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

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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.