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.

A Crystallin Fold in the Interleukin-4-inducing Principle of Schistosoma mansoni Eggs (IPSE/α-1) Mediates IgE Binding for Antigen-independent Basophil Activation

“…For backbone and side chain assignment CBCA(CO)NH, CBCANH, and (H)CCH-TOCSY (19), spectra were recorded (20). Distance information was obtained from 15 N-and 13 Cedited NOESY spectra with a mixing time of 70 ms. 15 N R 1 and R 2 relaxation rates and { 1 H}-15 N heteronuclear NOE data were measured at 750 MHz proton Larmor frequency and 298 K as described (21). Relaxation delays of 0.0216, 0.054, 0.27, 0.81, 1.08, 1.4, and 1.62 s were used to measure R 1 and 0.0144, 0.0288, 0.0576, 0.0864, 0.115, 0.173, and 0.173 were used to extract R 2 rates.…”

“…For preparation of uniformly 13 C, 15 N-and 15 N-labeled samples, bacteria were grown in M9 medium supplemented with 13 C-labeled glucose and/or 15 NH 4 Cl, respectively. For preparation of 2 H/ 15 N/ 13 C-labeled IPSE⌬NLS, the same protocol was employed but using [U- 2 H 13 C]glucose and growing bacteria in 100% D 2 O.…”

“…1C). 15 N NMR relaxation data showed that the polypeptide chain is rigid except for one loop (residues 62-69) that exhibits increased conformational flexibility (Fig. 1D).…”

“…1D). The tumbling correlation time c Ϸ7 ns estimated from 15 N R 2 /R 1 relaxation rates indicates that IPSE⌬NLS (12 kDa) is a monomer in solution. This is in line with earlier studies demonstrating that the C-terminal cysteine (Cys-132) absent in IPSE⌬NLS is required for covalent dimerization of wild-type IPSE/␣-1 (16).…”

“…IPSE⌬NLS lacks a stretch of 10 amino acids at the C terminus comprising a putative nuclear localization sequence (NLS) 7 (15) and Cys-132, which mediates a disulfide bridge for dimer formation of the native molecule (16). The structure consists of two symmetrically arranged greek key motifs and exhibits high structural similarity with members of the ␤␥-crystallin superfamily.…”