title: Interleukin-4-inducing principle from Schistosoma mansoni eggs contains a functional C-terminal nuclear localization signal necessary for nuclear translocation in mammalian cells but not for its uptake.
abstract: Interleukin-4-inducing principle from schistosome eggs (IPSE/alpha-1) is a protein produced exclusively by the eggs of the trematode Schistosoma mansoni. IPSE/alpha-1 is a secretory glycoprotein which activates human basophils via an IgE-dependent but non-antigen-specific mechanism. Sequence analyses revealed a potential nuclear localization signal (NLS) at the C terminus of IPSE/alpha-1. Here we show that this sequence (125-PKRRRTY-131) is both necessary and sufficient for nuclear localization of IPSE or IPSE-enhanced green fluorescent protein (EGFP) fusions. While transiently expressed EGFP-IPSE/alpha-1 was exclusively nuclear in the Huh7 and U-2 OS cell lines, a mutant lacking amino acids 125 to 134 showed both nuclear and cytoplasmic staining. Moreover, insertion of the IPSE/alpha-1 NLS into a tetra-EGFP construct rendered the protein nuclear. Alanine scanning mutagenesis revealed a requirement for the KRRR residues. Fluorescence microscopy depicted, and Western blotting further confirmed, that recombinant IPSE/alpha-1 protein added exogenously is rapidly internalized by CHO cells and accumulates in nuclei in an NLS-dependent manner. A mutant protein in which the NLS motif was disrupted by triple mutation (RRR to AAA) was able to penetrate CHO cells but did not translocate to the nucleus. Furthermore, the uptake of native glycosylated IPSE/alpha-1 was confirmed in human primary monocyte-derived dendritic cells and was found to be a calcium- and temperature-dependent process. Live-cell imaging showed that IPSE/alpha-1 is not targeted to lysosomes. In contrast, peripheral blood basophils do not take up IPSE/alpha-1 and do not require the presence of an intact NLS for activation. Taken together, our results suggest that IPSE/alpha-1 may have additional nuclear functions in host cells.
authors.affiliation: School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK.
authors.forename: Ishwinder
authors.initials: I
authors.lastname: Kaur
authors.forename: Gabriele
authors.initials: G
authors.lastname: Schramm
authors.forename: Bart
authors.initials: B
authors.lastname: Everts
authors.forename: Thomas
authors.initials: T
authors.lastname: Scholzen
authors.forename: Karin B
authors.initials: KB
authors.lastname: Kindle
authors.forename: Christian
authors.initials: C
authors.lastname: Beetz
authors.forename: Cristina
authors.initials: C
authors.lastname: Montiel-Duarte
authors.forename: Silke
authors.initials: S
authors.lastname: Blindow
authors.forename: Arwyn T
authors.initials: AT
authors.lastname: Jones
authors.forename: Helmut
authors.initials: H
authors.lastname: Haas
authors.forename: Snjezana
authors.initials: S
authors.lastname: Stolnik
authors.forename: David M
authors.initials: DM
authors.lastname: Heery
authors.forename: Franco H
authors.initials: FH
authors.lastname: Falcone
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D004527:Egg Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015801:Helminth Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006790:Host-Parasite Interactions / Q000502:physiology*; D006801:Humans; D019913:Nuclear Localization Signals / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010063:Ovum / Q000378:metabolism; D016133:Polymerase Chain Reaction; D021381:Protein Transport / Q000502:physiology; D012550:Schistosoma mansoni / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012555:Schistosomiasis mansoni / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D004527:Egg Proteins; D015801:Helminth Proteins; C474807:IPSE protein, Schistosoma mansoni; D019913:Nuclear Localization Signals