Schistosoma mansoni: Gene structure and localization of the cysteine protease ER 60

Marion FINKEN-EIGEN and Werner KUNZ*

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

Abstract

Finken-Eigen, M., and Kunz, W. 1996. Schistosoma mansoni: Gene structure and localization of the cysteine protease ER 60. Experimental Parasitology.We have isolated and characterized a cDNA encoding the complete sequence of a homolog of the ER-luminal cysteine protease ER 60 in Schistosoma mansoni. The deduced protein of 484 amino acids contains two thioredoxin domains, each with the CGHC motif, that characterizes all members of the protein disulfide isomerase family. The corresponding gene exists as a single copy and consists of six exons, interrupted by five very small introns of 32 to 40 nucleotides. Immunohistology and in situ hybridization identify the sustentacular cells of the testes, the wall cells of the protonephridia, and the gastrodermis of the gut to be the major tissues of ER 60 gene expression.

Index Descriptors and Abbreviations: Schistosoma mansoni; Cysteine protease; ER 60; Thioredoxin domain; endoplasmic reticulum (ER); protein disulfide isomerase (PDI); sodium dodecyl sulfate (SDS). The sequence data reported in this paper have been submitted to the GenBankTM data base with the accession number Z-22934.

Introduction

In the blood fluke Schistosoma mansoni, we have recently characterized a homolog of protein disulfide isomerase (PDI) (Finken et al. 1994). In the course of this work we found that schistosomes also express a second member of the PDI family: ER 60. In mammals, ER 60 is a cysteine protease of the endoplasmic reticulum. It degrades selected proteins and hence prevents them from being transported into the Golgi compartment (Urade and Kito 1992). ER 60 is one of four members of the PDI family, a group of proteins characterized by two or three thioredoxin-like domains, each with the central CGHC motif. In addition to ER 60 and PDI, the proteins ER 72 and P5 belong to this family (reviewed in (Freedman et al. 1994)). Their functions are quite different. PDI catalyzes the formation of disulfide bonds in proteins as well as the hydroxylation of prolyl residues (Tasanen et al. 1988), whereas ER 60 (Urade and Kito 1992) and ER 72 (Urade et al. 1993) are cysteine proteases. ER 72 also is a calcium-binding protein (Mazzarella et al. 1990; Rupp et al. 1994), as are PDI (Lebeche et al. 1994) and P5 (Lundstrom Ljung et al. 1995). Therefore, in rats the homolog of mouse ER 72 is called the calcium-binding protein 2 (CaBP2), and the homolog of hamster P5 is termed CaBP1 (Rupp et al. 1994).

These members of the PDI family have been identified to be resident in the lumen of the endoplasmic reticulum (ER) and are abundantly found in secretory cells. We already have shown that PDI in S.mansoni is mainly expressed in the gastrodermis, the protonephridia and the sustentacular cells of the testes, indicating a secretory function of these tissues (Finken et al. 1994). To study the sites of expression of the ER 60 homolog in schistosomes, in situ hybridization and immunolocalization were carried out. ER 60 was found to be accumulated in exactly the same cells as PDI.

Materials and methods

Schistosoma stock. A S. mansoni stock from Liberia was cultured as described previously (Köster et al. 1988), except that Syrian hamsters were used as mammalian hosts.

Labelling of nucleic acids. DNA was radiolabelled with (alpha32P)dATP by random priming. Radioactive in vitro transcripts of recombinant pTZ18U were obtained with T7 RNA polymerase using (alpha32P)-UTP. Oligonucleotides were 5'end-labelled with (gamma32P)-dATP (kit from Boehringer-Mannheim). In vitro transcripts of recombinant pTZ18U were labelled with Digoxigenin-UTP following the protocol of the manufacturer (Boehringer-Mannheim).

Primer extension. 300 ng poly(A+)RNA were primed with 200 ng of a specific 15mer oligonucleotide, and the reaction was carried out for 2 h in 10 µl with 20 U AMV reverse transcriptase, 20 U RNasin, and 1 mM dNTPs at 42°C.

Southern and Northern blotting. DNA and RNA were prepared by the guanidinium thioisocyanate method (Chirgwin et al. 1979). Southern blotting was carried out by standard procedure, and hybridization with radiolabelled DNA probes was at 65°C in 2x SSC, 0.2% SDS and 1x Denhardt's solution (1x SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0). For Northern blotting, RNA was dissolved in 50% formamide, 6% formaldehyde, 1x MOPS buffer (20 mM 3-N-morpholino propanesulfonic acid, 5 mM sodium acetate, 1 mM EDTA, pH 7.2), and heated for 5 min at 65°C. RNA was electrophoresed on formaldehyde gels, transferred to HybondTM membranes and UV-crosslinked according to the manufacturer's protocol (Amersham). Hybridization was performed at 42°C in 50% formamide, 5x SSC, 50 mM sodium phosphate buffer, pH 6.5, and 1x Denhardt's solution.

Western blots. Anti-schistosome antisera were produced by immunization of rabbits with homogenates of schistosomes. Purification of antibodies to monospecificity was carried out on filter replicas of plate lysates of isopropyl-ß-D-thiogalactopyranoside-induced recombinant lambda gt11 amp3 clones (Köster et al. 1988). Proteins were separated on 7.5% SDS-polyacrylamide gels, blotted onto Immobilon PVDF membranes (Millipore) and incubated with the antibodies. Detection of bound antibodies was achieved with alkaline phosphatase-conjugated goat anti-rabbit antibody (Dianova), and colour was developed with naphthol-AS-MX phosphate and Fast Brown RR (Sigma) (West et al. 1990).

In situ hybridization. Adult worms 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 hybridized for 12 h at 50°C with 25 ng digoxigenin labelled in vitro transcripts in 30 µl of hybridization solution (2x SSC, 50% formamide, 1x Denhardt's solution, 100 µg ml-1 salmon sperm DNA, 100 µg ml-1 yeast tRNA, and 10% dextran sulfate) (Köster et al. 1988). Detection of hybrids was achieved with alkaline phosphatase conjugated anti-digoxigenin antibodies, naphthol-AS phosphate and Fast Blue BN (Höltke et al. 1992).

Results and discussion

A partial clone from an S.mansoni cDNA library in lambda gt11 (Finken et al. 1994), arbitrarily termed cN11, was found to have sequence homology with a cysteine protease of the endoplasmic reticulum of rat, ER 60 (Urade and Kito 1992). This enzyme is known to degrade ER-luminal proteins with improper structures (Urade et al. 1992). Originally, ER 60 of rat has been misidentified as form-I of phosphoinositide-specific phospholipase C (Bennett et al. 1988). To search for a full-length clone, the 0.76 kb insert of our cN11 clone was used to screen 150,000 plaques from a S.mansoni lambda ZAPII cDNA expression library (Menrath et al. 1995). Seven positives were identified. The clone with the largest insert of 1.6 kb (termed cN11.2) was subcloned into pTZ18U and subsequently sequenced by the dideoxy chain termination method. The sequence contains a complete open reading frame of 484 amino acids, starting with the initiator codon AUG (Fig. 1). There is a 3'untranslated region of 122 nucleotides between the terminator codon TAG and the poly-A tail, as well as a canonical polyadenylation signal AATAAA eleven bases upstream of the poly-A tail. A search in the EMBL data base revealed 41% identity to rat ER 60 (Bennett et al. 1988) and 29% identity to rat protein disulfide isomerase (PDI) (Edman et al. 1985) (Fig. 2). Rat ER 60, rat PDI, and the schistosome N11 sequence are characterized by the common feature of a duplicated domain which is homologous to bacterial thioredoxin (Holmgren et al. 1975) (Fig. 2). The thioredoxin domain is about eighty amino acids long and contains the putative redox site motif CGHC (Edman et al. 1985). A presumed cleavage site of a signal peptide for ER entry is located after the first 14 or 17 amino acids (Von Heijne 1984).

The COOH-terminus KSEL matches the consensus of the ER retention signal KDEL, a tetrapeptide sequence with some variation at the first and second position (Munro and Pelham 1987). The KDEL signal is sufficient to cause retention of a protein in the ER that otherwise would be transported through the secretory pathway. It has been suggested that a receptor might function in a distal compartment by recognizing the KDEL signal on escaping resident proteins and mediating their return to the ER (Pelham 1989).

To determine the transcription start, a primer extension experiment was carried out (Fig. 3). A 15mer primer 74 bases downstream from the 5'end of cDNA clone cN11.2 was used for reverse transcription of poly(A+)RNA from adult worms. The transcription start site, designated as No. 1 in Fig. 1, was mapped 133 nucleotides upstream of the initiator codon, that means 99 bases in front of the cDNA clone. These 99 bases were determined on a genomic subclone of 409 nucleotides which overlaps the 5'end of the cDNA clone. The existence of introns within this portion of the sequence is rather unlikely, because no consensus motifs were found that could function as splicing signals.

On Northern blots, the cN11 probe detects a single band of 1.8 kb in RNA of both sexes (Fig. 4a) agreeing with the sequence data. In addition, protein homogenates of adult female and male schistosomes were probed with antisera on Western blots (Fig. 4b). We used rabbit antisera against proteins of adult schistosomes, affinity purified on the expression products of clone cN11 (method as in Köster et al. 1988). The antibodies detected a single band with an apparent molecular weight of 53 kD which corresponds to the size deduced from the N11 sequence. This demonstrates that the predicted PDI homolog does really exist in schistosomes. As on Northern blots, no differences were found in the level of expression between females and males. The same band has also been detected in proteins of miracidia and cercaria, the larval stages of schistosomes (data not shown).

To obtain genomic clones, a genomic S.mansoni library in EMBL3 (Finken et al. 1994) was screened with clone cN11. Out of 200,000, we found eight positive clones that appeared to be identical. The insert of one of these clones (termed gN11) was 15 kb. This was mapped using cN11.2 as a hybridization probe to detect transcribed regions. 2.3 kb of gN11 containing the entire transcribed region of the gene were subcloned in pTZ18U and sequenced (Fig. 1). Six exons were identified by a comparison with the cDNA clone cN11.2. The sequences of the exons were completely identical with the cDNA. All five introns are very small: their sizes are 40, 32, 34, 34 and 34 nucleotides (Fig. 1).

Genomic Southerns show fragments that correspond exactly with the restriction map of clone gN11 (Fig. 5). Since all fragments include introns and flanking regions of the gene which are not expected to be conserved, we conclude that N11 is a single copy gene.

In situ hybridizations were carried out with antisense transcripts of clone cN11. As a positive control, we used antisense transcripts of an eggshell precursor mRNA (Köster et al. 1988) resulting, as expected, in a specific labelling of the vitellarium. Heterologous mRNA from the Dig RNA labelling kit was used as a negative control and did not show any reaction. The major sites of ER 60 expression were the sustentacular cells of the testes (Otubanjo 1981) and the genital opening behind the ventral sucker in the male (Fig. 6a), as well as the epithelium of the protonephridia (Fig. 6b) and the gastrodermis surrounding the gut in females and males, whereas the parenchyma cells were only weakly labelled. Essentially the same tissues have been identified by immunohistological localization with the anti-N11 antibodies. No signals were visible in negative controls, where primary antibodies have been omitted. In positive controls with anti-eggshell precursor antibodies (Köster et al. 1988) only the vitellarium was labelled, as expected (data not shown).

Recent studies on the fate of proteins resident in the ER indicated the existence of a selective pre-Golgi degradation pathway. Not only unassembled proteins, but also intact proteins, such as HMG-CoA reductase, are degraded through this pathway (Inoue et al. 1991). It is possible that the endoplasmic reticulum resident proteins in mammals, ER 60 and ER 72, are components of this pre-Golgi degradation system. Both have been identified as novel cysteine proteases distinct from those of the cytosol and lysosomes (Urade and Kito 1992). In vitro, among ER proteins, specifically PDI and calreticulin are degraded by ER 60.

It is remarkable that the ER 60 homolog in schistosomes is located in exactly the same tissues that have been shown to be abundantly enriched in PDI (Finken et al. 1994): the sustentacular cells of the testes, the gastrodermis and the lining of the protonephridia. Being an ER-luminal protein, accumulation of PDI characterizes secretory cells that are rich in endoplasmic reticulum. This colocalization of PDI and ER 60 supports the assumption that ER 60 in schistosomes has a luminal ER-localization, similar to the ER 60 in vertebrates. It is therefore possible that the ER 60 homolog in schistosomes also has a similar function in protein degradation.

Acknowledgments

We thank Karin Wildhagen for outstanding photographic work. This investigation received financial supports from the Deutsche Forschungsgemeinschaft (grant Ku 282/13-2) and from the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases.

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FIGURE LEGENDS

FIG. 1. Nucleotide sequence and predicted amino acid sequence of the genomic clone gN11. The first nucleotide is the transcription start as determined by primer extension. The primer used is indicated by underlinement. The arrow at nucleotide position 100 designates the 5'end of cDNA clone cN11.2. The 5'- and 3`-untranslated regions and the five introns are presented in lower case letters. The active sites CGHC of the two thioredoxin domains are underlined. The putative ER retention signal KSEL and the putative signal for polyadenylation AATAAA are marked by double underlinement.

FIG. 2. Alignement of the deduced amino acid sequences of S.mansoni N11 rat ER 60. Dashes are inserted to maximize alignement and dots indicate identity with the rat sequence. The two thioredoxin domains are boxed, and the active sites CGHC are underlined.

FIG. 3. Determination of the transcription start by primer extension and parallel sequencing of a genomic subclone, using the same primer. The longest primer extension product (arrow) identifies an adenine as transcription start (underlined).

FIG. 4. (a) Northern blot of 20 µg of total RNA of adult schistosomes, hybridized with the 32P labelled transcript of clone cN11. Ethidium bromide stained RNA is shown on the left. (b) Western blot of 10 µg of protein homogenates of adult schistosomes, incubated with anti-N11 antibodies. High molecular weight standard is shown on the left.

FIG. 5. Southern blots of 20 µg of Eco RI (E) and HindIII (H) digested genomic DNA, hybridized with 32P labeled probes. A restriction map of the entire gene is shown below. Hybridization probes used were an Eco RI/HindIII fragment of 1.2 kb (a, c) and an HindIII/TaqI fragment of 0.4 kb (b). The fragments recognized on the blot are indicated below the map. Hatched regions represent exons. T = TaqI.

FIG. 6. Localization of ER 60 mRNA in tissue sections of adult male schistosomes by in situ hybridization. (a) is a longitudinal section in the region of the ventral sucker (VS); the sustentacular cells of the testes (T) and the lining of the genital opening (GO) are labelled. (b) is a longitudinal section through the lateral part of the body showing the labelled protonephridia. Scale bar = 50 µm.