Báo cáo khoa học: Structural analysis of the N-glycans of the major cysteine proteinase of Trypanosoma cruzi Identification of sulfated high-mannose type oligosaccharides doc

Structural analysis of the N-glycans of the major cysteine
proteinase of Trypanosoma cruzi
Identification of sulfated high-mannose type oligosaccharides
Mariana Barboza1, Vilma G. Duschak2, Yuko Fukuyama3,*, Hiroshi Nonami3, Rosa Erra-Balsells4,
Juan J. Cazzulo1 and Alicia S. Couto4
1
2
3
4

´
Instituto de Investigaciones Biotecnologicas-INTECH, Universidad Nacional de Gral. San Martin, Buenos Aires, Argentina
´
´
Instituto Nacional de Parasitologıa ‘Dr Mario Fatala Chaben’, ANLIS, Ministerio de Salud y Ambiente, Buenos Aires, Argentina
College of Agriculture, Ehime University, Matsuyama, Japan
´nica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
´
CIHIDECAR (CONICET) Departamento de Quımica Orga
Argentina

Keywords
Cruzipain; nor-harmane; sulfated
oligosaccharides; Trypanosoma cruzi; UVMALDI-TOF MS
Correspondence
A. S. Couto, CIHIDECAR (CONICET)
´nica,
´
Departamento de Quımica Orga
Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Buenos Aires,

CP 1428, Argentina
Fax: +54 11 45763346
Tel: +54 11 45763346
E-mail:
*Present address
Koichi Tanaka Mass Spectrometry Research
Laboratory, Shimadzu Corporation,
1 Nishinokyo-Kuwabaracho, Nakagyo-ku,
Kyoto 604-8511, Japan
(Received 8 April 2005, accepted 23 May
2005)
doi:10.1111/j.1742-4658.2005.04787.x

Trypanosoma cruzi, the parasitic protozoan that causes Chagas disease,
contains a major cysteine proteinase, cruzipain. This lysosomal enzyme
bears an unusual C-terminal extension that contains a number of posttranslational modifications, and most antibodies in natural and experimental infections are directed against it. In this report we took advantage of
UV-MALDI-TOF mass spectrometry in conjunction with peptide N-glycosidase F deglycosylation and high performance anion exchange chromatography analysis to address the structure of the N-linked oligosaccharides
present in this domain. The UV-MALDI-TOF MS analysis in the negativeion mode, using nor-harmane as matrix, allowed us to determine a new
striking feature in cruzipain: sulfated high-mannose type oligosaccharides.
Sulfated GlcNAc2Man3 to GlcNAc2Man9 species were identified. In
accordance, after chemical or enzymatic desulfation, the corresponding
signals disappeared. In addition, by UV-MALDI-TOF MS analysis (a) a
main population of high-mannose type oligosaccharides was shown in the
positive-ion mode, (b) lactosaminic glycans were also identified, among
them, structures corresponding to monosialylated species were detected,
and (c) as an interesting fact a fucosylated oligosaccharide was also detected. The presence of the deoxy sugar was further confirmed by high performance anion exchange chromatography. In conclusion, the total number
of oligosaccharides occurring in cruzipain was shown to be much higher
than previous estimates. This constitutes the first report on the presence of
sulfated glycoproteins in Trypanosomatids.

Trypanosoma cruzi, the parasitic protozoan that causes
the American Trypanosomiasis or Chagas disease, contains a major cysteine proteinase (CP), cruzipain. This
enzyme is present in the epimastigote, amastigote, metacyclic and tissue culture trypomastigote forms [1]. It
has been reported to be placed in the lysosomal compartment [2–4], but it seems to be also located at the

cell surface. Accordingly, plasma membrane-bound
isoform(s) of CPs have been shown in the different
developmental stages of T. cruzi [5].
Cruzipain is encoded by numerous genes which contain no introns, encoding a signal peptide, a propeptide and a mature enzyme. As for all Type I CPs
from trypanosomatids, the protein presents a catalytic

Abbreviations
CP, cysteine proteinase; HPAEC-PAD, high pH anion exchange chromatography with pulsed amperometric detection; PNGase F, peptide
N-glycosidase F.

FEBS Journal 272 (2005) 3803–3815 ª 2005 FEBS

3803


Structure of the N-glycans in cruzipain

moiety and a characteristic C-terminal domain [6], the
latter being the most characteristic structural feature of
this protein [7]. As in the CPs of Trypanosoma rangeli
and Crithidia fasciculata but in contrast to similar CPs
from Leishmania mexicana and Trypanosoma brucei,
cruzipain C-terminal is retained in the natural mature
form of the enzyme [8]. This extension is 130 aminoacid residues long [9], and most antibodies in natural
and experimental infections are directed against it [10].

Cruzipain is purified from epimastigotes as a complex
mixture of isoforms [8]. This microheterogeneity is
probably due to the presence of a number of posttranslational modifications [11] including carbohydrate
heterogeneity [12] as well as some point mutations
leading to amino-acid replacements, most if not all
present in the C-terminal domain [9,11].
It is known that the single N-glycosylation site in the
C-terminal domain (Asn255) as well as the first potential
N-glycosylation site in the catalytic moiety (Asn33)
are glycosylated in vivo [13]; the latter bears only highmannose type oligosaccharides. It had been suggested
that the C-terminal domain of cruzipain presents either
a high-mannose oligosaccharide, a hybrid monoantennary or a complex biantennary oligosaccharide chain
[12]. In vivo labeling of the parasites with 32Pi discounted
the presence of Pi in the mature enzyme [11].
Recently, two types of post-translational modifications involving carbohydrates have been described: a
complex N-glycosidic oligosaccharide bearing sialic
acid and single N-acetyl-glucosamine residues with an
O-glycosidic linkage [14]. The fact that cruzipain is a
complex mixture of isoforms with a great diversity in
the N-linked structures made the separation of species
very difficult. We took advantage of UV-MALDI-TOF
mass spectrometry in combination with enzymatic digestions and complemented with high pH anion exchange
chromatography (HPAEC) analysis to characterize the
N-linked glycans present in the protein.
In this paper we report, for the first time, the presence of sulfate in the N-linked oligosaccharides of
cruzipain. This structural feature was confirmed in the
unique N-glycosidic site present in the C-terminal
domain; a major population of high-mannose type
oligosaccharides, as well as lactosaminic, fucosylated
and sialylated complex type glycans in a minor extent,

were shown. The diversity of structures present in the
C-terminal domain might account for the microheterogeneities found in natural cruzipain.

Results
In an attempt to perform a structural study of the
oligosaccharide chains localized in the C-terminal
3804

M. Barboza et al.

66
45

29

kDa

1

2

3

4

5 6 7 8 9 10 11

Fig. 1. Analysis by SDS ⁄ PAGE followed by silver stainning of C-terminal domain purification. Lane 1, natural cruzipain; lane 2, self-proteolysed cruzipain; lanes 3–11 are fractions corresponding to the Bio
Gel P-30 columm. Lanes 6–11 correspond to purified C-terminal.
Molecular mass markers are indicated (in kDa) at the left side of the

figure.

domain of cruzipain, the protein was subjected to selfproteolysis and the C-terminal was further purified via
Bio Gel P-30 column chromatography [14]. Fractions
containing purified C-terminal (Fig. 1, lanes 8–11) were
joined, freeze-dried and subjected to peptide N-glycosidase F (PNGase F) treatment. The released oligosaccharides were separated from the polypeptide by
Ultrafree McFilters (MW 5000). A fraction of these
oligosaccharides was reduced with NaB3H4 and desalted by Biogel P-2 as already described [23]. Labeled
oligosaccharides included in the column corresponded
to neutral N-linked oligosaccharides as shown by
HPAEC (Fig. 2A). The acidic glycans already reported
[14] were recovered in the excluded fraction. Under
conditions where acidic glycans are resolved, the
HPAEC profile of the excluded fraction showed four
major peaks (Rt ¼ 8, 13, 25 and 32.5 min) (Fig. 2B).
Mild acid hydrolysis of this fraction to release sialic
acid and further analysis showed the absence of the
peak with Rt ¼ 25 min (Fig. 2C). When this desialylated fraction was analysed under conditions where
neutral glycans are resolved, a peak coincident with a
standard of a biantennary complex-type oligosaccharide was obtained (Fig. 2E). The fact that not all the
acidic glycans were sensitive to the mild acid hydrolysis
strongly suggested that another acidic group could be
present. Thus, a digestion with sulfatase was performed and the profile obtained by HPAEC (Fig. 2D)
showed the disappearance of the peak with Rt ¼
32.5 min. More evidence of the presence of sulfated
species in cruzipain was achieved using anion-exchange
chromatography [24]. A sample of the labeled oligosacFEBS Journal 272 (2005) 3803–3815 ª 2005 FEBS


M. Barboza et al.

Fig. 2. HPAEC analysis of the oligosaccharides released by PNGase
F treatment from the C-terminal domain of cruzipain. (A) Total neutral fraction; (B) total acidic fraction; (C) acidic fraction treated with
mild acid to release sialic acid; (D) acidic fraction treated with sulfatase; (E) same as (C). (A) and (E), analysis under ‘conditions a’ for
neutral glycan; (B), (C) and (D), analysis under ‘conditions b’ for acidic glycans. Standards: 1, Man3GlcNAc2OH; 2, Man5GlcNAc2OH; 3,
Man6GlcNAc2OH; 4, Gal2GlcNAc2Man3GlcNAc2OH; 5, Man9GlcNAc2OH; 6, monosialylated; 7, disialylated; 8, trisialylated oligosaccharides.

charides (12 000 cpm) was applied to a QAE-Sephadex
column equilibrated with 2 mm Tris-base. Although a
major fraction (9000 cpm) passed through, 25% (3000
cpm) of the label bound to the resin and was eluted
with 2 mm Tris ⁄ 20 mm NaCl correlating with the presence of another acidic group in addition to sialic acid.
To determine the structural identity of the N-glycans
present in cruzipain, a UV-MALDI-TOF MS analysis was performed. Figure 3A shows the positive
UV-MALDI spectrum of the whole oligosaccharide
fraction released from cruzipain by PNGase F digestion. Figure 3B shows the spectrum corresponding to
the analogous fraction obtained from the purified
C-terminal domain. The m ⁄ z-value, composition and
structure of the detected glycans are listed in Tables 1
and 2. Molecular ions were determined as monosodium adducts [M + Na]+. Although the quality of
the spectra (signal ⁄ noise ratio) obtained for each sample was different, both spectra showed major peaks
at m ⁄ z-values: 933.2 (933.7), 1094.8 (1096.1), 1259.1
(1258.3), 1420.4 (1420.4), 1581.7 (1582.7), 1744.5
(1744.8) and 1906.6 (1907.6) (Table 1, Fig. 3). These
signals correspond to high-mannose glycans containing
from 3 to 9 mannose residues (GlcNAc2Man3 to GlcNAc2Man9) (Table 1). Interestingly, signals at m ⁄ zvalues 2029.5 (2029.9), 2069.1 (2069.3) and 2395.4
(2395.1) compatible with lactosaminic type oligosaccharides, not reported so far as components of cruzipain, were found in both spectra (Table 1, Fig. 3).
The C-terminal spectrum also showed signals with
m ⁄ z-values 1013.9; 1176.4; 1338.7; 1500.6; 1663.6 and
1825.1 (Fig. 3B) compatible with sulfated high-mannose species (Table 2). Some of these peaks were also

observed in the cruzipain spectrum. In addition, a signal at m ⁄ z 1809.6 was compatible with a fucosylated
oligosaccharide (Fig. 3B, Table 1). As this deoxy-sugar
had not been previously identified as component
of cruzipain, its presence was investigated by HPAECPAD analysis. The sugar was released from the C-terminal domain by specific a-l-fucosidase treatment,
separated through Ultrafree McFilters (MW 5000),
labeled by reduction with NaB3H4 and analysed under
FEBS Journal 272 (2005) 3803–3815 ª 2005 FEBS

Structure of the N-glycans in cruzipain

A 400

1 23

4

5

CPM
300
200
100

B 500
0

5

CPM
400

1
6

7

8

300
200
100

C 500
CPM
400
300
200
100

D 500
CPM
400
300
200
100

E 425
CPM

4

325
225
125

0

5

10

15
20
25
Retention time (min)

30

35

40

3805


Structure of the N-glycans in cruzipain

M. Barboza et al.

A 100

1906.6
1744.5

90
80
1581.7

Relative abundance

70

933.2

1013.9
1420.4

60
1094.8
1541.6

1259.1

50

1217.0

1379.2

1923.2

1703.5

1338.7

40

1663.5

1843.1

2069.1
2029.5

30
2395.4

20
10

602

976

B 100

1350

2099

1725

2474

1258.3

90
1096.1

80

1420.4

Relative abundance

933.7

2029.9

70

1968.6
1977.8

2069.3

2395.1

60
1582.7

50
40

8X

1744.8

*

30

1500.6
1338.7

20

1054.8
1013.9

1176.4
1217.0
1200.0

1809.6
1663.6

1379.7

10

1825.1

881

1199

1517

1907.6

1815.1

1835

1968.6 2029.9
2069.3

2395.1

2153

2471

m/z
Fig. 3. UV-MALDI-TOF MS analysis of the released oligosaccharides in the lineal positive mode using GA as matrix. (A) oligosaccharides
obtained from cruzipain, m ⁄ z range: 600–2474 Da. (B) oligosaccharides obtained from C-terminal, m ⁄ z range: 881–2471 Da. Inset corresponds to expanded m ⁄ z range: 1950–2460 Da. Structures are detailed in Tables 1 and 2.

conditions c (Fig. 4). The same peak was obtained
when fucose was released by acid hydrolysis (not

shown). Also minor signals at m ⁄ z 1815.1 and 1977.8
corresponding to biantennary sialylated oligosaccharides were detected in the C-terminal spectrum
(Fig. 3B, Table 1).
3806

b-Carbolines have proven to be effective matrices for
the detection of sulfated carbohydrates in the negative
ion mode [20–22]. That is the reason why we carried out
a UV-MALDI-TOF MS analysis using nor-harmane as
matrix to confirm the presence of sulfated species. The
spectrum of the whole oligosaccharide fraction obtained
FEBS Journal 272 (2005) 3803–3815 ª 2005 FEBS


M. Barboza et al.

Structure of the N-glycans in cruzipain

Table 1. m ⁄ z-Value, composition and structure of the high mannose and complex type glycans of cruzipain and C-terminal domain. h,
N-Acetylglucosamine; d, mannose; , galactose; b, sialic acid; ,, fucose.
Calculated
m ⁄ z a [M+Na]+

Measured
m ⁄ z b [M+Na]+

Proposed
composition

933.31

933.78
1095.37
1095.92
1257.42
1258.06

933.2
933.7
1094.8
1096.1
1259.1
1258.3

HexNAc2Man3

1419.47
1420.21

1420.4
1420.4

HexNAc2Man6

1581.53
1582.35

1581.7
1582.7

HexNAc2Man7

1743.58
1744.49

1744.5
1744.8

HexNAc2Man8

1905.63
1906.63

1906.6
1907.6

HexNAc2Man9

1809.66
1810.56

–
1809.6

HexNAc4Man3Gal2Fuc

1815.61
1816.53

–
1815.1

HexNAc4Man3Gal1SA1

1977.67
1978.67

–
1977.8

HexNAc4Man3Gal2SA1

2028.71
2029.73

2029.5
2029.9

HexNAc5Man3Gal3

2069.74
2070.78

2069.1
2069.3

HexNAc6Man3Gal2

2393.85
2395.06

2395.4
2395.1

HexNAc6Man3Gal4

Structure

HexNAc2Man4
HexNAc2Man5

a

Upper number indicates the average mass; lower number indicates the monoisotropic mass. b Upper number indicates the m ⁄ z value
obtained for oligosaccharides from cruzipain; lower number indicates the m ⁄ z value obtained for oligosaccharides from the purified C-T
domain.

from the C-terminal domain in the negative ion mode
is shown in Fig. 5A. Signals with m ⁄ z 990.0, 1152.8,
1314.1, 1476.6, 1639.4, 1801.5 and 1963.7 were assigned
to sulfated high-mannose oligosaccharides as [M-H]–
ions (Table 2). No signals corresponding to neutral
oligosaccharides were detected. Noticeably, the major
population corresponded to signals with m ⁄ z 1007.6,
1170.5, 1332.4, 1494.7, 1656.9, 1819.1 and 1981.5 compatible with [M + H2O-H]– ions of the same glycans
(Table 2; Fig. 5A). Similarly, cationic adducts attaching
FEBS Journal 272 (2005) 3803–3815 ª 2005 FEBS

water in the positive ion mode were detected among
C-terminal oligosaccharides using GA as matrix
(Fig. 3B, peaks at: m ⁄ z 1054.8, 1217.0 and 1379.7;

Table 2). In order to discard the complex nature of the
acidic oligosaccharides, treatment of another sample of
the C-terminal oligosaccharides with endo-b-galactosidase was performed. The spectra obtained (Fig. 5B),
showed a pattern of signals similar to that of the
untreated sample (Fig. 5A), assuring the high-mannose
nature of the sulfated glycans.
3807


3808

1176.4

1338.7

1500.6

1663.6

1825.1

1175.9

1338.1

1500.2

1662.4

1824.5

a

1980.7

1865.5

1703.4

1541.2

1379.1

1216.9

1054.8

Calculated m ⁄ z a
[M+H2O+Na]+

Sulfates were present as the sodium salt.

–

1013.9

1013.8

1961.5

Measured
m ⁄ z [M+Na]+

Calculated
m ⁄ z [M+Na]+

–

–

1703.5

1541.6

1379.7

1217.0

1054.8

Measured m ⁄ z a
[M+H2O+Na]+

1800.5

HexNAc2Man8 + SO4

1962.7

1638.4

HexNAc2Man7 + SO4

HexNAc2Man9 + SO4

1476.2

HexNAc2Man6 + SO4

1314.1

HexNAc2Man5 + SO4

989.8

Calculated
m ⁄ z [M–H]–

1151.9

Structure

HexNAc2Man4 + SO4

HexNAc2Man3 + SO4

Proposed
composition

1963.7

1801.5

1639.4

1476.6

1314.1

1152.8

990.0

Measured
m ⁄ z [M–H]–

1980.7

1818.5

1656.4

1494.2

1332.1

1170.0

1007.0

Calculated m ⁄ z
[M+H2O–H]–

Table 2. m ⁄ z-Value, composition and structure of sulfated oligosaccharides of cruzipain and C-terminal domain. h, N-Acetylglucosamine; d, mannose ; q -SO4.

1981.5

1819.1

1656.9

1494.7

1332.4

1170.5

1007.6

Measured m ⁄ z
[M+H2O–H]–

Structure of the N-glycans in cruzipain
M. Barboza et al.

FEBS Journal 272 (2005) 3803–3815 ª 2005 FEBS


M. Barboza et al.

Structure of the N-glycans in cruzipain

1

A

2

25000
20000
15000

Radioactivity (CPM)

10000
5000
0
4

8

12

16

B

25000
20000
15000

m ⁄ z 1656.2 assigned to sulfated HexNAc2Man7 in relation with signal at m ⁄ z 1310.1 decreased more than
60% in the solvolysis treated sample and completely
disappeared after the enzymatic treatment (Fig. 6B). It
is interesting to point out that although the background signals shown in Fig. 6B were not assigned,
they were used as internal reference to express relative
abundance of the 1493.9, 1656.2, 1818.5 and 1980.3
peaks. In addition, the released sulfate ion was identified by ion chromatography using conductivity detection (Fig. 6D, as an inset in Fig. 6B).
Altogether, these data confirm the presence of sulfated high-mannose type oligosaccharides in cruzipain
and in its C-terminal domain. This is the first report of
the use of nor-harmane as matrix for structural characterization of N-linked oligosaccharides of glycoproteins.

10000

Discussion

5000
0
4

8

12

16

Time (min)
Fig. 4. HPAEC analysis of the a-L-fucose released from the C-terminal domain of cruzipain. (A) Purified C-terminal domain was
digested with a-L-fucosidase. The released sugar was labeled by
reduction with NaB3H4 desalted and analysed by HPAEC under

conditions c. (B) Same as (A) without enzyme. Standards: 1, fucitol;
2, sorbitol.

In another experiment, oligosaccharides obtained
from cruzipain were analysed using nor-harmane as
matrix in the negative ion mode (Fig. 6). Accordingly,
signals corresponding to sulfated glycans compatible
with [M + H2O-H]– ions were shown (m ⁄ z 1170.6,
1331.8, 1493.9, 1656.2, 1818.5 and 1980.3) (Table 2,
Fig. 6A). The corresponding adducts in the positive
ion mode, [M + H2O + Na]+, were also detected
when cruzipain oligosaccharides were analysed using
GA as matrix (Fig. 3A, peaks at m ⁄ z 1217.0, 1379.2,
1541.6 and 1703.5; Table 2). In order to confirm the
nature of the substitution present in glycans obtained
from cruzipain, desulfation was carried out using sulfatase. The UV-MALDI-TOF spectra of the treated
sample, obtained in the negative ion mode using
nor-harmane as matrix, showed the complete disappearance of the aforementioned signals (Fig. 6B).
Furthermore, when desulfation of the released oligosaccharides was performed by solvolysis, although
treatment was not complete a significant reduction of
those signals was observed (Fig. 6C). It should be
noted that the relative abundance of the major peak at
FEBS Journal 272 (2005) 3803–3815 ª 2005 FEBS

In this study we have examined the structures of
the N-linked oligosaccharides present in cruzipain.
HPAEC analysis of the total neutral oligosaccharide
fraction obtained from the C-terminal domain
showed a mixture of different types of carbohydrate
chains as previously reported [12,14]. However, when

the acidic glycans were analyzed the results obtained
pointed to the presence of sulfated oligosaccharides
in addition to the sialylated glycans previously reported [14]. The fact that part of the labeled oligosaccharides were bound to a QAE-Sephadex column
and eluted with 2 mm Tris-base ⁄ 20 mm NaCl also
agreed with the presence of sulfate groups in this
fraction [24].
UV-MALDI-TOF MS analysis demonstrated that
the overall glycosylation pattern of cruzipain is characterized by a remarkable structural diversity. There is
no doubt that oligosaccharides of cruzipain are mainly
of the high-mannose type. However, it is interesting to
note that lactosaminic glycans bearing from 2 to 4
lactosamine units are also present, a feature that had
not been detected before as component of T. cruzi glycoproteins. The fact that these signals were found in
the C-terminal UV-MALDI-TOF mass spectra confirms their localization in this domain as previously
suggested [12]. Taking into account that cruzipain
bears sialic acid in N-linked structures [14] which can
only be acquired at the cell surface through the action
of trans-sialidase [25], the finding that polylactosaminic
units are also present in this enzyme triggers the possibility that cruzipain would be transported via an endocytic recycling and ⁄ or lysosomal transport pathway as
proposed for T. brucei [26,27].
3809


Structure of the N-glycans in cruzipain

A 100

M. Barboza et al.

1494.7

1656.9

Relative abundance (%)

1332.4

1819.1

1476.6
1170.5

1639.4

1314.1

50

1801.5

1007.6 1152.8
990.0

1981.5

1963.7

Relative abundance (%)

B 100

1494.3
1332.1

50
1152.1 1312.1
1172.1

1656.8

1476.2

1818.3

1639.3
1800.9

1981.1
1962.1

800

1200

1600

2000
Mass (m/z)

In addition, a signal compatible with a fucosylated
structure was also found in the C-terminal domain.

The existence of the a-l-fucose unit in C-terminal was
further supported by HPAEC analysis. Up to now,
two surface glycoproteins of T. cruzi have been described containing a-l-fucose in their structure: Gp-72,
isolated from epimastigote forms [28,29] and the trypomastigote stage specific glycoprotein belonging to the
Tc-85 family [30]. However, in T. cruzi, no fucosyl
transferase has been reported so far.
Although their low abundances, signals corresponding to monosialylated oligosaccharides could be detected in the positive-ion mode without derivatization
using GA as matrix compatible with [M + Na]+
adducts. The main proposed structure was assigned to
a biantennary complex oligosaccharide, in accordance
with the results obtained by HPAEC. However, relative abundances of these peaks showed variations from
batch to batch.
It is known that a serious limitation in the study of
sulfated oligosaccharides are the few reliable analytical
methods of structural characterization [31]. For that
reason, in the last years, the development of new matrices have made UV-MALDI-TOF MS a suitable tool for
3810

2400

2800

Fig. 5. UV-MALDI-TOF MS analysis of the
oligosaccharides released from C-terminal
domain by PNGase F treatment in the linear
negative-ion mode using nor-harmane as
matrix. (A) Analysis of the total oligosaccharide fraction, m ⁄ z range: 800–2800 Da.
(B) Same as (A) after endo-b-galactosidase
treatment. Structures are detailed in
Table 2.

their analysis [20,32,33]. Nor-harmane was optimal for
the analysis of sulfated N-linked oligosaccharides in
negative ion mode because it provided not only good
detection sensitivity, but also no interference with the
matrix adduct ions. The conditions used allowed the
production of intense signals without any desulfation.
Signals corresponding to sulfated GlcNAc2Man3 to
GlcNAc2Man9 glycans were identified. Interestingly,
the major signals were attributed to [M + H2O-H]–
adducts. The water adducts (M + H2O) were also
found in the positive ion mode cationized by sodium
(M + H2O + Na)+ using either GA or nor-harmane
as matrix, in different samples (oligosaccharides from
C-terminal domain or from the whole protein) and using
different equipment. Therefore, the retention of water
can be explained taking into account its strong interaction with the negative charge site of the sulfated analytes
[33,34]. The fact that the resulting signals were totally
resistant to endo-b-galactosidase digestion allowed us to
discard the complex structure of the sulfated species. On
the other hand, considering that when the core glycan
structure is substituted, the modifications are present on
the NAc-glucosamine unit, the detection of the signals
at m ⁄ z-value 990 and 1007.6 in the C-terminal spectrum
FEBS Journal 272 (2005) 3803–3815 ª 2005 FEBS


M. Barboza et al.

Structure of the N-glycans in cruzipain

1656.2

A 100
90

Relative abundance

80
1818.5

70
60
50

1493.9

40
30

1331.8
1170.6 1310.8

20

1980.3
1598.6
1893.6

10

0

1000

1200

1400

1600

1800

2000

2200

2400

B 100
90

D

60
50
40
30

1310.1
1195.8

20
10
1000

1200

1400

1531.0
1622.0 1750.3

1600

1800

SO4

4.18

70

0

PO4

Conductivity(µs)

Relative abundance

80

Time (min)

2000

2200

2400

2200

2400

C 100
90

Fig. 6. UV-MALDI-TOF MS analysis of the
oligosaccharides released from cruzipain
domain by PNGase F treatment in the linear
negative-ion mode using nor-harmane as
matrix. (A) Analysis of the total oligosaccharide fraction, m ⁄ z range: 900–2500 Da. (B)
Same as (A) after sulfatase treatment. (C)
Same as (A) after solvolysis. (D) Ion chromatography analysis of sulfate released from
oligosaccharides obtained from cruzipain.

Relative abundance

80
70

60
1656.9

50
40

1310.1
1195.8

30

1818.5
1893.6

20
10
0
1000

and the growing sulfated high-mannose series suggest
that the sulfate group should be located on the chitobiosyl core (Fig. 5A).
The presence of sulfate groups in N-linked oligosaccharides has been reported in virus [35] and especially in
mammalian cells [36–40]. However, these reports are
mostly based on the results of radioisotope labeling and
there are only a few reports on the detailed structure
of these sulfated glycans. Such oligosaccharides usually

FEBS Journal 272 (2005) 3803–3815 ª 2005 FEBS

1493.9

1598.6

1200

1400

1600

1800
m/z

2000

sulfated on galactose, mannose, N-acetyl galactosamine,
N-acetyl glucosamine or glucuronic acid residues have
been implicated in several specific molecular recognition
processes [41,42]. In T. cruzi, sulfated structures have
been described as part of glycolipids [43,44]. The present
study constitutes the first report on the presence of
sulfated oligosaccharides in glycoproteins of T. cruzi.
Sulfated high-mannose type glycans have only
been described as component of glycoproteins from
3811


Structure of the N-glycans in cruzipain

Dictyostelium discoideum [24]. Likewise, these glycoproteins are localized in lysosome, however, Man6-SO4
accounts for the majority of the sulfated sugar.
In conclusion, the results obtained provide evidence

of the nature of the glycans present in the unique N-glycosylation site present in the C-terminal domain of cruzipain (Asn255). This site is mainly occupied by neutral
or sulfated high-mannose type oligosaccharides. To a
minor extent, biantennary lactosaminic chains, some of
them bearing sialic acid, or fucose are also present. The
finding of sulfated glycans indicates the activity of a
sulfotransferase which has not been described in T. cruzi
to date. In the present study, the precise location of
the sulfate group and its biological significance remain
to be established. Studies to address these questions are
currently in progress in our laboratory.

Experimental procedures
Materials
All solvents used were of analytical or HPLC grade. Ultra
free-MC centrifugal filter units Amicon Bio separations
were from Millipore Corporation (Bedford, MA, USA).
Radioactivity was determined in a 1214 Rackbeta Wallac
liquid scintillation counter using Optiphase’Hisafe 3 scintillation cocktail (LKB). Lowry’s method [15] was used to
quantify protein content. Neutral glycan standards were
obtained from Oxford Glyco System (Abingdon, UK) and
sialylated oligostandards obtained from fetuin were from
Dionex Corporation (Dionex Corporation, Sunnyvale, CA,
USA). All chemicals used in UV-MALDI-TOF MS analysis
were ACS grade or higher.

Purification of the C-terminal domain
Highly purified cruzipain was obtained by a procedure
using chromatography on Con-A Sepharose and Mono Q
[8]; fractions strongly bound to the anionic resin, eluted
with 0.25–0.50 m NaCl were used. The C-terminal domain

was obtained by self-proteolysis of cruzipain in sodium
acetate buffer pH 6.0 at 40 °C for 48 h. The C-terminal
domain was purified by gel filtration in a Bio Gel P-30
column (1.5 · 100 cm) eluted with Tris ⁄ HCl buffer pH 7.6
containing 50 mm NaCl. Fractions of 1 mL were collected
and monitored by measuring UV absorption at
280 ⁄ 230 nm. A sample of each fraction was analysed by
SDS ⁄ PAGE followed by silver staining or electroblotting,
developing with anticruzipain polyclonal antibody [14].

Mild acid hydrolysis
Sialic acid was hydrolysed with 0.01 m HCl for 20 min at
100 °C and freeze-dried. For fucose analysis hydrolysis was

3812

M. Barboza et al.

performed with 0.1 m HCl for 2 h at 100 °C. Samples were
freeze dried, dissolved in water (0.5 mL), the solution was
adjusted to pH 8 and labeled with NaB3H4 (0.12 mCi) for
3 h at room temperature. Reduction was completed with
NaBH4 for 2 h more and the reaction was stopped by addition of acetic acid to pH 5.

Solvolysis
Samples were passed over 0.5 mL of AG50W-X8 resin
(H+) and the column was washed with water (2 mL). Pyridine (0.015 mL) was added to the sample, which was then
lyophilized, dissolved in dimethylsulfoxide ⁄ methanol (9 : 1,
v ⁄ v; 0.2 mL), adjusted to pH 4 with dilute HCl, heated at
100 °C for 2 h and freeze-dried [16].

High pH anion exchange chromatography
(HPAEC)
For HPAEC analysis the released monosaccharides or
oligosaccharides were labelled by reduction with NaB3H4.
Boric acid was removed by repeated coevaporations with
methanol and the labelled oligosaccharides were desalted
by passage through a Bio-Gel P-2 column [14]. A
DX-300 Dionex BioLC system (Dionex Corporation) with
a pulse amperometric detector was used. The following
columns and conditions were employed: (a) Carbopack
PA-100 column equipped with a PA-100 precolumn; gradient elution with 50 mm NaOH and 0–50 mm sodium
acetate during 40 min. The flow rate was 0.6 mLỈmin)1;
(b) Carbopack PA-100 column equipped with a PA-100
precolumn; isocratic elution with 100 mm NaOH ⁄ 50 mm
sodium acetate for 5 min, followed by a gradient elution
with 100 mm NaOH and 50–170 mm sodium acetate during 60 min. The flow rate was 1 mLỈmin)1; and (c) Carbopack MA-1 column equipped with a MA-1 precolumn
and an isocratic elution with 25% solution A (NaOH
200 mm), 75% solution B (water). The flow rate was
0.4 mLỈmin)1.
Ion chromatography analysis was performed on a Dionex
AS4A column using 1.8 mm Na2CO3 ⁄ 1.7 mm NaHCO3
as eluent, with postcolumn in-line anion micromembrane
suppression and conductivity detection. The flow rate was
2 mLỈmin)1 [17].

Enzymatic digestions
PNGase F digestion was performed in 10 mm Tris ⁄ HCl
buffer pH 8.3, containing PNGase F (New England Biolabs
Inc., Beverly, MA, USA) (15 mU). The oligosaccharides

were separated from the protein by Ultrafree McFilters
(MW 5000).
Endo-b-d-galactosidase digestion was performed in
50 mm sodium acetate pH 6.0 with 8 mU of B. fragilis

FEBS Journal 272 (2005) 3803–3815 ª 2005 FEBS


M. Barboza et al.

endo-b-d-galactosidase (Oxford Glyco System, Abingdon,
UK) containing 250 lgỈmL)1 bovine serum albumine and
1 mm NaCl.
a-l-Fucosidase digestion was performed in 30 mm
sodium phosphate pH 5.6 containing 14 mU of b-l-fucosidase from bovine kidney (Sigma-Aldrich Co., St Louis,
MO, USA).
Sulfatase digestion was performed in 50 mm sodium acetate pH 5.0 with sulfatase from Abalone entrailis (Type VII.
Sigma-Aldrich Co., St Louis, MO, USA) (25 mU).
All digestions were performed for 18 h at 37 °C.
Before UV MALDI-TOF MS analysis, the oligosaccharides were treated with 150 mm acetic acid for 3 h at room
temperature [18] and desalted through a microcolumnn
containing AG-50 (H+) [19].

UV-MALDI-TOF mass spectrometry
Measurements were performed with an Applied Biosystems
Voyager DE-STR (Applied Biosystems, Foster City, CA,
USA) laser-desorption time-of-flight mass spectrometer
equipped with UV-nitrogen laser (337 nm) and an AXIMA-CFR plus (Shimadzu Biotech, Manchester, UK). All
spectra were acquired in the linear and reflectron modes, in
both positive- and negative-ion modes at an accelerating

voltage of 20 kV, grid voltage 95%, guide wire 0.05 and
delay time 400 ns; 30–150 shots were averaged for each
spectrum. Gentisic acid (2,5-dihydroxybenzoic acid, GA),
nor-harmane (9H-pirido-[3,4]-b-indole) [20–22] were used as
matrices. The best results were obtained in positive-ion
mode with GA and in negative-ion mode with nor-harmane. Matrix solutions were made by dissolving 2 mg of
the selected compound in 1 mL of MeOH ⁄ H2O (1 : 1, v ⁄ v).
Analyte solutions were freshly prepared by dissolving the
carbohydrates in water. The analyte-matrix deposit was
prepared following the thin-film layer method; 0.5 lL of
the matrix solution was placed on the sample probe tip and
the solvent removed by blowing air at room temperature.
Subsequently, 0.5 lL of the analyte solution was placed on
the same probe tip covering the matrix and partially dissolving it, and the solvent was removed by blowing air. Then,
two additional portions (0.5 lL) of the matrix solution
were deposited on the same sample probe tip producing a
partial dissolution of the previously deposited thin-film
matrix and analyte layers. The matrix to analyte ratio was
3 : 1 (v ⁄ v) and the matrix and analyte solution loading
sequence was matrix, analyte, matrix, matrix.
External calibration was performed with: insulin
(10 pmolỈlL)1 in H2O 0.1% trifluoroacetic acid) and ribonuclease [10 pmolỈlL)1 in H2O 0.1% (v ⁄ v) trifluoroacetic
acid] using sinapinic acid (1 mg per 0.04 mL MeCN plus
0.06 mL H2O 0.1% (v ⁄ v) trifluoroacetic acid as matrix];
b-cyclodextrine (10 pmolỈlL)1 in H2O) using nor-harmane
as matrix [2 mgỈmL)1 MeOH ⁄ H2O (1 : 1, v ⁄ v)]; bradykinin
[10 pmolỈlL)1 in H2O 0.1% (v ⁄ v) trifluoroacetic acid] and

FEBS Journal 272 (2005) 3803–3815 ª 2005 FEBS

Structure of the N-glycans in cruzipain

angiotensin II [10 pmolỈlL)1 in H2O 0.1% (v ⁄ v) trifluoroacetic acid] using CHCA [10 mgỈmL)1 in 50% (v ⁄ v)
MeCN ⁄ H2O 0.1% (v ⁄ v) trifluoroacetic acid], in positive
and negative ion modes. The following glycans were used
as standards: Man6GlcNAc2 (Oxford Glyco System) and
oligosaccharides obtained from urine follicle-stimulating
hormone (uFSH) (Massone S.A., Argentina).

Acknowledgements
The authors are indebted to the National Research
Council of Argentina (CONICET, PIP ⁄ 904 and
PIP ⁄ 447), the University of Buenos Aires (UBA, 022
´
and 218), Agencia Nacional de Promocion Cientı´ fica y
´
Tecnologica, Argentina (ANPCyT, BID 1201 ⁄ OC-AR
Pict 12312 and 06–06545) and UNDP ⁄ World Bank ⁄
WHO Special Programme for Research and Training
in Tropical Diseases (TDR; Project ID 970629) for
partial financial support. J. J. Cazzulo, A. S. Couto,
V. G. Duschak and R. Erra-Balsells are Research
Members of CONICET. M. Barboza has a fellowship
from the same Institution. UV-MALDI-TOF MS
experiments were performed: (a) as part of the Academic Agreement between REB (FCEyN-UBA, Argentina) and HN (CA-EU, Japan) with the facilities of the
High Resolution Liquid Chromatography-integrated
Mass Spectrometry System of the United Graduated
School of Agricultured Sciences (EU, Japan) and (b)
thanks to the Shimadzu Corporation with the facilities
of the Koichi Tanaka Mass Spectrometry Research

Laboratory (Kyoto, Japan).

References
1 Franke de Cazzulo BM, Martinez J, North MJ, Coombs GH & Cazzulo JJ (1994) Effects of proteinase inhibitors on growth and differentiation of Trypanosoma
cruzi. FEMS Microbiol Lett 124, 81–86.
2 Bontempi E, Martinez J & Cazzulo JJ (1989) Subcellular localization of a cystein proteinase from Trypanosoma cruzi. Mol Biochem Parasitol 33, 43–47.
3 Murta ACM, Persechini PM, de Souto-Padron T, De
Souza W, Guimaraes JA & Scharfstein J (1990) Structural and functional identification of GP57 ⁄ 51 antigen
of Trypanosoma cruzi as a cystein proteinase. Mol Biochem Parasitol 43, 27–38.
´
4 Souto-Padron T, Campetella O, Cazzulo JJ & de Souza
W (1990) Cystein proteinase in Trypanosoma cruzi:
immunocytochemical localization and involvement in
parasite–host cell interaction. J Cell Sci 96, 485–490.
5 Parussini F, Duschak VG & Cazzulo JJ (1998) Membrane-bound cysteine proteinase isoforms in different
developmental stages of Trypanosoma cruzi. Cell Mol
Biol 44, 513–519.

3813


Structure of the N-glycans in cruzipain

6 Coombs GH & Mottram JC (1997) Trypanosomiasis
and Leishmaniasis (Hide G & Mottram JC, Coombs
GH & Holmes PH, eds.), pp. 177–197. CAB International, Oxford, UK.
7 Cazzulo JJ, Stoka V & Turk V (1997) Cruzipain, the
major cystein proteinase from the protozoan parasite
Trypanosoma cruzi. Biol Chem 378, 1–10.
8 Cazzulo JJ, Labriola C, Parussini F, Duschak VG, Martinez J & Franke de Cazzulo BM (1995) Cystein proteinases in Trypanosoma cruzi and other Trypanosomatid

parasites. Acta Chimica Slovenica 42, 409–418.
9 Campetella O, Henriksson J, Aslund L, Frasch ACC,
Pettersson U & Cazzulo JJ (1992) The major cystein
proteinase (cruzipain) from Trypanosoma cruzi is
encoded by multiple polymorphic tandemly organized
genes located on different chromosomes. Mol Biochem
Parasitol 50, 225–234.
10 Martinez J, Campetella O, Frasch AC & Cazzulo JJ
(1993) The reactivity of sera from chagasic patients
against different fragments of cruzipain, the major
cystein proteinase of Trypanosoma cruzi, suggests the
presence of defined antigenic and catalytic domains.
Immunol Lett 35, 191–196.
11 Cazzulo JJ, Martı´ nez J, Parodi AJ, Wernstedt C & Hellman U (1992) On the post-translational modifications at
the C-terminal domain of the major cystein proteinase
(cruzipain) from Trypanosoma cruzi. FEMS Microbiol
Lett 100, 411–416.
12 Parodi AJ, Labriola C & Cazzulo JJ (1995) The presence of complex- type oligosaccharides at the C-terminal domain glycosylation site of some molecules of
cruzipain. Mol Biochem Parasitol 69, 247–255.
13 Metzner SI, Souza MC, Hellman U, Cazzulo JJ &
Parodi AJ (1996) The use of UDP-Glc: glycoprotein
glucosyltransferase for radiolabelling protein-linked high
mannose-type oligosaccharides. Cell Mol Biol 42,
631–635.
14 Barboza M, Duschak VG, Cazzulo JJ, Lederkremer RM
& Couto AS (2003) Presence of sialic acid in N-linked
oligosaccharide chains and O-linked N-acetylglucosamine
in cruzipain, the major cysteine proteinase of Trypanosoma cruzi. Mol Biochem Parasitol 126, 293–296.
15 Lowry OH, Rosebrough NJ, Farr AL & Randall RJ
(1951) Protein measurement with Folin phenol reagent.

J Biol Chem 193, 265–275.
16 Freeze HH, Yeh R, Miller AL & Kornfeld S (1983)
Structural analysis of the Asparagine-linked oligosaccharides from free lysosomal enzymes of Dictyostelium
discoideum. J Biol Chem 258, 14874–14879.
17 Bousfield GR, Baker VL, Gotschall RR, Butnev VY &
Butnev VY (2000) Carbohydrate analysis of glycoprotein hormones. Methods 21, 15–39.
18 Papac DI, Briggs JB, Chin ET & Jones AJS (1998) A
high throughput microscale method to release N-linked
oligosaccharides from glycoproteins for matrix-assisted-

3814

M. Barboza et al.

19

20

21

22

23

24

25

26

27

28

29

30

laser desorption ⁄ ionization time of flight mass spectrometric analysis. Glycobiology 8, 445–454.
Kuster B, Wheeler SF, Hunter AP, Dwek RA & Harvey DJ (1997) Sequencing of N-linked oligosaccharides
directly from protein gels: in-gel deglycosylation
followed by matrix-assisted laser desorption ⁄ ionization
mass spectrometry and normal-phase high-performance
liquid chromatography. Anal Biochem 250, 82–101.
Nonami H, Fukui S & Erra-Balsells R (1997) b-carboline alkaloids as matrices for matrix-assisted ultraviolet
laser desorption time-of-flight mass spectrometry of proteins and sulfated oligosaccharides: a comparative study
using phenylcarbonyl compounds, carbazoles and classical matrices. J Mass Spectrom 32, 287–296.
Nonami H, Tanaka K, Fukuyama Y & Erra-Balsells R
(1998) B-carboline alkaloids as matrices for UV-matrixassisted laser desorption ⁄ ionization time of flight mass
spectrometry in positive and negative ion modes. Analysis of proteins of high molecular mass, and of cyclic and
acyclic oligosaccharides. Rapid Commun Mass Spectrom
12, 285–296.
Erra-Balsells R & Nonami H (2002) Nor-Harmane
(9H-pyrido [3,4-b]indole) as outstanding matrix for UVmatrix-assisted laser desorption ⁄ ionization time-of-flight
mass spectrometry analysis of synthetic and biopolymers. Environ Control Biol 40, 55–73.
Duschak VG, Barboza M & Couto AS (2003) Trypanosoma cruzi: partial characterization of minor cruzipain
isoforms non-adsorbed to concanavalin A-Sepharose.
Exp Parasitol 104, 122–130.
Freeze HH & Wolgast D (1986) Structural analysis of
the N-linked oligosaccharides glycoproteins secreted by

Disctyostelium discoideum. Identification of mannose-6sulfate. J Biol Chem 261, 127–134.
Frasch ACC (2000) Functional diversity in the transsialidase and mucin families in Trypanosoma cruzi.
Parasitol Today 16, 282–286.
McConville MJ, Mullin KA, Ilgout SC & Teasdale RD
(2002) Secretory pathway of trypanosomatid parasites.
Microbiol Mol Biol Rev 66, 122–154.
Nolan DP, Geuskens M & Pays E (1999) N-linked
glycans containing linear polyN-acetyllactosamine as
sorting signals in endocytocis in Trypanosoma brucei.
Curr Biol 9, 1169–1172.
Snary D, Ferguson MAJ, Scott MT & Allen AK (1981)
Cell surface antigens of Trypanosoma cruzi: use of
monoclonal antibodies to identify and isolate an epimastigote specific glycoprotein. Mol Biochem Parasitol
3, 343–358.
Ferguson MAJ, Allen AK & Snary D (1983) Studies
on the structure of a phosphoglycoprotein from the
parasitic protozoan Trypanosoma cruzi. Biochem J 213,
313–320.
Couto AS, Colli W, Goncalves MF & Lederkremer.
¸
RM (1990) The N- linked carbohydrate chain of the

FEBS Journal 272 (2005) 3803–3815 ª 2005 FEBS


M. Barboza et al.

31

32

33

34

35

36

37

Tc-85 glycoprotein from Trypanosoma cruzi. trypomastigotes contains sialyl, fucosyl and galactosyl a-(1–3)
galactose units. Mol Biochem Parasitol 39, 101–108.
Dai Y, Whittal RM, Bridges CA, Isogai Y, Hindsgaul
O & Li L (1997) Matrix-assisted laser desorption ionization mass spectrometry for the analysis of monosulfated
oligosaccharides. Carbohydr Res 304, 1–9.
Wheeler SF & Harvey DJ (2001) Extension of the
in-gel release method for structural analysis of neutral
and sialylated N-linked glycans to the analysis of sulfated glycans: application to the glycans from bovine
thyroid-stimulating hormone. Anal Biochem 296,
92–100.
Fukuyama Y, Ciancia M, Nonami H, Cerezo A, Erra
Balsells R & Matulewicz MC (2002) Matrix-assisted
ultraviolet-laser desorption ⁄ ionization and electrospray
ion ionization time-of-flight mass spectrometry of
sulfated neocarrabiose oligosaccharides. Carbohydr Res
337, 1553–1562.
Karas M, Gluckmann M & Schafer J (2000) Ionization
in matrix-assisted laser desorption ⁄ ionization: singly
charged molecular ions are the lucky survivors. J Mass

Spectrom 35, 1–12.
Bernstein HB & Compans RW (1992) Sulfation of the
human immunodeficiency virus envelope glycoprotein.
J Virol 66, 6953–6959.
Van Rooijen JJ, Kamerling JP & Vliegenthart JF (1998)
Sulfated di-, tri- and tetraantennary N-glycans in human
Tamm-Horsfall glycoprotein. Eur J Biochem 256,
471–487.
Kawasaki N, Haishima Y, Ohta M, Itoh S, Hyuga M,
Hyuga S & Hayakawa T (2001) Structural analysis of

FEBS Journal 272 (2005) 3803–3815 ª 2005 FEBS

Structure of the N-glycans in cruzipain

38

39

40

41

42
43

44

sulfated N-linked oligosaccharides in erythropoietin.
Glycobiology 11, 1043–1049.

Green ED, van Halbeek H, Boime I & Baenziger JU
(1985) Structural elucidation of the disulfated oligosaccharide from bovine lutropin. J Biol Chem 260, 15623–
15630.
Noguchi N & Nakano M (1992) Structure of the acidic
N-linked carbohydrate chains of the 55-kDa glycoprotein family (PZP3) from porcine zona pellucida. Eur J
Biochem 213, 39–56.
Pierce M & Arango J (1986) Rous sarcoma virus-transformed baby hamster kidney cells express higher levels
of asparagine-linked tri- and tetraantennary glycopeptides containing [GlcNAc-beta(1,6)Man-alpha(1,6)Man]
and poly–N–acetyllactosamine sequences than baby
hamster kidney cells. J Biol Chem 261, 10772–10777.
Kawasaki N, Ohta M, Hyuga S, Hashimoto O & Hayakawa T (2000) Application of liquid chromatography ⁄ mass spectrometry and liquid chromatography with
tandem mass spectrometry to the analysis of the sitespecific carbohydrate heterogeneity in erythropoietin.
Anal Biochem 285, 82–91.
Honke K & Taniguchi N (2002) Sulfotransferases and
sulfated oligosaccharides. Med Res Rev 22, 637–654.
Petry K, Nudelman E, Eisen H & Hakomori S (1988)
Sulfated lipids represent common antigens on the surface of Trypanosoma cruzi and mammalian tissues. Mol
Biochem Parasitol 30, 113–121.
Uhrig ML, Couto AS, Zingales B, Colli W & Lederkremer RM (1992) Metabolic labelling and partial characterization of a sulfoglycolipid in Trypanosoma cruzi
trypomastigotes. Carbohydr Res 231, 329–334.

3815