The Schistosoma mansoni protein Sm16/SmSLP/SmSPO-1 is a membrane-binding protein that lacks the proposed microtubule-regulatory activity

Abstract

Sm16/SmSLP/SPO-1 (Sm16) has been identified as a developmentally regulated protein that is released from specific glands of the Schistosoma mansoni parasite during skin penetration. Sm16 has been ascribed both anti-inflammatory activities and a functional similarity with the conserved cytosolic tubulin-binding protein stathmin/Op18. Here we used a cell line to confirm signal peptide-dependent secretion and to define the secreted form of Sm16 for production in E. coli. We present evidence from both in vitro experiments and studies on transfected human cells that refute any functional similarity with stathmin/Op18. Instead of an Op18-like activity, we found that targeting of Sm16 to the cytosol of human cells, which was achieved by ectopic expression of Sm16 lacking the signal peptide, results in a caspase-dependent apoptotic response. Interestingly, by analysis of recombinant preparations we found that the secreted form of Sm16 is a lipid bilayer-binding protein that efficiently binds to the surface of diverse cell types by a polyanion-independent mechanism, which results in uptake by endocytosis. While the significance of the pro-apoptotic activity exerted by cytosolic Sm16 remains unclear, the present findings on cell-surface-binding properties of Sm16 seems likely to be of functional relevance during skin penetration of the parasite.

Introduction

The parasitic helminth Schistosoma mansoni is the causative agent of schistosomiasis and has a complex life cycle involving human and snail hosts, as well as two free-living water-borne larval stages [reviewed in 1]. Transformation between developmental stages is associated with major changes in size and physiological features. These developmental stages include the miracidium (which is derived from ova excreted in human feces and is infectious to snails), the sporocyst (a parasitic form in the snail), cercaria (released by snails and infectious to humans by penetration of skin), the schistosomulum (the stage after penetration of human skin), and the adult worm (a long-term parasitic form in humans).

Medium-chain free fatty acids in lipids on human skin stimulate holocrine release of adhesive secretions from the acetabular glands of the cercariae [reviewed in 2]. In addition to the well-characterized cercarial proteases, which are by far the most abundant components [3], these secretions have been proposed to possess various immunomodulatory activities [reviewed in 4]. The best characterized of these activities is the Sm28GST protein, which has prostaglandin D₂ synthase activity and thereby provides a strategy for the schistosome to evade host immune defenses [5]. Another activity present in cercaria secretions, termed S. mansoni apoptosis factor, has been proposed to induce apoptosis specifically in CD4⁺ T-lymphocytes [6]. Finally, a 16.8-kDa secreted cercaria protein, designated Sm16, has been ascribed anti-inflammatory activities [7], [8], and the corresponding cDNA was subsequently cloned [9]. A cDNA-encoding Sm16 has also been isolated based on preferential mRNA expression at the sporocyst-stage and termed SmSPO-1 [10]. Subsequent proteomic studies have revealed that Sm16 is an abundant protein in the secretions of cercariae and correspond to 3–4% of total proteins in these secretions [3].

Sm16 has also been identified as an abundant protein during the late sporocyst stage that disappears a few days after penetration of the skin of the mammalian host [11]. Based on the reported tubulin heterodimer-binding activity and a limited sequence homology with the tubulin dimer-binding protein stathmin/Op18, Sm16 was designated S. mansoni stathmin-like protein (SmSLP). Stathmin/Op18 is a highly conserved microtubule destabilizing protein [reviewed in 12], which bind two tubulin heterodimers via two imperfect helical repeats to form a complex with two tubulin heterodimers aligned head-to-tail [13], [14]. Multiple cell cycle and signal transduction-regulated kinase systems phosphorylate stathmin/Op18 to attenuate its microtubule destabilizing activity, which provides a mechanism to regulate microtubule polymerization in many cell types [15], [16].

Previous studies on Sm16 have been limited to the protein isolated from cercaria secretions, which imposes limitations on the both the quantity and the purity of the protein. To evaluate the reported functional similarity between stathmin/Op18 and Sm16, we here prepared purified recombinant proteins and analyzed these side by side in various in vitro systems. We also expressed these two proteins in the cytosol of human cells, i.e., the site of action of stathmin/Op18, to compare biological properties in intact cells. Our results refuted any functional similarities between Sm16 and the microtubule-regulatory protein stathmin/Op18. Instead we found that Sm16 has the potential to exert a potent pro-apoptotic activity if expressed as a cytosolic protein in human cell lines and that the secreted form of Sm16 is a lipid-binding protein that efficiently binds to the surface of human cells.