Characterization of fucosyllactose determinant-bearing glycoproteins probed by a Biomphalaria alexandrina lectin in Schistosoma mansoni cercariae

Developmental and

ComparativeImmunology,Vol. Copyright 0 1996 Else+

20, No. 2, pp. 87-%,I996 All rights rc8cm-d Printed ill Great Britain 0145-305X/96 $15.00+0.00

scienceLtd.

Pergamon

CHARACTERIZATION OF FUCOSYLLACTOSE DETERMINANT-BEARING GLYCOPROTEINS PROBED BY A Biomphalaria alexandrina LECTIN IN Schistosoma mansoni CERCARIAE Hoda I. Negm Department

of Zoology,

Faculty of Science, Monoufia

University, Shebin El Kom, Egypt

(Submitted September 1995; Accepted November 1995)

qAbstract-Utilizing a Biomphaluriu alexan&in+derived lectin (BaSII) of proven specilicity to a Schistosoma mansoni-associated fucosyllactose [(Fuc al-2) Gal fll-4 Glc] determinant-bearing determinant, two glycoproteins of 40 and 37 kDa were found to be synthesized by the cercarial stage of the parasite. The two glycoproteins were isolated by BaSII a&&y column chromatography from extracts of cercarlae metabolically radiolabelled with 3sS-methlonine. Treatments with endoglycosidases, alkaline borohydride, as well as concanavalin A column chromatography and analysis by two-dlmensional gels indicated that the two glycoproteins are synthesixed as a single 33 kDa polypeptide backbone that ls differentially glycosylated with one and/or two determlnant-bearing N-linked complex-type glycan units of either the biantennary, or, to a lesser extent, the tri- or tetra-antennary types. The two glycoproteins lack other conventional high mannose-type or O-linked glycans, and the distinct structures of the complex-type oligosaccharides accounted solely for the expression of three isomorphs for each determlnant-bearing glycoprotein. Based on the structural relatedness of the fucosyllactose determinant to the antigenlc mammalian blood group H trlsaccharide, our observations may have impllcatlons in mechanisms of host-parasite interactions as well as immunoprophylaxls. Copyright 6 19% Elsevier Science Ltd Address correspondence to: Dr H. I. Negm, Department of Zoology, Faculty of Science, Monoufia University, Shebin El-Kom, Egypt.

qKeywords-Biomphalaria alexandrina; Pectin; Schistosoma mansoni; Fuco8yllacto8e determinant; Complex-type oligosaccharides; Blood group H.

Nomenclature BSA FITC TCA 2-D SDS-PAGE

Con A Fuc Gal Glc GlcNAc

bovine serum albumin fluorescein isothiocyanate trichloroacetic acid twodimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis concanavalin A

fucose galactose glucose N-acetylglucosamine

Introduction Released and membrane-bound oligosaccharides are important structural constituents that contribute to the immunogenicity of developing schistosomes (l-5). Thus, there has been continued interest in the mechanism of expression and fate of glycoconjugates associated with the different stages of the parasite (6-10). Recently, structurallydefined oligosaccharide determinants of immunoprophylatic potential have been reported in the egg (1 l-l 3) schistosomular (14,15) as well as the adult (16-20) stages of Schistosoma mansoni. On the

88

other hand, limited attention has been directed towards oligosaccharides synthesized by the cercarial stage of the parasite (21-23). We have recently reported on a schistosome-associated fucosyllactose determinant, constituted by terminal fucose linked al,2 to the galactose residue of lactose, and specifically recognized by an oligomorphic family of Biomphalaria alexandrina-derived lectins (20,24,25). Multiple copies of this determinant were expressed in the miracidial stage by heterogeneous acidic glycoproteins of distinct charge and weight (24). In the adult worm, the same determinant was expressed in the outer chain of a single complex-type N-linked glycan unit associated with a 37 kDa (Sm 37) glycoprotein (25). Here we demonstrate, with the use of fluorescent staining, in vitro metabolic radiolabelling and B. alexandrina lectin affinity chromatography, that S. mansoni cercariae synthesize a single 33 kDa polypeptide that is differentially glycosylated by one and/or two fucosyllactose determinant-bearing complex-type glycan units. Our observations on the expression of a specific oligosaccharide structure in different developmental stages of S. mansoni may have implications in regard to mechanisms of host-parasite interactions.

H. I. Negm

PuriJication of Fucosyllactose-binding Lectin From B. alexandrina Hemolymph A purified fraction of the snail lectin (BaSII) specific for the schistosomeassociated fucosyllactose-determinant was prepared from 450 mL of hemolymph by affinity chromatography on a column of equimolar mixture of D- and L-glucose and elution by 0.3 M L-fucose as described previously (24). Purified BaSII (4 mg) was desalted and separated from monosaccharides by chromatography on a column of Bio Gel PlO (1.5 x 20 cm) in 20 mM Tris-HCl, pH 7.3, containing 100 mM NaCl (TBS), aliquoted, freeze-dried and stored at - 20°C.

Direct Fluorescence

Labeling

Fractions of purified BaSII or bovine serum albumin (BSA) (250 pg/mL) in 250 mM sodium carbonate, pH 9.0, containing 100 mM NaCl were conjugated with fluorescein isothiocyanate (FITC, Sigma Chem. Comp., St. Louis, MO) as described (26). The conjugated lectin or BSA were freed of unbound fluorochrome by desalting on a Bio Gel P 10 column (1.5 x 20 cm) pre-equilibrated and eluted with TBS, pH 7.3 containing 0.05% BSA and 10 mA4 Ca2+ (TBS-BSA). As measured by the ODzsa/ODsts ratio, the protein/fluorescein ratio of both conjuMaterials and Methods gates was approximately 2: 1. Schistosoma mansoni cercariae were fixed in ice-cold Animals and Parasites acetone for 5 min, washed five times in Naturally-infected and uninfected spe- TBS-BSA and stained for 30 min at room cimens of the snail Biomphalaria alexan- temperature with 25 pg/mL of FITCconjugated BaSII or BSA in the absence drina were provided by the Schistosome Biological Supply Program (SBSP, or presence of 50 mM L-fucose. After three washes in TBS, schistosomes were Theodor Bilharz Research Institute, Cairo, Egypt). Cercariae of Schistosoma mounted on slides in 75% glyccrol/TBS and examined by incident light fluoresmansoni (Egyptian strain) were collected cence using a Leitz microscope equipped from infected B. alexandrina, concenwith a mercury vapour lamp. Microtrated by spinning at 100 x g for 2 min photographs were taken with a Wild and supplied by SBSP.

Fucosyllactose determinants on S. mansonicercariae photoautomat MPS 55 exposure meter and Kodak Tri-X-Pan films.

Metabolic ~adiolabelling S. manson Cerceriae

of

Approximately 1 x lo4 carcariae were radiolabelled in vitro with 0.5 mCi/mL of 35S-methionine (Amersham, Cairo, Egypt) in Dulbecco’s modified Eagles medium without-methionine (GIl3CO) supplemented with 5% fetal calf serum and 1O/ ~~cil~n~treptomycin solution (10,000 units of penicillin GfmL and 10 mg of StreptomycinfmL in 0.9% NaCl) in a final volume of 2 mL for 48 h. Incubations were conducted at 37°C in a humidified incubator containing 5% COZ. At the end of the incubation, cercariae were motile and viable and cultures were devoid of bacterial and fungal contamination. Labelled cercariae were suspended in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 m&f NaCl, 2 mM phenylmethyls~fonyl fluoride, 1% triton X-100, 1 mM p-chloromercuribenzoic acid, 1 mM o-phenanthroline, 1 mM iodoactetamine) and solubilized by sonic treatment and three cycles of freezing and thawing (20,24). Solubilized proteins were separated by centrifugation at 100,000 x g for 1 h. Incorporation of 35S-methionine into protein was determined by scintillation counting of trichloroacetic acid (TCA)precipitable radioacti~ty using an automated liquid scintillation counter (Taurus, Micromedic Systems, Inc., USA).

BaSII Afinity Column Chromatography of Labelled Cercarial Glycopro teins A fraction of the purified BaSII was dialyzed against coupling buffer (100 mM sodium bicarbonate, pH 7.8, 150 mM NaCI) and coupled to CNBr-activated Sepharose 4B (Pharmacia Fine Chem.,

89

Sweden) at a concentration of 2 mg/mL of beads follo~ng the manufacturer’s ins~ctions. The coupled gel (1.5 mL with a coupling efficiency of 85%) was equilibrated in TBS, pH 7.3 containing 0.1% Triton X-100,150 mM NaCl, 1 mM CaCb 1 mM MgC12 (TBS-Triton X), and the labelled cercarial extract (7.5 mL, 1 x 10’ cpm) cycled through the column six times at 15°C. The gel was extensively washed with TBS-Triton X buffer and bound glycoproteins eluted with 300 mM L-fucose in TBS-Triton X buffer. Fractions of 2 mL were collected from the column and aliquots of each fraction mixed with ScintiVerse I (Fisher) for the determination of radioactivity in a liquid scintillation counter. Eluted glycoproteins were freed of monosaccharides by chromatography on a column of Bio Gel PlO (1.5 x 20 cm) in TBS-Triton X buffer, aliquoted, freeze-dried and stored at - 20°C until used.

Gel Electrophoresis Two-dimensional (2-D) gel electrophoresis was conducted essentially as described by O’Farrell (27). Freeze-dried samples were reconstituted in O’Farrell’s sample buffer incubated at 37°C for 4 h and applied to the isoelectric focusing gels for 18 h (8~0 V-h). Samples were resolved in the second dimension with 10% slab sodium dodecyl sulfat*polyacrylamide gel electrophoresis (SDSPAGE) under both non-reducing and reducing conditions (28) along with Bio Rad low molecular weight standards. Gels were tied, washed, and dried comparably. Radioactive 3SS-labelled proteins in the gels were detected by fluoro~aphy using En 3Hance (NEN, Boston, MA) and Kodak X-Omat XAR5 films.

90

Glycosidase and Alkaline 3orohydride Treatments The analysis of N-linked glycosylations was conducted by reconstituting aliquots of radiolabelled S. mansoni glycoproteins in either 50 pL of 100 mM sodium phosphate, pH 6.1, 50 m&4 EDTA, 1% NIV.0 and 100 mU of Endo-F @do+N-a~tyl~ucosa~~~e F, from F&ocoterie men~gose~tic~ 600 U/mg, Sigma Chem. Comp., St. Louis, MO) or 50 uL of 50 mM sodium citrate/sodium mono-hydrogen phosphate buffer, pH 5.5, 1% NP-40 and 10 mU of Endo-H acetylglucosaminidase H, (Endo-B-N from Streptomyces lividans, 25 U/mg, Sigma Chem. Comp., St. Louis, MO). Samples were incubated for 18 h at 37”C, precipitated with TCA, washed with cold acetone and dried under nitrogen gas before analysis by 2-D SDS-PAGE and fluoro~phy. The potential association of fucosyllactose determinants with O-linked was investigated by oligosaccharides reconstituting aliquots of radiolabelled fractions in 100 pL of 50 mM NaOH with or without the addition of 1 M NaBH4 and incubated at 45°C for 24 h. The treatment was terminated by freezedrying followed by analysis by 2-D SDS-PAGE and ffuorography.

H. 1.Negm

Results Expression of B. alexandrina BaSIIreactive Fucosyllactose Determinants in S. mansoni Cercariae Based on the proven specificity of B.

alexanclrinaBaSII lectin in selectively binding a fucosyllactose sequence on the miracidial stage as well as adult male worms of S. Toni ~20,24,25), the possible expression of this dete~nant by the cercarial stage was investigated by direct fluorescence microscopy. Incubation of acetone-fixed cercariae with a purified fraction of BaSII (25 pg/mL) conjugated with FITC resulted in an intense fluorescence labelling of the body and tail regions of the schistosome (Fig. 1 A,B). No positive labelling was observed with FITC-BSA or with FITC-BaSII incubated with sc~stosomes in the presence of 50 mM L-fucose.

Isolation of Cercarial Glycoproteins Expressing Fucosyllactose Determinants

The identity of glycoproteins bearing the BaSII-r~ctive fucosyllactose determinant in S. m~oni cercariae was investigated by subjecting 35S-labelled solubilized cercarial proteins to BaSII Concanavalin A Afinity affinity chromatography. As shown in Chromatography Figure 2A, the BaSII-bound fraction, Radiolabelled glycoproteins were frac- eluted with 300 mM L-fucose, represented tionated on a 2 mL column (0.7 x 5 cm) 15% of the 35S-labelled schistosome components. Of the total cercarial proof concanavalin A (Con A)-Sepharose (Sigma Chem. Comp., St. Louis, MO) teins (Fig. 2B), the BaSII-bound fraction equilibrated with TBS-Triton X buffer at was constituted in both reducing and nonreducing 2-D SDS-PAGE by two deterroom temperature. Bound glycoproteins were sequentially eluted with 10 rn&# 01- mina~t-~a~ng acidic glycoproteins of 40 methylglucoside followed by 100 mM tc- and 37 kDa, each being composed of three isomorphs (Fig. 2C). The estimated methylmannoside in the same buffer. pIs of the constituent charge variants of Radioactivity was monitored by scintillaeach of the two glycoproteins were 5.5, tion counting of aliquots of 2 mL of post5.8 and 6.0. column fractions, as indicated above.

Fucosyllactose determinants on S. mansonicercariae

91

Figure 1. Direct fluorescence visualization of the Biomphalaria alexandrina lectin (BaSII)reactive fucosyllactose determinant in Schistosoma mansoni cercariae. Positive FITC-BaSII fluorescent labelling was associated with the body and tail regions of the schistosome (A, x 250; 8, x 400, bar = 75 pm). No labelling was observed with FITC-BSA or with FITC-BaSII incubated with schistosomes in the presence of 50 mM L-fucose.

Diperential N-linked Glycosylations of the Cercarial Fucosyllactose Determinant-bearing Glycopro teins It has been established that, as in mammals, the pathway of N-glycosylation of proteins in schistosomes involves the transfer of a GlqMangGlcNAcz precursor from a dolichol pyrophosphate carrier to an asparagine residue in the nascent polypeptide, to be variably processed to either high mannose-type chains of variable sizes (uncharged) or different complex-type branches (possibly carrying various charged moieties) (17,18). Thus, the nature of the oligosaccharide side-chains expressing the BaSIIreactive fucosyllactose determinants, as well as the structural inter-relationship of determinant-bearing glycoproteins, was investigated by testing the susceptibility of purified “S-labelled post-BaSII column fractions to Endo-F, Endo-H and mild alkaline borohydride treatments and analyses by 2-D SDS-PAGE. As shown in Figure 3, treatment with Endo-F collapsed the heterodisperse pattern of untreated glycoproteins (Fig. 3A) into a single homogeneous spot with an obvious shift in both molecular weight as well as charge (Fig. 3B). The Endo-F-treated material focused as a single 33 kDa spot with a pI of 6.1. On the other hand, patterns observed with Endo-H or alka-

line borohydride-treatments, were essentially similar to that observed with the untreated glycoproteins (Fig. 3A), with no significant alterations in either the molecular weight or charge of the constituent isomorphs. Given the known specificity of Endo-F in cleaving linkages in the core of both complex- and high mannose-type Nlinked glycans (29) (changing the size and possibly the charge of glycoproteins), and based on the resistance of the BaSIIreactive glycoproteins to Endo-H treatment (selectively cleaves high mannosetype glycans (30), and may thus alter only the size of glycoproteins), the shift of about 7 and 4 kDa towards a basic p1 observed with the 40 and 37 kDa components with Endo-F was consistent with the removal of two and one complex-type glycan units, respectively. Inasmuch as both glycoproteins may lack conventional O-linked glycans, as evidenced by their resistance to mild alkaline borohydride treatment, a single polypeptide backbone of 33 kDa seemed to be subjected to differential post-translational additions of one and/or two complex-type units of distinct structures, which accounted for the molecular weight and charge microheterogeneity of the glycosylated protein constituting the BaSII-reactive fraction. To further assess the structure of the complex-type glycans that express the fucosyllactose determinants, the 35S-

92

H. 1.Negm

BaS 11 300 mM Fuc

“: 0 -

1

G

6

0

4

5.4

6.5

r

I

I

7.4 4

5.4

6.5

7.4

1

(B)

97 66 -

45 -

Mr

31 -

21 -

Figure 2. isolation of Schistosoma mansoni cercariai glycoproteins bearing the fucosyiiactose determinant. Soiubiiized 3bS-iabeiled cercariai giycoproteins were subjected to BaSli affinity chromatography and bound giycoproteins eiuted wtih 300 mA4L-fucose (A). Radioactivity was monitored by liquid scintiifation counting and more than 90% of the radioactivity applied to the column was recovered. 35S-iabelied giycoproteins in the total cercariai extract (B) and the Basil-bound fraction (C) were analyzed by 2-D SDS-PAGE and fluorography. The positions of molecular weight standards f x 10 -3) are indicated.

labelled post-BaSII column fraction was subjected to a Con A-Sepharose affinity column and its fractionation profile is shown in Figure 4. Con A-Sepharose does

not bind to highly branched tri- and tetraantennary N-linked oligosaccharides but does bind to complex-type bi-antennary glycans, which can be eluted by 10 mM LX-

93

Fucosyllactose determinants on S. mansoni cercariae

PH 1.4 4

i

I

I

I

I

I

I

I

(B)

(A)

97 66 -

4.5 -

31 -

21 -

Figure 3. Fluorograms of Z-D SDS-PAGE of glycosylated and deglycosylated “‘S-labelled fucosyllactose determinant-bearing glycoproteins. Isolated 35S-labelled BaSILreactive glycoproteins were either untreated (A) or treated with Endo-F (6) and analysed by 2-D SDS-PAGE and fluorography using 10% slab gels in the second dimension. The positions of molecular weight markers ( x 10e3) are indicated.

Con A

10 mM Ok +

10

IOOmMMan

1

.

20

30

Fraction

Figure 4. Concanavalin A affinity chromatography of 3sS-labelled fucosyllactose determinant-bearing glycoproteins. Isolated 3eSlabelled BaSll-reactive glycoproteins were applied to a Con A-Sepharose column and the bound fractions sequentially eluted with 10 m&4o- methylglucoside and 100 mMa- methylmannoside and monitored for radioactivity as indicated in the Materials and Methods.

methylglucoside, as well as high marmosetype N-linked oligosaccharides, which require 100 mM rx-methylmannoside for elution (31-33). A minor portion (9%) of the 35S-labelled post BaSII column fraction did not bind to the Con A-column, whereas a significant portion (91%) of the glyco~roteins bound and selectively eluted with 10 mM ~-methylglucoside, with no further displacement of radioactivity observed upon the sequential elution with 100 mM cc-methylmannoside. This observation was complementary to data obtained with glycosidase treatments indicating the absence of high mannose-type glycans in BaSII-reactive glycoproteins, and further confirmed their differential glycosylation with distinct complex-type oligosaccha~de structures. Evidently, fucosyll~to~ dete~nants were associated with complex-type units that were either of the bi-antennary type, or to a

94

lesser extent, of the tri- or tetra-ant~na~ type.

Discussion A salient structural alteration that marks the developmental transformation of S. mansoni cercariae to schistosomula involves the molecular ~onstit~nts synthesized by the parasite (l-3,5-7). ~ncomi~nt with this stru&ral modulation, the developing schistosome converts to a relatively immune-resistant state, thus implicating its molecular make-up in the possible development of immune evasive strategies within infected hosts Nonetheless, information (21,22,34). regarding the nature as well as the biosynthetic program of structurallydefined determinants associated with the cercarial stage is still limited (21-23). We have recently established that lectins derived from B. aiexandrina, the snail vector of the Egyptian strain of S. mansoni, express selectivity in binding a blood group H-related oligosaccharide determinant constituted by a (Fuc al-2) Galj314Glc sequence and expressed by the miracidial and adult worm stages of the parasite (20,24,25). We have now demonstrated with the use of fluorescent staining, in vitro metabolic ra~olabel~ng and B. ale~~dri~a lectin affinity chromatography that the fucosyllactose determinant is synthesized and expressed in the cercarial stage of the parasite. Unlike S. mansoni miracidia, in which fucosyllactose determinants are expressed in multiple copies among acidic glycoproteins that display a wide range of molecharge cular weight and extensive heterogeneity (24), determinant expression in cercariae was restricted to two acidic glycoproteins of 40 and 37 kDa, each being composed of three isomorphs. Treatments with glycosidases and analysis by 2-D gels were consistant with the determinant-bearing glycoproteins being synthesized as a single 33 kDa polypep-

H. 1.Negm

tide backbone, which lacks conventional high mannose-type or O-linked glycans, but is differentially glycosylated with one and/or two units of N-linked complextype glycans of either the bi-antennary type or the tri- or tetra-antennary type. Based on Con A binding, these distinct structures of the complex-type glycans were not synthesized in equal proportions, the majority being the bi-antennary type, and accounted for the microheterogeneous pattern of the determinantbearing glycoproteins. In accordance with these observations, the expression of fucosyllactose determinants in adult male worms has been also shown to involve a single polypeptide backbone, which is differentially glycosylated with one complex-type glycan unit of the bi-, tri- or tetra-antennary types (25). Although the functional significance of the di~erential glycosyla~on of determinant-bearing gly~oproteins is still to be resolved, the apparent change of determinant presentation in the different developmental stages of the parasite implies that, in the absence of possible host interference, a programed mechanism involving the formation and processing of N-linked oligosaccharides may be operative in schistosomes. Recent studies on st~ct~ally-de~ned glycan sequences actively s~thesized by schistosomes have indicated the remarkable capacity of the parasite to synthesize mammalian-type oligosaccharides (1419), which may be implicated in antigenic disguise in selective hosts. These included the series of fucose-containing glycospbingolipids constituted by FuczGlcNAc repeating units and expressed by the different developmental stages of the parasite (1 l-13), the trisaccharide determinant SSEA-1 associated with schistosomula (15), as well as the complex-type glycans having terminal polyfucosyiated polylactosarnine chains containing the Lewis X antigenic blood group that are synthesized by adult male worms (19). Our present observations on the expres-

Fucosyllactose determinants

on S. mansonicercariae

sion of the trisaccharide sequence (Fuc ~1-2) Galpl-4Glc by cercariae lend support to these obse~atio~s, and furthermore establish the conservation of a sequence related to the antigenic blood group H, which has the trisaccharide sequence (Fuc ~1-2) Galpl-4GlcNAc (35), in the different stages of the parasite. It is still to be elucidated whether the schistosome oligosaccharides reported here and elsewhere (14-19) would prove useful as targets for mechanistic studies

95

on host-parasite i~~oprophyla~s

interactions as well as in sc~stosomiasis.

Acknowledgements-This study was supported by a USAID-funded Schistosomiasis Research Project (262-0140.2) grant award # 07-01-62. As a co-principal investigator to this grant, the author would like to thank Dr M. H. Mansour (principal investigator) as well as the entire administrative staff of SRP for their efforts in facilitating this work.

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