or stained directlywith Hid/silver for the detection of carbohydrate. Themolecular weight of standard proteins is indicated.

The screening of hybridoma supernatants against amembrane extract of L. mexicana promastigotes led to theisolation of a line secreting mAb L3.13. This antibodyreacts with neither LPG from cells nor LPG from theculture supernatant of L. mexicana (Fig. 1).

mAbs AP3, L7.25 and L3.13 recognize the secreted acidphosphataseAlthough mAbs AP3 and L3.13 can be clearly differen-tiated in their reactivity with LPG, they both precipitate aprotein of about 100K from the supernatant of [^Slmeth-ionine-labeled L. mexicana promastigotes (Fig. 2B). Thisprotein is also bound by ConA or mAb WIC 108.3. Nodistinct high molecular weight proteins can be precipi-tated by these antibodies from the detergent extract ofcells (A). The precipitation of the surface protease, gp63,by mAb L3.8 (Medina-Acosta et al. 1989) was included aa apositive control.

Immunoprecipitations of the culture supernatant of[3H]galactose-labeled cells reveal a complex pattern ofcomponents (Fig. 2C). First, L3.13, AP3, WIC 108.3 andConA give rise to a [3H]galactose-labeled band at about100K, which corresponds to the protein band shown inFig. 2B. Second, AP3, WIC108.3 and ConA but not L3.13precipitate culture supernatant LPG, which is located as abroad smear at the front of the gel. Finally, all threeantibodies and the lectin sediment high molecular weight,[3H]galactose-labeled material found in the region of themarker protein myosin (205K). In the corresponding gelregion of part B no obvious [^SJmethionine-labeledproteins are detectable.

mAb AP3 or L7.25 quantitatively precipitate the acidphosphatase activity from the culture supernatant(Table 1). The same is observed for mAb L3.13, providedthat the precipitation is not performed in the presence ofBSA. The mechanism for this unusual inhibition remainsto be elucidated.

In summary, these findings suggest that all threeantibodies recognize the secreted acid phosphatase, aprotein of apparent MT100 000: mAb AP3 and L7.25 via anepitope also present on LPG, and L3.13 via an epitopeabsent from LPG. The cross-reactivity of mAbs AP3 and

< " co "O CO Q. > c

2 0 5 - | | ff"

9 7 -

66-45-

20- i!CO

co. C O CO

B205-

97-

66-

45-

20-

Oco a . *

f ••'

2 0 5 -

97-

66-45-

20- 1 IIFig. 2. Immunoprecipitation of [^SJmethionine-labeledproteins from a detergent extract of promastigotes (A) and theculture supernatant (B) or from the culture supernatant of[3H]galactose-labeled promastigotes (C). Proteins wereprecipitated with ConA or the indicated monoclonal antibodiescoupled to Sepharose and subjected to SDS-PAGE andautoradiography. Molecular weight standards are indicated(xlO~3). Filled arrows locate the surface protease, gp63; openarrows indicate the position of the 100xl03Mr protein,subsequently identified as secreted acid phosphatase.

L7.25 can be explained in two ways. First, LPG could betightly but non-covalently bound to the enzyme, theprecedent being the complex of LPG and BSA (King et al.1987). Alternatively, the enzyme could be covalentlymodified by LPG epitopes. Similarly, the high molecularweight carbohydrate and the phosphatase could beindependently secreted components or they could be

Glycoprotein modification in Leishmania 177

Table 1. Immunoprecipitation of secreted acidphosphatase

mAb

AP3L7.25*L3.13L3.13+1%BSA#137

Activity in supernatant(%)

«5*5

=£105B9O

3590

A sample of culture supernatant containing 2.5 mi.u. acid phosphataseactivity in 200 fi 20 m i Tns-HCl, pH8.0, was incubated for 2h with2fi] ascites fluid containing the antibodies listed. #137, an antibodyagainst Trypanosoma brucei procyclin (subclass IgGi; Richardson et al.(1988)), served as a control. After addition of 50/(I protein G-Sepharoseand a further 2 h incubation with agitation, the enzyme activity wasdetermined in the supernatant. Data were normalized to a controlwithout antibody.

*This precipitation was performed with antibody coupled toSepharose.

associated non-covalently in a complex. Differentiationbetween these possibilities required purified phosphatase.

Reaction of purified acid phosphatase with monoclonalantibodiesThe secreted acid phosphatase from L. mexicana waseither purified by conventional ion exchange, gel filtrationand hydrophobic interaction chromatography (Ilg et al.unpublished experiments) or, in analytical amounts, on anmAb L3.13 affinity column in a yield of 53 %. As shown inlanes 1 and 2 of Fig. 3, both procedures lead to ahomogeneous protein of 100K, confirming the results ofthe immunoprecipitation (Fig. 2B). Importantly, the pro-tein purified by either procedure strongly reacts with mAbAP3 or L7.25 (not shown) as well as L3.13 on immunoblots(lanes 3-6). In addition, both antibodies react withmaterial in the 200K region, which has no detectablecounterpart in the gels stained for protein with

— 13 -A •x1( 3

3

11697

67

43

29

Fig. 3. Secreted acid phosphatase stained on 10 % SDS-PAGEgels with permanganate/silver or on immunoblots using mAbsAP3, L3.13 or WIC108.3. Lanes 1 and 2 refer to the enzymepurified by column chromatography or on an L3.13 affinitycolumn, respectively. The purification leads to a single protein(app. Mr 100000), which reacts with AP3 (lanes 3 and 4), L3.13(lanes 5 and 6) but not with WIC108.3 (lane 7). A co-purifyingcomponent (app. Mr ~200 000) is detected by all antibodies butcan only be stained by KMnO4/silver on overloaded gels. Thearrow points to traces of the heavy chain of the IgGi antibodyL3.13.

permanganate/silver. In contrast, WIC 108.3 only reactswith the high molecular weight material but not with the100K protein. Finally, judging by the absence of AP3-reactive material at the front of the blots, the enzymepurified by either procedure is free of LPG.

Other Leishmania proteins are modified by LPG repeatepitopesIn addition to the secreted acid phosphatase, it appearedpossible that other proteins in promastigotes could bemodified by LPG epitopes. This is demonstrated in Fig. 4together with several controls. Proteins of a cell lysate orpurified LPG were separated by SDS-PAGE and electro-phoretically transferred to PVDF and Zeta Probe mem-branes arranged in a sandwich. The PVDF membrane wasstained for proteins using Ponceau S stain (Fig. 4A), andthen processed for immunostaining using mAb L7.25(Fig. 4B); the Zeta Probe membrane (Fig. 4C) was onlyimmunostained with mAb L7.25. This experiment led tothe following conclusions: first, promastigote lysatescontain a variety of proteins that react with mAbs L7.25 orAP3 (not shown) and, therefore, carry carbohydratesmodified by LPG epitopes. The proteinaceous nature ofthese reactive components is evident from their proteinaseK sensitivity, which leaves only the smear of LPG at thefront of the gel (lanes at the right side of Fig. 4A and B).Second, the antibody does not bind to proteins of themacrophage cell line J774, nor does added LPG associatewith these proteins during electrophoresis (compare highmolecular weight regions in Fig. 4B and C for J774 lysatewith or without LPG and LPG alone). Therefore, theseresults suggest that proteins in the range of 30K to >200Kare covalently modified by epitopes likewise present onLPG. However, it should be borne in mind that theseproteins have been detected by a highly sensitivetechnique (compare with the absence of prominentproteins precipitated by mAb AP3 in Fig. 2A), and that aquantitative approach will be required to estimate theirabundance. Significantly, neither the purified surfaceprotease, gp63 (unpublished results, see also Fig. 2A), nora recently purified membrane-bound acid phosphatase(Menz et al. 1991) reacts with the monoclonals.

Discussion

The observation that an antibody (AP3) initially definedby its reaction with a cellular as well as the secreted acidphosphatase of L.mexicana promastigotes, and then shownto react with LPG, was in itself not sufficient forpostulating a novel post-translational modification of theenzyme because LPG can form a tight complex with atleast one protein, bovine serum albumin (King et al. 1987).Therefore, the secreted acid phosphatase of L.mexicanawas purified by two independent approaches and identifiedas a protein with an apparent molecular weight of 100Kthat reacts in immunoblots with the anti-LPG antibodies.Strong evidence that this protein corresponds to thepurified enzymatic activity comes from a comparison of itsN-tenninal sequence with that of a membrane-bound acidphosphatase purified from L. mexicana promastigotes(app. Mr 70 000-72 000; Menz et al. 1991). These sequencesare similar, showing that the two enzymes are coded byrelated genes. The AP3-reactive phosphatase in promasti-gotes (Menz et al. 1991), most probably the precursor of thesecreted form of the enzyme, is present in very lowabundance and has not yet been purified. This low

178 T. Ilg et al.

116

116

14.3

/+2 1.5 1HQ LPG

0.5 J774 LysatePlus

5 2 1.5 1 0.5^g LPG

116

29

14.3

t tPromastigotecell lysate

Promastigotecell lysate+proteinase K

J774 Lysate

Fig. 4. Modification of promastigote proteins by LPG epitopes.The following samples were loaded on an 8 % to 16 % SDS-PAGE gel (from right to left): a lysate of L. mexicanapromastigotes in sample buffer (100 /<g protein); the sameamount of lysate previously treated with proteinase K; a celllysate of the macrophage cell line J774 (100 ^g protein); J774lysate (100/ig protein) plus 0.5-5 fig LPG; 0.5-5 ̂ g LPG. Afterelectrophoresis, the gel was blotted to a PVDF and a ZetaProbe membrane arranged in tandem. (A) The protein patternon the PVDF membrane shown by Ponceau S staining. Boththe PVDF (B) and the Zeta Probe membranes (C) wereimmunostained using mAb L7.25. The PVDF membraneretained all proteins but only part of the LPG, the rest beingrecovered on the cationized nylon membrane. In the absence orpresence of the J774 proteins, LPG moved as a smear to thefront of the gel, no staining was observed in the highmolecular weight region. In contrast, the antibody detectedpromastigote proteins in the 30 to >200 (xlO3)Mr range.

abundance can explain why the antibody does notprecipitate a readily identifiable protein from [^Sjmethio-nine-labeled promastigote lysates (Fig. 2A).

Recently, two other groups have proposed that the acid

phosphatase and LPG share related phosphate-containingcarbohydrate chains. First, mild acid hydrolysis known tocleave the phosphodiester bonds connecting the LPGrepeats, released most of the covalently bound phosphatefrom the L.donovani enzyme (Bates et al. 1990). Second,Jaffe et al. (1990) described the purification of a phos-phorylated secreted phosphatase from L.tropica(Mr>200 000) using an anti-LPG affinity column.

The secreted phosphatase purified either by columnchromatography or by L3.13 affinity chromatographycontains an additional high molecular weight componentthat can be readily labeled by radioactive galactose orstained in immunoblots with monoclonal antibodies(Figs 2 and 3). On the other hand, no obvious proteinscorresponding to this component were detectable (Figs 2Aand 3). Further analysis must show whether this materialis a phosphoglycan or a proteophosphoglycan. However,the fact that this component co-purifies with the acidphosphatase suggests that we are dealing with a highmolecular weight complex composed of several com-ponents.

mAbs AP3, L7.25 and WIC108.3 appear not to bedirected against the lyso alkylphosphatidylinositol core ofLPG because they do not react with the isolated core aftercleavage of the phosphosaccharide repeats by mild acidhydrolysis (T.Ilg, unpublished results). Therefore, theantibodies must be directed against the phosphosacchar-ide repeats or the sugar residues forming the cap at thenon-reducing end. The data shown in Fig. 3 clearlysuggest that mAbs AP3/L7.25 and WIC108.3 are directedagainst different epitopes because the former two anti-bodies react with both the 100K protein and the highmolecular weight component of the secreted phosphatasewhile WIC 108.3 recognizes only the high molecularweight component. In addition to the secreted acidphosphatase, other promastigote proteins appear to bemodified by epitopes recognized by mAbs L7.25 or AP3(Fig. 4). However, neither the promastigote surfaceprotease nor a mostly membrane-associated promastigoteacid phosphatase carries these epitopes. This posesinteresting questions regarding the signals determiningthis type of modification, considering that all threeproteins are likely to pass through the Golgi complexduring biosynthesis.

Recent evidence suggests that mAb L3.13 recognizes acarbohydrate epitope rather than the acid phosphatasepolypeptide chain. This epitope is present on both thephosphatase and the high molecular weight component ofthe complex but not on LPG. Furthermore, this antibodyreacts only with a few distinct components in the highmolecular weight region of immunoblots of promastigotelysates (unpublished experiments). Therefore, this epitopehas a more restricted distribution than that of mAbs AP3or L7.25. In the accompanying paper, the epitopesrecognized by these antibodies have been localized inpromastigotes and infected macrophages by immunofluor-escence and immunoelectron microscopy (Stierhof et al.1991).

This work was supported by a grant from the DeutscheForechungsgemeinschaft to P.O. and R.E. and by grants from theMacArthur Foundation, from the World Health Organisation,and a Whitehead Presidential fellowship awarded to D.G.R.

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Glycoprotein modification in Leishmania 179

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(Received 28 August 1990 - Accepted 21 September 1990)

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