A female-specific cDNA sequence of Schistosoma mansoni encoding a mucin-like protein that is expressed in the epithelial cells of the reproductive duct

Marion MENRATH, Anja MICHEL and Werner KUNZ

Institute of Genetics and Biological-Medical Research Center, Heinrich-Heine-University, Universitäts-Str.1, D-40225 Düsseldorf, Germany.

The sequence data reported herein has been submitted to EMBL Data Library and assigned the accession number Z 27403.

SUMMARY

Seven cDNA clones of Schistosoma mansoni containing the C-terminal part of the deduced sequence of a mucin-like protein have been identified. The protein contains 28% threonines, 20% serines, and has a pI of 3.4. On Northern blots of RNA of adult worms, the cDNA clones detect two transcripts of 1.65 and 4.2 kb which are expressed only in female worms. The tissue of gene expression, as revealed by in-situ hybridization, is the epithelium surrounding the female reproductive duct proximal to its entrance into the ootype. Accumulation of N-glycosylation sites suggests that the protein, like other mucins, might form a protective layer, coating the lining of the duct. Regarding its acidic pI, we hypothesize a role in preventing premature eggshell formation. This is the first female-specifically transcribed sequence, hitherto known in S. mansoni that is not expressed in the vitellaria.

INTRODUCTION

The sexually dimorphic schistosomes are an exception in the generally hermaphroditic plathyhelminthes. Development and fertility of the female schistosome is totally dependent on continuous pairing with the male (Clough, 1981). Unpaired females remain stunted and sexually immature (Armstrong, 1965). In an effort to shed more light on the molecular mechanism underlying this phenomenon, several groups have been engaged in the isolation and cloning of female-specifically expressed genes. All these genes hitherto sequenced are expressed selectively in the vitelline cells of mature, egg-laying females and, except for fs800, have been identified to code for egg-shell precursor proteins (Bobek et al., 1986; Johnson, Taylor & Cordingley, 1987; Reis et al., 1989; Kunz et al., 1995). Almost nothing is known about the genes that are active in the other female tissues, for example the ovary and the reproductive ducts.

To screen for cDNA clones that include even transcripts from moderately expressed genes, we utilized a subtractive cDNA library enriched for RNA of female parasites (Michel et al., 1995). One of the cDNA clones thus isolated was chosen for molecular characterization and histological localization. The extensive accumulation of serines and threonines reveals a similarity with mucins, a group of proteins with the function to protect epithelia (Strous & Dekker, 1992).

MATERIALS AND METHODS

Parasites

A Liberia strain (Köster et al., 1988) of S. mansoni was maintained in our laboratory using Biomphalaria glabrata snails and golden hamsters as hosts. Adult worms were harvested by hepatoportal perfusion (Duvall & DeWitt, 1967) seven weeks after infection and gently separated using fine paint-brushes. The worms were sorted according to gender and stored in liquid nitrogen.

Plasmids and bacterial strains

Bacterial host for recombinants of pBluescript clones was E.coli strain XL1-Blue; pTZ18U and pGEM subclones were cultured in DH5a.

Synthesis of S. mansoni cDNA libraries in lambda ZAPII vector

5 µg of poly(A+)RNA were prepared separately from male and female schistosomes and used for cDNA synthesis following the instructions of the ZAP cDNA synthesis kit (Stratagene). cDNA of each sex was ligated separately with lambda ZAPII EcoRI-XhoI treated vector and packaged using Gigapack Gold packaging extracts (Stratagene). The resulting male library contained 1.5x10e6 independent clones, the female library had 1.4x10e6 clones.

Preparation of phagemids from the lambda ZAPII specific library

Large scale phagemid and plasmid preparations from the S. mansoni cDNA libraries were done as described by Schweinfest et al. (Schweinfest et al., 1990). Phagemids were isolated from the female lambda ZAPII cDNA library in an in vivo excision step as described by the manufacturer (Stratagene). A total of 3x10e7 recombinant phages were allowed to infect 3x10e8 XL-1 Blue cells, coinfected with 1.5x10e9 R408 filamentous helper phages. At least 1x10e7 phagemids were then used to inoculate 50 ml of fresh XL-1 Blue cells. After shaking for 2 h at 37°C, 500 ml of prewarmed LB (containing 6.25 µg/ml of tetracyclin) were added and incubated for further 5 h before the addition of ampicillin (25 µg/ml). After shaking overnight at 37°C, bacterial cells were pelleted by centrifugation, and the phagemids were precipitated from the supernatant with PEG 6000 (final concentration 5%) in 3.5 M ammonium acetate for 30 min at room temperature. Single stranded phagemid DNA was purified by phenol extraction and ethanol precipitation.

Synthesis of biotinylated transcripts

Male DNA was recovered in form of plasmids from the pellet of bacterial cells following an in vivo excision step, infection and overnight growth, as described above. Plasmid DNA was prepared and linearized with XhoI. RNA synthesis was carried out in 62.5 mM DTT, 20 U T3 RNA polymerse, 3 U RNAsin, 0.8 mM each of rATP, rCTP, rGTP and bio-dUTP for 90 min at 37°C. Thereafter 2 U of RNAse-free DNAse were added and incubation continued for further 30 min at 37°C. Transcription was stopped by adding 2 volumes of ethanol and 1/10 volume of 2.5 M ammonium acetate and the precipitated RNA was redissolved in TE (10 mM TrisHCl, pH 8 and 1 mM EDTA).

Enrichment of a female-specific library by subtractive hybridization

In order to enrich for female specific cDNAs, biotinylated sense transcripts from the male library were hybridized with antisense female phagemids, according to Schweinfest et al. (1990) and Swaarop et al. (1991). Experimentally, 55 µg of male transcripts were coprecipitated with 1.3 µg of single stranded female phagemid DNA. The pellet was resuspended in 5 µl HE buffer (10 mM Hepes, 1 mM EDTA, pH 7.5), 5 µl polyA (0.5 µg/µl) in HE buffer and 10 µl 2x hybridization solution (1.5 M NaCl, 50 mM HEPES, 10 mM EDTA, 0.2% SDS, pH 7.5). The hybridization mix was heated to 100°C for 1 min and left to hybridize at 68°C for 48 h. Thereafter, the mixture was diluted with 180 µl HE buffer and heated for 5 min at 55°C. The mix was adjusted to 1 M NaCl, 20 mM HEPES, pH 7.5. Streptavidin agarose (Sigma) (250 µl) equilibrated in the above buffer was added and the mix was left for 15 min at 37°C. After a brief centrifugation, the supernatant containing non-hybridized female specific cDNA strands was collected. The matrix was washed twice with 200 µl each of 15 mM NaCl, 1.5 mM HEPES and 0.2% SDS. Supernatants were precipitated and resuspended in 10 µl of 20 mM Tris, pH 7.5, 50 mM NaCl containing 30 pg M13-sense primer. Following an annealing step of 15 min at 70°C and two cooling steps of 30 min at 37°C and of 15 min at room temperature, second strand synthesis was allowed to proceed in the presence of 0.1 mM each of dNTP, 10 U Klenow polymerase, 2 mM DTT, 25 mM MgCl2, 20 mM Tris/HCl pH 7.5 (total volume of 25 µl) for 2 h at 37°C. An aliquot of 5 µl was used to transform competent XL-1 Blue cells. White colonies were picked and left to grow overnight at 37°C in 50 µl of LB containing tetracyclin (25 µg/ml) in microtiter plates. An equal volume of 100% glycerine was added to the wells, left to incubate for a further hour and stored at -70°C.

RNA isolation and Northern blot analysis

Total RNA was isolated by the guanidium isothiocyanate method (Chirgwin et al., 1979). Northern blotting and hybridization were carried out as described in Dietzel et al. (1992).

DNA sequencing

Nucleotide sequencing was performed by the dideoxy chain termination method using the T7 sequencing kit from Pharmacia. Primers used were M13, M13 reverse (Pharmacia), T7 and SK- (Stratagene). Amino acid sequences were analyzed using the computer programs GENEPRO version 5.0, 1990 and PC/GENE version 6.6, 1991.

Tissue sections, in situ hybridization and immunohistology

Adult worm pairs from 8 week infections were fixed in Bouin's solution (picric acid/acetic acid/formaldehyde 15:1:5) and embedded in paraplast. 5 µm sections were treated as described in Köster et al. (1988). Digoxigenin-labelled in vitro transcripts were made according to the protocol of the manufacturer (Boehringer-Mannheim). Immunohistology and in situ hybridization were carried out following the procedure of Finken et al. (1994). Detection of hybrids or antibodies was achieved with alkaline phosphatase conjugated anti-digoxigenin antibodies, naphthol-AS-MX phosphate and Fast Red TR (West, Schröder & Kunz, 1990).

Western blotting

Total proteins prepared from S. mansoni (equivalent to three female or one and a half male worms per lane) or from IPTG-induced bacterial cultures expressing ß-galactosidase fusion proteins (equivalent to 0.5 ml culture) were separated on 7% - 17.5% SDS-polyacrylamide gradient gels and transferred onto nitrocellulose membranes. Expression of the fusion protein was tested with anti-ß-galactosidase antiserum. Detection of bound antibodies was achieved with alkaline phosphatase-conjugated goat anti-rabbit antibodies (Dianova), naphthol-AS-MX phosphate and Fast Brown RR (Sigma) (West, Schröder & Kunz, 1990).

RESULTS

For the production of a subtractive library enriched for cDNAs encoding female specific proteins, we carried out the steps, as described by Schweinfest et al.(1990) and Swaarop et al. (1991). A total of 2547 white colonies were obtained, which were propagated in microtiter plates, altogether giving 27 plates.

In order to isolate clones that are female specific, we screened more than 600 clones on triplicate filters. The first filter was hybridized to radioactively labelled cDNA prepared from male parasites, the second to cDNA from female parasites and the third to DNA prepared from three clones previously characterized as female specific. This third filter served as a control to avoid analyzing previously characterized clones. Eight clones which reacted with the female specific probe but not with the male specific probe nor with the three female specific clones were selected. The insert of one of these clones, termed B7 and having a length of 580 bp, was labelled with a32P-dATP by random priming and used to rescreen the subtractive library. A group of six overlapping clones was identified with insert lengths ranging from 860 bp (clone A1) to 1.25 kb (clone A11). The insert of B7 was used to probe a Northern blot with total RNA, which was prepared separately from adult male and female schistosomes. Two transcripts of 4.2 kb and 1.65 kb could be detected (Fig. 1). Both were female-specifically expressed. Even after a 5 week exposure of the autoradiograph, no transcripts were observed in the male RNA. The hybridization intensity of the larger band was about ten times stronger than that of the smaller band. These same results were later repeated with both the 5'-terminal 580 bp EcoRI/SalI or the 3'-terminal 620 bp SalI/XhoI fragment of the longest cDNA clone A11 as a hybridization probe.

Clones B7 and A11 were selected for sequencing using subclones in pTZ18U. The terminal sequences of the other five clones were also determined. No sequence differences among these independent cDNA clones were found. The exact length of the cDNA sequence of clone A11 totals 1236 bp. The insert starts within an open reading frame (ORF) that ends with a stop codon at nucleotide position 1078 (Fig. 2). A 3'-untranslated region of 138 bp follows, until the sequence terminates with a poly(A) tail of 18 nucleotides. Close to the 3'end, two direct tandem repeats of 120 bases follow each other (nt 765-885, 886-1005) which differ by ten base exchanges (Fig. 2). The predicted amino acid sequence is 359 amino acids long and consists of 28% threonines, 20% serines, 7% asparagines and 7% prolines. The sequence contains neither arginine, cysteine, phenylalanine nor tryptophan. Containing 10% acidic amino acids, but only 1.1% basic amino acids, the deduced polypeptide is acidic, having a pI of 3.4. There are 15 putative N-glycosylation sites. The hydropathic profile of the A11 sequence documents a predominantly hydrophilic protein with no subdivision into distinct domains (data not shown).

Comparison of the deduced polypeptide of the A11 sequence to several sequence data banks revealed similarity to a number of threonine-rich proteins or protein domains. This includes the Sgs-3 glue protein of the salivary glands of Drosophila (Martin, Mayeda & Meyerowitz, 1988), a mucin of Xenopus laevis, formerly termed spasmolysin (Hoffmann, 1988) and the human mucin Muc2 (Toribara et al., 1991). Highest homology was found to the human mucin (22.5%). In all three proteins, the homology with A11 is restricted to a central threonine-rich domain. The N- and C-terminal regions of the Sgs-3 protein and the Xenopus and human mucins are not particularly enriched in threonines, in contrast to A11 where the threonine accumulation extends to the carboxy terminus.

In order to elucidate the role of the A11 protein, we localized the sites of gene expression by in situ hybridization. For this purpose, the 5'-terminal 580 bp EcoRI/SalI fragment was subcloned in pGem2 and used to generate digoxigenated anti-sense transcripts. Sense transcripts of the respective subclone in pGem1 served as a negative control. With the pGem1 probe, no signals were visible. As a positive control, an eggshell precursor transcript (Köster et al., 1988) was used as hybridization probe. As expected, it reacted exclusively with the vitellaria (not shown).

The antisense transcript of the EcoRI/SalI fragment of the A11 clone hybridized with the epithelium surrounding the female reproductive duct along a limited section that is adjacent to its entrance into the ootype (Figs.3 A and C). A serial section stained with acetocarmine (Figs.3 B and D) shows the twisted winding of this duct, filled with red-orange vitellocytes, clearly identifying this portion of the duct to be the vitelloduct (Gönnert, 1955). Right in the figures, the ootype containing an egg is visible. No expression of the A11 gene was found along the entire other part of the vitelloduct oriented versus the vitellarium; neither are ovary, vitellarium or ootype labelled.

To localize the A11 protein on Western blots and immunocytochemically, we raised antibodies against an A11 b-galactosidase fusion protein. The antiserum recognized the blotted A11 fusion protein, even after a dilution of 1:20,000. On Western blots of adult schistosome protein extracts and on tissue sections, however, no specific detection of any schistosome protein was observed, neither with the crude antiserum nor with antibodies which were purified on the fusion protein (data not shown). This is probably explained by posttranslational glycosylation (see Discussion).

DISCUSSION

This paper reports a sequence, named A11, that is exclusively expressed in female schistosomes. All female-specifically transcribed genes, hitherto known in S. mansoni are expressed in the vitellaria. The transcripts of A11, however, have been localized in the lining of the reproductive duct close to its junction with the ootype.

The A11 protein belongs to the group of mucin-like molecules. Mucins are secreted by many epithelia serving as a protective physical barrier between the plasma membrane and the environment of the cells. Mucins are dominated by the five amino acids serine, threonine, alanine, glycine and proline, which account for more than 50% of the total amino acid residues (Strous & Dekker, 1992). Particularly serines and threonines together comprise 25 to 40% of total amino acids. The deduced amino acid composition of our A11 sequence also contains a high percentage of serines (20%), threonines (28%) and prolines (7%), whereas alanines and glycines are not above average.

In mucin-like molecules, almost all serine and threonine residues bear O-linked glycans (Strous & Dekker, 1992). This abundance of closely packed glycans protects a large part of the mucin polypeptide with the consequence that it is not recognized by antisera raised against anti-peptide epitopes (Gum et al., 1989). This would explain why our antibodies against the A11 fusion protein did not recognize the native schistosome protein on Western blots or on tissue sections.

A general feature of most mucins is an up to 100fold repetition of an internal sequence of 9 to about 80 amino acids (Strous & Dekker, 1992). However, there are also examples of mucin-like molecules without such repeated regions, like GlyCAM-1, the endothelial ligand for L-selectin in human lymph nodes (Lasky et al., 1992). The deduced amino acid sequence of A11 has also no extensive repetition, but contains a segment of 40 amino acids that is repeated only once.

In analogy to the functional role of mucins, the A11 protein may also form a protective layer, coating the lining of the reproductive duct. Furthermore, the acidic pI of the protein might be crucial for its function, since acidic conditions are essential to prevent premature eggshell formation (Wells & Cordingley, 1991). The precursors of the eggshell proteins are synthesized in the vitelline cells and stored in these cells in large secretory vesicles (Köster et al., 1988). It has been shown that the content of the eggshell vesicles in the vitelline cells is acidic (Wells & Cordingley, 1991). As eggshell crosslinking can be triggered experimentally in these vesicles, it is concluded that the crosslinking enzyme is already present in the vesicles (Bennett, Seed & Boff, 1978; Seed, Boff & Bennett, 1978). The enzyme which probably is a phenol oxidase, remains inactive in an environment of low pH (Wells & Cordingley, 1991). We suggest that our A11 protein which is secreted into the reproductive duct close to its junction into the ootype is reponsible for maintaining this low pH. When eggshell vesicles fuse together at the periphery of the ootype, they come in contact with a layer of higher pH. This, according to the model of Wells and Cordingley (1991), activates the phenol oxidase and causes sclerotization of the outer surface of the forming eggshell.

At the present, four different genes are known in S.mansoni that are expressed selectively in the vitelline cells of mature, egg-laying females: p14 (Bobek et al., 1986; Köster et al., 1988; Pena et al., 1990), p48 (Johnson, Taylor & Cordingley, 1987), fs800 (Reis et al., 1989), and p19 (Kunz et al., 1995). Sequence information on the 5'-regulatory region is available for three of these genes, p14, p48, and fs800 (Johnson, Taylor & Cordingley, 1987; Kunz et al., 1987; LoVerde & Chen, 1991). Putative enhancer boxes have been described that are conserved among these genes and among different Schistosoma species. The question arises whether these sequence elements are characteristic for female-specific or tissue-specific expression.

The A11 sequence is the first female-specifically expressed gene which is not transcribed in the vitellaria. It is therefore highly desirable to obtain sequence information of the 5'-regulatory region of this gene. Further investigation is in progress to analyze the regulatory signals of female-specifically regulated genes that are expressed in non-vitellarian tissues.

Preliminary data show that the A11 expression is dependent on continuous pairing with a male. In an in-vitro culture, paired worms maintain a high level of A11 expression for several days, whereas females that have been separated from males show a marked decrease in expression already after 1 day of culture and a total repression after three days (Sommer, unpublished).

We wish to thank Gabriele Sommer for helpful discussions, Lutz Gohr for computer work, Dr. J. D'Haese for immunization of rabbits, and Karin Wildhagen for photographic work. This investigation received financial support of the Deutsche Forschungsgemeinschaft (Ku 282/13-2). MM was supported by the Graduiertenförderung of North-Rhine Westfalia.

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FIGURE LEGENDS

Fig. 1. Northern blot of 20 µg of total RNA of adult female (f) and male (m) schistosomes, hybridized with the 32P labelled insert of clone B7 (b). The arrows designate the two female-specific transcripts. Ethidium bromide staining in panel (a) demonstrates equal RNA amounts on both lanes. mw = 10 µg of 1 kb ladder (BRL).

Fig.2. Nucleotide sequence of the cDNA clone A11. The amino acid sequence (given in the single-letter code) of the translated ORF is shown below the nucleotide sequence. The 15 putative N-glycosylation sites are marked with asterisks. The two direct tandem repeats of 120 bp are underlined and their ends are indicated by arrowheads. The polyadenylation site is marked in bold.

Fig.3. Localization of A11 mRNA in a longitudinal tissue section of a schistosome pair by in situ hybridization (A, C), in comparison with a serial section stained with acetocarmine (B, D). (A, B) overview showing the ovary (ov) at the left, the twisted vitelloduct (vd) in the center, and the ootype containing a spined egg (e) at the right. (C, D) The same section demonstrating the vitelloduct at a higher magnification. (A, C) The arrows point to the limits of the positively stained portion of the vitelloduct. The red stain of the azo dye detection is light grey, whereas the yellowish colour of the unstained vitellocytes appears in a darker grey in this black and white photograph. in, intestine; scale bar = 50 µm.