IPSE/alpha-1: A major immunogenic component secreted from Schistosoma mansoni eggs☆
Introduction
Infection with the parasitic trematode Schistosoma mansoni is dominated by a pronounced T helper type 2 (Th2) response with elevated IgE synthesis [1]. The first weeks of infection, however, are characterized by a Th1 response directed to worm antigens. From 6 to 8 weeks after infection, coincident with the onset of egg deposition, the Th1 response is down-regulated and replaced by a Th2 response primarily directed to egg antigens. Together with the observation that a Th2 response does not develop during infection with single-sex male or radiation-attenuated cercariae, i.e. in the absence of mature female worms and egg deposition [2], [3], these findings suggested that the Th2 response in S. mansoni infection is egg-dependent. Further proof for this notion was provided by the observation that injection of live eggs or administration of water-soluble S. mansoni egg antigens (SmEA) led to a Th2 response in naïve animals [4], [5], [6], [7]. Schistosome eggs and SmEA can thus be deemed to contain one or more constituents with Th2-inducing capacity.
SmEA is a complex mixture of both structural and excretory/secretory antigens. As the Th2 response starts very early after egg laying, it seems likely that the egg components responsible for Th2 induction are excretory/secretory products (rather than structural components of the egg) which react with the immune cells that are recruited around the egg to form peri-oval granulomas.
Recently we identified a protein in SmEA, which stimulates human basophils rapidly to release large amounts of IL-4 [8]. The protein was purified, and its gene cloned and sequenced. It is a dimeric glycoprotein forming a double band of about 40 kDa in non-reducing SDS-PAGE and of about 20 kDa under reducing conditions. The molecule was named IPSE (for IL-4-inducing principle of S. mansoni eggs). We found that IPSE is a general activator of human basophils, triggering their degranulation as well as the release of IL-13 and histamine, and that it is the only molecule present in SmEA responsible for these effects.
Nearly 25 years ago, Dunne et al. [9] described and characterized several antigens contained in SmEA based on their electrophoretic mobility and recognition by sera from mice with chronic patent infections. Two of the antigens, termed alpha-1 and omega-1, were co-purified from complete SmEA by cation exchange chromatography in a salt-eluted fraction termed CEF6. SDS-PAGE revealed that alpha-1 is a dimer of two bands of 41 and 36 kDa under non-reducing conditions and two bands of 22 and 18 kDa under reducing conditions [10]. A function for this molecule was not found at that time. Since IPSE and alpha-1 are both cationic glycoproteins with similar apparent molecular weights, we asked whether they were identical. To study this question, N-terminal sequencing and Western blots were performed, and the results obtained clearly indicate that alpha-1 and IPSE are indeed the same molecule.
Two further issues were addressed in the present work: (1) since the Th2-inducing capacity of S. mansoni is restricted to its egg stage, the putative schistosomal Th2 trigger molecule should be likewise restricted to this developmental stage. Investigation of the transcriptional and translational expression of IPSE/alpha-1 in different life cycle stages of this parasite indicated that it was synthesized exclusively in the egg stage of S. mansoni; (2) IPSE/alpha-1 was found to be secreted from the egg and to come into contact with circum-oval immune cells. Consistent with this, we found that S. mansoni-infected mice and humans develop a pronounced antibody response against IPSE/alpha-1.
Together with its capacity to induce the release of IL-4 from human basophils, these findings are in line with our hypothesis that this potent immunogen might be involved in inducing the Th2 response characteristic of S. mansoni infections.
Section snippets
Antigens
SmEA was prepared from S. mansoni eggs isolated from the tissues of mice infected percutaneously with 250 cercariae approximately 42 days previously. Methods were as described by Doenhoff et al. [11].
CEF6 was purified from SmEA by cation exchange chromatography using a single-step adaptation [12] of a method described earlier by Dunne et al. [9].
S. mansoni egg culture supernatant (SmECS) was prepared essentially as previously described by Ashton et al. [13]. Approximately 0.5 million S. mansoni
IPSE is identical with alpha-1
The two proteins IPSE and alpha-1 exhibited very similar characteristics in SDS gels with respect to their apparent molecular weight, forming a double band, and sensitivity to reducing agents. We, therefore, asked whether these two proteins were identical.
To address this question, a polyclonal rabbit antiserum raised against alpha-1 and a monoclonal antibody raised against recombinant His-tagged IPSE (74 4B7) were tested in parallel for their reactivity against SmEA (S. mansoni soluble egg
Discussion
Here, we show that IPSE, a secreted egg glycoprotein with basophil IL-4-inducing activity [8], is identical with the S. mansoni egg antigen alpha-1, originally described nearly 25 years ago [9]. Moreover, we demonstrate that IPSE/alpha-1 is exclusively expressed by the egg stage and acts as a potent immunogen, which possibly plays a role in initiating a Th2 response.
The putative identity between IPSE and alpha-1 had first become apparent when their purification protocols, molecular weights and
Acknowledgements
The authors like to thank PD Dr. Arnd Petersen for N-terminal sequencing and Stefan Sroka for technical assistance.
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