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Vol. 265, No. 32, Issue of November 15, pp. 19611-19623,199O
0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
Structure of the Lipophosphoglycan from Leishmania major*
(Received for publication, June 1, 1990)
Malcolm J. McConvilleS, Jane E. Thomas-Oatesg, Michael A. J. Ferguson, and Steven W. Homans
From the Department of Biochemistry, The University, Dundee, DDl 4HN, United Kingdom
The major cell surface glycoconjugate of the parasitic protozoan Leishmania major is a
heterogeneous lipophosphoglycan. It has a tripartite structure, consisting of a phosphoglycan
(&& 5,000-40,000), a variably phosphorylated hexasaccharide glycan core, and a lysoalkyl-
phosphatidylinositol (lysoalkyl-PI) lipid anchor. The structures of the phosphoglycan and the
hexasaccharide core were determined by monosaccharide analysis, methylation analysis, fast
atom bombardment-mass spectrometry, one- and two-dimensional 500-MHz (correlated spec-
troscopy (COSY), homonuclear Hartmann-Hahn spectroscopy (HOHAHA)) ‘H NMR spectros-
copy, and exoglycosidase digestions. The phosphoglycan consists of eight types of phosphorylated
oligosaccharide repeats which have the general structure,
[POa-GGalp(fll-4)Manpal-1,
I
3
Ii
where R = H, Galp(Bl-3), Galp(gl-3)Galp(Bl-3), Arap(al-2)Galp(/31-3), Glcp(j31-3)Galp@l-
3), Galp@l-3)Galp(@l-3)Galp(/31-3), Arap(al-2)Galp@l-3)Galp@l-3), or Arap(cul-2)Galp@l-
3)Galp(Bl-3)Galp(Bl-3), and where all the monosaccharides, including arabinose, are in the D-
configuration. The average number of repeat units/molecule (n) is 27. Data are presented which
suggest that the nonreducing terminus of the phosphoglycan is capped exclusively with the
neutral disaccharide Manp(cyl-2)Manpcwl-. The structure of the glycan core was determined to
be,
s
I
PO,-6Galp(al-6)Galp(al-3)Galf(~l-3)Manp(~l-3)Man~(~l-4)GlcN~(~l-6)~yo-inositol
where approximately 60% of the mannose residues distal to the glucosamine are phosphorylated
and where the inositol is part of the lysoalkyl-PI lipid moiety containing predominantly 24:0
and 26:0 alkyl chains. The unusual galactofuranose residue is in the &configuration, correcting
a previous report where this residue was identified as aGalf. Although most of the phosphoryl-
ated repeat units are attached to the terminal galactose 6-phosphate of the core to form a linear
lipophosphoglycan (LPG) molecule, some of the mannose 6-phosphate residues may also be
substituted to form a Y-shaped molecule. The L. major LPG is more complex than the previously
characterized LPG from Leishmania donovani, although both LPGs have the same repeating
backbone structure and glycolipid anchor. Finally we show that the LPG anchor is structurally
related to the major glycolipid species of L. major, indicating that some of these glycolipids may
have a function as precursors to LPG.
The protozoan parasite Leishmania major is the etiologic phosphatidylinositols (GPIs).’ Three distinct classes of GPI
agent of human cutaneous leishmaniasis. It occurs as an have been identified; those that are linked to polysaccharide
extracellular promastigote in the alimentary canal of the to form the lipophosphoglycans (LPGs) (Handman and God-
sandfly vector and as an obligate intracellular amastigote in ing, 1985; McConville et al., 1987), those that act as membrane
the phagolysosomal compartment of macrophages in the anchors for cell surface glycoproteins (Bordier, 1987; Murray
mammalian host. The cell surface of L. major promastigotes et al., 1989), and a family of low molecular weight glycoinos-
is coated by a complex glycocalyx which is rich in glycosyl- itolphospholipids (GIPLs) that are not attached to either
protein or polysaccharide (McConville and Bacic, 1989,199O;
* This work was supported by the Wellcome trust. The costs of McConville et al.. 1990).
publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked “adver- ’ The abbreviations used are: GPI, glycosylphosphatidylinositol;
tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate LPG, lipophosphoglycan; GIPL, glycoinositolphospholipid; AHM,
this fact. 2,5anhydromannitol; GC-MS, gas liquid chromatography-mass spec-
$ Recipient of an Australian National Health and Medical Re- trometry; COSY, correlated saectrosconv; HOHAHA. homonuclear
search Council C. J. Martin Fellowship. To whom corresoondence Hartmann-Hahn spectroscopy; HPLC, high performance liquid chro-
should be addressed. matography; FAB-MS, fast atom bombardment-mass spectrometry;
§ Beit Memorial Research Fellow. Pent, pentose; Hex, hexose.
19611
This is an Open Access article under the CC BY license.
19612 Lipophosphoglycan of Leishmania major
LPG is the major cell surface macromolecule and plays a ter linkages. This treatment released a series of phosphoryl-
key role in determining parasite virulence and survival in the ated oligosaccharides (see below), which no longer bound to
mammalian macrophage (Handman et al., 1986; McConville octyl-Sepharose and two major glycolipid species which bound
et al., 1987, McConville and Bacic, 1990; Elhay et al., 1990). to octyl-Sepharose and were eluted with a gradient of l-
It appears to be involved in facilitating the initial attachment propanol (Fig. 2). The relative yields of carbohydrate in the
of promastigotes to macrophages and their subsequent uptake unbound and bound fractions were 92 and 8%, respectively.
into the phagolysosome. Uptake of the parasites may occur Eight phosphorylated oligosaccharides were resolved by ion
following direct binding of LPG to macrophage receptors exchange HPLC using gradient program b (Fig. 3). After
(Handman and Goding, 1985; Russell and Wright, 1988) or preparative HPLC the purified fractions were homogeneous
after opsonization of surface LPG by complement compo- when rechromatographed on the CarboPac column. They also
nents (C3b, C3bi) (Puentes et al., 1988, da Silva et al., 1989). migrated as single components after alkaline phosphatase
LPG-like molecules are also expressed on the cell surface of dephosphorylation and ion exchange HPLC using gradient
the amastigotes and may be necessary for parasite survival in program a (results not shown). Using this program all the
the macrophage phagolysosome compartment (Handman et dephosphorylated oligosaccharides could be resolved (Fig. 4b).
al., 1984, 1986; Chan et al., 1989; McNealy and Turco, 1990). The elution positions of the dephosphorylated glycans on Bio-
In addition, LPG may be involved in the induction of a host Gel P4 chromatography suggested that there was one phos-
protective response and has been used to vaccinate susceptible phorylated disaccharide (P2), one phosphorylated trisaccha-
mice strains against cutaneous leishmaniasis (Handman and ride (P3), three phosphorylated tetrasaccharides (P4a, P4b,
Mitchell, 1985, McConville et al., 1987). There is evidence P~c), two phosphorylated pentasaccharides (P5a, P5b), and
that this protective response may be due to the specific one phosphorylated hexasaccharide (P6) (Fig. 4). A single
recognition of LPG by T-cells (Moll et al., 1989). neutral oligosaccharide (N2’) was also released by mild acid
The LPG from L. major has been partially characterized as hydrolysis of LPG. This species migrated as a disaccharide
a polymer (Mr 5,000-40,000) of repeating phosphorylated di-, on Bio-Gel P4 chromatography and was eluted just after
tri-, and tetrasaccharides that contain mannose, galactose, dephosphorylated P2 (N2) when the HPLC column was eluted
glucose, and arabinose (McConville et al., 1987). By contrast, with a low salt gradient (Fig. 4b). The relative yield of each
the structurally similar LPG which has been characterized oligosaccharide was determined from the peak areas of the
from Leishmania donovani, the etiologic agent of visceral HPLC elution profiles (Table I). In the following sections,
leishmaniasis, only contains the phosphorylated disaccharide the structures of the oligosaccharide repeat units and the
P04-GGal(@l-4)Manol- as the repeating units (Turco et al., glycolipid anchors are described. In each case, determination
1987). However, both LPGs are anchored to the surface of the absolute configuration of the constituent monosaccha-
membrane by an unusual lysoalkyl-PI containing 24:0 and rides showed that all the monosaccharides were in the D-
26:0 alkyl chains (Orlandi and Turco, 1987; McConville et al., configuration.
1987) and may have the same hexaglycosyl glycan core (Turco Disaccharide Repeat (P2)-Positive ion FAB-MS of per-
et al., 1989; McConville and Bacic, 1990). The site of attach- methylated P2 gave an (M + H)’ molecular ion at m/z 549,
ment of the repeat units to the core glycan have not been corresponding to PO,. Hex2 and an A+-type fragment ion at
determined in either structure.
m/z 313, corresponding to PO,.Hex’, which defines the po-
In this study we report the complete structure of L. major
sition of the phosphate on the nonreducing hexose (Table II).
LPG. The structure of Leishmuniu mexicanu LPG will be
Monosaccharide analysis showed mannose and galactose 6-
reported elsewhere.* These results indicate that the LPGs of
phosphate as major components (Table III). Methylation
different species have common architectural elements. In
analysis of the dephospohorylated fraction (N2) identified the
particular, they all have the same backbone sequence of
disaccharide to be Galp-(1-4)-Man (Table IV). Finally, one-
repeating PO,-GGal(pl-4)Manal- units which may either be
dimensional 500-MHz ‘H NMR spectroscopy revealed that
unsubstituted (as in the L. donouuni LPG) or substituted in
the galactose was in the P-configuration; the spectrum (not
a species-specific manner with saccharide residues. In this
depicted) showed a doublet for an anomeric protein at 6 =
regard, the L. major LPG was found to be more complex than
4.45 ppm with a coupling constant of J1,2 = 7.8 Hz (Table V).
either the L. donovuni or the L. mexicana LPGs, as the
From these results, P2 has the structure, PO,-GGalp@l-
majority of the galactose residues in the backbone sequence
4)Man.
were substituted with a diverse array of galactose-, arabin-
Trisaccharide Repeat (P3)--Positive ion FAB-MS of per-
ose-, and glucose-containing side chains. The results also show
methylated P3 gave an (M + H)’ molecular ion at m/z 753
that the glycan core and lipid anchor are highly conserved in
corresponding to Hexs. PO, (Fig. 5A, Table II). Fragment ions
these molecules.
of the A+-type were observed at m/z 219 (Hex+) and 517
(Hex. (PO,)Hex+) which define the position of the phosphate
EXPERIMENTAL PROCEDURES AND RESULTS3
on the internal hexose. Monosaccharide analysis (Table III)
LPG was extracted from delipidated promastigotes with l- indicated that the phosphorylated hexose was galactose and
butanol-saturated water and purified to homogeneity by octyl- that the phosphate was located on the C-6 position. Methyl-
Sepharose chromatography as described previously (Mc- ation analysis of the dephosphorylated trisaccharide, N3,
Conville et al., 1987). The scheme for the characterization of either as the nonreduced (Table IV) or the reduced saccharide
LPG is shown in Fig. 1. The purified LPG was depolymerized (results not shown), defined the structure Galp-(1-3)-Galp (l-
with mild acid, under conditions that hydrolyze phosphodies- 4)-Man. From the NMR analysis, both galactose residues
were in the /?-configuration (Table V), consistent with the
2 T. Ilg, R. Etges, P. Overath, J. Thomas-Oates, M. J. McConville, finding that @-galactosidase treatment of N3 converted it to
S. W. Homans, and M. A. J. Ferguson, manuscript in preparation. a single peak that comigrated with hexoses on HPLC. The
’ Portions of this paper (including “Experimental Procedures,” results of the NMR analyses were also consistent with the
Tables I-X, and Figs. 2, 4, 6, 7, 9, and 11) are presented in miniprint
presence of phosphate on the C-6 position of the internal
at the end of this paper. Miniprint is easily read with the aid of a
standard magnifying glass. Full size photocopies are included in the galactose residue (Gal-2; for numbering of residues see Table
microfilm edition of the Journal that is available from Waverly Press. V). Comparison of the COSY spectra of N3 and P3 showed
Lipophosphoglycan of Leishmania major 19613
LPG
(1) TFA
FIG. 1. Scheme for the character-
ization of L. major LPG. Mild acid I (2) Octyl-Sepharose chromatography
hydrolysis of LPG released a series of r I
phosphorylated oligosaccharides (P2-
unbound glycolipid anchors
P6) and a neutral oligosaccharide, N2’ (GPI-A, GPI-B)
which no longer bound to octyl-Sepha-
Ion exchange HPLC I I
rose. Ion exchange HPLC was used to
fractionate the phosphorylated oligosac- 1
neutral- phosphorylated-oligosaccharide repeats
charides and their dephosphorylated de-
rivatives (NZ-N6). The glycolipid an- I N2’ ” P2 P3 P4a, b, c PSa, b P6a, b ’ 1
chors were eluted from the octyl-sepha- m Pm PI-PLC I
rose column with a gradient of l-
propanol. Treatment of the anchors with
PI-specific phospholipase C released the
+p
phosphosaccharide-inositolphosphate
moieties, whereas nitrous acid deamina- (1) Alkaline phosphatase (1) f-IN02
tion and reduction released glycan moie-
ties terminating in 2,&anhydromanni- (2) Bio Gel P4 chromatography / (2) NaB 3H 4
tol. Symbols: 0, Gal, Man, or Ara; 0, Ion exchange HPLC
myoinositol; n , glucosamine; 0, 2,5-an-
hydromannitol; P, phosphate; TFA, tri- N2
fluoroacetic acid. m Alkaline
phosphatase
“a corresponding to Pent. Hexs. PO, (Fig. 5B). The presence of
I
A+-type fragment ions at m/z 175, 379, and 677 indicate the
sequence Pent. Hex. (POJHex. Hex (Fig. 5B, Table II). Meth-
ylation analysis of the dephosphorylated oligosaccharide, N4a,
indicated the structure Arap-(l-2/3)-Galp-(l-2/3)-Galp-(1-4)-
Man (Table III). To resolve the ambiguity in the linkage
assignments, N4a was treated with mild acid (40 mM trifluo-
roacetic acid, 1 h, 100 “C) to remove the terminal arabinose
(confirmed by FAB-MS and HPLC (data not presented)).
Methylation analysis of the hydrolysate showed that all the
2-O-substituted galactose was converted to terminal galactose,
indicating that the arabinose was originally linked to the C-2
-
I position of the subterminal galactose. NMR analysis showed
0
that the galactose residues were in the @-configuration and
that the arabinose residue was in the a-configuration (6 =
FIG. 3. Fractionation of the phosphorylated oligosaccharide 5.35, Jl,z = 3.5) (Fig. 6, Table V). NMR analysis of P4a,
repeats of L. major LPG. LPG was depolymerized with 40 mM showed that the chemical shift of the H-4 of Gal-2 was shifted
trifluoroacetic acid (8 min, 100 “C) and fractionated by octyl-sepha-
down field by 0.06 ppm, compared with the chemical shifts of
rose chromatography. Oligosaccharides in the unbound fraction were
H-4 of Gal-2 in N4a (Table VI), consistent with the phosphate
chromatographed by ion exchange HPLC using gradient program b.
being located on Gal-2. The phosphate was assigned to the C-
6 position from the absence of heteronuclear splitting on Hl-
that the H-4 proton of Gal-2 was shifted down field by 0.06
H4 and the presence of galactose 6-phosphate in the compo-
ppm in the phosphorylated trisaccharide (Table VI). By con- sitional analyses (Table III). These results reveal that P4a
trast, the chemical shifts of the protons in the terminal has the structure,
galactose residues of both N3 and P3 were identical. The
phosphate was assigned to the C-6 position of Gal-2 from the
absence of heteronuclear splittings in the resonances corre-
6
sponding to Hl-H4. From these results P3 has the structure,
Arap(ol1-2)Galp(/31-3)Galp@l-4)Man
PO4
Positive ion FAB-MS of permethylated P4b gave an (M +
I
H)’ molecular ion at m/z 957 and A+-type fragment ions at
6
Galp(fll-3)Galp(@l-4)Man m/z 219,423, and 721 (Fig. 5C, Table II) corresponding to the
sequence Hexp. (POJHex . Hex. Methylation analysis of re-
Unexpectedly, the terminal galactose of this phosphorylated duced and nonreduced N4b (Table IV) defined the sequence
oligosaccharide was not removed by P-galactosidase. Gal-(l-3)-Gal-(l-3)-Gal-(1-4)-Man. This structure is consist-
Tetrasaccharide Repeats (P4a, P4b, P4c)-After alkaline ent with the data obtained from the NMR COSY spectrum
phosphatase treatment, three of the oligosaccharide repeat (Fig. 6, Table V), which also showed that all the galactose
units (P4a, P4b, and P4c) migrated as tetrasaccharides on residues were in the P-configuration, and the finding that N4b
Bio-Gel P4 chromatography. Positive ion FAB-MS of per- was completely digested to hexoses with fi-galactosidase. The
methylated P4a gave an (M + H)’ molecular ion at m/z 913 location of the phosphate on the 6 position of Gal-2 was also
19614 Lipophosphoglycan of Leishmania major
confirmed by comparison of the COSY spectrum of P4b and
I
MW N4b (Table VI) and compositional analysis (Table III). These
data define the structure of P4b as,
PO,
I
6
As noted for P3, purified P4b was also resistant to calf
intestine &galactosidase.
The positive ion FAB mass spectrum of permethylated P4c
F; 689
contained an (M + H)’ molecular ion at m/t 957 and A+-type
1
888 fragment ions at m/z 219,423, and 721 corresponding to the
sequence Hex2 s( POI)Hex. Hex (Table II). This is consistent
with the methylation analysis of the dephosphorylated oligo-
saccharide, N4c (Table IV) which defined the sequence Glc-
(l-3)-Gal-(l-3)Gal-(l-4)-Man. The /3-configuration for the
glucosidic linkage was determined by one-dimensional 500
MHz ‘H NMR spectroscopy (not shown) from the presence
of a doublet at 4.68 ppm with a coupling constant of J1,2 = 7.8
Hz. The coupling constants of the galactose residues were all
7.8 Hz, indicating that they were in the /I-configuration,
Together with the monosaccharide analysis (Table III), these
results show that P4c has the structure,
PO4
s
Glcp(@l-3)Galp(@l-3)Galp(~l-4)Man
67 7 Pentasaccharide Repeats (P5a, P5b)-Two of the oligosac-
charide repeat units (P5a and P5b) migrated as pentasaccha-
I rides on Bio-Gel P4 chromatography after alkaline phospha-
tase treatment. The positive ion FAB mass spectrum of per-
methylated P5a contained an (M + H)’ molecular ion at m/
z 1117 and A+-type fragment ions at m/z 175,379, 583, and
881 consistent with the structure Pent. Hexz. (POJHex. Hex,
whereas the mass spectrum of P5b contained an (M + H)‘
molecular ion at m/z 1161 and fragment ions at m/z 219,423,
627, and 925 for the structure Hexa. (POI)Hex.Hex (Table
II). Methylation analysis (Table IV) and NMR analysis
(Table V), showed that the dephosphorylated pentasaccha-
rides, N5a and N5b, had the structures Arap(cul-S)Galp(@l-
3)Galp@l-3)Galp(B1-4)Man and Galp(pl-B)Galp(Bl-3)Galp
(/31-3)Galp(/31-4)Man, respectively. Taken together with the
monosaccharide analyses (Table III), these results indicate
that the phosphorlyated pentasaccharides, P5a and P5b, have
the structures,
I
M.H’ 6
957 Arap(al-2)Galp(~l-3)Galp(~l-3)Galp(~l-4)Man
and
PO4
6
FIG. 5. Positive ion FAB-mass spectra of permethylated
phosphorylated oligosaccharides. A, the spectrum of permethyl-
ated P3. The signal at m/z 753 corresponds to the M + H’ molecular respectively.
ion of Hex+(PO1)Hex.Hex, whereas the signals at m/z 219 and 517
Hexasaccharide Repeat (PG)--Positive ion FAB-MS analy-
correspond to A+-type fragment ions for Hex+ and Hex.Hex.PO:,
respectively. B, the spectrum of permethylated P4a. The signal at m/ sis of the permethylated and peracetylated derivates of P6
z 913 corresponds to an M + H+ molecular ion for Pent.Hexs.PO1. afforded molecular ions and A+-type fragment ions for the
The signals at m/z 175,379, and 677 correspond to A+-type fragment structure Pent. Hexa. (POJHex. Hex (Table II). Methylation
ions for Pent+, Pent. Hex+, and Pent. Hex. Hex. PO:, respectively. C, (Table IV) and NMR (Table V) analyses of the dephospho-
the spectrum of permethylated P4h. The signal at m/z 957 represents rylated hexasaccharide indicated the structure; Arap(crl-
the M + H+ molecular ion of Hexa. POI. The signals at 219, 423, and
2)Galp(/31-3)Galp(@1-3)Galp(fil-3)Galp(j31-4)Man. Finally,
721, correspond to A+-type fragment ions for Hex+, Hex;, Hexp.Hex.
monosaccharide analysis of P6 indicated that the phosphate
PO:, respectively. Signals from the glycerol matrix are marked G.
was located on the 6 position of the Gal-2 residue. The
Lipophosphoglycan of Leishmania major 19615
combined results indicate that P6 has the structure, 507
PO,
I
6 669
Arap(cul-2)Galp(~1-3)Galp(~l-3)Galp(~l-3)Galp(~l-4)Man
I
Anomeric Configuration of the Mannose Residues--The 697
finding that LPG was depolymerized after mild trifluoroacetic
acid hydrolysis or hydrofluoric acid treatment (McConville et
al., 1987) suggested that the oligosaccharide repeat units were
linked together by phosphodiester bonds. The structural data
described above are consistent with the phosphorylated oligo-
saccharide repeats being linked through C-l of the mannose
residue and C-6 of the Gal-2 residue, to form the repeating 830
backbone structure -[6Galp(/?l-4)Manl-PO&. The anomeric
configuration of the mannose residue in this sequence was
992
determined by one-dimensional 500-MHz NMR spectroscopy 1020
of the intact LPG. The spectrum (not depicted) showed a I I
doublet of doublets for the anomeric proton of the major
mannose resonance at 6 = 5.44 ppm with a coupling constant
J1.2 = -2 Hz, JH,p = 7.3 Hz, consistent with these residues
having the cu-configuration.
123L
Structure of the Glycolipid Anchor-Mild acid hydrolysis of ?-
LPG released two major inositol-containing glycolipid species
(GPI-A and GPI-B) which had very slow HPTLC mobility
(Fig. 2). Two additional minor glycolipid species (GPI-C and
GPI-D) were also released which were probably hydrolysis
products of the major species (see Supplemental Material).
Negative ion FAB-MS of the underivitized glycolipids gave
(M - H)- molecular ions at m/z 1800 and 1828 for (PO,).
Hexs.HexN.lyso-PI with 24:0 and 26:0 alkyl chains, respec-
tively, and at m/z 1720 and 1748 for (POJ . HexB. HexN. lyso-
PI with 24:0 and 26:0 alkyl chains, respectively (Fig. 8).
Structurally informative fragment ions were formed by /3-
cleavage (Dell, 1987) as shown in Fig. 8. In particular, the
1720
presence of the ion at m/z 1478 indicated that the terminal
M-H.
hexose was phosphorylated in the monophosphorylated spe-
cies, whereas those at m/z 992, 1234, and 1558 showed that
the diphosphorylated species had the sequence (PO1)Hex.
Hex. Hex. (POJHex. Hex. HexN. lyso-PI (Fig. 8). GPI-A and
GPI-B were purified by HPTLC and subjected to monosac-
charide analysis. Both glycolipids contained galactose, man-
nose, glucosamine, and galactose 6-phosphate, whereas GPI-
A also contained mannose 6-phosphate (Table VII). Methyl-
ation analysis of dephosphorylated deaminated GPI-A and
-B revealed that both glycans contained 3 galactose residues/
mol (1 terminal galactopyranose, 1 6-O-substituted galacto-
pyranose, and 1 3-O-substituted galactofuranose), 2 3-O-sub-
stituted mannose residues, and 14-O-substituted glucosamine r- ;- r-
:nal ; 1478 , I,16 rllw :sGz ,tm r1-6 69 ;si,
(Table VIII). Treatment of the dephosphorylatedglycans with l(174lll ( usoq f1344~1~1182)l llom ,ftlR) 1f wm I fS3R
coffee bean cw-galactosidase removed the 2 galactopyranose 1 I I 0 I
GFI-n ’ f.~lel:0-llcll0-llerl0-llel(0-)~cll~-HelN1I*)0-lnosilollP-ARylllycerol
residues and gave a major peak at 4.3 glucose units after Bio-
Gel P4 chromatography. This product was resistant to jack FIG. 8. Negative ion FAB-mass spectrum of the glycolipid
anchors of L. major LPG. The glycolipid anchors were released
bean cu-mannosidase digestion, but was hydrolyzed with 40
after hydrolysis of LPG in 40 mM trifluoroacetic acid (8 min, 100 “C)
mM trifluoroacetic acid (100 “C, 60 min) under conditions and purified by octyl-Sepharose chromatography. The proposed frag-
which hydrolyze hexofuranosidic but not hexopyranosidic mentation scheme of the major components, GPI-A and GPI-B, is
bonds, to give a single peak eluting at 3.2 glucose units. shown. All the ions contained an alkylglycerol species with 24:0 or
Treatment with jack bean Lu-mannosidase converted this com- 26:0 (in brackets) alkyl chains. The ions at m/z 1154 and 1234 are
ponent to a peak at 1.7 glucose units on Bio-Gel P4 chroma- more intense than would be expected of fragment ions (c./ m/z 992,
1154, 1234, 1316, and 1396) as they also correspond to the (M - H)-
tography, corresponding to free 2,5-anhydromannitol. These
molecular ions of the minor components GPI-C and GPI-D, respec-
results indicate that the dephosphorylated glycans of GPI-A
tively, that arise due to cleavage of the acid labile glycosidic linkage
and -B contain the sequence Galp(cul-G)Galp(otl-3)Galf(l- of the internal galactofuranose residue (see Miniprint).
3)Manp(al-3) Manp(al-4)GlcNp.
GPI-A and -B were treated with PI-specific phospholipase inositol and define the anomeric configuration of the galac-
C and the released phosphosaccharide-inositolphosphate tofuranose residue. The ‘H NMR chemical shifts for residues
moieties analyzed by two-dimensional 500-MHz ‘H NMR in these moieties are listed in Table IX and the spectrum for
spectroscopy to identify the linkage between glucosamine and the GPI-A glycan shown in Fig. 9. Similar chemical shifts
19616 Lipophosphoglycan of Lekhmania major
were obtained for both glycans, although there were differ-
A
ences in the H-l and H-2 resonances of some residues (cf. S ma I :I:
GlcN, Man, and Galf residues), reflecting the presence of
I F m m..i
phosphate on the mannose residue distal to the glucosamine
in the GPI-A glycan which was absent in the GPI-B glycan.
From previous studies on the GIPLs of L. major (McConville
et al., 1990), the cross-peak at w1 = 5.69 ppm and wz = 3.31
ppm in the GPI-B glycan is diagnostic of non-l\r-acetylated
glucosamine linked al-6 to myo-inositol 1,2-cyclic phosphate
(Table IX). The /3-anomeric configuration of Galf in GPI-A
and -B was concluded from comparison of the coupling con-
stants of H-l (J1,* = 1.6 Hz) with those of the anomeric signals
of the synthetic disaccharide Galf(fil-3)Manpal-Az (Jl,Z = 1.4
Hz) and the reported values (Gerwig et al., 1989) for methyl
P-D-Galf (J1,p = 2.0 Hz) and methyl cY-D-Galf (cJ~,~ = 4.0 Hz)
(Table X). Galactofuranose residues also occur as constituents
in the GIPLs of L. major and were tentatively assigned the
a-configuration in a recent study (McConville et al., 1990).
However, comparison of the coupling constants of the Galf
H-l and H-2 in the GIPLs showed that they are identical to
those in the LPG anchor, indicating that they are also in the
D. 3.5 1.0
@-configuration (Table X).
Homonuclear (‘H) HOHAHA spectroscopy of the diphos-
B
phorylated glycan core was used to infer the location of the LH-l
phosphate on the mannose distal to the glucosamine (Fig. 10).
A cross-section parallel to wi = 4.30 ppm gave an essentially
complete one-dimensional spectrum of Man-2 (Fig. 10B) from
which two broad doublets corresponding to the C-6 protons
at w1 = 3.81 and 4.22 ppm could be observed. The broad nature
of H6 and H6’ due to multiplicity and anomalous downfield
shifts of H5, H6, and H6’ suggests that this residue is substi-
tuted at position 6 with phosphate. These results indicate
that the glycolipid anchors of LPG have the following struc-
tures;
PO4
I
6
GPI-A PO,-6Galp(~1-6)Galp(al-3)Galf(pl-3)Manp(~l-3) FIG. 10. Two-dimensional HOHAHA spectrum of the di-
phosphorylated glycan core of L. major LPG. HOHAHA spec-
Manp(Lul-4)GlcNp(al-G)lyso-PI
trum of the phosphosaccharide-inositolphosphate moiety released
GPI-B PO~-6Galp(oll-6)GaIp(cxl-3)Galj(~1-3)Manp(al-3) from GPI-A by PI-specific phospholipase C (A). Subspectrum of the
HOHAHA spectrum at 6 = 4.30 ppm, showing resonances of the
Manp(oll-4)GlcNp(cYl-G)lyso-PI Man-2 H-l-H-6 protons (B).
The relative proportions of GPI-A and GPI-B in the intact
LPG were approximately 62 and 58%, respectively (see Min- residues in the core were substituted in the intact chain and
iprint). that most of the mannose 6-phosphate residues in the di-
Site of Attachment of the Oligosaccharide Repeat Units to phosphorylated core were unsubstituted. However, the resist-
the Core-Cleavage of the oligosaccharide repeat units from ance of 35% of the mannose B-phosphate residues to alkaline
the glycan core with mild acid suggests that they are attached phosphatase suggests that some of them may also be substi-
to the core via a phosphodiester bridge. In the monophospho- tuted with saccharide residues.
rylated core, the repeat units can only be attached to the LPG Is Capped with Dimannoside-To determine whether
terminal galactose 6-phosphate, whereas in the diphosphoryl- any of the phosphorylated oligosaccharides were present at
ated core there are two possible sites of attachment, namely the nonreducing terminus of the phosphoglycan chains, intact
the terminal galactose 6-phosphate and/or the internal man- LPG was treated sequentially with alkaline phosphatase and
nose 6-phosphate. The attachment of oligosaccharides to the then galactose oxidase/NaB3H4. The galactose oxidase/Na-
core was probed by FAB-MS (see Supplemental Material) B3H4 treatment labels all the dephosphorylated oligosaccha-
and by determining the susceptibility of the core phosphates ride repeat units, which all contain either terminal, 2-, or 3-
in the intact chain to alkaline phosphatase treatment. Intact substituted galactose with a free 6 hydroxyl (results not
LPG was labeled at the reducing terminus, by nitrous acid shown). However, none of the repeat units in the intact LPG
deamination, and NaB3H4 reduction and digested with alka- were dephosphorylated and labeled by this procedure, sug-
line phosphatase. HPLC analysis of the mild acid-released gesting that the phosphoglycan chains were capped by other
cores showed that while the phosphate on the galactose was moieties. It is likely these chains are capped with the neutral
completely resistant to alkaline phosphatase, approximately oligosaccharide N2’, which was released from LPG by mild
65% of the phosphate on the mannose was removed (Fig. 11). acid and was present in approximately 1 mol/mol LPG. The
Extended digestion over 3 days with repeated addition of positive ion FAB mass spectrum of permethylated N2’ af-
fresh enzyme did not remove any more phosphate. These forded (M + H)’ and (M + NH4)+ molecular ions at m/z 455
results suggested that all the terminal galactose 6-phosphate and 472, corresponding to Hexz. Monosaccharide (Table III)
Lipophosphoglycan of Leishmania major 19617
and methylation analysis (Table IV) identified N2’ as Man- tion of the galactose in this sequence is either unsubstituted
(1-2)-Man. The mannose was present in the Lu-configuration or substituted with galactose or linear saccharide chains con-
from the coupling constant (J1,2 = 1.9) (Table V). These taining from 2 to 4 saccharide residues. Some of these side
results define the structure of N2’ as Manp(cYl-2)Man. N2’ chains are capped by the highly unusual a-D-arabinopyranose.
was present at a nonreducing terminus as jack bean a-man- As far as we are aware, this residue has not been reported
nosidase treatment of intact LPG released approximately 1.6 previously in other eukaryotic glycoconjugates, although there
mol of mannose/mol of LPG. Moreover, after mannosidase is evidence that it may occur in a partially characterized
treatment, N2’ was no longer detected in the mild acid hy- arabinogalactan from the insect parasite Crithidia fasiculata
drolysate of LPG. Taken together, these results indicated that (Gorin et al., 1979). Labeling with galactose oxidase/NaB3H4
the nonreducing terminus of the phosphoglycan chains were indicates that none of the phosphorylated oligosaccharides
capped with the sequence Manp(cYl-2)Mancul-P04. are located at the nonreducing terminus of the phosphoglycan,
Molecular Weight of L. major LPG-The average molecular and it is likely that this terminus is capped with the sequence
weight of the L. major LPG, obtained from stationary phase Man(Lul-B)Manal-Pod. The phosphoglycan chain is attached
promastigotes, was esimated from the molar ratio of hexose/ to a linear phosphosaccharide core that has the sequence
pentose to myo-inositol determined by GC-MS. Triplicate PO,-6Galp(cul-6)Galp(cul-3)Galf(~l-3)Man~(~l-3)Manp(~l-
determinations revealed a ratio of 90:1, suggesting that the 4)GlcNp(al-6) myo-inositol, where approximately 60% of the
glycan moiety has an average molecular weight of approxi- mannose residues distal to the glucosamine are phosphoryl-
mately 15,000. This is in good agreement with the estimated ated on the 6 position. Furthermore, the site of attachment
number of repeats in LPG, determined from the one-dimen- of the repeat units appears to be predominantly through the
sional 500-MHz ‘H NMR spectrum of intact LPG. Integration terminal galactose 6-phosphate residue, suggesting that the
of the anomeric proton peaks of the 4-O-substituted u-man- LPG is organized as a linear molecule. However the resistance
nose from the oligosaccharide repeat units and the 6-O-sub- of some of the mannose 6-phosphate residues to alkaline
stituted a-galactopyranose from the glycan core gave a ratio phosphatase digestion raises the possibility that a small pro-
of 27:l. These results suggest that the mean number of repeat portion (<20%) of the LPG molecules have two saccharide
units per LPG is 27. chains branching from the core to form a Y-shaped LPG
molecule. As shown previously, all these molecules are an-
DISCUSSION chored to the membrane by a novel lysoalkylphosphatidyli-
The lipophosphoglycans of L. major are a heterogeneous nositol lipid moiety that contains predominantly 24:0 and
family of molecules which have a tripartite structure, consist- 2&O alkyl chains (McConville et al., 1987).
ing of a phosphoglycan (Mr 5,000-40,000), a variably phos- It is now possible to compare the structures of LPG from
phorylated glycan core, and a lysoalkyl-PI lipid moiety. The L. major (this study), L. donouani (Orlandi and Turco, 1987;
proposed structure of L. major LPG is shown in Fig. 12. The Turco et al., 1987, 1989), and L. mexicana.’ These studies
phosphoglycan is built up of at least eight different oligosac- indicate that all LPGs have the same tripartite structure. A
charide repeat units which are linked together in linear array notable feature of these molecules is the presence of a common
by phosphodiester bonds. There are on average 27 repeat repeating backbone sequence, P04-GGalp(bl-4)Manpal-, in
units/molecule. Analysis of the phosphorylated oligosaccha- all the phosphoglycan moieties, which may be variably sub-
ride suggests that these chains have a repeating backbone stituted, on the 3 position of the galactose residues, with other
structure of -Pod-GGalp(@l-4)ManpLul- and that the 3 posi- sugars. In the L. donouani LPG, there is negligible substitution
i p 6 I
I 0-~-0-6Galal-6Galal-3Gal~~1-3I;lanal-3Manal-4GlcN~l-6 ;
io.H \ , / i
FIG. 12. Structure of L. major li- Mmal-2Manal- lyso -Alkyl-PI
pophosphoglycan. All sugarsa re in the
D-configuration and the pyranose ring / \
/ \
form except where indicated. Approxi- l’ / \ \
mately 60% of the mannose residuesd is- /’ \
tal to the glucosaminea re phosphoryl- i
ated. Although most of the repeat units
are attached to the core via the galactose
g-phosphate, repeat units may also be
attached to the mannose B-phosphate in
a small proportion (20%) of the mole-
cules.
R=H F”
=Gal PI- 31
=Amal-ZGalpl- 29
=Gal bl-3G.d f31- 13
= Glc pl-3 Gal PI- 1
=Amal-2Gal~l-3Gal~l- 5
=Gal~1-3Gal~l-3Gal~l- 3
=~raal-2GalBl-3Galpl-3GalBl- 1
“average = 27
19618 Lipophosphoglycan of Leishmania major
of the backbone sequence (Turco et al., 1987), whereas in the a nondividing infectious “metacyclic” stage. The LPGs of
L. mexicana LPG, approximately 25% of the galactose resi- metacyclic cells have a higher average molecular weight than
dues are substituted with pGlc residues.2 The L. major LPG those of logarithmic phase cells and no longer bind the lectin
is the most complex LPG to be characterized as approximately peanut agglutinin (Sacks and da Silva, 1987). They may also
87% of the galactose residues in the backbone sequence are express epitopes not detectable in the LPGs from logarithmic
substituted with a diverse array of different side chains. The cells (Sacks and da Silva, 1987). As the LPG characterized in
presence of a common backbone structure which is variably this study was derived from cultures containing a mixed
elaborated with species-specific side chains is consistent with population of both actively dividing and metacyclic promas-
serological studies which indicate the presence of both con- tigotes, it is probable that it is a mixture of at least two chain
served and species-specific epitopes (Handman et al., 1984). types. One chain type, produced by actively dividing promas-
The glycolipid anchor moiety is the most highly conserved tigotes, is likely to be enriched in oligosaccharide repeat units
region of Leishmaniu LPGs. All the LPGs contain the same containing terminal /3Gal residues (peanut agglutinin-posi-
hexasaccharide core sequence which is characterized by hav- tive), whereas the second type, produced by metacyclic pro-
ing the unusual 3-O-substituted galactofuranose residue. This mastigotes, is likely to be enriched in repeat units containing
residue was assigned the ,&configuration in L. major LPG, terminal aAra or @Glc residues (peanut agglutinin-negative).
from the results of NMR analysis and by comparison with an Whether changes in the composition of the repeat units of
authentic standard. Although this residue was identified as LPG are involved in increased infectivity of metacyclic pro-
LyGalf in a recent study on the L. donovani LPG (Turco et al., mastigotes has not been established.
1989), reanalysis by NMR spectroscopy indicates that it is LPG probably forms a highly antigenic capsular network
also in the /3-configuration.4 Another feature of the glycan on the surface of L. major promastigotes. This is suggested by
core is that the terminal galactose residue is always phos- ultrastructural studies which show that the cell surface is
phorylated on the 6 position. By contrast, phosphorylation of coated by a glycocalyx and that the thickness of this layer
the internal mannose residue may either be partial, as in the increases in metacyclic promastigotes, coincident with an
L. major LPG, or complete, as in the L. donouani LPG (Turco increase in the average molecular weight of LPG (Pimenta et
et al., 1989). At present it is not known whether the LPGs of al., 1989). This is also consistent with estimates of the cellular
other Leishmania are also predominantly linear molecules or copy number (approximately 5 x lo6 molecules/cell), which
whether in some LPGs the mannose 6-phosphate residue of indicate that LPG is the major macromolecule on the cell
the core is more highly substituted or even the sole site of surface (McConville and Bacic, 1990). Furthermore, prelimi-
phosphoglycan attachment. nary molecular modelling studies (Homans, 1990) of the phos-
The phosphoglycan moieties of Leishmania LPGs are com- phoglycan moiety indicate that the phosphorylated disaccha-
pletely novel structures for eukaryote glycoconjugates. How- ride backbone sequence exists in an extended configuration
ever, the glycolipid anchors of LPG show limited structural with a helical pitch (-6 repeats/turn) and that the oligosac-
homology to the protein-linked GPI anchors in containing charide side chains are directed away from the main axis.
the sequence Manoc(l-4)GlcNa(l-G)myo-inositol-l-PO4 (Fer- These studies predict that the LPG will extend away from
guson et al., 1988, Homans et al., 1989, Schneider et al., 1990), the plasma membrane for some distance and that it will cover
suggesting that this motif may be conserved in all eukaryotic a larger proportion of the cell surface than previously esti-
glycosylated phosphoinositides that function as membrane mated (-25%) (McConville and Bacic, 1990) due to its large
anchors for surface macromolecules. The glycolipid anchors cross-sectional area (Homans, 1990). There is indirect evi-
of LPG also show structural homology to the major glycolipids dence that this surface network may form a macromolecular
of L. major (McConville and Bacic, 1989; McConville et al.,
diffusion barrier in metacyclic promastigotes, as there is a
1990). In particular, all the GIPLs contain the same core progressive decrease in the ability to detect the low molecular
sequenceGalf(l-3)Man(Lul-3)Man(al-4)GlcN(al-6)myo-ino-
weight glycolipids on the cell surface by immunofluorescence
sitol (McConville et al., 1990). In this previous study we
as promastigotes progress from logarithmic to stationary
identified the galactofuranose as aGalf from comparison with
growth (Elhay et al., 1988).
the glycan core of L. donouani LPG. We now show that the
Cell surface LPG may be important for both infectivity and
galactofuranose residues in these glycolipids are also in the p-
parasite survival in the sandfly vector and mammalian host.
configuration. Some of these glycolipids have the same glycan
LPG appears to be the major acceptor for complement (pre-
structure as the LPG core and are selectively deacylated in
dominantly C3b) and consequently may be involved in facil-
uiuo, suggesting that they may function as biosynthetic pre-
itating phagocytosis of opsonized promastigotes by macro-
cursors to LPG (McConville and Bacic, 1990; McConville et
phages via the CR1 receptor (Puentes et al., 1988; da Silva et
al., 1990). In L. major, the build up of phosphoglycan occurs
al., 1989). The finding that L. major LPG is capped with a
predominantly or exclusively on these abundant glycolipids.
dimannoside may be significant in this regard as a mannose-
However, recent studies suggest that in other species of Leish-
binding protein has been found in various mammalian sera
mania similar phosphoglycans may also occur on some para-
which activates complement through the classical pathway
site glycoproteins (Bates et al., 1990, Jaffe et al., 1990).
(Ohta et al., 1990). Activation of complement at the nonre-
The heterogeneous nature of the L. major LPGs, compared
ducing terminus of the LPG chain, at some distance from the
with the LPGs of L. donouani and L. mexicana, raises the
cell surface, would not only ensure that complement frag-
question of whether all the LPG chains contain a random
ments are accessible to complement acceptors, but may also
selection of repeat units or whether they are a mixture of
contribute to complement resistance by preventing stable
different chains that show restricted heterogeneity. The pos-
insertion of the C5b-9 complex into the plasma membrane of
sibility that different cells may produce LPGs with different
phosphoglycan compositions is indicated by recent studies on the parasite. This is consistent with the observation that
while both logarithmic growth phase promastigotes and
the LPG of L. major promastigotes as they undergo sequential
development from an actively dividing noninfectious stage to metacyclic promastigotes bind the same amount of comple-
ment to their cell surface, metacyclic promastigotes produce
larger LPG chains on average and are more resistant to
‘J. Thomas, J. Thomas-Oates, M. J. McConville, S. J. Turco, M.
A. J. Ferguson, S. W. Homans, manuscript in preparation. complement mediated lysis (Franke et al., 1985; Sacks and da
Lipophosphoglycan of Leishmania major 19619
Silva 1987). LPG may also bind directly to macrophage recep- Franke, E. D., McGreevy, P. B., Katz, S. P., and Sacks, D. L. (1985)
tors (Handman and Goding, 1984; Russell and Wright, 1988), J. Immunol. 134,2713-2718
Gerwig, G. J., Kamerling, J. P., and Vliegenthart, J. F. G. (1978)
although the significance of this interaction in promastigote
Carbohydr. Res. 62, 349-357
phagocytosis is unclear. It is also unknown whether structural
Gerwig, G. J., de Waard, P., Kamerling, J. P., Vleigenthart, J. F. G.,
differences in the LPGs of different species contribute to the Morgenstern, E., Lamed, R., and Bayer, E. A. (1989) J. Biol. Chem.
pronounced tissue tropism of different L.eishmaniu species. 264, 1027-1035
Once inside the macrophage, the LPG may protect the para- Gorin, P. A. J., Previato, J. O., Mendonca-Previato, L., and Travassos,
L. R. (1979) Protozool. 26. 473-478
site cell surface from hydrolytic enzymes. In this regard it is
Handman, E., Greenblatt, Cl L., and Goding, J. W. (1984) EMBO J.
of interest that the phosphorylated oligosaccharides are re-
3,2301-2306
sistant to calf intestine /3-galactosidase. LPG has been shown Handman, E., and Goding, J. W. (1985) EMBO J. 4,329-336
previously to inhibit p-galactosidase (El-On et al., 1980), and Handman, E., and Mitchell, G. F. (1985) Proc. Natl. Acad. Sci. U. S.
the resistance of the phosphorylated oligosaccharides suggests A. 83,5988-5991
Handman. E.. Schnur. L. F.. Suithill. T. W.. and Mitchell. G. F.
that they may be acting as competitive inhibitors of this
(1986) J. Immunol. i37, 3608’3613’ ’
enzyme. Finally, LPG may be important in protecting the Homans, S. W., Ferguson, M. A. J., Dwek, R. A., Rademacher, T. W.,
parasite from the oxidative burst of the host macrophage, Anand, R., and Williams, A. F. (1988) Nature 333,269-272
either by inhibiting the protein kinase C involved in the Homas, S. W. (1990) TZGG 2, 144-155
Jaffe, C. L., Perez, M. L., and Schnur, L. F. (1990) Mol. Biochem.
activation of the burst or by acting as an efficient scavenger
Parasitol. 4 1,233-240
of oxygen free radicals (McNealy et al., 1989; Chan et al., Marion, D., and Wuthrich, K. (1983) Biochem. Biophys. Res. Com-
1989; McNealy and Turco, 1990). mun. 117,967-974
McConville, M. J., and Bacic, A. (1989) J. Biol. Chem. 264, 757-766
Acknowledgments-We wish to thank Dr. Bacic for helpful discus- McConville, M. J., and Bacic, A. (1990) Mol. Biochem. Parasitol. 38,
sions, Drs. J. Thomas and W. Masterson for critical review of the 57-68
manuscript, Dr. A. Mehlert for assistance with the NMR experiments, McConville, M. J., Bacic, A., Mitchell, G. F., and Handman, E. (1987)
and Dr. B. Caudwell for assistance with the amino acid analyses. We Proc. Natl. Acad. Sci. U. S. A. 84,8941-8945
McConville, M. J., Homans, S. W., Thomas-Oates, J. E., Dell, A.,
also thank Dr. M. Low for providing the purified PI-specific phos-
and Bacic, A. (1990) J. Biol. Chem. 265,7385-7394
pholipase C and Dr. P. Gorin for providing the synthetic oligosaccha-
McNeely, T. B., and Turco, S. J. (1990) J. Zmmunol. 144,2745-2750
ride.
McNeely, T. B., Rosen, G., Londner, M. V., and Turco, S. J. (1989)
Biochem. J. 259,601-604
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19620 Lipophosphoglycan of Leishmania major
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