Figure 1.
Domain structures of S. mansoni integrin receptors.
Schematic structure of the schistosome α- and β-integrin receptors. Amino acid positions of the predicted conserved domains: Smα-Int1: 1273 aa; Int α 255–334, 359–418, 449–496; TM 1212–1234; Smα-Int2: 1492 aa; Int α 404–472, 476–546, 552–603; SP 1–30, 1296–1318; Smα-Int3: 1091 aa; Int α 257–316, 345–399; TM 980–1008; Smα-Int4: 1411 aa; SP 1–36; Int α 419–477; TM 1292–1314; Smβ-Int1: 865 aa; INB 36–470; EGF 567–604; TM 794–816;. (EGF: epidermal growth factor-like domain, Int α: integrin α domain (beta-propeller repeats), INB: integrin β domain, SP: signal peptide, TM: transmembrane domain).
Figure 2.
Phylogenetic analyses showing the unique status of plathyhelminth α-integrins.
Phylogram of the analysis of the full-length sequences of the S. mansoni α-integrin receptors Smα-Int1, Smα-Int2, Smα-Int3, and other α-integrin receptors using CLUSTAL X (www.clustal.org) and TreeViewX. The phylogenetic relationship was deduced using the Bootstrap Neighbour-Joining (N–J) method and the bootstrap values were generated based on 1000 bootstrap trails with a random number generator seed of 100. Sequences were obtained from the National Centre for Biotechnology Information using the WWW Entrez Browser (www.ncbi.nlm.nih.gov), Swiss-Prot (www.uniprot.org), GeneDB (www.genedb.org), and the Schmidtea mediterranea Genome Database (http://smedgd.neuro.utah.edu/). The corresponding protein numbers are: Sha a1 (α-integrin 1 receptor, S. haematobium; Sha_102401), Sm a1 (α-integrin 1 receptor, S. mansoni; FR749887), Sjp a1 (α-integrin 1 receptor, S. japonicum; Sjp_0037690), Cs a5 (α-integrin 5 receptor, Clonorchis sinesis; GAA56616.1), Em a1 (α-integrin 1 receptor, Echinococcus multilocularis; EmuJ_000215000 ), Sm a4 (α-integrin 4 receptor, S. mansoni; Smp_1735401, Smp_181010), Sha a4 (α-integrin 4 receptor, S. haematobium; Sha_104436, Sha_106831), Sjp a4 (α-integrin 4 receptor, S. japonicum; Sjp_0046780, Sjp_0046790), Cs a4 (α-integrin 4 receptor, Clonorchis sinesis; GAA28731), Em a4 (α-integrin 4 receptor, Echinococcus multilocularis; EmuJ_000573500), Sha a2 (α-integrin 2 receptor, S. haematobium; Sha_106921), Sm a2 (α-integrin 2 receptor, S. mansoni; FR749888), Sjp a2 (α-integrin 2 receptor, S. japonicum; Sjp_0069490), Cs a-ps (α-integrin-ps receptor, Clonorchis sinesis; GAA54095, GAA49531, GAA49530), Em a2 (α-integrin 2 receptor, Echinococcus multilocularis; EmuJ_000192500 ), Smed a3 (α-integrin 3 receptor, Schmidtea mediterranea; lcl|mk4.000046.14.01), Smed a1 (α-integrin 1 receptor, Schmidtea mediterranea; lcl|mk4.001411.00.01), Smed a2 (α-integrin 2 receptor, Schmidtea mediterranea; lcl|mk4.003797. 00.01), Sha a3 (α-integrin 3 receptor, S. haematobium; Sha_102914), Sm a3 (α-integrin 3 receptor, S. mansoni; FR749889, Smp_156610, Smp_156620), Sjp a3 (α-integrin 3 receptor, S. japonicum; Sjp_0063430, Sjp_0063420), Cs a7 (α-integrin 7 receptor, Clonorchis sinesis; GAA52225.1), Em a3 (α-integrin 3 receptor, Echinococcus multilocularis; EmuJ_000782500), Sp aP (α-integrin P receptor, Strongylocentrotus purpuratus, AF177914), Dm aPS2 (α-integrin PS2 receptor, Drosophila melanogaster, Q24247), Mm a2b (α-integrin 2b receptor, Mus musculus; EDL34136.1), Hs a2b (α-integrin 2b receptor, Homo sapiens; EAW51595.1), Xl a2b (α-integrin 2b receptor, Xenopus laevis; NP_001088223.1), Mm a5 (α-integrin 5 receptor, Mus musculus; CAA55638.1), Rn a5 (α-integrin 5 receptor, Rattus norvegicus; NP_001101588.1), Hs a5 (α-integrin 5 receptor, Homo sapiens; NP_002196.2), Xl a5 (α-integrin 5 receptor, Xenopus laevis; NP_001081072.1), Hs aV (α-integrin V receptor, Homo sapiens; P06756), Hs a8 (α-integrin 8 receptor, Homo sapiens; P53708), Ce a-pat2 (α-integrin pat-2, Ceanorhabditis elegans; P34446), Gc a (α-integrin receptor, Geodia cydonium; X97283), Hs a1 (α-integrin 1 receptor, Homo sapiens; P56199), Hs a2 (α-integrin 2 receptor, Homo sapiens; P17301), Hs a10 (α-integrin 10 receptor, Homo sapiens; O75578), Hs a11 (α-integrin 11 receptor, Homo sapiens; Q9UKX5), Hs aD (α-integrin D receptor, Homo sapiens; Q13349), Hs aX (α-integrin X receptor, Homo sapiens; P20702), Hs aM (α-integrin M receptor, Homo sapiens; P11215), Hs aL (α-integrin L receptor, Homo sapiens; P20701), Hs aE (α-integrin E receptor, Homo sapiens; P38579), Hs a4 (α-integrin 4 receptor, Homo sapiens; P13612), Hs a9 (α-integrin 9 receptor, Homo sapiens; Q13797), Mm a7 (α-integrin 7 receptor, Mus musculus; AAA16600.1), Rn a7 (α-integrin 7 receptor, Rattus norvegicus; NP_110469.1), Hs a7 (α-integrin 7 receptor, Homo sapiens; EAW96822.1), Hs a6 (α-integrin 6 receptor, Homo sapiens; P23229), Hs a3 (α-integrin 3 receptor, Homo sapiens; P26006), Dm aPSI (α-integrin PSI receptor, Drosophila melanogaster, Q24247), and Ce a-ina1 (α-integrin ina1, Ceanorhabditis elegans; Q03600).
Figure 3.
Phylogenetic analyses showing the unique status of plathyhelminth β-integrins.
Phylogram of the analysis of the full-length sequences of the S. mansoni β-integrin receptor Smβ-Int1 and other β-integrin receptors using CLUSTAL X (www.clustal.org) and TreeViewX. The phylogenetic relationship was deduced using the Bootstrap Neighbour-Joining (N–J) method and the bootstrap values were generated based on 1000 bootstrap trails with a random number generator seed of 100. Sequences were obtained from the National Centre for Biotechnology Information using the WWW Entrez Browser (www.ncbi.nlm.nih.gov), Swiss-Prot (www.uniprot.org), GeneDB (www.genedb.org), and the Schmidtea mediterranea Genome Database (http://smedgd.neuro.utah.edu/). The corresponding protein accession numbers are: Dm bPS (β-integrin PS, Drosophila melanogaster; P11584), Bg b (β-integrin, Biomphalaria glabrata; AF060203), Ce b-pat3 (β-integrin pat-3, Ceanorhabditis elegans; Q27874), Sp bL (β-integrin L subunit, Strongylocentrotus purpuratus; NP_999731), Sp bG (β-integrin G subunit, Strongylocentrotus purpuratus; NP_999732), Sp bC (β-integrin C subunit, Strongylocentrotus purpuratus; AF0559607), Mm b1 (β-integrin 1 receptor, Mus musculus; NP_034708.1), Rn b1 (β-integrin 1 receptor, Rattus norvegicus; NP_058718.2), Hs b1 (β-integrin 1 receptor, Homo sapiens; P05556), Gg b1 (β-integrin 1 receptor, Gallus gallus; NP_001034343.2), Xl b2 (β-integrin 2 receptor, Xenopus laevis; NP_001080017.1), Hs b2 (β-integrin 2 receptor, Homo sapiens; NP_000202.2), Hs b7 (β-integrin 7 receptor, Homo sapiens; NP_000880.1), HS b3 (β-integrin 3 receptor, Homo sapiens; P05106), Hs b5 (β-integrin 5 receptor, Homo sapiens; P18084), Hs b6 (β-integrin 6 receptor, Homo sapiens; P18564), Hs b 8 (β-integrin 8 receptor, Homo sapiens; P26012), Gc b (β-integrin receptor, Geodia cydonium; O97189), Am b (β-integrin, Acropora millepora; AF005356), Hs b4 (β-integrin 4 receptor, Homo sapiens; P16144), Sha b1 (β-integrin 1 receptor, S. haematobium; Sha_105750), Sm b1 (β-integrin 1 receptor, S. mansoni; FR749886), Sjp b1 (β-integrin 1 receptor, S. japonicum; Sjp_0081260), Cs b1 (β-integrin 1 receptor, Clonorchis sinesis; GAA31131.2), Em b1 (β-integrin 1 receptor, Echinococcus multilocularis; EmuJ_000528400), and Smed b1 (β-integrin 1 receptor, Schmidtea mediterranea; lcl|mk4.001280.01.01).
Figure 4.
In situ-hybridization localized transcripts of Smβ-Int1, Smα-Int1- Smα-Int4 in the gonads and gonad-associated tissues of adult S. mansoni.
Representative sections (5 µm) of adult schistosome couples are shown (males and females are indicated), which were hybridized with DIG-labeled antisense-RNA probes of Smβ-Int1 (A–C), Smα-Int1 (D–F), Smα-Int2 (G, H), Smα-Int3 (I), Smα-Int4 (K) and for control with a DIG-labeled sense-RNA probe of Smα-Int3 (J), Smα-Int4 (L), or Smβ-Int1 (M, N). mRNA transcripts of Smβ-Int1 were detected in the ovary (o), the ootype-surrounding area (ot) anterior the ovary, the vitellarium (v), the subtegument (st), the testes (te), and the parenchyma (p) of both genders. Transcripts of Smα-Int1 were also detected in the ovary (o), the ootype-surrounding area (ot), and the vitellarium (v) of the female, the testes of the male, and the parenchyma (p) of both genders. Transcripts of Smα-Int2 were exclusively detected in the ootype-surrounding area (ot) anterior the ovary (o). Antisense and sense transcripts of Smα-Int3 and Smα-Int4 were only detected in the ovary (o). No signals were detected using sense transcripts of Smβ-Int1 (K, L), Smα-Int1, and Smα-Int2 (unpublished). dp: dorsal part; vp: ventral part; vs: ventral sucker; scale bars as indicated.
Figure 5.
Interaction studies confirmed binding of Smβ-Int1 to the schistosome cellular kinases SmTK3, SmTK6, and SmTK4. A:
For binding studies in the YTH system, yeast cells (strain AH109) were co-transformed with the prey plasmid Smβ-Int1-C-term pACT2 together with the baits SmTK3-SH4SH3 pBridge, SmTK3-SH3 pBridge, SmTK6-SH4SH3 pBridge, SmTK6-SH3 pBridge, and SmTK4-SH2SH2 pBridge. Yeast clones were selected on Trp−/Leu−/His− media (T−/L−/H−) for interactions between bait and prey proteins, and β-galactosidase colony lift filter assays were performed for detection of LacZ expression. B: Comparative β-galactosidase liquid assays were performed with the yeast clones from A to determine the relative binding strengths. As control, untransformed yeast cells (AH109) were used (control). The statistical evaluation of seven independent measurements of β-Gal activity (n = 7) is shown (error bars are indicated). C: Co-Immunoprecipitation of HA-Smβ-Int1 and V5-SmTK4 expressed in Xenopus oocytes. Anti-HA antibodies immunoprecipitated Smβ-Int1 together with SmTK4 upon co-expression in oocytes. Inversely, anti-V5 antibodies immunoprecipitated SmTK4 with Smβ-Int1, when they were expressed together in oocytes.
Figure 6.
RNAi knocking-down Smβ-Int1 and SmDia led to morphological changes in the ovary of S. mansoni females. A:
Using a combination of four siRNAs specific for Smβ-Int1 (β-Int1 siRNAs 1–4), a Smβ-Int1-transcript reduction down to 58% was determined by semi-quantitative RT-PCRs (n = 2) compared to control worms (no siRNAs), or worms electroporated with Smα-Int1-specific siRNAs (α-Int1 siRNA 1–4). B: The combination of two dsRNAs specific for SmDia led to a SmDia-transcript reduction down to 43% compared to control worms (no dsRNAs), which was determined by semi-quantitative RT-PCRs (n = 3). C: Confocal scanning laser microscope images of carmine red-stained whole-mount preparations of S. mansoni couples treated with siRNA specific for Smβ-Int1 (A, B), Smα-Int1 (C), without si/dsRNA (D), or with SmDia-specific dsRNAs (E, F). io: immature oocytes, mo: mature oocytes, arrows: poly-nucleated oocytes; scale bars 50 µm.
Figure 7.
Model for integrin receptor and RTK-induced signaling pathways in S. mansoni.
The Syk kinase SmTK4, but also the Src kinase SmTK3, and the Src/Abl kinase SmTK6 are able to bind to the intracellular part of Smβ-Int1. Results of previous studies had already indicated that these three kinases interacted with each other and with SmVKR1, and all co-localized in the ovary of females [23], [27], [29]. Furthermore, SmTK3 was found to interact with the diaphanous homolog SmDia [71], which is a binding partner of the Rho-GTPase SmRho1. Both SmDia and SmRho1 were suggested to organize the actin cytoskeleton within the gonads of schistosomes [71]. As downstream partners of SmTK4, MAPK-activating protein (PM20/21) and mapmodulin were found, which may be involved in cytoskeleton reorganization and mitosis [29]. SmDLG as a binding partner of SmTK6 [27] may become activated upon complex formation and may subsequently interact with SmLGL and Scribble to control processes of cell growth and/or cell polarity (Buro et al., unpublished).