An Isoform of Ferritin as a Component of Protein Yolk Platelets in Schistosoma mansoni

Peter SCHÜßLER 1, Elke PÖTTERS 1, Ralf WINNEN 1, Werner BOTTKE 2, and Werner KUNZ 1

1 Institut für Genetik and Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universität, D-40225 Düsseldorf, and 2 Institut für Allgemeine Zoologie und Genetik, Universität Münster, D-48149 Münster, Germany

Keywords: Parasite, gender-specific gene expression

ABSTRACT

Schistosoma mansoni possesses two isoforms of the iron storage protein ferritin, Fer1 and Fer2. At the mRNA level as well as at the protein level, Fer1 is much more abundant than Fer2; females contain an about 15fold excess of Fer1 compared with males. In contrast, nearly equal amounts of Fer2 occur in both sexes. By electron microscopy we identify ferritin as a component of electron dense membrane-bound bodies in cells of the vitellarium. The mode of formation of these inclusions (as inferred from electron microscopy) and the abundance of phospholipid multilayered membranes suggest that these bodies are of a lysosomal nature. Here we interpret these ferritin-containing inclusions as protein yolk platelets. To date, most of the literature does not contain any hints for the existence of protein yolk in trematodes. The possible function of ferritin for embryonic development is discussed.

INTRODUCTION

Iron is essential for many enzymatic and other biochemical processes. However, free iron is toxic for cells. For this reason, most organisms possess an iron storage protein, ferritin (reviewed in Theil, 1987; Andrews et al., 1992).

The fluke worm Schistosoma mansoni lives endoparasitically in the blood veins of man where it ingests hundreds of thousands of erythrocytes per hour. Therefore, it incorporates huge amounts of iron. Mature females take up eight times more red blood cells than males (Lawrence, 1973). This observation is supported by spectrometric measurements demonstrating that female schistosomes contain a relatively higher concentration of iron than males (WoldeMussie and Bennett, 1982). It has also been shown that iron has a stimulatory effect on early growth and development of schistosomes (Clemens and Basch, 1989).

The massive uptake of iron by the females as compared with males suggests a possible role in egg formation and development. Therefore, we investigated Schistosoma for the presence of ferritins and their cellular localization. In a previous paper, we reported the sequences of two ferritin isoforms, Fer1 and Fer2 (Dietzel et al., 1992). Interestingly, both ferritin types share about 50% sequence identity with ferritin H chains of vertebrates. In the course of these investigations, evidence has been obtained for a female-specific expression of one of the two isoforms, Fer1. This prompted the present study in which this ferritin isoform is further characterized. In addition, we localized ferritin in membrane-bound cytoplasmic inclusions of vitelline cells, interpreted as protein yolk platelets. Thus, ferritin may exert an important function in early embryogenesis of schistosomes.

MATERIALS AND METHODS

Parasites and Hosts

S.mansoni of Liberian origin was maintained as described previously (Dietzel et al., 1992).

RNA and Protein Extraction

Preparation of RNA, DNA, and protein extracts from adult female and male schistosomes based on the RNA extraction procedure by Chirgwin et al. (1979) with modifications. Here the tissue was homogenized in liquid nitrogen and solubilized in a guanidinium thiocyanate buffer (4M Gu-SCN, 25 mM Na-citrate, 0.5% N-laurylsarcosine, 0.7% ß-mercaptoethanol) at 65°C for 10min. After removing the cell debris by a centrifugation step (12,000g, 4°C, 20min), the supernatant was carefully loaded onto a CsCl cushion (2ml 5.7M CsCl/0.1M EDTA pH7.4, 2ml 4.5M CsCl/0.1M EDTA pH7.4), and centrifuged at 35,000rpm for 20hr in a SW40 rotor (Beckman). Bands representing protein (top), DNA (center), and total RNA (bottom) can be recovered. The protein fraction is dialyzed against PBS buffer (150mM NaCl, 10mM NaH2PO4 pH7.2) at 4°C for 24hr followed by precipitaion with trichloracetic acid (5%, solid) and resuspension in PBS. The RNA is solubilized from the bottom of the tube with DEPC treated dH2O at 50°C for 1hr and recovered by precipitation with 0.1 vol sodium acetate (3M, pH4.5) and ethanol. This procedure yields in 30mg protein and 600mg total RNA per 1000 worms.

Northern Blotting

Total RNA of female and male schistosomes was dissolved in 50% formamide, 6% formaldehyde, 1x Mops buffer (20mM 3-N-morpholino propanesulfonic acid, 5mM sodium acetate, 1mM EDTA, pH7.2), and heated for 5min at 65°C. 20mg samples of RNA per lane were electrophoresed through 1.5% agarose/2.2M formaldehyde gels and transferred to HybondTM membranes according to the manufacturer's protocol (Amersham). The filters were baked for 2hr at 80°C.

In Vitro Transcription and Hybridization

cDNA sequences, subcloned in pTZ18U/19U or pGEM3Z, were transcribed in vitro using T7/SP6 RNA polymerase. 0.5mg of recombinant plasmid DNA were linearised, and transcription was carried out at 37°C for 1hr in standard buffer, 5mM DTT, 10u RNasin, 0.5mM ATP, CTP, GTP each, and 10mCi (a32P)UTP. The template DNA was removed by DNase, RNase-free. Hybridization on Northern blots was performed overnight at 42°C in 50% formamide, 5x SSC, 50mM sodium phosphate buffer pH6.5, and 1x Denhardt's solution. Quantification of hybrids on X-ray films was carried out with a Soft Laser Densitometer Scanner SL-504-XL by integration.

Antibodies

The complete Fer1 cDNA sequence has been cloned in pEMBLex2 vector and expressed in bacterial strain GC382. Following the procedure of Levi et al. (1987), we have isolated pure Fer1 protein which has been used for immunization of rabbits and production of anti-Fer1-antibodies by standard methods. The complete Fer2 cDNA sequence was expressed as a fusion protein which was used to produce antibodies. These were purified by incubation with Fer1 protein.

SDS-PAGE and Immunoblotting

Protein suspensions in PBS (see above) were further treated for gel electrophoresis and Western blotting as described earlier (Köster et al., 1988). Electrophoretically separated proteins were blotted onto Immobilon PVDF membranes (Millipore). Detection of bound antibodies was achieved with alkaline phosphatase-conjugated goat anti-rabbit antibody (Dianova), and colour was developed with naphthol-AS phosphate and Fast Brown RR (Sigma) (West et al., 1990).

Electron Microscopy

Adult schistosomes were fixed by immersion in 2% glutaraldehyde and post-fixed in 1% OsO4. Tissues were dehydrated in graded acetone and embedded in Araldite. Unstained thin sections were inspected in a Zeiss EM 900 electron microscope. For reference, sections post-stained with uranyl acetate and lead citrate were inspected in parallel.

RESULTS

Fer1 is Preferentially Expressed in Females

In a previous paper we have reported the complete coding regions of the cDNAs of two ferritin subunits from S.mansoni named Fer1 and Fer2 (Dietzel et al., 1992). The Fer1 isoform has been shown to be expressed preferentially in females.

To quantify gender specific differences in ferritin mRNA amounts, we hybridized Northern blots of male and female RNA with three probes. Two of them consisted of the entire coding regions of Fer1 and Fer2 (Fig.1A).

The Fer1 transcript was found to be about 15 times more abundant in females, whereas the amount of the Fer2 transcript was nearly equal in both sexes (Fig.1B,C). Since it has not been possible to carry out all the hybridization experiments under exactly the same conditions, we were not able to quantify the frequency of Fer1 in relation to Fer2 expression. We therefore constructed as a third probe for hybridization a chimeric recombinant plasmid containing portions of both Fer1 and Fer2 sequences of equal length (Fig.1A). On Northern blots, the signal of hybridization with this probe was about ten times more intense in females than in males (Fig.1B,C). From these data it has been calculated that in S.mansoni the Fer1 transcript is expressed about 28 times more frequently than the Fer2 transcript, and it is mainly the female that transcribes Fer1.

At the protein level, Fer1 was documented on Western blots using monospecific antibodies. These antibodies react with a single band of about 21 kDa (Fig.2). This lies in the size range of vertebrate ferritin subunits. Densitometric measurements of the amounts of antibodies bound reveal an approximately 15fold excess of Fer1 protein in females. In contrast, antibodies against Fer2 detect nearly equal amounts of Fer2 protein in the two genders (data not shown). These values are consistent with the Northern blot data, documenting again the female preferential expression of Fer1.

Ferritin is Deposited in Vitelline Cells

The abundance of Fer1 in females suggests the functional importance of this ferritin type for oogenesis and/or embryogenesis. Therefore, glutaraldehyde-fixed and osmicated but otherwise unstained thin sections of Schistosoma were inspected for the presence of electron dense ferritin cores. Small amounts of ferritin cores were found randomly dispersed in different tissues of both sexes. However, massive concentrations of ferritin were detected in the vitelline cells where it accumulated in membrane-bound cytoplasmic inclusions, but not in the cytosol (Fig.3A-D). Ferritin occurred in the form of randomly dispersed particles and in paracrystalloid arrangements (Fig.3B-D). Some ferritin cores formed curved arrays, probably as a result of their association with phospholipid membranes (Fig.3C).

In contrast to the two major types of inclusion bodies of the vitelline cells, egg shell protein platelets and lipid droplets, the membrane-bound inclusions are irregular in shape (Fig.3A). Staining of sections with uranyl acetate and lead citrate revealed that these inclusions contained whorls of membranes thus giving them the appearance of myelinosomes (Fig.3B). In a later part of this paper, we interpreted these myelinosomes as being protein yolk platelets (see Discussion).

DISCUSSION

The oocytes of oviparous organisms contain large amounts of protein yolk which is utilized by the growing embryo. Here vitellogenesis involves massive heterosynthetic synthesis of vitellogenins and their deposition in the oocytes (Postlethwait and Jowett, 1980; Wallace, 1985; Wahli, 1988). It is known that these exogenously derived yolk protein precursors are synthesized in the liver of vertebrates (Tata, 1988), in the digestive gland of snails (Bride et al., 1992), in the fat body as well as the follicle cells in insects (Postlethwait and Jowett, 1980; Brennan et al., 1982), and in the gut epithelium of nematodes (Kimble and Sharrock, 1983). Exogenous yolk is then incorporated into the oocyte by receptor-mediated endocytosis (Ferenz, 1993).

Mainly on the basis of electron microscopical work, it has been postulated that some animal groups like amphibia (Kress, 1982), ticks (ElShoura et al., 1989), Crustacea (ScanabissiSabelli and Tommasini, 1990), Annelida (Pfannenstiel and Grunig, 1982) and molluscs (Bottke and Tiedtke, 1988) produce a second type of yolk, the so-called autosynthesized or endogenously formed yolk. Morphological observations suggest that in these cases the rough endoplasmic reticulum and the Golgi apparatus are involved in yolk production. However, while it is clear that oocytes of these animal groups synthesize proteins during vitellogenesis (Bottke and Tiedtke, 1988), none of the putative yolk proteins has been further characterized and no evidence for participation of specific yolk protein encoding genes, e.g. vitellogenin genes has been obtained.

Up to now, there exists only one hint in the literature for a possible existence of protein yolk in trematodes (Bunke, 1981). In contrast to almost all other oviparous animals hitherto investigated, schistosomes are not characterized by predominant protein bands in polyacrylamide gels that distinguish mature females from males or immatures (Ruppel and Cioli, 1977). From this, it could be concluded that schistosomes either do not have specific yolk protein coding genes or their expression remains at a low level.

This conclusion is supported by observations at the cytological level. In contrast to most other oviparous animals, oocytes of trematodes do not contain protein yolk platelets. In the cells of the vitellarium which is considered to be a sister organ of the ovary, egg shell protein vesicles and lipid droplets are the dominant inclusion bodies. Definitive protein yolk platelets are not mentioned (Erasmus, 1973).

However, Erasmus made some most important observations in mature vitelline cells (1973; 1975). He described in detail the formation of "ribosomal complexes" or "cytosegresomes" and presented evidence that these structures are formed from ribosomes and elements of the rough endoplasmic reticulum by a process of autolysis resulting finally in the formation of myelin-like membranous bodies. From our observations, it is clear that these structures are identical with the ferritin-containing inclusion bodies here described as myelinosomes. In analogy to findings with other animal groups, e.g. molluscs (Bottke et al., 1982) and in coincidence with a suggestion of Bunke (1981), we tentatively identify these inclusion bodies as protein yolk platelets.

So far, the source of the vitellogenic ferritin is not known. Since we failed to detect isolated ferritin particles in the cytosol of the vitellocytes, synthesis of major amounts of the protein in these cells is unlikely. Instead, preliminary results are more in favour of the synthesis of Fer1 in somatic tissue and of the transport of the protein into the vitelline cells via endocytosis (Winnen et al., in prep.). This classifies the putative yolk platelets of schistosomes to be of mixed origin, combining endogenously formed components with exogenously synthesized ferritin. Formation of yolk platelets with proteins of both endogenous and exogenous origin is also found in some amphibia (Kress, 1982).

In both sexes Fer2 is only present in small amounts. In contrast, Fer1 is 28times more abundant and highly enriched in females. It follows that the ferritin found in the vitellarium is probably the Fer1 isoform. In schistosomes, Fer1 may well be a yolk ferritin, in analogy to a parallel situation in the snail Lymnaea where ferritin was first discovered to exist as a separate isoform that functions as the principal yolk component (Von Darl et al., 1994). The presence of a ferroxidase center in the sequence of Fer1 (Dietzel et al., 1992) suggests that it is capable of accelerating iron oxidation. Thus it appears to be stored on account of its iron sequestering properties rather than as a metabolically inert amino acid supply for the embryo. Two different functions for yolk ferritin are possible. It may either be essential as an iron source for the developing embryo or it may protect the embryo and the larva against bacterial growth, since iron is the only known nutritional factor of general importance for enteropathogenic bacteria (Winkelmann et al., 1987). Ferritin has also been shown to occur in amphibian oocytes (Kandror et al., 1992). In egg white protein of chicken, ovotransferrin has been identified as an iron transport protein (Stevens, 1991), and another metalloprotein, ovohemerythrin, is a major yolk component in the leech (Baert et al., 1992).

Vertebrates and other higher eukaryotes regulate their ferritin gene expression at the post-transcriptional level. This regulation is based on the interaction between the iron-responsive element (IRE) within the 5'untranslated region of the ferritin mRNA and the IRE-binding protein (IRE-BP) (Casey et al., 1988). In the snail Lymnaea, only the ferritin isoform expressed in somatic tissue is regulated by the IRE, whereas the yolk ferritin does not possess an IRE (Von Darl et al., 1994). In schistosomes, both ferritin isoforms also have no IRE (Schüßler et al., in prep.). We therefore conclude that they are not controlled by an IRE/IRE-BP-mechanism, but that regulation is solely at the transcriptional level as known for lower eukaryotes and plants (Rothenberger et al., 1990; Lobreaux et al., 1993). Fer1 is expressed in a developmentally regulated manner. It is present in abundance only in mature, egg-laying females, whereas immature females like males only show a low level of expression (Winnen et al., in prep.).

ACKNOWLEDGEMENTS

We thank Dr. Jutta Dietzel and Jörg Hirzmann for supplying us with the Fer2 and Fer1 clones. We also thank Jörg Hirzmann, Dr. Markus Maniak, and Dr. Anja Michel for helpful hints and discussions. 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. Quantification of the mRNAs of the two ferritin isoforms in males and females. (A) Restriction enzyme maps of the three hybridization probes. (B) Lanes M, 1 and 2: ethidium bromide- stained RNA gels; lanes 3-8: Northern blots of the RNAs of lanes 1 and 2. M: 20mg of 1kb ladder; 1, 3, 5, and 7: female RNA; 2, 4, 6, and 8: male RNA; 3 and 4: Fer1 probe; 5 and 6: Fer2 probe; 7 and 8: Fer1+2 probe. Weak signal at 2kb results from crosshybridization with rRNA. (C) Densitometric quantification of the Northern blots of (B): Fer1 profile relates to lanes 3 and 4, Fer2 profile relates to 5 and 6, and Fer1+2 profile relates to 7 and 8. Integration of the areas below the curves yields male-female mRNA relation.

Fig.2. Immunoblotting: 40mg of S.mansoni protein were separated on a 15% SDS-polyacrylamide gel, blotted for 2hr and incubated with anti-Fer1 antibody, 1:500. Lane M: low molecular weight Biorad marker, lane 1: female protein, lane 2: male protein.

Fig.3. Electron microscopical localization of ferritin in putative yolk platelets in vitelline cells. (A) Overview of the three types of inclusion bodies in a vitelline cell. Bar = 1mm. (B) Yolk platelet from Fig.3A at higher resolution. Arrows designate ferritin paracrystalloids in part associated with whorls of phospholipid membranes. Bar = 1mm. (C) and (D) Ferritin paracrystalloids and randomly dispersed ferritin particles in yolk platelets. Ferritin paracrystalloids in (C) are associated with myelin-like phospholipid membrane whorls. Bar = 0.25 mm. E, egg shell protein platelet, L, lipid droplet, M, phospholipid membranes, Y, ferritin-containing yolk platelet.