(Received for publication, November 23, 1994; and in revised form, January 9, 1995)
From the
We have investigated the structure of the glycosylphosphatidylinositol (GPI) anchor and the O-linked glycan chains of the 40/45-kDa glycoprotein from the cell surface of the protozoan parasite Trypanosoma cruzi. This glycoconjugate is the major acceptor for sialic acid transferred by trans-sialidase of T. cruzi Y-strain, epimastigote form. The GPI anchor was liberated by treatment with hot alkali, and the phosphoinositol-oligosaccharide moiety was characterized and shown to have the following structure.
The flagellate protozoan Trypanosoma cruzi, the causative agent of Chagas' disease in man, infects several million people throughout the Americas. Characterization of cell surface glycoconjugates in the various developmental stages of the parasite (1) is important because these compounds mediate a number of biological processes related to parasitism and to the pathogenesis of this infection. Glycoproteins have been implicated in the invasion of host cells(2, 3) , parasite escape from endosomes into the cytoplasm of infected cells(4) , morphological transitions(5) , and induction of protective lytic antibodies(6) . In a previous paper we demonstrated sialylation of galactose residues in cell surface macromolecules of T. cruzi by a novel trans-sialidase reaction, rather than by the more usual CMP-sialic acid-dependent sialyltransferase(7) . The biological significance of the trans-sialidase reaction emerged when it was shown that sialylation of T. cruzi cell surface components was necessary for invasion of host cells(3, 8) .
In
trypomastigote forms of T. cruzi the sialic acid residues are
transferred predominantly to O-linked oligosaccharide chains
of 60-200-kDa GPI()-anchored glycoproteins,
collectively designated the F2/3 complex(6) . These
glycoproteins are equivalent to the highly glycosylated proteins
carrying the Ssp-3 epitope which is recognized by monoclonal antibody
3C9(3) . In epimastigotes (9) and in metacyclic
trypomastigotes (10) derived from axenic cultures, which do not
express the Ssp-3 epitope, sialic acid residues are transferred to O-linked carbohydrate chains of GPI-anchored glycoproteins
which migrate on polyacrylamide gel electrophoresis as a broad band of
apparent molecular masses in the 35-50-kDa range. Although there
is considerable evidence for the participation of sialylated epitopes
in the attachment and invasion of mammalian cells by the trypomastigote
forms of T. cruzi, until recently the structures of the sialic
acid acceptors were unknown. We recently showed (11) that the
sialic acid acceptors of epimastigote forms of T. cruzi G-strain were oligosaccharides glycosidically linked to threonine
and/or serine residues mainly via N-acetylglucosamine rather
than the more usual N-acetylgalactosamine.
In the present paper we extend our analyses to O-linked oligosaccharides isolated from the major GPI membraneanchored glycoprotein in epimastigote forms of the Y-strain of T. cruzi (the 40/45-kDa glycoprotein), which differ from the oligosaccharides of strain G. We have also characterized the GPI anchor of this glycoprotein, which differs from previously characterized anchors in that it contains glucosamine substituted with 2-aminoethylphosphonate. Also unusual is the fact that in a proportion of molecules the third sugar unit distal to glucosamine, in the tetramannose chain, is substituted by aminoethylphosphonate rather than by the more typical ethanolamine phosphate.
Sample were peracetylated for FAB-MS by treatment for 10 min at room temperature with a 2:1 (v/v) mixture of trifluoroacetic anhydride and acetic acid. The reagents were removed in a stream of nitrogen, and the residue was dissolved in 1 ml of chloroform and washed three times with an equal volume of water. The chloroform was evaporated under nitrogen, and the peracetylated oligosaccharides were dissolved in 5 µl of methanol prior to analysis.
Analysis by SDS-polyacrylamide gel electrophoresis of a Bio-Gel P-100-purified phenol water extract of T. cruzi cells revealed two Schiff positive bands at 40/45 and 20/30 kDa. The 40/45-kDa components were purified to apparent homogeneity by selective extraction and affinity chromatography on a concanavalin A-Sepharose column.
Chemical analysis of the purified 40/45-kDa glycoprotein revealed the presence of neutral sugars (45%), protein (7%), hexosamine (10%), phosphorus (1%), and lipids. Galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc) were detected by carbohydrate analysis in the molar ratio of 3.0:1.5:1.0:0.1, together with traces of inositol (Ins) and sialic acid. Amino acid analysis indicated that the predominant amino acids were threonine, alanine, and aspartic acid, which together comprised about 65% of the total, the balance being mainly glutamic acid, glycine, proline, and lysine.
On acid
methanolysis the glycoprotein released methyl palmitate, methyl
stearate, and a monoalkylglycerol in the molar ratio of 0.9:0.2:1.0.
The monoalkylglycerol was trimethylsilylated and identified by GC-MS as
a hexadecylglycerol. The presence of fragment ions at m/z 205
(CHOSi(CH)
-CH
OSi(CH
)
)
in the mass spectrum, together with the absence of m/z 218 and
191, suggested that the alkyl chain is exclusively located on the sn-1-position of glycerol. GC and GC-MS analyses of the
products obtained by digestion of 40/45-kDa glycoprotein with
PI-phospholipase C, following partition between chloroform and water,
revealed that all glycerolipid moiety was present only in the
chloroform layer. In the non-enzymatic control, the glycerolipid moiety
was present only in the aqueous layer. The molar ratio of alkylglycerol
and polypeptide chain (calculated by amino acid analysis) in the
40/45-kDa glycoprotein was 0.9:1.0.
Figure 1:
P NMR spectroscopy of
PI-oligosaccharide. The 202-MHz
P NMR spectrum of the
PI-oligosaccharide from the GPI anchor of T. cruzi 40/45-kDa
glycoprotein is shown.
Four major signals were observed
in the anomeric region of the one-dimensional 500-MHz proton NMR
spectrum of the PI-oligosaccharide (Fig. 2) in an approximate
area ratio 1:2:1:2. A lower field anomeric resonance (5.711 ppm) was
recognized as -GlcN H-1 by its coupling to a high-field H-2
resonance and by the pattern of cross-peaks in the TOCSY spectrum (Fig. 3). An additional
-GlcN spin system observed in the
TOCSY spectrum was attributed to the
(1
4)GlcN
(1-6)-Ins-2-phosphate resulting from phosphate
migration during base cleavage; this was also the reason for the
presence of two inositol phosphate spin systems. The other major spin
systems were assigned as
-Man residues. The anomeric
configurations of the mannose residues were determined from the values
of
J
measured from the HMQC(35, 36) spectrum. These varied between 172 and 174 Hz and are
characteristic of equatorial anomeric protons (i.e.
-linked D-Man residues)(37) . At a higher
field, characteristic resonances were observed attributable to AEP (at
3.22 and 2.04 ppm) and to EtNP (at 3.29 ppm). These assignments are
summarized in Table 1.
Figure 2:
H NMR spectroscopy of
PI-oligosaccharide. The 500-MHz one-dimensional proton NMR of the
PI-oligosaccharide from GPI anchor of 40/45-kDa glycoprotein of T.
cruzi is shown. The resonances are labeled according to the
convention used in the text and Table 1.
Figure 3:
TOCSY
spectrum of the PI-oligosaccharide. The partial 500-MHz TOCSY spectrum
of the PI-oligosaccharide from the GPI anchor of T. cruzi 40/45-kDa glycoprotein is shown. This region of the spectrum shows
the correlations between the anomeric protons and the other sugar
residue protons. The arrows indicate the traces on the H-1
resonances of the major residues. The boxed area indicated the
approximate region where the H-1/H-2 cross-peak of a
(13)Man
(1-4)GlcN or
(1
3,6)Man
(1-4)GlcN is expected. The low intensity
``cross-peak'' present is not symmetrical about the diagonal
and is considered to be artifactual.
Inter-residue NOEs were observed in the
ROESY experiment between the -GlcN H-1 and H-1 and H-6 of
Ins-1-phosphate (Fig. 4), and between
-Man(2) H-1 and
-GlcN H-4. In the HMQC spectrum, the anomeric proton of
-Man(3) correlated to a relatively high-field C-1 resonance (99.22
ppm), diagnostic of a (1, 2, 3, 4, 5, 6) -linkage.
The NOEs to resonances at 4.001, 3.837, and 3.767 ppm of
-Man(3)
were not rigorously assigned, but were consistent with inter-residue
NOEs to H-5, H-6, and H-6` of the adjacent residues, as observed in
other systems(38) , including GPI anchor
structures(39) . The value of the chemical shift of the Man(2)
H-2 (4.063 ppm) is similar to that found in the corresponding residue
of the GPI anchor from yeast (39) and the
lipopeptidophosphoglycan (LPPG) of T. cruzi(40) rather than to its value (typically 4.23 ppm) when
present in (1
3)- or (1
3,6)-linked
structures(34, 38, 41) , indicating a linear
rather than a branched oligosaccharide. None of the minor
-Man
H-1/H-2 cross-peaks in the TOCSY spectrum (Fig. 3) suggested the
presence of either branched (1
3,6)Man
(1-4)GlcN or
linear (1
3)Man
(1-4)GlcN substructures. In the HMQC
spectrum the H-2 of
-Man(3) correlated to a low-field C-2
resonance (79.38 ppm) and inter-residue NOEs were observed between
-Man(1) H-1 and H-1 and H-2 of
-Man(3). Similarly,
-Man(1) H-2 correlated with a low-field C-2 resonance (79.18 ppm),
and inter-residue NOEs were assigned as indicating that
-Man(4)
H-1 is close in space to both
-Man(1) H-1 and H-2. The low-field
chemical shift of the
-GlcN H-5 is consistent with the presence of
a phosphorylated substituent at O-6 position, probably AEP by
analogy with LPPG(42) , leading to the following partial
structure.
Figure 4: Scheme showing the chemical shift and inter-residue NOE data used to assign linkages and sequence in the PI-oligosaccharide from GPI anchor of T. cruzi 40/45-kDa glycoprotein.
A weak cross-peak was observed in the ROESY spectrum between two
resonances at 5.05 and 5.308 ppm assigned to the H-1s of two residues
arbitrarily designated -Man(4a) and
-Man(1a). This cross-peak
could be due to either a minor tetramannose species in which the
subterminal mannose is substituted with AEP rather than EtNP (i.e. Man(4a)
(1-2)Man(6AEP) (1a) (
1
), or
alternatively, to an
(1-2) extension to the tetramannose
chain by Man(4a) as in the yeast GPI anchor(39) . A molecular
ion consistent with the presence of the latter was detected by mass
spectrometry (see below).
The location of the EtNP substituent was
not apparent from the NMR data and could not be established by
methylation analysis, since GPI structures containing four hexoses and
two phosphate ester of EtNP or 2-AEP are difficult to methylate. ()Methylated derivatives eventually obtained from this
preparation originated from a co-purified high mannose contaminant,
derived from the 40/45-kDa glycoprotein. In agreement with this
observation, the NMR spectrum showed minor resonances corresponding to
a high mannose oligosaccharide such as would arise from elimination of
an Asn-linked chain(43) . The terminal
-Manp units of this contaminant contribute to the intensity of the
resonance at 5.05 ppm (Fig. 2).
The most significant feature
in the FAB mass spectrum of the PI-oligosaccharide was a cluster of
signals in the m/z 1300-1400 region. These were assigned
as multiply cationized forms of two GPI species, both with the
composition (hexose)-hexosamine-inositol-phosphate, but
varying in substitution pattern. The major species ([M +
H]
= 1300.4, [M +
Na]
= 1322.4, [M - H +
Na
]
= 1344.3) has one AEP and
one EtNP substituent, whereas the other ([M +
H]
= 1284.3, [M +
Na]
= 1306.4, [M - H +
Na
]
= 1328.4) has two AEP
substituents. The calculated values of the protonated molecules of
these two species are, respectively, 1300.339 and 1284.345. A minor
signal at m/z 1484.5 may correspond to the sodium-cationized
molecule ([M + Na]
) of a homologue of
the major species (m/z 1300) containing an additional hexose
residue.
Because of high levels of salt contamination, and the
presence of high mannose species, the signal to noise ratio of the
spectrum was poor, and it was consequently difficult to assign with
confidence any fragment ions. The characteristic Y fragment
ion(34, 41, 44, 45) , which (in
salt-free samples) is diagnostic of the substituent on glucosamine, was
not observed at m/z 529, 545, or 422; however an ion at m/z 551 (549 in the negative ions spectrum) may correspond to
a sodium-cationized analogue of m/z 529, in which case an AEP
substituent is located on glucosamine. The signals at m/z 689,
851, and 1013, although differing by the residue mass of hexose, could
not be rationalized as fragmentation products of the
PI-oligosaccharide, although they could correspond to sodium-cationized
molecules of oligohexoses. This was verified by FAB-MS after treatment
with trifluoroacetic anhydride/acetic acid, which resulted in a
spectrum containing peracetylated oligohexose ions. In order to improve
the quality of the FAB-MS data, the sample was desalted with Dowex
50W-X8 and reanalyzed. Two protonated molecules were now obtained, at m/z 1300.3 (major) and 1284.3 (minor), as expected. An
abundant Y
fragment ion at m/z 529 confirmed that
the glucosamine was substituted primarily with AEP. The mass increment
of 285 between the fragments assigned as Y
(m/z 1138) and Y
(m/z 853) suggests that the EtNP
substituent of the major species is located on the third mannose distal
to glucosamine. A corresponding signal at m/z 1122 indicated
that this residue is substituted with AEP in the analogue containing
two AEP groups.
The
-eliminated saccharide-alditols were methanolyzed,
trimethylsilylated, and analyzed by GC. Fractions II, III, and IV
contained galactose, GlcNAc-ol and GalNAc-ol in molar ratio of
1.0:1.0:0.09, 2.0:1.0:0.1, and 3.0:1.0:0.08, respectively, whereas
fraction I contained only GlcNAc-ol. The protonated molecules (M +
H)
obtained by FAB-MS identified Fractions II (m/z 386), III (m/z 548), and IV (m/z 710) as,
respectively, N-acetylhexosaminitol-containing mono-, di-, and
trisaccharides. The products obtained in the methylation analysis of
the monosaccharide alditol (fraction II) corresponded to terminal
galactose, 3-O- and 4-O-substituted GlcNAc-ol in the
molar ratio 1.0:0.4:0.6. The proton NMR spectrum of this fraction (Fig. 5A) contained two major anomeric resonances in a
6:4 ratio at 4.502 and 4.494 ppm (
J
about 8
Hz), typical of
-Galp units and two major N-acetyl methyl resonances near 2.0 ppm from the GlcNAc-ol
residues. The 80-ms TOCSY spectrum, the
C NMR assignments,
and comparison with the spectra of oligosaccharides isolated from T. cruzi G-strain (11) enable an almost full
assignment of the proton NMR spectrum of this fraction (Table 2).
These data define the monosaccharide alditol fraction as a mixture of
oligosaccharides of structures Galp
1-3GlcNAc-ol and
Galp
1-4GlcNAc-ol.
Figure 5:
H NMR spectroscopy of the O-linked oligosaccharides. 500-MHz
H NMR spectra
of O-linked oligosaccharide from 40/45 kDa glycoprotein of T. cruzi are shown. A, monosaccharide-alditols; B, disaccharide-alditols; and C,
trisaccharide-alditols.
The proton NMR spectrum of the
disaccharide-alditol (fraction III) differed from that of fraction II
in several respects (Fig. 5B). Two -Galp anomeric resonances were present in an approximate 6:4 ratio;
although 0.06-0.09 ppm downfield of those in the previous
spectrum, their chemical shifts and those of the ring protons were
still consistent with a nonreducing terminal location. A third anomeric
resonance was present at 4.450 ppm (
J
about 8
Hz) with approximately the combined intensity of the other two
anomerics. This signal was assigned to an additional nonreducing
terminal
-Galp residue by means of an 80-ms TOCSY
experiment. The location of this residue was deduced from the following
arguments: (a) the proton (Fig. 5B) and carbon
chemical shifts of the additional anomeric resonance (4.450 ppm
H and 104.41 ppm
C) (Table 2) are very
similar to those of the Galp
1-6GlcNAc-ol system
found in the T. cruzi G-strain oligosaccharides (4.431 and
104.41 ppm, respectively(11) ); (b) the resonances
assigned as GlcNAc-ol H-6 and H-6` are shifted downfield compared with
the corresponding signals in the monosaccharide-alditol; (c)
the high-field resonances corresponding to C-6 of GlcNAc-ol in the
monosaccharide-alditol (at 63.53 and 63.27 ppm) are replaced by signals
at 70.83 and 70.92 ppm (Table 2). These data suggest that the
disaccharide-alditol is a 4:6 mixture of
Galp
1-3(Galp
1-6)GlcNAc-ol and
Galp
1-4(Galp
1-6)GlcNAc-ol and
agree with the detection in the methylation analysis of nonreducing end
units of galactose, 3,6- and 4,6-di-O substituted GlcNAc-ol in
a molar ratio of 2.0:0.4:0.6.
In the methylation analysis of the
trisaccharide-alditol (fraction IV), in addition to 3,6-di-O-
and 4,6-di-O-substituted GlcNAc-ol (in a molar ratio of
0.4:0.6), derivatives corresponding to nonreducing terminal galactose
and 2-O-substituted galactose were detected in 2:1 molar
ratio. The one-dimensional proton spectrum of the trisaccharide-alditol (Fig. 5C) was complex and lacked the anomeric doublet
assigned as Galp1-6GlcNAc-ol in the
disaccharide-alditol, consistent with the addition of a
-galactose
residue to that branch. These data show that the trisaccharide-alditol
is a mixture of two compounds:
Galp
1-2Galp
1-6(Galp
1-3)GlcNAc-ol
and
Galp
1-2Galp
1-6(Galp
1-4)GlcNAc-ol.
The structures of the major O-linked GlcNAc oligosaccharides
from the 40/45-kDa glycoprotein of T. cruzi Y-strain are
summarized Table 3.
Figure 6:
Formation of mono- and
disialyl-oligosaccharide-alditols from -eliminated
oligosaccharides of T. cruzi 40/45-kDa glycoprotein. Upper
panel, the labeled mono- (A), di (B), and
trisaccharide-alditols (C) were incubated with sialyllactose
and T. cruzitrans-sialidase and run on paper
electrophoresis in pyridine-acetate buffer, pH 6.5. The charged
compound in A, the slow migrating oligosaccharide in B, and the fast migrating compound in C were eluted,
incubated with C. perfringens neuraminidase, and run on paper
electrophoresis as above (D, E, and F, respectively). Lower panel, the slow migrating compounds obtained in upper panels B and C were eluted and reincubated with trans-sialidase and sialyllactose and run on paper
chromatography (lower panels A and B,
respectively).
In the present study we have determined the structure of the GPI anchor of the 40/45-kDa glycoprotein of T. cruzi epimastigotes and of the oligosaccharides O-linked to the protein. We have also shown that these latter oligosaccharides act as acceptors for sialic acid transferred via the trans-sialidase reaction(7) .
The PI-oligosaccharide of the anchor was
characterized by NMR spectroscopy, FAB mass spectrometry, and
compositional analysis. Our results show that the major GPI species
contains an EtNP group attached to a mannotetraose backbone of sequence
Man(1-2)Man
(1-2)Man
(1-6)Man
(1
.
This tetramannosyl chain is (1
4)-linked to a non-acetylated GlcN
residue substituted at O-6 by AEP, a feature that has not been
reported previously in a GPI-protein anchor. The GlcN unit is
glycosidically linked to the O-6 position of a myo-inositol phospholipid, containing
1-O-alkyl-2-O-acyl-sn-glycerol. The EtNP was
shown by FAB-MS to be located on the third mannose distal to inositol,
in agreement with the structure of all GPI-protein anchors
characterized so for(46) .
Apart from the AEP substituent, the structure of this compound is identical with that of the GPI anchor of the 1G7 antigen isolated from metacyclic forms of T. cruzi(47) . The presence of AEP in the 1G7 anchor cannot, however, be excluded, because the structure was determined after dephosphorylation with aqueous hydrofluoric acid(47) .
Interestingly, T. cruzi epimastigotes synthesize a free glycoinositolphospholipid called LPPG(48) , the PI-oligosaccharide core of which (42) is similar to the GPI anchors of T. cruzi glycoproteins. It differs, however, in that the tetramannosyl chain is further substituted by galactofuranosyl residues and by the absence of EtNP. This suggests that T. cruzi may use the same set of glycosyltransferases for the synthesis of both LPPG and glycoprotein-linked GPI anchors. The lipid moiety of LPPG, however, is a ceramide (42, 49) and thus diverging from the protein-linked GPI anchors.
It has been suggested that GPI-containing glycoconjugates, including LPPG from T. cruzi, the heterogeneous lipophosphoglycan of Leishmania species, and the free glycoinositolphospholipids of various trypanosomatids(50) , may have evolved from primitive GPI anchors linked via EtNP to protein. In the 40/45-kDa GPI anchor, however, FAB mass spectra showed that EtNP can be partially replaced by AEP. An alternative hypothesis therefore is that the GPI anchors of ancestral Kinetoplastida might have contained AEP, which was replaced by EtNP in the course of evolution.
The O-linked
oligosaccharides of 40/45-kDa glycoproteins of the Y-strain of T.
cruzi differ from those of the G-strain (11) in several
aspects. Although linked to threonine and/or serine via GlcNAc, they
are shorter and do not contain -galactofuranosyl units. They form,
after
-elimination, two series of mono-, di-, and
trisaccharide-alditols in which the
-galactopyranosyl branch is
either (1
3)- or (1
4)-linked. The short oligosaccharide
chains of the 40/45-kDa glycoprotein of Y-strain epimastigotes
described in this paper differ from the analogous structures of
cell-derived trypomastigotes of the same strain(6) . In the
latter glycoprotein (called F2/3 or Ssp-3), the shortest O-linked oligosaccharide is the trisaccharide
Galp
(1-3)Galp
(1-4) GlcNAc, but
chains as long as 10 sugar units have been detected. In contrast to the
highly glycosylated components of epimastigotes (from both the G- and
the Y-strains) and of metacyclic trypomastigotes, the corresponding
glycoproteins from cell-derived trypomastigotes are unique in that they
contain terminal
-Galp units(6) . The 3-O substitution of GlcNAc described in the present study seems to
occur only in the oligosaccharides of epimastigotes. In cell-derived
trypomastigotes, a high proportion of the O-linked
oligosaccharide chains not containing
-Galp are
substituted by sialic acid units, giving rise to an epitope recognized
by monoclonal antibody 3C9. This monoclonal antibody, however, does not
react with the glycoproteins of epimastigotes and metacyclic
trypomastigotes of the Y-strain(3) . This suggests that sialic
acid substitution of O-linked oligosaccharides in these
molecules is not sufficient per se to generate the epitope
characteristic of the Ssp-3 (or F2/3) glycoproteins of cell-derived
trypomastigotes which is recognized by the 3C9 antibody. Differences in
the glycosylation of these molecules in T. cruzi parallels the
varying patterns of O-linked glycosylation observed as a
function of cell lineage and stage of differentiation in mammalian
cells(51) .
It has been demonstrated previously that most of the sialic acid transferred by the trans-sialidase reaction from fetal calf serum donor molecules is incorporated into the 40/45-kDa glycoprotein of T. cruzi (Y-strain) epimastigotes(7) . This is another example from a eukaryotic system of a transglycosylation reaction that is independent of nucleotide phosphate sugars; oligosaccharide biosynthesis via similar reactions have previously been noted in fungi (52, 53) .
Non-sialylated molecules were obtained
by culturing epimastigotes in the absence of serum; these were used as
acceptors for in vitro sialic acid transfer by trans-sialidase from trypomastigotes, with sialyllactose as a
donor substrate. The oligosaccharide acceptors specificity of T.
cruzitrans-sialidase has been studied
previously(54, 55) . Only -linked terminal
galactosyl units are susceptible to sialylation by the enzyme. Internal
-linked or terminal
-linked residues cannot serve as
acceptors. Fucosylation of the residues adjacent to an otherwise
susceptible galactopyranose also abolishes sialylation. Although it has
been claimed that the trans-sialidase reaction is unaffected
by chain length and the nature of (unbranched) vicinal residues, it
seems clear from the present data that sialylation of one residue in T. cruzi glycans modulates the susceptibility of nearby sites
to sialylation, as shown by the kinetics of in vitro sialic
acid incorporation into the oligosaccharides of the 40/45-kDa
glycoprotein. These results suggest that the potential use of the
trypanosome trans-sialidase as an effective and economical
replacement of the
(2-3) sialyl transferase for in vitro sialylation (56) may be limited by an inability to
efficiently sialylate complex multiantennary substrates.
Because of the low activity of the trans-sialidase in epimastigotes, some workers (56, 57) have suggested that sialic acid is absent from the surface of these forms; however, it should be noted that the 40/45-kDa glycoprotein isolated from epimastigotes grown in the presence of fetal calf serum has similar composition and properties to the recently characterized ``sialylated lipophosphoglycan-like molecules'' from epimastigote forms of T. cruzi(58) .
It is unclear whether the structural differences in the mucin-like molecules of epimastigotes from different strains of T. cruzi are a common feature of this species or whether they are in any way related to the infectivity of the corresponding trypomastigote stage. Further analyses with additional strains of this parasite should answer this question.