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

The UV-MALDI-TOF MS analysis in the negative-ion mode, using nor-harmane as matrix, allowed us to determine a new striking feature in cruzipain: sulfated high-mannose type oligosaccharid

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 Cazzulo1and Alicia S Couto4

1 Instituto de Investigaciones Biotecnolo´gicas-INTECH, Universidad Nacional de Gral San Martin, Buenos Aires, Argentina

2 Instituto Nacional de Parasitologı´a ‘Dr Mario Fatala Chabe´n’, ANLIS, Ministerio de Salud y Ambiente, Buenos Aires, Argentina

3 College of Agriculture, Ehime University, Matsuyama, Japan

4 CIHIDECAR (CONICET) Departamento de Quı´mica Orga´nica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina

Trypanosoma cruzi, the parasitic protozoan that causes

the American Trypanosomiasis or Chagas disease,

con-tains a major cysteine proteinase (CP), cruzipain This

enzyme is present in the epimastigote, amastigote,

meta-cyclic and tissue culture trypomastigote forms [1] It

has been reported to be placed in the lysosomal

com-partment [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 con-tain no introns, encoding a signal peptide, a propep-tide and a mature enzyme As for all Type I CPs from trypanosomatids, the protein presents a catalytic

Keywords

Cruzipain; nor-harmane; sulfated

oligosaccharides; Trypanosoma cruzi;

UV-MALDI-TOF MS

Correspondence

A S Couto, CIHIDECAR (CONICET)

Departamento de Quı´mica Orga´nica,

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: acouto@qo.fcen.uba.ar

*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 post-translational modifications, and most antibodies in natural and experimen-tal infections are directed against it In this report we took advantage of UV-MALDI-TOF mass spectrometry in conjunction with peptide N-gly-cosidase F deglycosylation and high performance anion exchange chroma-tography analysis to address the structure of the N-linked oligosaccharides present in this domain The UV-MALDI-TOF MS analysis in the negative-ion 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 detec-ted The presence of the deoxy sugar was further confirmed by high per-formance 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

Abbreviations

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

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

amino-acid 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

post-translational 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

high-mannose type oligosaccharides It had been suggested

that the C-terminal domain of cruzipain presents either

a high-mannose oligosaccharide, a hybrid

monoanten-nary or a complex biantenmonoanten-nary oligosaccharide chain

[12] In vivo labeling of the parasites with32Pidiscounted

the presence of Piin the mature enzyme [11]

Recently, two types of post-translational

modifica-tions 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

diges-tions 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

pres-ence 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

microhetero-geneities found in natural cruzipain

Results

In an attempt to perform a structural study of the

oligosaccharide chains localized in the C-terminal

domain of cruzipain, the protein was subjected to self-proteolysis 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-glycosi-dase F (PNGase F) treatment The released oligo-saccharides were separated from the polypeptide by Ultrafree McFilters (MW 5000) A fraction of these oligosaccharides was reduced with NaB3H4and

desalt-ed by Biogel P-2 as already describdesalt-ed [23] Labeldesalt-ed 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 (Rt¼ 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 desialyl-ated fraction was analysed under conditions where neutral glycans are resolved, a peak coincident with a standard of a biantennary complex-type oligosaccha-ride 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 per-formed and the profile obtained by HPAEC (Fig 2D) showed the disappearance of the peak with Rt¼ 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

66

45

29

kDa

Fig 1 Analysis by SDS ⁄ PAGE followed by silver stainning of C-ter-minal domain purification Lane 1, natural cruzipain; lane 2, self-pro-teolysed 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.

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

pres-ence 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

ana-lysis was performed Figure 3A shows the positive

UV-MALDI spectrum of the whole oligosaccharide

fraction released from cruzipain by PNGase F

diges-tion 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

mono-sodium adducts [M + Na]+ Although the quality of

the spectra (signal⁄ noise ratio) obtained for each

sam-ple 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

Glc-NAc2Man9) (Table 1) Interestingly, signals at m⁄

z-values 2029.5 (2029.9), 2069.1 (2069.3) and 2395.4

(2395.1) compatible with lactosaminic type

oligosac-charides, not reported so far as components of

cruzi-pain, 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-man-nose species (Table 2) Some of these peaks were also

observed in the cruzipain spectrum In addition, a

sig-nal 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

HPAEC-PAD analysis The sugar was released from the

C-ter-minal domain by specific a-l-fucosidase treatment,

separated through Ultrafree McFilters (MW 5000),

labeled by reduction with NaB3H4and analysed under Retention time (min)

4

125 225 325

425

CPM

100 200 300 400

500

CPM

1

100 200 300 400

500

CPM

100 200 300 400

500

CPM

A

B

C

D

E

100 200 300

400

CPM

Fig 2 HPAEC analysis of the oligosaccharides released by PNGase

F treatment from the C-terminal domain of cruzipain (A) Total

neut-ral fraction; (B) total acidic fraction; (C) acidic fraction treated with

mild acid to release sialic acid; (D) acidic fraction treated with

sulfa-tase; (E) same as (C) (A) and (E), analysis under ‘conditions a’ for

neutral glycan; (B), (C) and (D), analysis under ‘conditions b’ for

aci-dic glycans Standards: 1, Man3GlcNAc2OH; 2, Man5GlcNAc2OH; 3,

Man6GlcNAc2OH; 4, Gal2GlcNAc2Man3GlcNAc2OH; 5, Man9

Glc-NAc 2 OH; 6, monosialylated; 7, disialylated; 8, trisialylated

oligosac-charides.

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

oligosaccha-rides were detected in the C-terminal spectrum

(Fig 3B, Table 1)

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

1013.9

1258.3

1096.1

1420.4 933.7

1582.7

1744.8

*

1500.6 1338.7

1176.4

2029.9 1809.6

8 X

2395.1 1968.6

2029.9

100

90

80

70

60

50

40

30

20

10

m/z

1906.6

1744.5

1581.7 1420.4

1013.9

1259.1

1843.1 1338.7

1541.6

1663.5

2029.5 1923.2 933.2

2069.1

2395.4

1217.0

1703.5 1379.2

1094.8

10

20

30

40

50

60

70

80

90

100

A

B

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 corres-ponds to expanded m ⁄ z range: 1950–2460 Da Structures are detailed in Tables 1 and 2.

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

com-patible with [M + H2O-H]– ions of the same glycans

(Table 2; Fig 5A) Similarly, cationic adducts attaching

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-galactosi-dase 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

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

933.31

933.78

933.2 933.7

HexNAc2Man3 1095.37

1095.92

1094.8 1096.1

HexNAc 2 Man 4

1257.42

1258.06

1259.1 1258.3

HexNAc2Man5

1419.47

1420.21

1420.4 1420.4

HexNAc2Man6

1581.53

1582.35

1581.7 1582.7

HexNAc 2 Man 7

1743.58

1744.49

1744.5 1744.8

HexNAc2Man8

1905.63

1906.63

1906.6 1907.6

HexNAc 2 Man 9

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

HexNAc 6 Man 3 Gal 2

2393.85

2395.06

2395.4 2395.1

HexNAc6Man3Gal4

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.

Calculated m

Proposed composition

Calculated m

O4

O4

O4

O4

O4

O4

O4

a Sulfates

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

sul-fatase The UV-MALDI-TOF spectra of the treated

sample, obtained in the negative ion mode using

nor-harmane as matrix, showed the complete

dis-appearance of the aforementioned signals (Fig 6B)

Furthermore, when desulfation of the released

oligo-saccharides 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

m⁄ z 1656.2 assigned to sulfated HexNAc2Man7in rela-tion 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 back-ground 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 identi-fied by ion chromatography using conductivity detec-tion (Fig 6D, as an inset in Fig 6B)

Altogether, these data confirm the presence of sul-fated 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 charac-terization of N-linked oligosaccharides of glycopro-teins

Discussion

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 repor-ted [14] The fact that part of the labeled oligosac-charides 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 charac-terized 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 gly-coproteins The fact that these signals were found in the C-terminal UV-MALDI-TOF mass spectra con-firms 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 possi-bility that cruzipain would be transported via an endo-cytic recycling and⁄ or lysosomal transport pathway as proposed for T brucei [26,27]

0

5000

10000

15000

20000

25000

Time (min)

0

5000

10000

15000

20000

25000

A

B

2 1

Fig 4 HPAEC analysis of the a- L -fucose released from the

C-ter-minal domain of cruzipain (A) Purified C-terC-ter-minal domain was

digested with a- L -fucosidase The released sugar was labeled by

reduction with NaB3H 4 desalted and analysed by HPAEC under

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

2, sorbitol.

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

des-cribed containing a-l-fucose in their structure: Gp-72,

isolated from epimastigote forms [28,29] and the

trypo-mastigote 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

correspond-ing to monosialylated oligosaccharides could be

detec-ted 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,

relat-ive 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

matri-ces have made UV-MALDI-TOF MS a suitable tool for

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 interac-tion 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

A

B

100

50

1963.7 1152.8

990.0

1007.6

1314.1

1476.6

1819.1

1494.7

50

100

1962.1

1332.1

1312.1

1494.3

1476.2 1656.8

1639.3 1818.3

1152.1

Mass (m/z)

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 oligosaccha-ride fraction, m ⁄ z range: 800–2800 Da (B) Same as (A) after endo-b-galactosidase treatment Structures are detailed in Table 2.

and the growing sulfated high-mannose series suggest

that the sulfate group should be located on the

chito-biosyl core (Fig 5A)

The presence of sulfate groups in N-linked

oligosac-charides 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

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

0

1980.3 1310.8

10 20 30 40 50 60 70 80 90 100

1656.2

1818.5

1493.9

1598.6 1331.8

1170.6

1893.6

A

B

C

0 10 20 30 40 50 60 70 80 90 100

1750.3

1310.1 1195.8 1531.01622.0

Time (min)

PO 4 SO 4

D

m/z

0 10 20 30 40 50 60 70 80 90 100

1656.9

1310.1

1598.6

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

oligosaccha-ride fraction, m ⁄ z range: 900–2500 Da (B)

Same as (A) after sulfatase treatment (C)

Same as (A) after solvolysis (D) Ion

chroma-tography analysis of sulfate released from

oligosaccharides obtained from cruzipain.

Dictyostelium discoideum[24] Likewise, these

glycopro-teins 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-gly-cosylation site present in the C-terminal domain of

cru-zipain (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

scintil-lation 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

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 NaBH4for 2 h more and the reaction was stopped by addi-tion 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) Pyri-dine (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; gra-dient elution with 50 mm NaOH and 0–50 mm sodium acetate during 40 min The flow rate was 0.6 mLÆmin)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 dur-ing 60 min The flow rate was 1 mLÆmin)1; and (c) Car-bopack 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 mLÆmin)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 mLÆmin)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