THE JOURNAL OF BIOLOG~CAL CHEMISTRY Vol. 269, No. 39, Issue of September 30, pp. 24073-24081, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

0- and N-Glycosylation of the Leishmania mexicana-secreted Acid Phosphatase CHARACTERIZATION OF A NEW CLASS OF PHOSPHOSERINE-LINKED GLYCANS”

(Received for publication, May 10, 1994, and in revised form, July 6, 1994)

Thomas IlgS, Peter OverathS, Michael A. J. FergusonOn, Trevor Rutherford§, David G. Campbell§, and Malcolm J. McConville§ll** From the Wax-Planck-Institut fur Biologie, Abteilung Membranbiochemie, Correnstrasse 38, D 72076 Tubingen, Federal Republic of Germany, the $Department of Biochemistry, University of Dundee, Dundee DD1 4HN, United Kingdom, and the lllepartment of Biochemistry, University of Melbourne, Parkville 3052, Australia

The protozoan parasite Leishmania mexicana se- cretes a heavily glycosylated 100-kDa acid phosphatase ( S A P ) which is associated with one or more polydisperse proteophosphoglycans. Most of the glycans in this com- plex were released using mild acid hydrolysis conditions that preferentially cleave phosphodiester linkages. The released saccharides were shown to consist of mono- meric mannose and a series of neutral and phosphoryl- ated glycans by Dionex high performance liquid chro- matography, methylation analysis, exoglycosidase digestions, and one-dimensional ‘H NMR spectroscopy. The neutral species comprised a linear series of oligo- saccharides with the structures [Manal-B],,Man. The phosphorylated oligosaccharides were characterized as P04-6Galfi14Man and PO,-6[Glc~1-3lGalfil-4Man. The attachment of these glycans to the polypeptide backbone via the linkage, Manal-PO,-Ser, is suggested by: 1) the finding that more than 60% of the serine residues in the polypeptide are phosphorylated and 2) the resistance of the phosphoserine residues to alkaline phosphatase di- gestion unless the S A P was first treated with either mild acid (to release all glycans) or jack bean a-mannosidase (to release neutral mannose glycans). Analysis of the par- tially resolved components of the complex indicated that the most of the 0-linked glycans on the 100-kDa phos- phoglycoprotein comprised mannose and the mannose- oligosaccharides. In contrast the major 0-linked glycans on the proteophosphoglycan were short phosphoglycan chains, containing on average two repeat units per chain. In addition to the 0-linked glycans, both components in the SAP complex contained N-linked glycans. The N-gly- canase F-released glycans were characterized by Bio-Gel P4 chromatography and exoglycosidase digestions to be the biantennary oligomannose type with the structures Glc,Man,GlcNAc, and Man,GlcNAc,. The 0-linked gly- cans of the S A P complex are similar to those found in the phosphoglycan chains of the abundant surface lipophos- phoglycan, but differ in having much shorter phospho- glycan chains and a more diverse series of mannose cap oligosaccharides. These data suggest that there are marked differences in the ability of different glycosyl- transferases to utilize peptide-linked uersus glycolipid- linked acceptors.

* This work was supported by the Deutsche Forschungsgemeinschaft and the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 Howard Hughes International Scholar. ** Wellcome Trust Senior Research Fellow. To whom correspondence

should be addressed: Dept. of Biochemistry, University of Melbourne, Parkville 3052, Australia. Tel.: 61-3-344-5681; Fax: 61-3-347-7730.

Protozoan parasites of the genus Leishmania are the causa- tive agent of a variety of diseases in the tropics and subtropics where they represent a major health problem (Moddaber, 1987). They have a digenetic life cycle that alternates between the extracellular promastigote form living in the digestive tract of sandflies and the intracellular amastigote form residing in the phagolysosome of mammalian macrophages (Alexander and Russell, 1992). Cell surface and secretory antigens of the parasite form an interface between the parasite and the host and are likely to play key roles in the survival of Leishmania parasites in these hostile host environments. While the major cell surface molecules of these parasites have been relatively well characterized (cf . for reviews, McConville, 1991; Turco and Descoteaw, 1992; Medina-Acosta et al., 1993; McConville and Schneider, 1993; McConville and Ferguson, 1993) less informa- tion is available concerning the secreted antigens. Amajor com- ponent in the culture supernatant is the lipophosphoglycan (LPG)’ which is also the major macromolecule on the cell sur- face. The structures of the cell surface LPGs have been char- acterized from several species. They all contain a polydisperse phosphoglycan chain, made up of repeating phosphorylated oli- gosaccharides that contain the backbone sequence PO,- 6Galpl-4Man (reviewed in Turco and Descoteaux, 1992; Mc- Conville and Ferguson, 1993). The phosphoglycan chains are capped at the nonreducing terminus with short mannose-con- taining oligosaccharides and are anchored to the plasma mem- brane via a complex glycosylphosphatidylinositol glycolipid. Marked species- and stage-specific differences occur in the chain length of the phosphoglycan moiety and in the nature of the mono- and oligosaccharide side chains that substitute the repeating disaccharide backbone. LPG molecules, either retain- ing or lacking the glycolipid anchor, have been characterized in the culture supernatant of Leishmania promastigotes (Ilg et al., 1992; Greis et al., 1992). While the origin of the latter species is unknown, the former species are probably derived from surface expressed LPG which is passively and continuously shed from the plasma membrane (Handman et al., 1984; King et al., 1987; Ilg et al., 1992).

Leishmania parasites also secrete a number of protein-con- taining antigens. In particular, all species, except Leishmania major secrete an acid phosphatase ( S A P ) into the culture me- dium (Lovelace and Gottlieb, 1986). The Leishmania donouani S A P has been Characterized in detail over the last decade by Dwyer and colleagues as a heterogeneous phosphoglycoprotein which is substituted with both N-linked and abundant acid-

’ The abbreviations used are: LPG, lipophosphoglycan; S A P , secreted acid phosphatase; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody; HPLC, high performance liquid chromatography.

24073

24074 Glycans of Leishmania-secreted Acid Phosphatase

labile glycans (Gottlieb and Dwyer, 1982; Lovelace et al., 1986; Lovelace and Gottlieb, 1987a, 1987b; Bates and Dwyer, 1987; Bates et al., 1987, 1988, 1989, 1990). Chemical (Bates et al., 1990) and immunological studies (Jaffe et al., 1990; Ilg et al., 1991a, 1993) suggest that the acid-labile glycans of S A P and other Leishmania proteins are structurally related to the phos- phoglycan chains and neutral cap oligosaccharides of the LPG from the same species. Moreover, species-specific differences are evident in the macromolecular organization of the S A P

molecules. In L. donouani, S A P is present in culture as mono- mers and oligomers of the phosphoglycoprotein, whereas in L. mexicana, S A P is present in extended filaments which comprise multiple units of the 100-kDa phosphoglycoprotein and a poly- disperse proteophosphoglycan (Ilg et al., 1991a, 1991b).

In this study we report the structural analysis of the acid- labile glycans and N-linked glycans of the L. mexicana S A P complex. We present evidence for the attachment of cap oligo- saccharides and capped phosphosaccharides to phosphoserine residues of serine-rich polypeptide chains which establishes a novel type of polypeptide 0-glycosylation.

EXPERIMENTAL PROCEDURES MaterialsAack bean a-mannosidase and Aspergillus saitoi a-man-

nosidase were from Oxford Glycosystems. Flavobacterium meningosep- ticum N-glycosidase F was from New England Biolabs. Glucosidase I1 was a generous gift from Dr. Terry Butters (Department of Biochemis- try, University of Oxford).

Cell Culture-L. mexicana (MNYC/BZ/62/M379) promastigotes were grown as described previously (llg et al., 1992).

Purification of SAP-". mexicana S A P was purified as described (Ilg et aE., 1991b) except for additional ultracentrifugation (100,000 xg , 3 h) after DE52-cellulose chromatography. The enzyme was recovered in the ultracentrifugation pellet. To separate the L. mexicana SAP subcompo- nents, lyophilized samples were dissolved in 6 M guanidinium chloride, 50 mM TridHC1, pH 7.5,5 mM EDTAand applied to a Superose 6-column (Pharmacia, Freiburg, Federal Republic of Germany) equilibrated in the same buffer (0.2 ml/min). Elution of proteins was monitored by continuous measurement of the absorbance at 226 nm. Fractions (0.5 ml) were collected and analyzed by two-site ELISA, ELISA plates were coated with 50 p1 of mABAP3 (an IgM (20 pg/ml) in 50 mM NaHCO,, 100 mM NaCl, 16 h, 4 "C), nonspecific binding sites were blocked with 5% non-fat dried milk powder (5% MP), phosphate-buffered saline (137 mM NaC1, 2.7 mM KCl, 8 mM Na,HPO,, 1.4 mM KH,PO,, pH 7.2, 1 h, 37 "C) followed by an incubation with 100 p1 of MOO dilutions of the Superose 6 fractions in 2% bovine serum albumin in phosphate-buffered saline. After three washings, the plates were incubated with mouse hybridoma supernatants (LT8.2, IgG2a; LT15, IgG1; LT17, IgG1; 1 h, 37 "C) fol- lowed by three further washing steps and an incubation with a 1:lOOO dilution of goat anti-mouse IgG (7-chain-specific) coupled to alkaline phosphatase (Sigma, Deisenhofen, Federal Republic of Germany) in 5% MP, phosphate-buffered saline (100 pl, 1 h, 37 "C). The plates were washed four times with "is-buffered saline (10 mM Tris-HC1, 140 mM NaC1, pH 7.5) and developed with 5 mMp-nitrophenyl phosphate in 1 M diethanolamineEIC1, pH 9.8, 1 m~ MgCl, and continuous measurement of the absorbance at 405 nm. The specificity of the monoclonal antibod- ies AP3, LT8.2, LT15, and LT17 as well as SDS-PAGE and Stains-all staining of S A P have been described previously (Ilg et al., 1993).

Release and Analysis of 0-Glycans-SAP was hydrolyzed using con- ditions which selectively cleave hexose-1-P linkages (40 m~ trifluoro- acetic acid, 100 "C, 12 min) (McConville et al., 1990), before or after N-glycosidase F digestion. The acid was removed by evaporation in a Speed-vac concentrator. The acid-released mono- and oligosaccharides were purified by HPLC on a Dionex BioLC Carbohydrate analyzer (Dionex Corp., Sunnyvale, Ca) equipped with a pulsed amperometric detector and self-regenerating suppressor. Neutral and phosphorylated glycans were separated on a CarboPac PA1 column (250 x 4 mm inner diameter) eluted with 0.15 M NaOH and two linear gradients of NaOAc. The concentration of NaOAc was maintained at 0.0125 M for 1 min, then increased to 0.05 M over 30 min, followed by a second linear increase to 0.25 M over 20 min, and maintained at 0.25 M for a further 20 min (program 1). For separation of neutral saccharides alone the concentra- tion of NaOAc was maintained at 0.0125 M for 1 min, then increased to

by passage down a column ofAG50 X12 (H') overAG3 X8 (OH-). When 0.33 M over 60 min. Fractions containing neutral glycans were desalted

the purification of phosphorylated glycans was desired, the suppressor unit was taken out of line and fractions desalted by passage down AG50 X12 (H+), lyophilization and flash evaporation with toluene to remove residual acetic acid.

Release and Analysis of N-Glycans-N-Linked glycans were released from SAP with IC: meningosepticum N-glycanase (100 units) in 20 pl of 0.5 M sodium phosphate, pH 7.5, containing 1% Nonidet P-40 and 15 mM dithiothreitol. Incubations were performed at 37 "C over 24 h with addition of an extra 50 units after 16 h. To precipitate the protein and stop the reaction, bovine serum albumin (10 mg/ml in water, 80 pl) was added, followed by methanol (400 pl) and chloroform (100 pl). After vortex mixing, water (300 pl) was added, mixed, and the sample een- trifuged to separate the two phases. The upper aqueous phase was removed and methanol (300 pl) added to the lower phase which was mixed and centrifuged to pellet the protein. The aqueous phase, con- taining the released glycans, was dried and then resuspended in 1 M NH,OH (15 pl) and reduced with the addition of NaB3H, (15 pl, 30 mM, 20 Ci/mmol). The reduction was stopped with 1 M acetic acid and the mixture passed down a column ofAG50 X12 (H+). Radiochemical impu- rities were removed by chromatography on a column (4 ml) of micro- granular cellulose (Sigma) eluted sequentially with 1-butanol/ethanol/ water (4:1:1, v/v) (30 ml), methanol (1 ml), then 200 mM NaOAc (5 ml). The labeled glycans which eluted with the NaOAc wash were desalted by passage down a column ofAG5O X12 (H') and flash evaporation with toluene. After separation by Dionex HPLC, purified glycans were se- quenced on a GlycoMap 1000 (Oxford Glycosystems, Abingdon, United Kingdom). Separations were carried out on a glycan sizing column (480 x 10 mm inner diameter) packed with Bio-Gel P4 and maintained at 55 "C. The column was eluted with water a t an initial flow rate of 60 pl/min for 11 ml, after which the flow rate increased linearly to 200 pl/min over 27 ml. Samples were injected together with a partial dex- tran hydrolysate to calibrate the size of the labeled glycan, which were expressed in glucose units. Labeled glycans were detected with a radi- oisotope flow monitor, whereas the dextran oligomers were detected with a refractive index monitor.

Enzyme Digestions-The following incubation conditions were used for the exoglycosidase digestions: jack bean a-mannosidase, 30 unitdml in 0.1 M sodium acetate, pH 5.O;A. saitoi a-mannosidase, 2 mg/ml in 0.1 M sodium acetate, pH 5.0; a-glucosidase 11, 50 milliunits/ml in 0.1 M sodium phosphate, pH 7.0, containing 0.1 M NaCl and 10% glycerol. Calf intestine alkaline phosphatase digestions were performed in 0.5 M NH,HCO,, pH 8.5. All incubations were performed a t 37 "C for 18 h in presence of toluene vapor. Incubations were stopped by boiling for 5 min and the samples desalted by passage down a column of AG50 X12 (H') over AG3 X8 (OH-).

Monosaccharide Analysis-Neutral monosaccharides were analyzed and quantified by gas chromatography-mass spectrometry as their tri- methylsilyl derivatives after methanolysis (0.5 M HCl in methanol, 80 "C, 16 h) as described previously (McConville et al., 1990). Phospho- rylated monosaccharides were either analyzed as the trimethylsilyl derivatives of the corresponding hexosephosphate dimethylesters (Fer- guson et al., 1988) or as neutral monosaccharides after dephosphoryla- tion with alkaline phosphatase or 50% hydrofluoric acid (0 "C, 48 h). Hydrofluoric acid was removed by evaporation.

Methylation Analysis-Methylation analysis on dephosphorylated samples was performed as described previously (McConvilIe et al., 1990) except for using trifluoroacetic acid (2 M, 2 h, 100 "C) for acid hydrolysis of the permethylated oligosaccharides. The permethylated alditol ac- etates were analyzed on an SE54 column (30 m x 0.25 mm, Chrompack).

Amino Acid and Phosphoamino Acid Analysis-L. mexicana S A P

(1-2 pg) was mixed with a norleucine internal standard, subjected to total acid hydrolysis (16 h, 110 "C, 6 M HCl in the vapor phase), and dried. The samples were dissolved in 30 pl water:ethanol:triethylamine (2:2:1, v/v), dried, and derivatized by the addition of 20 pl of water/ ethanol/triethylamine/phenylisothiocyanate (7:1:1:1, v/v). The samples were dried extensively and analyzed using a Pico-Tag system with the suppliers recommended buffer systems (Waters Associates). For the detection of phosphoamino acids, S A P samples (2-4 pg) were subjected to partial acid hydrolysis (2 h, 110 "C, 6 M HCl). The partial hydroly- sates were analyzed as described above except for changing the recom- mended pH of the elution buffer from pH 6.4 to pH 5.6 to improve the separation of phosphoserine and phosphothreonine from aspartic acid and glutamic acid. The identity of the derivatives was confirmed by coelution with authentic standards. The absorbance at 254 nm of the phenylthiohydantoin-derivatives of phosphoserine and phosphothreo- nine was very similar to the absorbance of their respective nonphos- phorylated counterparts. Standards of phosphoserine and phospho- threonine were included in each set of partial acid hydrolysis to correct

Glycans of Leishmania-secreted Acid Phosphatase 24075

A

c u c N \ 1.6 -

E 1.4 E 1.2

w 0.8 -

= 0.6 u

4

0.4 - 0

m cn 0.2 e 0.0

F R A C T I O N

B I

2 0 5 -\ 116 -( 97 - 67 - 43 - 29 - 2 L -

S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2L 1 POOL1 1 POOL2 I POOL3 I POOL4 I FIG. 1. Gel filtration of denatured L. mexicana S A P . A, the pu-

rified S A P complex was chromatographed on a column of Superose 6 in 6 X I guanidinium hydrochloride. Fractions were monitored for protein (OD,,,) and for reactivity to the mAbs, LT8.2 (anti-peptide), LT15 (anti- phosphodisaccharide), and LT17 (anti-phosphotrisaccharide) in a two- site ELISA and pooled as indicated. The void vohme (dextran blue, VJ and the elution positions of human transferrin (80 kDa) and lysozyme (14.3 kDa) are also indicated. B , SDS-PAGE (7.5%-20%) analysis of the Superose 6 column fractions and detection of phosphosaccharides with the Stains-all dye. S = starting material. The molecular masses of standard proteins in kilodaltons are indicated.

for the partial hydrolysis of the phosphomonoester bonds. Calculations on the relative abundance of serine and phosphoserine were done under the assumption of identical release rates of phosphorylated and non- phosphorylated serine from the S A P polypeptide and identical hydrol- ysis sensitivity of free and polypeptide-bound phosphoserine. In some analyses, S A P samples were alkaline phosphatase-treated before and after mild acid hydrolysis and subsequently subjected to phosphoamino acid analysis.

500 M H z ' H N M R Spectroscopy-The S A P complex was exchanged with deuterium oxide (99.96% 'H, Aldrich) by repeated evaporation. All spectra were aquired on a Bruker AM-500 spectrometer with a nominal probe temperature of 27 "C. The one-dimensional spectrum was aquired with 8000 complex data points, zero-filled to 16,000 before transforma- tion, covering a spectral width of 8064 Hz. The recycle time per tran- sient was 11.0 s, allowingcomplete relaxation between successive scans. The two-dimensional COSY-90 spectrum was aquired in magnitude mode and processed with unshifted sine-bell weighting in both dimen- sions. The digital resolution of the processed data was 1.0 Hzlpoint in f2 and 2.0 Hdpoint in fl.

RESULTS

Fractionation of the S A P Complex-L. mexicana S A P was pu- rified as a complex of a 100-kDa glycoprotein and several high molecular weight polydisperse proteophosphoglycans. These components were partially fractionated by Superose 6 chroma- tography in the presence of guanidinium hydrochloride (Fig. 1). The fractions were analyzed by SDS-PAGE and Stains-all stain- ing and by two-site ELISA using mAb AP3 as binding antibody and mAbs LT8.2, LT15, and LT17 as detection antibodies. AP3 recognizes the LPG mannose caps and binds to all the compo- nents in the complex. LT8.2 binds to a peptide domain of the

100-kDa subunit of the complex, whereas LT15 and LT17 bind to the phosphorylated di- and trisaccharides of L. mexicana LPG, respectively (Ilg et al., 1993). The first protein peak (pool l), which eluted near the void volume of the Superose 6 column displayed the lowest electrophoretic mobility, was not recog- nized by LT8.2, but was strongly recognized by LT15 and LT17. The second protein peak (pool 2) comprised material which mi- grated as a diffuse band near the 200-kDa marker as well as some slower migrating material. This peak was recognized by all three antibodies. Components in pools 1 and 2 are referred to as high molecular weight proteophosphoglycans. The majority of the protein eluted in the third peak (pools 3 and 4). The major component in these fractions was a 100-kDa phosphoglycopro- tein that carries the enzymatic activity, although small amounts of contaminating proteophosphoglycan were also detected by SDS-PAGE. Amino acid and monosaccharide analysis indicated that pools 1 and 2 were more heavily glycosylated than pools 3 and 4 and that they also contained a significantly higher level of serine, which was the major amino acid in all fractions (Table I). This peak bound strongly to LT8.2 but weakly to the anti- repeat antibodies. The analysis of the glycans from the unfrac- tionated complex and individual components is described below.

The Major Glycans of S A P Are Not N-Linked-N-Glycosidase F digestion of the S A P complex released approximately 7% of the total carbohydrate. The released glycans contained Man, GlcNAc, and Glc in the molar ratio 5:O.l:l. The remainder of the glycans were quantitatively released from the protein by hydrolysis with trifluoroacetic acid, using conditions (40 mM, 12 min, 100 "C) that quantitatively cleave hexose-1-PO,, linkages, while leaving glycosidic linkages intact (McConville et al., 1990). Monosaccharide analysis indicated that these glycans contained only Man, Glc, Gal-6-P04, and trace amounts of ga- lactose. After hydrofluoric acid dephosphorylation, the molar ratio of Man:Gal:Glc was 6.7:3.4:1

Structure of the Acid-released Glycans-Glycans released by mild acid hydrolysis of the S A P complex were chromatographed on a Dionex HPLC using a gradient program which resolves both neutral and phosphorylated oligosaccharides. These analyses resolved seven neutral (Nl-N6) and two charged (P1 and P2) components, which were purified for characterization (Fig. 2). N1 eluted with monomeric hexose and was shown to contain only mannose by direct analysis of the trimethylsilyl derivative without prior methanolysis. N2 contained two par- tially resolved components which coeluted with the disaccha- ride cap structures, Mancul-2Man and Galpl-4Man from L. mexicana LPG. The presence of these disaccharides is consist- ent with the methylation analysis (Table 11) and with the re- sults of the exoglycosidase digestion which showed that the later eluting peak in this doublet was susceptible to jack bean a-mannosidase (Fig. 3). Methylation analysis of N3-N6 indi- cated the presence of linear oligosaccharides containing from 3-6 2-linked mannose residues (Table 11). These oligosacchar- ides were degraded by jack bean a-mannosidase, demonstrat- ing that all the mannose residues were in the a-configuration.

The phosphorylated S A P oligosaccharides, P2 and P3, coe- luted with the phosphorylated di- and trisacharides from L. mexicana LPG, respectively. Monosaccharide analysis showed that both oligosaccharides contained mannose and galactose 6-phosphate, whereas P2 also contained glucose. From the methylation analysis of the dephosphorylated oligosaccharides, P1 and P2 have the structures P04-6Gall-4Man and PO,- 6[Glcl-3]Gall-4Man, respectively (Table 11).

The anomeric proton region of the intact one-dimensional NMR spectrum is shown in Fig. 4. On the basis of the small (-3 Hz) axial-equatorial J2,, spin-coupling constants determined from the two-dimensional COSY spectrum (not shown) the res-

Glycans of Leishmania-secreted Acid Phosphatase

TABLE I Amino acid analysis of the SAP complex and Superose 6-fractionated components

Amino acid S A P complex Pool

1 2 3 4

Asn + Asp 10.7 5.1 10.5 11.7 Gln + Glu 6.9

11.2 4.1

Ser 19.0 6.9

42.0 7.1 7.6

GlY 5.8 22.5 15.0

3.4 19.5

His 1.9 0.6 6.3 5.7 1.4

6.5 2.2

Arg 4.1 1.7

Thr 13.7 1.9 0.1" 1.0" 4.8

Ala 7.2

9.8 21.6 17.6

11.7 15.0

9.6 Pro

9.6 6.4

9.1

Tyr 1.8 10.0 4.2 5.9 5.7 0.5

Val 0.8 1.7

6.1 2.8 5.0 6.5 1.4

Met 1.6 1.6 5.1

Ile 2.1 0.8 1.7 1.0

1.2 Leu 5.8

1.5 2.3 3.3

1.9

Phe 4.2 6.4

2.6 1.6 4.3

2.0 LY 2.0 1.0 1.5 2.1

2.9 2.1 2.2

Ratio monosaccharide/amino acid (nmolhmol) 12.3 2.8 1.3 1.3 The peaks for Arg and Thr were incompletely separated in the analyses of pools 2 and 3 so that the Arg values represent minimum estimates.

P1

Hex

I N2 P2

-1- I

60

Time (rnin)

FIG. 2. Dionex HPLC profile of glycans released with mild acid hydrolysis from L. mezicana SAP. The S A P complex was hydrolyzed with 40 mM trifluoroacetic acid (100 "C, 12 min) and the protein separated from the released glycans by solvent precipitation. The neutral and phosphorylated glycans were resolved using the gradient program 1.

idue with an H-1 resonance at 4.47 ppm (Jl,2 = 7-8 Hz) was assigned as the Gal residue in the repeat units which is in the P-configuration. The residue with the lower intensity H-1 peak at 4.55 ppm was also in the galacto-configuration, and the chemical shifts of H-1 through H-4 were assigned from the COSY spectrum. The chemical shifts were the same as those of the 3-substituted PO,-GGalpl residue in the LPG phosphogly- cans (McConville et al., 1990) with H-2 through H-4 resonances shifted 0.3-0.4 ppm downfield of the corresponding resonances in the free monosaccharide (Jansson et al., 1989). All reso- nances in the PGlc configuration spin system (H-1 = 4.69 ppm, Jl0 = 7-8 Hz) were within 0.1 ppm of those in the free monosac- charide (Jansson et al., 1989) and were assigned to an unsub- stituted PGlc. In all other H-1-H-2 COSY cross-peaks the H-2 multiplicity was consistent with the mannose residues. 'H NMR chemical shifts reported for Manal-2Manal-0-Ser (He- lander et al., 1992) are in close agreement with those for the H-1 peaks at -5.06 ppm and their corresponding H-2 reso-

9 Hex

I

0 10 20 30 40

Time (min) FIG. 3. Digestion of the neutral acid-released glycans with jack

bean a-mannosidase. The neutral acid-released glycans from the S A P complex were analyzed by Dionex HPLC before (A) and after ( E ) diges- tion with jack bean a-mannosidase.

nances. The chemical shift of the H-1 signal at 5.31 ppm and its associated H-2 (at 4.12 ppm) are consistent with previous as- signments for unsubstituted aMan-1-PO, (Nikolaev et al., 1989). Two partially overlapping doublets (JHp -8 Hz) appear at 5.65 and 5.67 ppm, -0.3 ppm downfield of the H-1 signals from unsubstituted Mana-1-PO,. Since downfield shifts of -0.3 ppm have been reported for aMan H-1 signals upon substitu- tion at the 2-position with a-Man (Nikolaev et al., 1989; He- lander et al., 19921, the two doublets at 5.6 ppm were assigned to the 2-substituted aMan-1-PO, in the cap structures. Two

Glycans of Leishmania-secreted Acid Phosphatase 24077

TABLE I1 Methylation analysis of oligosaccharides released by mild acid hydrolysis of the S A P complex

PMMA Origin N2 N3 N4 N5 N6 P1 P2

Mannitol 2,3,4,6-Tetra-O-methyl Terminal Man 1.0 1.0 1.0 1.0 1.0 3,4,6-Tri-O-methyl 2-0-Substituted Man 0.9 2.0 3.1 3.6 4.1 2,3,6-Tri-O-methyl 4-0-Substituted Man 0.3 1.1 6.8

2,3,4,6-tetra-O-methyl Terminal Gal 0.4 1.0 2,4,6-Tri-O-methyl 3-0-Substituted Gal 0.7

2,3,4,6-Tetra-O-methyl Terminal Glc 1.0

Galactitol

Glucitol

516 5 1 4 5 . 2 5 . 0 4 . 8 4 . 6 P P m

FIG. 4. One-dimensional 600 M H z ‘H NMR of the L. mzicana SAP complex. The anomeric region of the one-dimensional spectrum of the native S A P complex is shown and the relative peak integrals indi- cated in parentheses.

mannose H-1 multiplets (J,,, = -7-8, Jl,2 = -2 Hz) were over- lapped at 5.44 ppm, identical to the signal for H-1 of aMan-1- PO, substituted with p-Gal at the 4-position (McConville et al., 1990). These data suggest that the mannose residues in the Galpl-4Man disaccharde are linked to phosphate in the a-con- figuration in the native molecule.

Differences were evident in the relative abundance of the released oligosaccharides in the different S A P pools. Pools 1 and 2 containing the high molecular weight proteophosphogly- can were enriched for the phosphorylated repeat units. In con- trast, pools 3 and 4, which contained mainly the 100-kDa phos- phoglycoprotein, were enriched for the mannose/mannan oligosaccharides (Table 111). It is possible that the repeat units in this latter fraction are linked to the proteophosphoglycans that were not completely separated from the 100-kDa phospho- glycoprotein by gel filtration (see Fig. 1).

The Major Glycans Are 0-Linked to Phosphoserine-The use of mild acid hydrolysis conditions, that selectively hydrolyzes hexose-1-PO, linkages, to quantitatively release the major S A P

oligosaccharides suggested that these oligosaccharides were linked to the protein by phosphodiester linkages. Moreover, sequential treatment of S A P with N-glycanase followed by mild acid hydrolysis removed all the carbohydrate from the protein, indicating that this linkage was directly to the polypeptide backbone and not via a acid-resistant (0- or N-linked) glycan core. This type of linkage was supported by phosphoamino acid analysis which showed that approximately 60% of the major amino acid, serine, was phosphorylated. In contrast no phos- phothreonine was detected. These phosphate groups are resis- tant to alkaline phosphatase digestion in the native and control treated (20 mM NaC1, 10 min, 100 “C) molecule, indicating that the phosphate groups are not present as simple monoesters (Fig. 5A). However, after mild acid hydrolysis to quantitatively remove the 0-linked glycans, most of the phosphoserine be- came susceptible to alkaline phosphatase. Mild acid hydrolysis alone did not remove the phosphate groups (Fig. 5 A ) . These results suggest that the phosphoserine residues are substituted with oligosaccharides. This was further probed by sequential treatment of the 100-kDa phosphoglycoprotein (pool 3) with

TABLE I11 Relative abundance of neutral cap oligosaccharides and

phosphorylated repeat oligosaccharides released with mild acid hydrolysis of Superose 6-purified SAP fractions

Component Pool

1 2 3 4

mol% Caps

Man 20.7 20.2 21.5 37.3 N2 6.7 7.3 11.7 9.0 N3 2.2 3.6 8.8 7.0 N4 N5

3.4 4.6 11.7 8.4 2.6 2.8 6.5

N6 6.4

1.4 1.0 2.7 1.8 Repeats

P1 P2

44.6 41.8 25.5 23.3 16.0 15.0 6.0 5.2

Ratio capdrepeats 0.6 0.7 1.7 2.0

jack bean a-mannosidase followed by alkaline phosphatase. The major 0-linked glycans in this fraction are the mannose- containing oligosaccharides which should be completely re- moved by the a-mannosidase treatment without effecting the conformation of the polypeptide backbone. Jack bean a-man- nosidase treatment removed approximately 80% of the mannose/mannan oligosaccharides (see below), with a concom- itant increase in the sensitivity of the phosphoserine residues to alkaline phosphatase (Fig. 5B). These results indicate that the mannose residues are linked directly to the phosphoserine residues and that they are in the a-configuration.

Size Distribution of the Phosphoglycan Chains in the 100- kDa Protein-The above data suggested that most of the man- nose and mannans in pool 3 were linked directly to phospho- serine residues in the protein. However, some of these glycans may also cap chains of phosphorylated repeat units. This was suggested by the low relative abundance of Galpl-4Man to P04-6Galpl-4Man in the total acid-released mixture, where Galpl-4Man is the expected terminal oligosaccharide of phos- phoglycan chains that are not capped with mannose or man- nans (Figs. 2 and 6A). In order to distinguish between the possiblities that this fraction contained a small number of long phosphoglycan chains or a larger number of short phosphogly- can chains, and hence the relative distribution of protein, uer- sus phosphoglycan-linked mannose/mannans, pool 3 material was digested first with jack bean a-mannosidase and second with alkaline phosphatase as described above. This procedure removes the capping mannose structures and dephosphory- lates the first subterminal repeat unit. The extent to which the phosphorylated repeat units were dephosphorylated was deter- mined by Dionex HPLC analysis of the acid-released oligosac- charides. As shown in Fig. 6B, this procedure converted more than half of the phosphorylated disaccharide to Galpl-4Man. These results indicate that more than half of the disaccharide repeat units were substituted with neutral cap oligosaccha- rides and suggest that the phosphoglycan chains are very short, consisting of two repeat units per chain, on average.

24078 Glycans of Leishmania-secreted Acid Phosphatase

A r

Acid - + t f +

B

4" + BI AP + i-

FIG. 5. Susceptiblity of phosphoserine residues to alkaline phosphatase digestion. The susceptibility of the phosphoserine resi- dues in the SAP complex to alkaline phosphatase (AP) digestion, before and after mild acid hydrolysis (to cleave hexose-1-PO, linkages), was determined by phosphoamino acid analysis (A). The susceptibility of the phosphoserine residues to alkaline phosphatase after digestion of the 100-kDa phosphoglycoprotein (pool 3) with jack bean a-mannosidase was also determined (B) . In the latter case, values in the mannosidase- treated samples are expressed as percent of the control sample (incu- bated with denatured mannosidase).

Based on these findings it can be estimated that 70% of the mannose or mannose oligosaccharides are linked directly to the protein, whereas the remaining 30% cap the short phosphogly- can chains. A similar conversion of P04-Galp14Man to Galpl- 4Man was observed when pool 2 was treated in the same way, suggesting that all fractions contain phosphoglycan chains with a similar degree of polymerization.

Structure of the N-Linked Glycans-The glycans released by N-glycosidase F from pool 3 were radiolabeled by reduction with NaB3H, and analyzed by Dionex HPLC. A major peak (80% of 3H-labeled oligosaccharides) which eluted at 3.0 Dionex units (Du, elution time relative to coinjected dextran oligomers) and a minor component which eluted at 4.8 Du were detected (Fig. 7A). Glycans in the same relative abundance were re- leased from the other S A P pools (results not shown). These glycans were sequenced by exoglycosidase digestion and the products analyzed by Bio-Gel P4. The major peak (G10) eluted at 9.88 glucose units on Bio-Gel P4, suggestive of a biantennary oligomannose structure (Fig. 7 B ) . Digestion with the al-2- specific mannosidase from A. saitoi gave a product which eluted a t 7.93 glucose units, indicating the loss of two al-2 linked mannose residues (Table IV). Digestion of this product with jack bean a-mannosidase resulted in a product which eluted at

A Hex P1

B N2 a

Pi

Hex

P2

U K L " , I 1 I 1

0 10 20 30 40 50 60 70

Time (min) FIG. 6. Uncapping of the phosphoglycan chains of 100-kDa

phosphoglycoprotein. The 0-linked glycans of pool 3 containing the 100-kDa phosphoglycoprotein were released by mild acid hydrolysis before (A ) and after sequentially treatment with jack bean a-mannosi- dase then alkaline phosphatase ( B ) and analyzed by Dionex HPLC using program 1.

5.69 glucose units, indicating the loss of three a1-3(6)-linked mannose residues (Table IV). This product migrates in the ex- pected position of the N-glycan core, Manpl4GlcNAcpl- 4GlcNAcol (Olafson et al., 1990). The minor peak (G11) eluted at 10.72 glucose units on Bio-Gel P4 (Fig. 7C), which is the same elution position as the glucosylated biantennary oligo- mannose from the L. mexicana metalloproteinase (Olafson et al., 1990). This glycan was resistant to A. saitoi a-mannosidase, consistent with the presence of a residue other than al-2 linked mannose, at the nonreducing terminus of the 3-arm (Table IV). However, it was digested with a-glucosidase I1 to a product that eluted at 9.88 glucose units, indicating the loss of a terminal a1-3 glucose residue. This product comigrated with the G10 fraction and was now susceptible to digestion with A. saitoi a-mannosidase to give a product a t 7.93 glucose units (Table IV). Taken together, these data are consistent with the S A P N-linked glycans having the glucosylated biantenarry oli- gomannose structures shown in Fig. 8.

DISCUSSION

Previous studies have shown that the secreted acid phospha- tase of L. mexicana promastigotes occurs as a filamentous com- plex which includes a 100-kDa phosphoglycoprotein and a high molecular weight proteophosphoglycan (Ilg et al., 1991b; Sher-

Glycans of Leishmania-secreted Acid Phosphatase 24079

0 10 20 30 40 50 0 10 20 30 40 50

Time (min)

200

160

I20

B 80

40

0 10 15 20

Volume (ml)

25 30 35

FIG. 7. Dionex HPLC and Bio-Gel P4 chromatography of the N-linked glycans from the 100-kDa phosphoglycoprotein. Oligo- saccharides were released from the 100-kDa phosphoglycoprotein (pool 3) with N-glycosidase F and radiolabeled by reduction with NaB3H,. Two labeled neutral glycans (GI0 and G11) were identified afier chro- matography on a CarboPac PA1 column using program 2 (A). The Di- onex-purified glycans, G10 (B) and G11 ( C ) were rechromatographed on a Bio-Gel P4 column, together with a series of dextran oligomers and their relative elution time given in glucose units (Gu).

hof et al., 1994). In this study, we have characterized the 0- and N-linked carbohydrate structures of this complex, which are shown in Fig. 8. We show that both components in the complex are highly substituted with short chains of O-linked phospho- glycans and phosphomannose/phosphomannans. These oligo- saccharides appear to be linked to serine residues in the polypeptide backbone and probably all contain the sequence Mana-l-PO,-serine. This is a novel type of O-glycosylation. A related, but distinct, type of protein glycosylation has only pre-

viously been detected in a lysosomal proteinase of Dictyostelium discoideum, which contains a single N-acetylglucosamine res- idue linked to phosphoserine (Gustafson and Milner, 1980; Gustafson and Gander, 1984; Finn and Gustafson, 1987). I t is likely that most of the serine residues, which account for 1540% of the S A P polypeptide are O-glycosylated, based on the stoi- chiometry of carbohydrate to protein and the determination of serine phosphorylation which provides a lower estimate. Inter- estingly, there appear to be pronounced differences in the 0- glycan structures of the proteophosphoglycan and the 100-kDa phosphoglycoprotein. While the O-linked phosphoglycan chains account for most of the acid-labile glycans in the proteophos- phoglycan, the 100-kDa phosphoglycoprotein contains a high proportion of O-linked phosphomannose/mannan. At least some of the repeat units in this latter fraction are probably derived from contaminating proteophosphoglycan, suggesting that the 100-kDa phosphoglycoprotein is highly enriched for the phosphomannose/mannans. I t was not possible to identify which domains of the L. mexicana S A P are O-glycosylated be- cause of the resistance of the complex to proteolysis. However, similar studies on the L. donovani S A P which lacks an associ- ated protoephosphoglycan, have indicated the presence of a dis- tinct O-glycosylated domain that could be separated from the catalytic domain after proteinase K treatment.'

The O-linked glycan structures of the S A P complex are sim- ilar to those found in the phosphoglycan chains of the abundant surface LPG of these parasites. However, the O-linked glycans of the S A P complex are distinct 1) in having much shorter phosphoglycan chains than the LPG (on average 2 versus 30) and 2) in having mannan oligosaccharides with longer chains (i.e. from Man, to Man,). In contrast the cap oligosaccharides of LPG are restricted to Man, and Man,, which may or may not contain an additional pGal residue (Ilg et al., 1992). These results suggest that there are marked differences in the ability of the different glycosyltransferases to transfer sugars to either glycolipid or protein bound oligosaccharides. In particular, they suggest that the protein-linked oligosaccharides are preferred substrates for the putative al-2 mannosyl transferase or al- ternatively that they are poor substrates for the transferases responsible for synthesis of the phosphorylated repeat units.

It is likely that the 100-kDa phosphoglycoprotein and the proteophosphoglycan in pool 2 have a common or related polypeptide backbone from their amino acid composition (see Table I) as well as their reactivity with mAb LT8.2. I t is likely that these components are the products of the two genes (SAP-1 and -2) that have recently been cloned and ~equenced.~ However, the proteophosphoglycan of pool 1 probably contains a different polypeptide chain as it has a much higher serine content and does not bind the monoclonal antibody LT8.2. A similar proteophosphoglycan, which is devoid of acid phospha- tase activity, has been purified and partially characterized from L. mexicana amastigotes (Bohr et al., 1993; Ilg et al., 199414

The 100-kDa phosphoglycoprotein and the high molecular weight proteophosphoglycans also contain N-linked glycans which account for approximately 7% of the protein glycosyla- tion. These glycans were characterized as the biantennary oli- gomannose structures, Man,GlcNAc, and Glc,Man,GlcNAc,. The same N-linked glycans have been found on the abundant surface metalloproteinase of L. mexicana amazonensis pro- mastigotes (Olafson et al., 19901, although in this protein the glucosylated species were the major N-linked glycans. In

T. Ilg, unpublished results. M. Wiese, T. Ilg, F. Lottspeich, and P. Overath, manuscript in prep-

T. Ilg, Y.-D. Stierhof, M. J. McConville, and P. Overath, submitted for aration.

publication.

24080 Glycans of Leishmania-secreted Acid Phosphatase

TABLE IV Summary of enzymatic sequencing data on the N-linked glycans from the 100-kDa phosphoglycoprotein

ASAM, A. saitoi a-mannosidase; JBAM, jack bean a-mannosidase; aGlc, glucosidase 11.

Proposed structure Treatment Size (Gu) Product

Manal -3Manal-6\

5.69 Man GtcNAc JBAM

7.93 Man4 GlcNAc ASAM

9.88 -

ManPl-4GlcNAc~1-4GlcNAcol / Manal -2Manal-2Manal-3

(GI 0)

- 10.72

Manal-3Manal-6\ ASAM Glcl Man6 GlcNAc2

7.93 Man4 GlcNAc2 aGlc / ASAM

9.88 Man GlcNAc aGlc

10.72 Man~l-4GlcNAc~l-4GlcNAcol

Glcal-3Manal-2Manal-2Manal-3/

(GI 1)

COOH \ I \

\ [Manal-21 Manal-P04-6Galp1 -4Manal -PO4 -6Galpl-4Manal -PO4 - Ser J \ \ \

0-5 I I

I \

+/- GlcPl-3 +/- Glcpl-3 \ - - - \ Q,

/ n \

\ [Manal-2]1-5 Manal-PO4 -Ser r - - = 0

Manal-P04 -Ser I / Y m" \ / 9

U Q Q, Q

I / Q, \ \ .- +

-

Manal-3Manul-6 1 % - \ / +/- ' Manpl-4GlcNAcpl-4GlcNAc -Asn "- / /

i- - - Glcal-3Manal-PManal-2Manal-3 I

/

/ /

/

NH2 1

0- and N-linked glycans phosphoglycoprotein / SAP complex proteophosphoglycan

FIG. 8. Summary of the N-linked and novel 0-linked glycan structures found on L. mexicana SAP. Monomers of the 100-kDa phosphoglycoprotein appear to aggregate to form extended filaments that are, in turn, coated by a high molecular weight proteophosphoglycan ( S A P complex). This study shows that both the 100-kDa phosphoglycoprotein and the proteophosphoglycan are heavily glycosylated with short chains of 0-linked phosphoglycans and phosphomannose/phosphomannans that are attached to serine residues in the polypeptide backbone and account for 93% of the protein glycosylation. Oligomannose N-linked glycans account for the remaining glycosylation.

Leishmania, glucosylation of N-glycans occurs after the oligo- mic reticulum (reviewed by Parodi, 1993). The retention of Glc saccharide has been transferred to protein and is normally on at least some of the N-glycans of both cell surface and se- transient, as the glucose is normally removed in the endoplas- creted Leishmania proteins suggests that the putative endo-

Glycans of Leishmania-secreted Acid Phosphatase 24081

plasmic reticulum glucosidase I1 is less active in this parasite than in other eukaryotes.

Based on antibody reactivity and structural analyses it is likely that the S A P S from four other Leishmania species (L. donovani, L. amazonensis, L. braziliensis, L. aethiopica, and L. tropica) carry cap and/or phosphosaccharide repeat units (Jaffe et al., 1990; Ilg et al., 1994h5 In L. amazonensis (Antoine et al., 1987) and L. mexicana (Ilg et al., 1991a) secretion of S A P is restricted to the promastigote stage, whereas it may occur in both promastigote and amastigote stages in L. donouani (Bates et a t , 1989). At present there is little information on the func- tion of S A P in either the sandfly vector or during promastigote infection of mammalian macrophages. I t does not appear to be required for parasite nutrition since secretion is not induced by phosphate starvation in L. donouani (Lovelace et al., 1986) and is strongly inhibited in L. mexicana at low phosphate concen- trations.' The abundant 0-glycans may stabilize the enzyme and protect it from proteases in the sandfly digestive tract or alternatively be functionally important themselves at particu- lar stages of parasite development. In this regard it has previ- ously been shown that nonhydrolyzable oligo- and polysaccha- rides and polyanionic compounds cause vacuolization of macrophages (Kielian et al., 1982; Kielian and Cohn, 19821, and it is possible that the S A P complex secreted by invading pro- mastigotes may contribute to the large parasitophorous vacu- oles which are characteristic of L. mexicana (Antoine et al., 1990). In addition, the high molecular weight S A P complexes may protect the intracellular promasigotes during their differ- entiation to amastigotes by disrupting antigen presentation by major histocompatibility complex class I1 molecules and by scavenging oxygen radicals liberated during the macrophage oxidative burst, as has been described previously for structur- ally related glycoconjugates (Leyva-Cobian and Unanue, 1988; Chan et al., 1989).

Acknowledgments-We thank Dr. Terry Butters for the purified a-glucosidase I1 and Dr. Nikolaev for helpful discussions in the inter- pretat ion of the NMR spectrum.

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