(Received for publication, June 19, 1995; and in revised form, August 28, 1995)
From the
The major acceptors of sialic acid on the surface of metacyclic
trypomastigotes, which are the infective forms of Trypanosoma cruzi found in the insect vector, are mucin-like glycoproteins linked to
the parasite membrane via glycosylphosphatidylinositol anchors. Here we
have compared the lipid and the carbohydrate structure of the
glycosylphosphatidylinositol anchors and the O-linked
oligosaccharides of the mucins isolated from metacyclic trypomastigotes
and noninfective epimastigote forms obtained in culture. The single
difference found was in the lipid structure. While the
phosphatidylinositol moiety of the epimastigote mucins contains mainly
1-O-hexadecyl-2-O-hexadecanoylphosphatidylinositol,
the phosphatidylinositol moiety of the metacyclic trypomastigote mucins
contains mostly (70%) inositol phosphoceramides, consisting of a
C
sphinganine long chain base and mainly C
and C
fatty acids. The remaining 30% of the
metacyclic phosphatidylinositol moieties are the same
alkylacylphosphatidylinositol species found in epimastigotes. In
contrast, the glycosylphosphatidylinositol glycan cores of both
molecules are very similar, mainly
Man
1-2Man
1-2Man
1-6Man
1-4GlcN.
The glycans are substituted at the GlcN residue and at the third
Man distal to the GlcN residue by ethanolamine phosphate or
2-aminoethylphosphonate groups. The structures of the desialylated O-linked oligosaccharides of the metacyclic trypomastigote
mucin-like molecules, released by
-elimination with concomitant
reduction, are identical to the structures reported for the
epimastigote mucins (Previato, J. O., Jones, C.,
Gonçalves, L. P. B., Wait, R., Travassos, L. R.,
and Mendoça-Previato, L.(1994) Biochem. J. 301, 151-159). In addition, a significant amount of
nonsubstituted N-acetylglucosaminitol was released from the
mucins of both forms of the parasite. Taken together, these results
indicate that when epimastigotes transform into infective metacyclic
trypomastigotes, the phosphatidylinositol moiety of the
glycosylphosphatidylinositol anchor of the major acceptor of sialic
acid is modified, while the glycosylphosphatidylinositol anchor and O-linked sugar chains remain essentially unchanged.
Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease in humans, has a complex life cycle alternating between the insect vector and the mammalian host. In the vector, it multiplies as noninfective epimastigotes that migrate to the hindgut and differentiate into infective metacyclic trypomastigotes. During the insect blood meal, the metacyclic trypomastigotes are deposited with the feces and urine near a skin wound, initiating the natural infection.
T. cruzi is unable to synthesize sialic acids
(SA), ()but it expresses a unique trans-sialidase
(TS), which transfers
2-3-linked SA from host glycoproteins
and glycolipids to acceptors containing terminal
-galactosyl
residues present on the parasite surface (reviewed in (1, 2, 3, 4) ). Several studies
characterizing the nature and structure of the SA acceptors have been
published. These acceptors are abundant on the parasite surface and
were first described as major surface glycoproteins of epimastigotes by
Alves and Colli(5) , who called them bands A, B, and C.
Subsequently, a similar cell surface glycoprotein complex, called GP24,
GP31, and GP37 was described by Ferguson et al.(6) ,
and Previato et al.(7) first described a 43-kDa SA
acceptor. More recently, they have been called 38/43
glycoconjugates(8) , and the so called epimastigote
lipophosphoglycan-like molecule could belong to the same family of
molecules(9) . In metacyclic trypomastigote forms, the SA
acceptors were reported originally as the 35/50-kDa antigens (10, 11) that were subsequently defined as mucin-like
glycoproteins(12) . In the trypomastigote forms found in
mammals, the SA acceptors were described as a group of molecules that
share the stage-specific epitope 3 (Ssp-3) (13) , an epitope
dependent on parasite sialylation(14) , and were also
identified as mucin-like molecules that appear larger than the
epimastigote and metacyclic mucins on SDS-polyacrylamide gel
electrophoresis(15, 16) . These trypomastigote mucins
also contain some terminal
-galactosyl residues(16) . In
summary, these mucin-like molecules are glycoproteins rich in threonine
and serine that are linked to the parasite membrane via a
glycosylphosphatidylinositol (GPI) anchor and that contain novel O-linked oligosaccharides. The O-linked
oligosaccharides are attached to the protein via GlcNAc residues and
act as SA acceptor sites for the parasite TS. The chemical structure of O-linked oligosaccharides of epimastigote mucins of G (8) and Y (17) strains have recently been elucidated.
They are quite similar but differ in their average size and in some of
the Gal linkages. The glycan structure of the GPI anchor of the
epimastigote mucin (Y strain) has also been reported(17) .
Several lines of evidence suggest that the 35/50-kDa mucins of metacyclic-trypomastigotes are involved in host cell invasion. Monoclonal antibodies directed against the mucin, and the purified molecule itself, are able to inhibit parasite entry(10, 18) , and the 35/50-kDa antigens are capped and locally released during invasion (12) . Epimastigotes are unable to enter mammalian cells but express large amounts of mucins with similar size recognized by the same monoclonal antibodies. Therefore, we decided to investigate possible subtle structural differences between the mucins of these two stages. We found that, after differentiation of epimastigotes into metacyclic forms, the lipid portion is modified, while the oligosaccharide chains and the glycan structure of the GPI are conserved. This lipid change might correlate with the increased infectivity of metacyclic forms and the ability of the parasite to shed the mucins upon invasion of the host cell(12) .
Purified oligosaccharitols obtained by fractionation on the
Glycoplex HPLC column were submitted to exoglycosidase
digestion, as described below, or to mild acid hydrolysis (200 µl
of 40 mM trifluoroacetic acid for 1 h at 100 °C) to
preferentially cleave Galf glycosidic bonds(25) ,
dried in a speed-vac, redried twice from 200 µl of water and
re-N-acetylated as described in (22) . The products
were analyzed by Bio-Gel P4 chromatography.
H
NMR-One-dimensional 500-MHz
H NMR spectra of the
individual oligosaccharitols were obtained using a Bruker AM 500
spectrometer equipped with a 5-mm triple resonance probe, and the
samples were dissolved in 0.5 ml of
H
O after
repeated exchange in
H
O. All experiments were
performed at 300 K, and chemical shifts were referenced externally to
acetone (2.225 ppm). Further assignments for oligosaccharitol c were deduced from two-dimensional
H-
H
experiments. Correlated spectroscopy and triple quantum-filtered
correlated spectroscopy experiments were performed using a sweep width
of 2,200 Hz, and 4,000 data points, and 512 increments were collected
in f1. The rotating frame Overhauser effect spectroscopy experiment
used 64 transients of 4,000 data points, and 1,024 increments were
collected in f1. The spectral width collected was 4,400 Hz in each
domain, and the mixing time was 500 ms. In the total correlation
spectroscopy experiment, 64 transients of 4,000 datapoints were
collected, and 512 experimental increments were collected in f1. The
sweep width was 1,500 Hz, and the mixing time used was 203 ms.
The mucins were purified from epimastigotes and metacyclic
trypomastigotes by solvent extraction and octyl-Sepharose
chromatography. The material recognized by the monoclonal antibody
10D8, specific for the sialic acid acceptors, eluted at 25% (v/v)
propan-1-ol and appeared as two bands with apparent molecular masses of
35 and 50 kDa on SDS-polyacrylamide gel electrophoresis, as shown
previously for the metacyclic trypomastigote sialic acid acceptors (12) . The significance of the double nature of the antigen is
unknown, but it may reflect the presence of at least two different (O-glycosylated/GPI-anchored) gene products. The purified
mucins were judged to be free of T. cruzi LPPG, which migrates
near the front of an SDS-polyacrylamide electrophoresis gel, by silver
staining and by Western blot analysis using an LPPG-specific antibody
(data not shown). Based on the myo-inositol content of the
recovered material (25 nmol/10 cells), the metacyclic
mucin is present at a minimum of 1.5
10
copies/parasite. The mucins eluted from the octyl-Sepharose
column were subjected to compositional analysis, showing that amino
acids (particularly Ser and Thr) and monosaccharides (Man, Gal, GlcNAc,
and SA) together with myo-inositol, ethanolamine,
1-O-hexadecylglycerol, and fatty acids were present in both
preparations in amounts similar to those reported
previously(8, 12) . In addition, a previously
unidentified peak in the amino acid analyses (with a retention time of
3.2 min) was shown to co-elute with an authentic standard of
2-aminoethylphosphonate (2-AEP). The molar ratio of ethanolamine to
2-AEP was approximately 1:1 for both preparations.
Figure 1: Negative ion electrospray mass spectra of the mucin PI moieties. The PI moieties released from the epimastigote and metacyclic mucins (panels A and B, respectively) by nitrous acid deamination were recovered by solvent extraction and analyzed by ES-MS. The identities of major species detected in the epimastigote (panel A, m/z 823 and 795) and metacyclic (panel B, m/z 892 and 780) PI fractions were corroborated by the collision-induced dissociation daughter ion spectra (panels C and D and panels E and F, respectively).
In contrast, the mucin from metacyclics showed, in
addition to the same alkylacyl-PI pseudomolecular ion species at m/z 795.5 and 823.6, abundant ions at m/z 892.7 and 780.6 (Fig. 1B). The
daughter ion spectra of these species (Fig. 1, E and F) define these ions as the [M-1] pseudomolecular ions of inositol phosphoceramides (ceramide-PIs).
In this case, the daughter ion spectra contain common ions at m/z 79, 97, 241, and 259, corresponding to
[PO
]
,
[H
PO
]
,
[inositol-1,2-cyclic-PO
]
, and
[inositol-1-HPO
]
, respectively.
The relatively stable amide bond of the ceramide prevents the formation
of the carboxylate ions seen previously for the alkylacyl-PI species.
The m/z values of the pseudomolecular ions at m/z 892.7 and 780.6 suggest that the ceramide
components are made up of a sphinganine (C
) long chain
base and C
and C
fatty acids,
respectively. The minor pseudomolecular ions at m/z 890.7 and 778.6 most likely represent ceramide-PIs containing
sphingosine (C
) or mono-unsaturated fatty acids. The
collisioninduced daughter ion spectra of the m/z 795.5 and 823.6 ions were identical to those shown in panels C and D (data not shown). The relative proportions of all
of different PI species and the tentative identification of some of the
very minor species are given in Table 1.
Figure 2:
Sequential exoglycosidase digestion and
partial acetolysis of GPI neutral glycans. A, neutral glycans
from epimastigotes (EPI) and metacyclic trypomastigote (META) mucins were obtained by nitrous acid deamination and
reduction with NaBH
, followed by aqueous HF
treatment, and microsequenced by exoglycosidase digestion and partial
acetolysis as described under ``Materials and Methods.'' The
labeled glycans were analyzed by HPTLC in solvent system A without
treatment (lanes 1 and 5) or after A. saitoi
-mannosidase (lanes 2 and 6), partial
acetolysis (Ac
O)(lanes 3 and 7) or jack bean
-mannosidase (lanes 4 and 8) digestions. On the right side are
indicated the migration positions of
Man
1-2Man
1-2Man
1-6Man
1-4AHM
(Man4-AHM*); Man
1-2Man
1-6Man
1-4AHM*
(Man3-AHM*); Man
1-6Man
1-4AHM* (Man2-AHM*);
Man
1-4AHM* (Man1-AHM*); and 2,5-anhydromannitol (AHM*)
standards obtained from T. cruzi LPPG. DEX is
H-reduced dextran hydrolysate standard. F corresponds to the solvent front and OR to the origin. Panel B shows an additional HPTLC of epimastigote neutral
glycan, with a longer exposure time.
Figure 3:
Location of phosphoryl/phosphonyl
substituents. HPTLC analysis of the mucins subjected to partial acid
hydrolysis followed by sequential exoglycosidase digestion and
dephosphorylation. Deaminated and reduced mucins from epimastigotes (EPI) and metacyclic trypomastigote (META) mucins
were subjected to partial acid hydrolysis (H) followed
by aqueous HF dephoshorylation (HF) (lanes 2 and 6), partial acid hydrolysis, jack bean
-mannosidase
digestion, and aqueous HF dephosphorylation (lanes 3 and 7) or to partial acid hydrolysis and passage through
QAE-Sephadex A-25 column (lanes 4 and 8). The
products were analyzed by HPTLC in solvent A and fluorography. The
migration of standards (DEX), and the glycans of T. cruzi LPPG (as described in Fig. 2) are shown in lanes 1 and 5.
Figure 4: Proposed GPI anchor structures of T. cruzi mucins. See text for details.
Figure 5:
Fractionation of mucin oligosaccharitols
released by reductive -elimination. Epimastigote (panel
A) and metacyclic trypomastigote (panel B) mucins were
submitted to mild alkaline
-elimination, with concomitant
reduction with NaB
H
, and separated by HPLC
using a Glycoplex
column, as described under
``Materials and Methods.'' C, the purity of
individual oligosaccharitols from metacyclic sample (lanes
a-f) versus the unfractionated oligosaccharitol
mixture (lane T) was accessed by HPTLC in solvent system B. Lane S contains a [1-
H]GlcNAc-ol
standard, and lane DEX contains the
H-reduced
dextran hydrolysate standard.
Figure 6: Proposed structures of the O-linked oligosaccharitols released from epimastigote and metacyclic trypomastigote mucins. The structures were deduced as described in the text, except that the sialylation patterns are inferred from the SA transfer experiments described in Fig. 7. The relative percentage of each structure was based on the recovery of each peak relative to the labeled material as shown in Fig. 5.
Figure 7: Sialylation of O-linked oligosaccharitols of metacyclic trypomastigote mucin accessed by Mono Q chromatography. Purified and labeled neutral oligosaccharitols c-f (panels A, B, C, and D) of metacyclic trypomastigotes were incubated with TS and sialyllactose, as described under ``Materials and Methods.'' After 3 h of incubation, the samples were diluted with 5 mM sodium acetate, pH 4.0, and loaded onto a Mono Q column. The number above each peak represents the amount of sialic acid residues per oligosaccharitol.
Peak a contained a component
that produced an [M-1] pseudomolecular ion
at m/z 223 in negative ion ES-MS and that contained
only [1-
H]GlcNAc-ol, as judged by GC-MS
composition analysis (Table 2). These data define peak a as the alditol GlcNAc-ol.
Peak b contained a component
that produced an [M-1] pseudomolecular ion
at m/z 385 in negative ion ES-MS (deutero-reduced
Hex-HexNAc-ol = 386 Da) and that contained only
[1-
H]GlcNAc-ol and Gal, as judged by GC-MS
composition analysis (Table 2). The GC-MS methylation analysis
revealed the presence of a terminal Galf residue and
4-O-substituted [1-
H]GlcNAc-ol (Table 2). The radiolabeled
(NaB
H
-reduced) form of this component had a
size of 3.5 Gu that was reduced to 2.6 Gu (the size of HexNAc-ol) after
cleavage of the Galf residue by mild acid hydrolysis (Table 4). The chemical shift (5.154 ppm; Table 2) and
extremely small J
coupling constant (data not shown), of
the Galf H-1 proton defined the galactofuranosidic linkage as
(8) . Taken together, these data define the peak b component as Galf
1-4GlcNAc-ol.
Peak c contained a component that produced an [M-1] pseudomolecular ion at m/z 547 in negative ion
ES-MS (deutero-reduced Hex
-HexNAc-ol = 548 Da) and
that contained only [1-
H]GlcNAc-ol and Gal, as
judged by GC-MS composition analysis (Table 2). The GC-MS
methylation analysis revealed the presence of a terminal Galf residue, a terminal Galp residue, and
4,6-di-O-substituted [1-
H]GlcNAc-ol. The
radiolabeled (NaB
H
-reduced) form of this
component had a size of 4.5 Gu that was reduced to 3.5 Gu after
cleavage of the Galf residue by mild acid hydrolysis or after
the cleavage of the
Galp residue with jack bean
-galactosidase (Table 4). The sensitivity of the structure
to jack bean
-galactosidase, which does not efficiently cleave
Galp
1-4GlcNAc-ol, and its resistance to bovine
testicular
-galactosidase, which does cleave
Galp
1-4GlcNAc-ol(16) , suggest that the
Galp residue is attached to the 6-position of the
GlcNAc-ol residue and that the peak c component has the
structure
Galp
1-6(Galf
1-4)GlcNAc-ol. The
chemical shifts of the protons of this component (Table 3), which
are identical to those described in (8) for the same structure,
confirm this assignment. The data in Table 3add some additional
assignments compared with (8) : (i) the H-5 resonance of the
Galp residue, which was assigned by chemical shift
arguments in (8) , was confirmed from the rotating frame
Overhauser effect spectroscopy experiment, and (ii) the H-6` proton of
the
Galf residue was assigned via the triple
quantum-filtered correlated spectroscopy experiment, which is selective
for the H-5, H-6, and H-6` protons.
Peak d contained a
component that produced an [M-1] pseudomolecular ion at m/z 709 in negative-ion ES-MS
(deutero-reduced Hex
-HexNAc-ol = 710 Da) and that
contained only [1-
H]GlcNAc-ol and Gal, as judged
by GC-MS composition analysis (Table 2). The GC-MS methylation
analysis revealed the presence of a terminal Galf residue, a
terminal Galp residue, a 3-O-substituted Galp residue, and 4,6-di-O-substituted
[1-
H]GlcNAc-ol. The radiolabeled
(NaB
H
-reduced) form of this component had a
size of 5.6 Gu that was reduced to 4.5 Gu after cleavage of the
Galf residue by mild acid hydrolysis and to 3.5 Gu after the
cleavage of 2
Galp residues with jack bean
-galactosidase (Table 4). Taking into account the
methylation data, the accessibility of both
Galp residues
to jack bean
-galactosidase suggests that they are linked together
in the form Galp
1-3Gal
1-. The bovine
testicular
-galactosidase enzyme was capable of removing the
terminal
1-3-linked Gal residue only, to yield a 4.5-Gu
product, again suggesting that the
Galp branch was
attached to the 6-position of the GlcNAc-ol residue. Taken together,
these data suggest that the peak d component has the structure
Galp
1-3Galp
1-6(Galf
1-4)GlcNAc-ol.
The chemical shifts of the anomeric protons of this component (Table 2), which are identical to those described in (8) for the same structure, confirm this assignment.
Peak e contained a component that produced an
[M-1] pseudomolecular ion at m/z 871 in negative ion ES-MS (deutero-reduced
Hex
-HexNAc-ol = 872 Da) and which contained only
[1-
H]GlcNAc-ol and Gal, as judged by GC-MS
composition analysis (Table 2). The GC-MS methylation analysis
revealed the presence of a terminal Galf residue, two terminal
Galp residues, a 2,3-di-O-substituted Galp residue, and 4,6-di-O-substituted
[1-
H]GlcNAc-ol. The radiolabeled
(NaB
H
-reduced) form of this component had a
size of 6.4 Gu that was reduced to 5.4 Gu after cleavage of the
Galf residue by mild acid hydrolysis (Table 4).
Interestingly, the structure was resistant to both jack bean
-galactosidase and bovine testicular
-galactosidase enzymes. (
)The chemical shifts of the anomeric protons of this
component (Table 2) are identical to those described in (8) for the structure
Galp
1-3(Galp
1-2)Galp
1-6(Galf
1-4)GlcNAc-ol,
and all of the above data are consistent with this structure.
Peak f contained a component that produced an
[M-1] pseudomolecular ion at m/z 1,033 in negative ion ES-MS (deutero-reduced
Hex
-HexNAc-ol = 1,034 Da) and that contained only
[1-
H]GlcNAc-ol and Gal, as judged by GC-MS
composition analysis (Table 2). The radiolabeled
(NaB
H
-reduced) form of this component had a
size of 7.1 Gu that was reduced to 5.5 Gu after mild acid hydrolysis,
suggesting that the structure contained either 2 Galf residues
or 1 Galf residue that is substituted by a Galp residue (Table 4). The structure was resistant to both jack
bean
-galactosidase and bovine testicular
-galactosidase
enzymes.
The chemical shifts of the anomeric protons of
this component (Table 2) are identical to those described in (8) for the structure
Galp
1-3(Galp
1-2)Galp
1-6(Galp
1-2Galf
1-4)GlcNAc-ol,
and all of the above data are consistent with this structure.
We have compared the structures of the O-linked oligosaccharides and the GPI anchors of the major mucin-like SA acceptors from metacyclic trypomastigote and epimastigote forms of T. cruzi. We found that when epimastigotes transform into metacyclic trypomastigotes the O-linked oligosaccharides and the GPI glycan core structures remain unchanged, whereas the lipid portion of the GPI anchor changes substantially from alkylacylglycerol-PI to mostly ceramide-PI. The ES-MS analyses showed that the epimastigote mucins contain alkylacyl-PI species, the principal component being 1-O-hexadecyl-2-O-hexadecanoyl-PI, whereas 70% of the metacyclic mucins contain inositol phosphoceramides (ceramide-PIs), with the principal ceramide components identified as lignoceroyl-sphinganine and palmitoyl-sphinganine. The remaining 30% of the metacyclic mucins contain the same alkylacyl-PI species as the epimastigote mucins. The lipids present in the epimastigote mucins of strain Y, identified as 40/45-kDa glycoconjugates, were also shown to contain 1-O-hexadecyl-2-O-palmitoylglycerol and 1-O-hexadecyl-2-O-stearoylglycerol(17) , in similar proportions to those found in G-strain epimastigotes in this study. The lipid portion of a molecule called lipophosphoglycan-like glycoconjugate, isolated from T. cruzi epimastigotes (Peru strain) also contains the same alkylacylglycerol-PI species(9) . No direct evidence for the presence of phosphosaccharide repeats, characteristic of Leishmania lipophosphoglycans, was presented in that study, and, given its similarity in composition and properties to the mucin-like molecules reported here and in Refs. 8, 12, 16, and 17, it should be considered as a member of the T. cruzi mucin family.
The GPI anchors
of both trypomastigote Tc-85 (30) and the metacyclic 90-kDa
1G7-antigen (26, 31) have been shown to contain mostly
1-O-hexadecyl-2-acyl-PIs, which are similar to those reported
here and in (9) and (17) for the epimastigote mucin
GPI membrane anchors. In the case of the metacyclic mucins, the GPI
anchor ceramide-PI structures are identical to the main types found in
LPPG, the major surface glycoconjugate of T. cruzi (Y strain)
epimastigotes(32, 33) ; i.e. they contain
palmitoyl-sphinganine and lignoceroyl-sphinganine. The LPPG molecule
also shares the same Man-GlcN core glycan structure as the
mucin GPI anchor and contains a 2-AEP group attached to the 6-position
of the GlcN residue(24, 32, 33) . Thus, it is
possible that these ceramide-PI anchors are biosynthetically related to
LPPG. The putative point of biosynthetic divergence would be in the
addition of the ethanolamine phosphate (or 2-AEP) bridge to
Man
-(2-AEP)GlcN-(ceramide-PI) to form the GPI anchor
precursor or the addition of two terminal Galf residues to
form LPPG. The relationship between the ceramide-PI type GPI structures
and the alkylacyl-PI structures is not clear. In the case of Saccharomyces cerevisiae, the GPI precursors are based on
diacyl-PIs that on most (but not all) glycoproteins are exchanged to
ceramide-PIs after transfer to protein in an as yet undefined
lipid-remodelling reaction(34, 35) . It is possible
that a similar mechanism may operate in T. cruzi, as discussed
in (26) . The observation that all of the early GPI
intermediates in T. cruzi epimastigotes are based on
alkylacyl-PI is consistent with this notion(36) .
Interestingly, a glycoinositolphospholipid (called GIPL A) with the
same glycan core structure as LPPG, but containing a lipid moiety
composed exclusively of
1-O-hexadecyl-2-O-palmitoylglycerol, has been
detected in early cultures of Y strain T. cruzi epimastigotes(37) . This glycoinositolphospholipid could
be the immediate precursor to LPPG. If so, the shift from alkylacyl-PI
to ceramide-PI seen in the mucins upon transformation of late
epimastigotes to metacyclic trypomastigotes may be similar to the shift
from GIPL A to LPPG seen upon the transformation from early to late
epimastigotes. Thus the change in the PI lipid structure of the mucin
molecules of T. cruzi appears to be under developmental
control. Interestingly, the ES-MS analysis of the metacyclic
1G7-antigen PI moieties also revealed that a small quantity (>15 mol
%) of the GPI anchors contained ceramide-PIs(26) . However, in
this case the ceramides were predominantly palmitoyl-sphinganine and
stearoyl-sphinganine, and the reason for the lack of
lignoceroyl-sphinganine is not clear. As Grace's medium was used
to induce metacyclogenesis in the 1G7-antigen work, whereas liver
infusion tryptose medium was used throughout this mucin study, it is
possible that the nutrient conditions might affect the fatty acid
content of the ceramide components.
The possibility that the
ceramide-PI content of the metacyclic trypomastigote mucin preparation
might be due to contamination with LPPG can be ruled out for the
following reasons: (i) the mucin fractions were eluted from the
octyl-Sepharose column between 23 and 27% propan-1-ol, while LPPG is
known to elute from this column at >40% propan-1-ol(33) ;
(ii) metacyclic trypomastigotes express about 10 times less LPPG than
epimastigotes(38) , suggesting that any LPPG contamination
would be greater in epimastigote preparations; (iii) no LPPG could be
detected in the mucin preparations by SDS-polyacrylamide gel
electrophoresis and silver staining and by Western blot with an
anti-LPPG antibody; (iv) the mucin samples were exhaustively
pre-extracted with butan-1-ol to remove glycolipid and phospholipid
contaminants prior to deamination; (v) the Man-AHM*
fragment, generated by partial acid hydrolysis of the deaminated
NaB
H
-reduced mucin, was completely protected
from jack bean
-mannosidase digestion by an aqueous HF-sensitive
substituent (Fig. 3, lane 7). The corresponding LPPG
Man
-AHM* fragment would be digested to AHM*.
The
structure of the major GPI glycan (shown to be as
Man1-2Man
1-2Man
1-6Man
1-4GlcN-myo-inositol)
was identical in epimastigote and metacyclic mucins. A small proportion
of the GPI glycans contained an additional unidentified sugar residue
attached to the
Man residue adjacent to the GlcN residue. Similar
results have been reported recently for the epimastigote mucin of Y
strain(17) . The major Man
GPI glycan structure
described above is also found in the 1G7-antigen(26) , and this
structure represents only a minor substitution of the conserved
Man
GPI glycan found in all GPI anchors characterized to
date(29) . Interestingly, the GPI glycan core of one surface
glycoprotein of infective trypomastigotes derived from mammalian cells
contained a glycan core composed a Man
-GlcN rather than a
Man
-GlcN(30) .
The mucins from epimastigotes and
metacyclic trypomastigotes also share the same O-linked
oligosaccharide chains, linked to the protein backbone via N-acetylglucosamine residues rather than N-acetylgalactosamine that is commonly found in vertebrate
mucins. In the case of T. cruzi mucins described here about
20% of the oligosaccharitols released by reductive -elimination
corresponded to non-substituted GlcNAc-ol. Most of the O-linked GlcNAc residues are substituted and might not be
accessible. These substitutions included chains formed by one to five
additional galactose residues, always with one
Galf residue linked to the 4-position of the GlcNAc. The remaining
Galp residues are mostly linked as linear and branched
side-chains to the 6-position of the GlcNAc. The O-linked
oligosaccharide structures determined in this study are identical to
those found in the epimastigote mucin of G strain(8) , except
that the Galf
1-4GlcNAc-ol structure was not
described by these authors. Since Galp
1-6GlcNAc-ol
was not observed in this or any other study, we suggest that during
biosynthesis the addition of the
Galf residue precedes
the addition of the
Galp residues.
Studies of TS
specificity have shown that the enzyme is able to sialylate unbranched
terminal Galp residues, and that substitutions near the
Gal decrease the extent of sialylation(39, 40) . We
have confirmed these findings by using the purified O-linked
oligosaccharides that are the natural acceptors on the parasite
surface. The branched oligosaccharide e, that has two terminal
Galp residues, can accept only one SA, while the
oligosaccharide f, that has one additional terminal
Galp residue, can accept a second SA. In agreement with
these data, a maximum of two SA are incorporated per oligosaccharide in vivo (data not shown). The reasons why the branched
oligosaccharide structure cannot accept two SA residues is unknown, but
it is likely to be due to steric effects since the two terminal
Galp residues are attached to adjacent oxygen atoms on
the penultimate Gal residue.
In summary, the major difference between epimastigote and metacyclic mucins is the acquisition of a high content of ceramide-PI in the metacyclic forms, in the place of alkylacyl-PI. Epimastigotes express large amounts of LPPG, which contains the same ceramide-PI species, indicating that acquisition of ceramide-PI in the mucin is not unique to metacyclic forms. The reason why the metacyclic mucins are anchored to the cell surface by a ceramide-PI, rather than an alkylacyl-PI, is unknown. However, it may be significant that the majority of the metacyclic mucins are shed by the parasite during host cell invasion, whereas the majority of the alkylacyl-PI anchored 1G7-antigen is retained on the parasite surface(12) . Thus, surface-stability during invasion may be modulated by lipid remodelling of specific molecules. The structural changes in the mucin molecules upon metacyclogenesis are clearly less profound than those observed in the LPGs of the Leishmania, where the lipids remain unchanged but the type and/or number of phosphosaccharide repeats change dramatically(41, 42) . Nevertheless, the mucin shedding phenomenon may make the subtle change in lipid structure equally important for the infectivity of T. cruzi.