Interleukin-4-Inducing Principle from Schistosoma mansoni EggsContains a Functional C-Terminal Nuclear Localization Signal
Necessary for Nuclear Translocation in MammalianCells but Not for Its Uptake�†
Ishwinder Kaur,1,8 Gabriele Schramm,2 Bart Everts,3 Thomas Scholzen,4 Karin B. Kindle,5Christian Beetz,6 Cristina Montiel-Duarte,5 Silke Blindow,2 Arwyn T. Jones,7
Helmut Haas,2 Snjezana Stolnik,8 David M. Heery,5and Franco H. Falcone1*
Immune Modulation Research Group,1 Gene Regulation Group,5 and Advanced Drug Delivery Group,8 School of Pharmacy,University of Nottingham, Nottingham, United Kingdom; Division of Cellular Allergology2 and Department of
Immunology and Cell Biology,4 Research Center Borstel, Borstel, Germany; Department ofParasitology, Leiden University Medical Center, Leiden, Netherlands3;
Uniklinikum, IKCL-FZL, Jena, Germany6; and Welsh School ofPharmacy, University of Cardiff, Cardiff, United Kingdom7
Received 29 September 2010/Returned for modification 2 December 2010/Accepted 3 January 2011
Interleukin-4-inducing principle from schistosome eggs (IPSE/alpha-1) is a protein produced exclusively bythe eggs of the trematode Schistosoma mansoni. IPSE/alpha-1 is a secretory glycoprotein which activates humanbasophils via an IgE-dependent but non-antigen-specific mechanism. Sequence analyses revealed a potentialnuclear 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 greenfluorescent protein (EGFP) fusions. While transiently expressed EGFP-IPSE/alpha-1 was exclusively nuclearin the Huh7 and U-2 OS cell lines, a mutant lacking amino acids 125 to 134 showed both nuclear andcytoplasmic staining. Moreover, insertion of the IPSE/alpha-1 NLS into a tetra-EGFP construct rendered theprotein nuclear. Alanine scanning mutagenesis revealed a requirement for the KRRR residues. Fluorescencemicroscopy depicted, and Western blotting further confirmed, that recombinant IPSE/alpha-1 protein addedexogenously is rapidly internalized by CHO cells and accumulates in nuclei in an NLS-dependent manner. Amutant protein in which the NLS motif was disrupted by triple mutation (RRR to AAA) was able to penetrateCHO cells but did not translocate to the nucleus. Furthermore, the uptake of native glycosylated IPSE/alpha-1was confirmed in human primary monocyte-derived dendritic cells and was found to be a calcium- andtemperature-dependent process. Live-cell imaging showed that IPSE/alpha-1 is not targeted to lysosomes. Incontrast, peripheral blood basophils do not take up IPSE/alpha-1 and do not require the presence of an intactNLS for activation. Taken together, our results suggest that IPSE/alpha-1 may have additional nuclearfunctions in host cells.
Interleukin-4-inducing principle from schistosome eggs(IPSE/alpha-1) is a glycoprotein specifically secreted by the eggstage of Schistosoma mansoni (22, 46), a helminthic parasitewhich infects more than 200 million people in the tropics andsubtropics. The pathology of schistosomiasis is caused mainlyby a granulomatous and fibrosing immune reaction in the liverand gut in response to the eggs produced by mature femalefertile worms (55). Recently, proteomic analyses identifiedIPSE/alpha-1 as a highly abundant protein in S. mansoni eggsecretions (9, 14, 28, 37). It was previously shown that IPSE/alpha-1 has immunoglobulin-binding properties and activatesbasophils of immunologically naïve donors, resulting in hista-
mine release and T-helper 2 (Th2)-type cytokine production(45). IPSE/alpha-1 also induces interleukin-4 (IL-4) secretionfrom murine basophils in vivo (47). However, since there areno known homologs of IPSE/alpha-1 in metazoans outside theSchistosoma genus, little is known regarding its potential func-tions in host cells or its role in basophil activation.
Our sequence analyses identified a putative monopartite nu-clear localization sequence (NLS) at the C terminus of IPSE/alpha-1. Monopartite NLS motifs comprise a small cluster ofbasic amino acids preceded by a proline residue, with theclassic example represented by the PKKKR motif in the simianvirus 40 (SV40) T antigen (31). In addition to the NLS, the Nterminus of IPSE/alpha-1 contains a classical hydrophobic se-cretory signal (CSS) sequence (45). CSS motifs are involved inthe transport of nascent secretory polypeptide chains into theendoplasmic reticulum (ER), in a process called cotransla-tional transport, particularly for proteins with lengths of morethan 100 amino acids (43). A signal peptidase removes thissignal once it has entered the ER lumen. Under such condi-
* Corresponding author. Mailing address: School of Pharmacy, Uni-versity of Nottingham, Science Road, Boots Science Building, Notting-ham NG7 2RD, United Kingdom. Phone: 44 115 84 66073. Fax: 44 11595 15102. E-mail: [email protected].
† Supplemental material for this article may be found at http://iai.asm.org/.
tions, a protein will normally not be targeted to the nucleuseven if an NLS is present. Thus, most known nuclear proteinsdo not possess CSS motifs. N-terminal sequencing of matureIPSE/alpha-1 confirmed that the CSS motif is removed duringexport (45). One known example of the rare molecules exhib-iting both CSS and NLS motifs on the same polypeptide chainis mouse fibroblast growth factor 3 (FGF-3), which displaysboth nuclear and extracellular localization (34). Given thatIPSE/alpha-1 is secreted in large amounts from the subshellarea of eggs (46), which are in close contact with the surround-ing host tissues, we hypothesized that the mature IPSE/alpha-1protein might be targeted to the nuclei of host cells. In thisstudy, we demonstrate that IPSE/alpha-1 contains a functionalNLS motif that is necessary and sufficient for translocation tomammalian cell nuclei and that also has a potential role inDNA binding. This suggests that IPSE/alpha-1 could have animportant role in modulating the immune response after en-tering host cell nuclei.
MATERIALS AND METHODS
Subcloning and PCR mutagenesis. Full-length IPSE/alpha-1 cDNA was am-plified by PCR as described previously (45). Since the putative NLS is locatedclose to the C terminus of IPSE/alpha-1, PCR mutagenesis was achieved byintroducing the desired mutations in the 3� primer. Oligonucleotide primersequences are available in Table S1 in the supplemental material. The primersintroduced an alanine substitution for each single amino acid in the putative NLS(125-PKRRRTY-131) (amino acid numbers refer to the full-length protein se-quence, including the signal peptide). PCR fragments were amplified usinghigh-fidelity proofreading Pfu Ultra DNA polymerase (Stratagene). The cyclingconditions used were as follows: 95°C for 2 min for 1 cycle; 95°C for 30 s, 58°Cfor 30 s, and 72°C for 1 min 30 s for 30 cycles; and 1 cycle at 72°C for 10 min. Theamplified fragment was purified using a QIAquick gel purification kit (Qiagen),digested with BglII and HindIII restriction enzymes, and subcloned into thepEGFP-C1 vector. To investigate whether the nuclear localization of full-lengthIPSE/alpha-1 is due to the putative monopartite NLS predicted in silico, we usedthe previously published plasmid pTetra-EGFP, which is an excellent tool formeasuring NLS activity (4).
Phosphorylated, annealed oligonucleotide pairs encoding the sequencesPKRRRTY (wild type), PARRRTY, and PKAAATY and containing GATCoverhangs were subcloned into the BglII site of the pTetra-EGFP vector (4). Thisinserts the test sequence between the third and fourth copies of a tetra-enhancedgreen fluorescent protein (tetra-EGFP) fusion protein which is normally cyto-plasmic, as it is too large to cross nuclear pores by passive diffusion. All con-structs were verified by DNA sequencing.
Cell culture. The human hepatocellular carcinoma cell line Huh7 D12(ECACC 01042712) and the human osteosarcoma cell line U-2 OS (ATCCHTB-96) were grown in T25 and T75 cell culture flasks (Nunclon, Denmark) at37°C in a humidified atmosphere of 95% air and 5% CO2 in Dulbecco’s modifiedEagle’s medium (DMEM) (GibcoBRL, United Kingdom) supplemented with10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 1 U/ml peni-cillin, and 1 mg/ml streptomycin. Chinese hamster ovary transferrin receptorvariant b cells (CHO-TrVb cells; designated CHO-TfR� cells in this study) andCHO-TrVb cells stably transfected with human transferrin receptor 1 (CHO-TrVb1 cells; designated CHO-TfR� cells herein) were cultured in Ham’s F12-HEPES (Autogen Bioclear, United Kingdom) supplemented with 5% heat-inactivated FCS, 2 mM L-glutamine, 1 U/ml penicillin, and 1 mg/ml streptomycin.The medium for CHO-TfR� cells additionally contained 200 �g/ml G418(Fisher Bioreagents, United Kingdom).
Transient expression. Transfections were performed using Transfast (Pro-mega, United Kingdom) as instructed by the manufacturer. All transfectionswere performed in 6-well plates (Nunc) on autoclaved glass coverslips seededwith cells 24 h prior to transfection (1 � 106 to 3 � 106 cells/well). The exper-imentally determined optimal conditions (24 h with a 1:2 DNA/Transfast ratio)were used for all transfections.
Fluorescence microscopy. Recombinant EGFP-IPSE/alpha-1 fusion proteinexpressed in transfected cells was visualized using fluorescence microscopy. Thetransfected Huh7 or U-2 OS cell line was washed twice with phosphate-bufferedsaline (PBS) and fixed in a 4% (wt/vol) paraformaldehyde-PBS solution for 10
min at room temperature. The cells were then washed 4 or 5 times with PBS andincubated with 0.5 �g/ml Hoechst 33258 stain for 10 min at room temperature.The Hoechst stain was removed by 4 or 5 washes with PBS. The coverslips werethen mounted on 10 �l of 90% glycerol in PBS, and the edges were sealed withnail polish. Images were taken on a confocal microscope (LSM510 Meta; Zeiss)using the provided software (Zeiss LSM Image Examiner v3.5).
Immunofluorescence. The antibody solutions used were spun at 14,000 � g for1 min prior to use, and all steps of incubation were performed at room temper-ature unless otherwise stated. CHO-TfR� and CHO-TfR� cells were seeded oncoverslips at a density of 0.5 � 105 cells for 48 h. Cells were then incubated with0.15 nM recombinant IPSE/alpha-1 in serum-free internalization medium(HEPES-buffered Ham’s F12 medium containing 10 mM NaHCO3 and 2 mg/mlbovine serum albumin [BSA; fraction V] [Biomol]) for 30 min at 37°C. Immu-nolabeling was performed as previously described (49). Briefly, cells were fixed ina 3% (wt/vol) paraformaldehyde-PBS solution for 15 min, washed three timeswith PBS, incubated in 50 mM NH4Cl for 10 min, washed three times again, andpermeabilized in 0.2% (vol/vol) Triton X-100. The cells were then incubated inblocking buffer (2% [vol/vol] FCS, 2% [wt/vol] BSA) for 30 min prior to additionof anti-IPSE monoclonal antibody culture supernatant at a 1:10 dilution for 30min. The cells were washed and then incubated with anti-mouse IgG secondaryantibody labeled with Alexa Fluor 594 (Invitrogen, United Kingdom) for 30 minat room temperature. The unbound secondary antibody was removed by re-peated washing in PBS, and the nucleus was stained by incubating the cells for 10min in 10 �g/ml Hoechst 33258. After being washed in PBS, the coverslips weremounted on 10 �l of 90% glycerol in PBS, and the edges were sealed with nailpolish.
Extraction of cytoplasmic and nuclear fractions. Recombinant IPSE/alpha-1was incubated with 5 � 106 CHO-TfR� and CHO-TfR� cells for 2, 6, 12, and24 h. After incubation, each cell line was trypsinized, washed twice in ice-coldPBS (Sigma, United Kingdom), and collected by centrifugation at 5,000 rpm(2,655 � g) for 2 min in a microcentrifuge tube (Eppendorf model 5417Rcentrifuge). To the cell pellets, 200 �l of buffer A (50 mM NaCl, 10 mM HEPES[pH 8.0], 500 mM sucrose, 1 mM EDTA, 0.2% Triton X-100) containing 1�protease inhibitors (Calbiochem, United Kingdom) was added and vortexedbriefly. The mixture was spun in a refrigerated microcentrifuge at 5,000 rpm(2,655 � g) for 2 min. The supernatant comprised the cytoplasmic extract andwas stored for Western blot detection. The cell pellets containing the nuclei wereresuspended and washed in 500 �l buffer B (50 mM NaCl, 10 mM HEPES [pH8.0], 25% glycerol, 0.1 mM EDTA) containing 1� protease inhibitors (Calbio-chem, United Kingdom). The supernatant was discarded, and cell pellets wereincubated on ice for 30 min with frequent agitation in 50 �l buffer C (350 mMNaCl, 10 mM HEPES [pH 8.0], 25% glycerol, 0.1 mM EDTA) containing 1�protease inhibitors (Calbiochem, United Kingdom). The mixture was spun at14,000 rpm (20,817 � g) for 15 min at 4°C. The supernatant was removedcarefully and stored as the nuclear extract for Western blot detection. Theextracts were run in standard 12% SDS-PAGE gels, blotted on nitrocellulose(Schleicher and Schuell Bioscience, Dassel, Germany [now Whatman/GEHealthcare]), stained with primary monoclonal antibody to IPSE/alpha-1 (1:2,500 for nuclear staining and 1:5,000 for cytosolic staining) and a secondaryanti-mouse IgG1 antibody (Invitrogen Western Breeze kit) (1:1,000), and devel-oped by a chemiluminescence detection method (Invitrogen Western Breezekit), using a Fujifilm luminescence image reader (LAS-4000) as directed by themanufacturer. A primary goat polyclonal antibody to histone H3 (Abcam,United Kingdom) (diluted 1:5,000) and a rabbit anti-goat IgG1–alkaline phos-phatase (AP) secondary antibody (1:5,000) for detection of H3, as well as amouse monoclonal antibody to beta-actin (Sigma, United Kingdom) (1:1,000)and the AP-linked anti-mouse IgG from a Western Breeze kit as a secondaryantibody, were used as control antibodies for subcellular extract specificity forboth cytosolic and nuclear fractions.
Sequence analysis. The IPSE/alpha-1 amino acid sequence (GenBank acces-sion no. AAK26170) was used for identification of sequence motifs such as CSSor NLS motifs by using Web-based bioinformatic tools, including Cello (57),ESLPred (6), LOCSVMPSI (56), LocTree (40), MultiLoc (26), NLSpredict (11),Proteome Analyst (51), PLOC (42), PSORTII (41), SignalP 3.0 (5), SherLoc(48), SubLoc (29), TargetP 1.1 (18), and Wolf Psort (27). All programs were usedwith default settings.
Basophil activation. Human basophils were obtained from peripheral blood ofhealthy donors by using a three-step purification protocol as described before(21). Yield and viability were determined by trypan blue exclusion, and basophilpurity was determined by staining cytospin preparations with May-Grunwaldstain. Purified basophils were incubated overnight in RPMI 1640 medium sup-plemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 1 U/ml penicillin,and 1 mg/ml streptomycin, with recombinant IPSE/alpha-1, IPSE/alpha-1 �NLS,
or IPSE/alpha-1 3R at different concentrations, at 37°C in a 5% CO2 humidifiedincubator. IL-4 was measured in supernatants by enzyme-linked immunosorbentassay (ELISA) as previously described (45).
Expression of recombinant IPSE/alpha-1. Recombinant IPSE/alpha-1, IPSE/alpha-1 �NLS, and IPSE/alpha-1 3R were expressed in Escherichia coli, purified,and refolded as described earlier (45). Glycosylated recombinant IPSE/alpha-1used for live-cell imaging of monocyte-derived dendritic cells (MDDCs) andbasophils was expressed in human embryonic kidney (HEK) cells transfectedwith the expression vector pSegTag2-IPSE/alpha-1. The pSegTag2 vector wasobtained from Invitrogen. Secreted recombinant HEK-IPSE/alpha-1 was se-quentially purified from the culture medium by immobilized metal-affinity chro-matography and affinity chromatography with monoclonal anti-IPSE antibodiescoupled to an NHS-HiTrap Sepharose column (GE Healthcare).
Labeling of recombinant HEK-IPSE/alpha-1 and live-cell imaging. Purifiedrecombinant HEK-IPSE/alpha-1 was fluorescently labeled with N-hydroxysuc-cinimide (NHS)-fluorescein (Pierce; now ThermoScientific) according to themanufacturer’s instructions. After protein labeling, nonreacted NHS-fluoresceinwas removed using Zeba Desalt spin columns (Pierce). Live-cell imaging wasperformed with a Leica TCS SP5 inverse confocal laser scanning microscope andanalyzed with LAS AF software. In detail, 2.5 � 104 MDDCs were added to achannel of an IV0.4 �-slide (Ibidi, Martinsried, Germany) and incubated for 2 hat 37°C and 6% CO2 with 1 �l fluorescein-labeled HEK-IPSE/alpha-1 (1 mg/ml).Nuclei and lysosomes of the cells were then counterstained with Hoechst 33342(1:10,000) and LysoTracker (1:20,000), respectively, for 30 min. A channel con-taining MDDCs without preincubation of fluorescein-labeled IPSE/alpha-1 wasstained with LysoTracker alone as a control.
Purification and labeling of native IPSE/alpha-1. For flow cytometry binding/uptake studies, IPSE/alpha-1 was purified from SEA via cation-exchange chro-matography and affinity chromatography using specific anti-IPSE/alpha-1 mono-clonal antibodies coupled to an NHS-HiTrap Sepharose column according to themanufacturer’s instructions (GE Healthcare), as described earlier (17, 45). Pu-rified IPSE/alpha-1 was concentrated and dialyzed. IPSE/alpha-1 was fluores-cently labeled with PF-647 by use of a Promofluor labeling kit (Promokine,Heidelberg, Germany) according to the manufacturer’s recommendations.
IPSE/alpha-1 binding/uptake by human MDDCs. Monocytes were isolatedfrom venous blood of healthy volunteers according to Institutional ReviewBoard-approved protocols by density centrifugation on Ficoll followed by aPercoll gradient, as described previously (15), and were cultured in RPMI 1640medium supplemented with 10% FCS, human recombinant granulocyte-mac-rophage colony-stimulating factor (rGM-CSF) (500 units/ml; a gift from Scher-ing-Plough, Uden, Netherlands), and human rIL-4 (250 units/ml) (R&D Sys-tems). On day 6, 10,000 immature DCs/well were seeded in a 96-well plate.Where indicated, cells were preincubated with 10 mM EGTA. Subsequently,cells were incubated with 500 ng/ml labeled IPSE/alpha-1 at 37°C or 4°C for 1 hand washed in ice-cold PBS or EGTA, where indicated, before analysis usingflow cytometry.
RESULTS
Translocation of IPSE/alpha-1 to the nucleus requires aC-terminal NLS. Sequence motif analysis software such asMultiLoc, PSORTII, and SherLoc predicted the presence of amonopartite NLS (125-PKRRRTY-131) at the C terminus ofIPSE/alpha-1 (see Table S2 in the supplemental material). Todetermine whether this sequence facilitates localization ofIPSE/alpha-1 to host cell nuclei, Huh7 or U-2 OS cells weretransiently transfected with pEGFP-IPSE/alpha-1 or a series ofEGFP-IPSE constructs in which the putative NLS was deletedor mutated. As shown in Fig. 1, EGFP fused to wild-typeIPSE/alpha-1 showed a strong and exclusively nuclear stainingin both cell lines (Fig. 1A and D), suggesting that IPSE/alpha-1contains a functional NLS motif. In contrast, EGFP-IPSE/alpha-1 �NLS, which lacks amino acids 125 to 134, was de-tected in both the nucleus and the cytosol (Fig. 1B and E) andshowed very similar staining to that of control EGFP alone(Fig. 1C). This result indicates that cytosolic IPSE/alpha-1 cantranslocate to the nuclei of mammalian host cells and suggeststhat the PKRRRTY motif at its C terminus may indeed func-
tion as an NLS. In the absence of a functional NLS, as seenwith the unfused EGFP control, the protein can also enter thenucleus, probably by diffusion, resulting in a mixed cytoplasmicand nuclear localization.
To explore this further, alanine scanning mutagenesis wasperformed on the C terminus of IPSE/alpha-1 to examine theeffects of single amino acid substitutions within the putativeNLS on subcellular localization in U-2 OS cells. Replacementof P125 (Fig. 1F), T130 (Fig. 1L), or Y131 (Fig. 1M) withalanine had no deleterious effect on the nuclear localization ofEGFP-IPSE/alpha-1. However, mutant EGFP-IPSE/alpha-1proteins harboring alanines at any one of the amino acids inthe 126-KRRR-129 region showed significant cytoplasmic lo-calization (Fig. 1G to J). Moreover, replacement of the threearginine residues with alanines (3R) (Fig. 1K) resulted in asevere disruption of the nuclear localization. These resultsstrongly suggest that the 126-KRRR-129 motif is a functionalNLS motif.
To perform a more quantitative analysis of the effects ofthese mutations, 100 transfected cells for each construct werescored for the percentage of cells showing complete nuclearlocalization of the EGFP fusion protein. As shown in Fig. 1N,while wild-type (100%), P125A (98%), and T130A (95%) con-structs were almost entirely nuclear, the K126A mutation (5%)drastically reduced the number of cells showing exclusively nu-clear staining. The single-arginine mutations R128A and R129Areduced the percentage of nucleus-only cells by approximately50%, whereas the R127A mutation had a stronger effect, withonly approximately 20% of cells showing strong nuclear local-ization. For the mutant with all three arginines replaced (IPSE3R), all cells examined were disrupted for nuclear localizationof the fusion protein. In summary, our results show that IPSE/alpha-1 contains an NLS that targets it to the nuclei of mam-malian cells, where it may interact with DNA, chromatin,and/or nuclear proteins.
The NLS is sufficient to target IPSE/alpha-1 or EGFP pro-teins to the nucleus. Having identified the KRRR residues asnecessary for nuclear translocation of IPSE, we next evaluatedwhether the putative NLS sequence was sufficient on its own todirect targeting of an unrelated protein to the nucleus. Since allEGFP-IPSE NLS mutants showed at least partial nuclearlocalization, it is possible that the fusion proteins are smallenough to undergo passive diffusion into the nucleus. Inter-actions with DNA or other proteins might permit retentionof EGFP-IPSE in the nuclear compartment. To circumventthis problem, IPSE NLS sequences were subcloned into thepTetra-EGFP plasmid construct, encoding a tetra-EFGP fu-sion protein of approximately 100 kDa that is largely excludedfrom the nucleus (Fig. 2A). Insertion of a canonical SV40 NLSbetween the third and fourth copies of the EGFP sequences onthe tetra-EGFP plasmid resulted in complete nuclear localiza-tion of the tetra-EGFP protein (Fig. 2B). Similarly, insertion ofthe IPSE PKRRRTY motif at this position also resulted incomplete nuclear localization, showing that this sequence issufficient to target large polypeptides to the nucleus. As ex-pected, mutation of the three arginines or K126 to alanineresulted in proteins that were exclusively cytoplasmic (Fig. 2Dand E), confirming our previous finding that the KRRR motifis a functional NLS.
To confirm that non-EGFP-fused IPSE/alpha-1 can localize
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to the host cell nucleus, wild-type or mutant mature IPSE/alpha-1 proteins (without EGFP) were transiently expressed intransfected U-2 OS cells. IPSE/alpha-1 proteins were detectedusing a specific monoclonal antibody raised against the matureprotein (45). As shown in Fig. 3, whereas the wild-type IPSE/alpha-1 protein localized entirely to the nucleus (Fig. 3A), theIPSE/alpha-1 �NLS (Fig. 3B) and IPSE/alpha-1 3R (Fig. 3C)mutants were entirely cytoplasmic. This provides further evi-dence that the NLS sequence is essential for translocation tohost cell nuclei.
Exogenous IPSE/alpha-1 protein translocates to the nucleiof mammalian cells. Since IPSE/alpha-1 is a secreted proteinwith a functional NLS, we investigated whether exogenousIPSE/alpha-1 can be internalized and translocate to mamma-lian cell nuclei. Our previous studies indicated that IPSE/al-pha-1 binds to serum transferrin (Tf) from humans or othermammals, suggesting the Tf/TfR pathway as a possible route of
entry into host cells (S. Blindow et al., unpublished data). Totest this hypothesis, CHO-TrVb cells, which do not express anyendogenous transferrin receptors (designated CHO-TfR� cells),and CHO-TrVb1 cells, which stably express human TfR1 (39)(designated CHO-TfR� cells), were incubated with 0.15 nMpurified recombinant IPSE/alpha-1 expressed in E. coli, andlocalization of the protein was assessed by immunocytochem-istry after 30 min. As shown in Fig. 4A to D, staining with theIPSE-specific monoclonal antibody (45) revealed that exoge-nous recombinant IPSE/alpha-1 rapidly entered both CHO-TfR� and CHO-TfR� cells and showed nuclear staining.These results do not point to a facilitating role of the humanTfR1 for cellular uptake of IPSE/alpha-1. Consistent with this,Western blot analysis of cytosolic and nuclear extracts pre-pared 24 h after addition of exogenous recombinant IPSE/alpha-1 revealed that in CHO-TfR� and CHO-TfR� extracts,IPSE/alpha-1 protein was also detected in the nuclear fraction
FIG. 1. Localization of EGFP-IPSE/alpha-1 in transiently transfected mammalian cell lines. (A) Huh7 cells transfected with pEGFP-IPSE/alpha-1, showing complete translocation of EGFP-IPSE/alpha-1 to the nucleus. (B) Huh7 cells transfected with pEGFP-IPSE/alpha-1 �NLS,lacking the C-terminal PKRRRTY NLS, showing mixed nuclear and cytoplasmic fluorescence. (C) Control transfection of U-2 OS cells withunmodified EGFP-C1 vector. (D to M) U-2 OS cell line transfected with pEGFP-IPSE/alpha-1 mutants with different alanine-substituted aminoacids in the NLS and counterstained with Hoechst stain (insets). (D) pEGFP-IPSE/alpha-1, showing exclusive localization in the nucleus.(E) pEGFP-IPSE/alpha-1 �NLS, showing mixed nuclear and cytoplasmic localization. (F, L, and M) pEGFP-IPSE/alpha-1 NLS mutants in whichP, T, and Y were replaced, showing no or little effect on nuclear localization. (H, I, and J) Single mutants partially decreasing nuclear localization.Replacement of all three arginines (K) or lysine (G) led to complete disruption of nuclear localization. (N) Summary of the effects of Ala mutationson subcellular localization of the EGFP-IPSE fusion protein. U-2 OS cells were transiently transfected with pEGFP-IPSE/alpha-1 and mutants.One hundred transfected cells were counted for each transfection, and the percentage of cells displaying exclusively nuclear fluorescence, asopposed to mixed nuclear and cytoplasmic fluorescence, was recorded.
(Fig. 4G). Exogenous addition of the 3R mutant, with a non-functional NLS, resulted in cellular uptake but not in nucleartranslocation (Fig. 4E and F). This suggests that while an intactNLS is needed for nuclear translocation, cellular uptake in thiscell type is independent from the NLS.
Native glycosylated IPSE/alpha-1 is taken up by human den-dritic cells in a Ca2�-dependent manner. To determine whethernative IPSE/alpha-1 is also internalized by primary cells, thecapacity of human MDDCs to take up IPSE/alpha-1 was as-sessed. Dendritic cells incubated for 1 h with glycosylatedIPSE/alpha-1 purified from S. mansoni eggs and labeled withPF-647 displayed a strong increase in fluorescence as deter-mined by flow cytometry (Fig. 5A). Since SEA, which alsocontains IPSE, is internalized by dendritic cells in a C-typelectin- and calcium-dependent manner (52), we tested the cal-cium dependency of these cells for recognition and internal-ization of IPSE/alpha-1. Indeed, pretreatment with EGTA, acalcium chelator, almost totally abolished the ability of thecells to bind and take up IPSE/alpha-1 (Fig. 5A). Finally, toascertain that IPSE/alpha-1 is truly internalized by MDDCs,the incubated cells were washed in EGTA to remove all sur-face C-type lectin-bound IPSE/alpha-1 (Fig. 5B). Importantly,the latter treatment did not reduce the fluorescence intensityof the cells, while the same treatment on cells incubated withIPSE/alpha-1 at 4°C, which prevents receptor-mediated up-take, did lower the fluorescence of the cells back to back-ground levels (Fig. 5B). This shows that native IPSE/alpha-1 isefficiently internalized by human dendritic cells, in a Ca2�- andtemperature-dependent manner. Interestingly, uptake by thedendritic cells was dependent on glycosylation, as recombinantIPSE/alpha-1 expressed in bacteria, in contrast to that taken upin CHO cells, was not internalized by the primary cells (Fig.5C). Together, the calcium and glycosylation dependencies ofuptake by monocyte-derived dendritic cells point to a C-typelectin-mediated mechanism.
IPSE/alpha-1 is not targeted to lysosomes. To rule out thepossibility that IPSE/alpha-1 undergoes lysosomal degradationafter uptake via endocytic mechanisms, live-cell imaging wasperformed with human monocyte-derived dendritic cells incu-bated for 2.5 h with fluorescein-HEK-IPSE/alpha-1 (green)and LysoTracker (red) and counterstained with Hoechst 33342(blue). The results shown in Fig. 5D to G suggest that inter-nalized IPSE/alpha-1 found in the cytosol of the cell does notcolocalize with lysosomes. The lack of nuclear localization inthese experiments was probably due to the interference offluorescein, which is conjugated via free amino groups in theprotein, including mainly the KRRR residues in the NLS, andthus very likely to affect its functionality, as suggested by theresults shown in Fig. 1. Since the uptake of this protein ismediated via carbohydrates, fluorescein labeling did not affectthe protein’s entry into MDDCs.
Neither a functional NLS nor cellular uptake is required forIPSE/alpha-1-mediated activation of human basophils. IPSE/alpha-1 is known to trigger IL-4 release from naïve human baso-phils (45), although the exact underlying mechanism, which isknown to involve immunoglobulin E binding (22, 45), is notfully understood. For example, and in light of our findings, it isnot known whether this phenomenon requires nuclear local-ization of the parasite protein after internalization. To inves-tigate this, we compared basophil activation by recombinantwild-type protein and the IPSE/alpha-1 �NLS or IPSE/alpha-13R mutant. As shown in Fig. 6, wild-type IPSE showed adose-dependent induction of IL-4 release from peripheralblood basophils, as shown previously (45). In contrast, IPSE/alpha-1 �NLS showed little or no ability to induce IL-4 pro-duction under the same conditions. However, basophil activa-tion was not affected in the NLS-defective IPSE/alpha-1 3Rmutant, suggesting that the NLS activity per se is not requiredfor this function. The apparent reduction in IL-4 productionwith IPSE/alpha-1 at concentrations above 100 �g/ml was pre-sumably due to stimulation in the supraoptimal concentrationrange, which can lead to Src homology 2 domain-containinginositol 5�-phosphatase (SHIP)-mediated downregulation ofmediator release (20). This was not observed with the IPSE/alpha-1 3R mutant, as indicated by the dose-response curve(Fig. 6A). The reason for the missing suppression of IL-4production at high concentrations of the IPSE/alpha-1 3R mu-tant resulting in IL-4 levels beyond those reached upon stim-
FIG. 3. Nuclear and cytoplasmic localization of non-EGFP-fusedIPSE/alpha-1. (Left) U-2 OS transfection with wild-type IPSE/alpha-1-encoding plasmid, showing exclusively nuclear localization of IPSE/alpha-1. (Middle) U-2 OS transfection with IPSE/alpha-1 �NLS-en-coding plasmid. (Right) U-2 OS transfection with IPSE/alpha-1 3R-encoding plasmid. Both the middle and right panels show exclusionfrom the nucleus. After 24 h, the cells were fixed, permeabilized,stained with a monoclonal antibody to IPSE/alpha-1 and an Alexa594-labeled secondary antibody, and counterstained with Hoechst nu-clear stain (insets).
FIG. 2. Nuclear translocation of tetra-EGFP. U-2 OS cells weretransfected with tetra-EGFP-NLS constructs and counterstained withHoechst stain (insets). (A) Control tetra-EGFP expressed only in thecytoplasm. (B) Tetra-EGFP fused with canonical SV40 NLS, showingcomplete nuclear localization. (C) NLS from IPSE/alpha-1 fused withtetra-EGFP, showing exclusively nuclear localization similar to thatwith SV40 NLS. (D) Tetra-EGFP-NLS constructs in which all thearginines (3R) were replaced with alanine, showing cytoplasmicdistribution, which confirms the disruption of nuclear localization.(E) Tetra-EGFP-NLS construct in which lysine (K) was replaced withalanine, also showing complete disruption of nuclear localization.
VOL. 79, 2011 NUCLEAR TRANSLOCATION OF S. MANSONI IPSE/ALPHA-1 1783
ulation of basophils via anti-IgE at optimal concentrations(data not shown) is presently under study.
The above results suggest that nuclear localization of IPSE/alpha-1 is not necessary for IgE binding and subsequent IL-4induction in human basophils. However, the results also indi-cate that deletion of the C terminus, including the NLS, abro-gates this function. As an explanation for this, our recentstudies have shown that dimerization of native IPSE/alpha-1requires a cysteine residue (C132) at the C terminus of IPSE,which forms an interchain disulfide bond (54). This cysteine isremoved in IPSE/alpha-1 �NLS but is present in the IPSE/alpha-1 3R mutant. Since IPSE/alpha-1 �NLS therefore occursas a monomer (Fig. 6B), we concluded that dimerization ofIPSE/alpha-1, but not nuclear localization, is necessary forits ability to induce IL-4 release by human basophils. Finally,live-cell imaging of basophils incubated with fluorescein iso-thiocyanate (FITC)-labeled HEK-IPSE/alpha-1 (Fig. 6C,showing successive sections along the z axis) depicts that whilethe parasitic protein aggregates on the surface, probably clus-tering the high-affinity IgE receptor via binding to IgE, it is notinternalized.
Altogether, these results show that while the NLS in IPSE/alpha-1 is functional, i.e., is able to translocate large proteinsfrom the cytosol to the nucleus, it is not needed for cellularuptake or for activation of peripheral blood basophils. Thisstudy demonstrates that a protein secreted by S. mansoni con-tains an NLS motif that is functional in host cells.
DISCUSSION
Several pathogens use host cell surface receptors to gainentry into host cells. For example, the mannose receptor (MR)(which is probably responsible, at least in part, for the uptakeof IPSE/alpha-1 by MDDCs) is used by Trypanosoma cruzi togain entry into cardiomyocytes (50), and cellular uptake hasalso been shown for the opportunistic pathogenic yeast Can-dida albicans (8). Dendritic cell-specific intercellular adhesionmolecule-3-grabbing nonintegrin (DC-SIGN) and MR on im-mature dendritic cells are also used by viruses such as humanimmunodeficiency virus (HIV), via gp120 (35), as well as bylarger pathogens such as Leishmania (12), for internalization.Because of their relative sizes, with a few exceptions, such asTrichinella spiralis and the whipworm Trichuris, most helminthparasites cannot enter host cells. However, it is possible thatthey use similar strategies involving hijacking host cell recep-tors for internalization and subsequent nuclear translocation ofswitch factors which may, e.g., affect host cell transcriptionalpatterns.
This study has demonstrated for the first time that a proteinsecreted by the human parasite S. mansoni contains an NLSmotif that is functional in host cells. IPSE/alpha-1 is producedin the subshell area of the egg and is not detectable in themiracidium, the parasitic larval stage present in mature eggs, aseither protein or mRNA (46). After secretion, IPSE/alpha-1comes into close contact with inflammatory cells recruited to
FIG. 4. Subcellular localization of exogenously added recombinant IPSE/alpha-1. CHO-TfR� (A and B) and CHO-TfR� cells (C and D) weretreated with recombinant purified IPSE/alpha-1 for 30 min at 37°C and stained with a monoclonal anti-IPSE/alpha-1 antibody and an Alexa594-labeled secondary antibody. The photographs demonstrate intracellular and nuclear localization of IPSE/alpha-1 in both cell lines. (E and F)Incubation of CHO-TfR� cells with IPSE/alpha-1 3R mutant, showing internalization but loss of nuclear translocation. Bars, 10 �m (A to D) and20 �m (E and F). (G) Western blots of nuclear and cytosolic extracts of CHO-TfR� (lanes 1 and 3) and CHO-TfR� (lanes 2 and 4) cells 24 hafter exposure to exogenous recombinant IPSE/alpha-1. Blots were stripped and reprobed with an antibody to beta-actin or histone H3 as a controlfor extract specificity. IPSE/alpha-1 was found in the cytosolic and nuclear fractions, but the nuclear extracts contained an additional, higher-molecular-weight band, suggesting that IPSE/alpha-1 could undergo modifications during nuclear translocation.
the vicinity of the egg surface (46). There is no evidence to datefor an intracellular or nuclear localization of IPSE/alpha-1 inschistosome tissues. Altogether, the data suggest that IPSE/alpha-1 may have functional interactions with host cells ratherthan in the parasite. In order to reach the nucleus, a secretedprotein has to be internalized back to the cytoplasm or, in thecase of IPSE/alpha-1, into the cytoplasm of host cells. Thismay involve endocytosis-dependent processes, as reportedfor angiogenin (23) and parathyroid hormone-related pro-tein (PtHrP) (3).
Using standard approaches, we have demonstrated thatIPSE/alpha-1 contains a monopartite functional NLS motif(PKRRRTY) that is necessary and sufficient for its transloca-tion to the nucleus. We have also demonstrated that this se-quence is functional in the context of other proteins, such asEGFP. The PKRRRTY sequence is in agreement with thebasic core consensus sequence K(K/R)X(K/R) of monopartiteNLS motifs (24, 32). Our results showed that different alaninemutations had different effects on disruption of NLS function-ality (which we have defined as exclusively nuclear localiza-tion), depending on the position. Mutations of 126K and 127Rappeared to have the strongest effects. This is in good agree-ment with the work of Hodel et al., who determined the energyprofiles for the binding of the SV40 NLS (PKKKRKV) toimportin alpha and, based on these, defined the relative im-portance of each residue in the NLS (25).
Native IPSE/alpha-1 released by schistosome eggs is a dimeras a consequence of an interchain disulfide bond involving theC-terminal cysteine residue C132 (54). Despite the relatively
low molecular mass (33 to 35 kDa) of IPSE/alpha-1 dimers, themutants defective for NLS activity were excluded from thenucleus (Fig. 3 and 4E and F). This may indicate retention ofIPSE/alpha-1 by cytosolic components or the presence of anuclear export signal (NES), although we were unable to iden-tify a consensus NES motif (36) within the IPSE/alpha-1 se-quence.
As stated earlier, examples of proteins that contain both CSS(secretory) and NLS motifs are relatively rare, with an excep-tion being the mouse FGF-3 protein (34). This raises the ques-tion of how proteins containing both motifs are secreted ratherthan targeted to the nucleus in the cell of origin. In FGF-3, theCSS cleavage site is adjacent to a bipartite NLS which is sep-arated by only six amino acids. Increasing the distance betweenthe signal sequence cleavage site and the NLS in FGF-3, byintroducing a 15-amino-acid neutral linker sequence, results inexclusively secretory pathway localization, favoring recognitionof the secretory signal over the NLS (34). In IPSE/alpha-1, thedistance between the two motifs is 104 amino acids. Thus, thisseparation of the CSS motif from the NLS may be important indirecting IPSE/alpha-1 for secretion from the producing struc-tures in schistosome eggs.
We have shown that introduction of E. coli-expressed IPSEprotein to the culture medium of growing CHO cells results inits internalization and subsequent targeting to the nucleus.Moreover, deletion of the C terminus of IPSE, including theNLS sequence, disrupted nuclear uptake (Fig. 3 and 4) but notinternalization (Fig. 4E and F). These findings raise questionsregarding which mechanisms IPSE/alpha-1 uses to gain access
FIG. 5. (A) Uptake/binding of IPSE/alpha-1 by human dendritic cells. Immature MDDCs were preincubated with EGTA, where indicated,followed by a 1-h incubation with PF-647-labeled IPSE/alpha-1 at 37°C. Uptake of antigens by MDDCs was evaluated by fluorescence-activatedcell sorter (FACS) analysis. (B) To determine that IPSE/alpha-1 is truly internalized by dendritic cells, incubated cells were washed in EGTA toremove all surface C-type lectin-bound IPSE/alpha-1. Cells incubated with IPSE/alpha-1 at 4°C, preventing receptor-mediated uptake, were usedas a control. Fluorescence intensity was set to 100% for conditions without EGTA treatment. (C) Uptake/binding of native purified glycosylatedIPSE/alpha-1 (nIPSE) in comparison with the nonglycosylated bacterial recombinant. (A to C) Data are shown as means plus standard deviations(SD) for duplicates. Data for one representative experiment out of two are shown. P � 0.001 for significant differences compared to the control(one-sided t test). (D to G) Live-cell imaging of MDDCs incubated with fluorescein-labeled HEK-IPSE/alpha-1 for 2 h at 37°C before staining ofthe nucleus and the lysosomes with Hoechst 33342 and LysoTracker, respectively. (D) Overlay image of panels E to G, showing that IPSE/alpha-1does not colocalize significantly with lysosomes. (E) Hoechst 33342 staining (blue). (F) Fluorescein-HEK-IPSE/alpha-1 (green). (G) LysoTrackerstaining (red). Bar, 10 �m.
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to the cytosolic and nuclear compartments of host cells. Wehave previously found that IPSE/alpha-1 binds mammalian Tfsof different species (human, mouse, rat, and goat) (Blindowet al., unpublished data), suggesting the Tf/TfR pathway as apossible route of entry. Our data suggest that the presence ofTfR1 is not a prerequisite for IPSE/alpha-1 entry into cells(Fig. 4). This is in good agreement with the findings of Cerviand coauthors showing that labeled SEA (a complex antigenicmixture also containing IPSE/alpha-1) does not colocalize withTfR in murine dendritic cells (10). Entry via this route wouldpose an additional problem, as this receptor is internalizedmainly through clathrin-coated pits and the ligand and recep-tor are recycled back to the plasma membrane (13). In order togain access to the host cell cytoplasm, IPSE/alpha-1 wouldhave to dissociate from the Tf-TfR complex and escape theendosomal recycling compartment.
Incubation of primary human MDDCs with native as well asHEK cell-expressed recombinant IPSE/alpha-1 showed that
uptake of IPSE/alpha-1 into these cells is mediated by acalcium-dependent mechanism. C-type lectins are carbohy-drate-binding receptors expressed on both immune and struc-tural cells and are known to depend on calcium for binding toand internalization of glycosylated molecules. Given that na-tive IPSE/alpha-1 is glycosylated (54), it is likely that in den-dritic cells and possibly other cell types, such as macrophages,IPSE/alpha-1 can gain access to the cytosolic and nuclear com-partments through C-type lectin-dependent internalization.Previous work demonstrated uptake of SEA (containing IPSE/alpha-1) by human MDDCs via the MR, macrophage galactose-type lectin (MGL), and DC-SIGN (52). However, that studyfailed to identify any uptake into the nucleus. In our view, thiswas due to the coupling technique used, which is likely to leadto a covalent modification of the NLS leading to a loss offunction. The lack of uptake by peripheral blood basophils(Fig. 6C) is in agreement with the lack of C-type lectin expres-sion on basophils. To the best of our knowledge, neither MR
FIG. 6. (A) Dose-response curve for interleukin-4 secretion by naïve purified human peripheral blood basophils. Basophils were stimulatedwith different concentrations of recombinant IPSE/alpha-1, IPSE/alpha-1 �NLS, and IPSE/alpha-1 3R in the presence of IL-3 (2.5 ng/ml) (n � 3independent experiments with cells from different donors). Values represent the mean percentages of maximum IL-4 release standard errorsof the means (SEM). (B) Nonreducing 12% SDS-PAGE of unglycosylated IPSE/alpha-1, IPSE/alpha-1 3R, and IPSE/alpha-1 �NLS. While thefirst two recombinant proteins appear as a double or single band with an apparent molecular mass of 32 to 36 kDa, the truncated mutant, in whichthe seventh and terminal Cys residue has been removed, appears as monomeric bands of approximately 16 to 18 kDa. (C) Live-cell imagingdepicting a human peripheral blood basophil (lobulated nucleus; stained with Hoechst 33342 [red]) incubated with 1 �l fluorescein-labeledHEK-IPSE/alpha-1 (1 mg/ml) for 60 min at 37°C and 6% CO2. Serial sections (1 to 10) along the z axis show that fluorescein-labeled IPSE/alpha-1(green) is not internalized but is seen in clusters on the plasma membrane in sections not containing the nucleus. Bar, 10 �m.
(CD206), DC-SIGN (CD209), nor MGL (CD301) has beendescribed as present on human basophils. The lack of uptake at4°C (Fig. 5B) points to an endocytic process and is compatiblewith a C-type lectin-mediated process, as is the complete lackof binding and uptake of unglycosylated recombinant IPSE/alpha-1 by MDDCs (Fig. 5C).
In the case of endocytic uptake, a cytosolic phase has to beassumed for subsequent translocation to the nucleus. Interest-ingly, the MR in particular has been shown to be involved inantigen cross-presentation (7), a process by which exogenousantigens are presented in association with major histocompat-ibility complex class I (MHC I) (33). While different mecha-nisms have recently been suggested to account for cross-pre-sentation in dendritic cells (1, 16), most include a cytosolictransit phase. Such a cytosolic phase would provide an oppor-tunity for an interaction of IPSE/alpha-1 with the nuclear im-port machinery and would result in nuclear translocation. Fur-thermore, while the work of van Liempt et al. (52) suggestedtargeting of SEA to the MHC II lysosomes of MDDCs, ourresults (Fig. 5D to G) clearly indicate that, at least for IPSE/alpha-1, these compartments are distinct. In addition to C-typelectin-dependent uptake, other mechanisms of uptake can beenvisaged. Dendritic cells can internalize antigens via Fc re-ceptors (44), and IPSE/alpha-1 is an immunoglobulin bindingfactor (45).
It remains to be established whether IPSE/alpha-1 uptake byCHO cells may rely on such a process as well. Since the CHOexperiments were carried out with unglycosylated recombinantprotein and MDDCs did not take up the unglycosylated form,underlying pathways appear to differ between cell types andmay reflect alternative surface receptor expression patterns.Alternatively, IPSE/alpha-1 entry into CHO cells in these ex-periments might be mediated by a receptor-independent path-way. Indeed, some proteins are able to enter cells due to thepresence of short, positively charged Arg/Lys-rich stretchesof amino acids, termed protein transduction domains or cell-penetrating peptides (CPPs), described, e.g., for a positivelycharged domain in HIV-1 Tat protein (GRKKRRQRRR)(53). It is now thought that CPPs can be internalized via severaldifferent endocytic mechanisms or via direct translocationthrough the plasma membrane (2, 30). Thus, the PKRRRTYNLS in IPSE/alpha-1 might fulfill a dual role as a CPP and anNLS, transporting this secretory protein directly from theextracellular environment into the nuclei of host cells sur-rounding the parasite’s eggs. However, the finding that therecombinant IPSE/alpha-1 3R mutant, with a strongly re-duced positive charge, was still able to enter mammalian cells(without translocating to the nucleus, due to the disruption ofthe NLS motif) and, to some extent, the finding that IPSE/alpha-1 is not taken up by basophils argue against such aCPP-like mechanism. Nevertheless, the CPP-like sequencemight still be necessary for endosomal escape and access to thecytosol, as it has been shown that CPPs such as that of HIV-1Tat use a mechanism requiring endosomal acidification in or-der to escape to the cytosol before translocating to the nucleus(19).
Regarding the potential roles of IPSE/alpha-1 in host cellnuclei, we have found that IPSE/alpha-1 has DNA bindingactivity and is also associated with DNA in SEA (G. Schrammet al., unpublished data). It is interesting to speculate that
binding of IPSE/alpha-1 to DNA or chromatin may have a rolein altering gene expression in its target host cells. DNA bindingexperiments (data not shown) indicated an overlap of the NLSand DNA binding activities, as deletion of the NLS also fullyablated DNA binding. This is in line with the work of Cokol etal. (11), who found an overlap between the NLS and DNAbinding regions for 90% of the proteins for which both theNLS and DNA binding regions were known. Our preliminaryexperiments using whole-genome DNA arrays point to dra-matic changes in transcriptional patterns in MDDCs treatedwith IPSE/alpha-1 (data not shown).
IPSE/alpha-1 has no clear sequence homology with anyother known protein, and its recently elucidated three-dimen-sional structure has yet to reveal more about its potentialfunctions (38). This study has demonstrated that the C termi-nus of IPSE appears to have multiple functions, as an NLS andin basophil activation, in addition to other potential roles de-scribed above. Future work will focus on further characterizingthe receptors enabling IPSE/alpha-1 to enter mammalian (pri-mary) cells, the potential mechanisms of endosomal escape,and the consequences of nuclear targeting of IPSE/alpha-1 inthese cells, e.g., via transcriptional profiling. The properties ofIPSE/alpha-1 described here also make it an interesting poten-tial vehicle for intracellular and nuclear delivery.
ACKNOWLEDGMENTS
We thank Max Paoli for initial help with the mutagenesis experi-ments and Daniela Barths for technical assistance with the basophilactivation experiments. The CHO-TrVb and -TrVb1 cell lines were akind gift from Timothy E. McGraw and Frederick R. Maxfield (WeillCornell Medical College, NY).
I.K. was involved in the design and performed most of the experi-ments described and wrote parts of the manuscript. G.S. and H.H.cloned IPSE and IPSE mutants into pSecTagII, expressed and purifiedrecombinant IPSE (mutants and wild type), and performed the baso-phil activation experiments. B.E. performed IPSE uptake experimentswith dendritic cells. S.B. was involved in characterizing Tf binding ofIPSE. K.B.K., C.M.-D., A.T.J., and D.M.H. were involved in confocalmicroscopy, project design, and writing the manuscript. T.S. performedlive-cell imaging with MDDCs and basophils. C.B. provided the tetra-EGFP plasmid, designed the oligonucleotides for NLS cloning intotetra-EGFP, performed some of the cloning, and sequenced the re-combinant plasmids. S.S. was involved in the project design, supervi-sion, and writing of the manuscript. F.H.F. devised and supervised theproject, wrote parts of the manuscript, and cloned the initial pEGFP-IPSE constructs from which the other mutants were obtained.
REFERENCES
1. Ackerman, A. L., C. Kyritsis, R. Tampe, and P. Cresswell. 2003. Earlyphagosomes in dendritic cells form a cellular compartment sufficient forcross presentation of exogenous antigens. Proc. Natl. Acad. Sci. U. S. A.100:12889–12894.
2. Amand, H. L., K. Fant, B. Norden, and E. K. Esbjorner. 2008. Stimulatedendocytosis in penetratin uptake: effect of arginine and lysine. Biochem.Biophys. Res. Commun. 371:621–625.
3. Amaya, Y., T. Nakai, K. Komaru, M. Tsuneki, and S. Miura. 2008. Cleavageof the ER-targeting signal sequence of parathyroid hormone-related proteinis cell-type-specific and regulated in cis by its nuclear localization signal.J. Biochem. 143:569–579.
4. Beetz, C., et al. 2004. Identification of nuclear localisation sequences inspastin (SPG4) using a novel tetra-GFP reporter system. Biochem. Biophys.Res. Commun. 318:1079–1084.
5. Bendtsen, J. D., H. Nielsen, G. von Heijne, and S. Brunak. 2004. Improvedprediction of signal peptides: SignalP 3.0. J. Mol. Biol. 340:783–795.
6. Bhasin, M., and G. P. Raghava. 2004. ESLpred: SVM-based method forsubcellular localization of eukaryotic proteins using dipeptide compositionand PSI-BLAST. Nucleic Acids Res. 32:W414–W419.
7. Burgdorf, S., V. Lukacs-Kornek, and C. Kurts. 2006. The mannose receptormediates uptake of soluble but not of cell-associated antigen for cross-presentation. J. Immunol. 176:6770–6776.
VOL. 79, 2011 NUCLEAR TRANSLOCATION OF S. MANSONI IPSE/ALPHA-1 1787
8. Cambi, A., et al. 2003. The C-type lectin DC-SIGN (CD209) is an antigen-uptake receptor for Candida albicans on dendritic cells. Eur. J. Immunol.33:532–538.
9. Cass, C. L., et al. 2007. Proteomic analysis of Schistosoma mansoni eggsecretions. Mol. Biochem. Parasitol. 155:84–93.
10. Cervi, L., A. S. MacDonald, C. Kane, F. Dzierszinski, and E. J. Pearce. 2004.Cutting edge: dendritic cells copulsed with microbial and helminth antigensundergo modified maturation, segregate the antigens to distinct intracellularcompartments, and concurrently induce microbe-specific Th1 and helminth-specific Th2 responses. J. Immunol. 172:2016–2020.
11. Cokol, M., R. Nair, and B. Rost. 2000. Finding nuclear localization signals.EMBO Rep. 1:411–415.
12. Colmenares, M., A. Puig-Kroger, O. M. Pello, A. L. Corbi, and L. Rivas.2002. Dendritic cell (DC)-specific intercellular adhesion molecule 3 (ICAM-3)-grabbing nonintegrin (DC-SIGN, CD209), a C-type surface lectin in hu-man DCs, is a receptor for Leishmania amastigotes. J. Biol. Chem. 277:36766–36769.
13. Conner, S. D., and S. L. Schmid. 2003. Regulated portals of entry into thecell. Nature 422:37–44.
14. Curwen, R. S., P. D. Ashton, D. A. Johnston, and R. A. Wilson. 2004. TheSchistosoma mansoni soluble proteome: a comparison across four life-cyclestages. Mol. Biochem. Parasitol. 138:57–66.
15. de Jong, E. C., et al. 2002. Microbial compounds selectively induce Th1cell-promoting or Th2 cell-promoting dendritic cells in vitro with diverse Thcell-polarizing signals. J. Immunol. 168:1704–1709.
16. Di Pucchio, T., et al. 2008. Direct proteasome-independent cross-presenta-tion of viral antigen by plasmacytoid dendritic cells on major histocompati-bility complex class I. Nat. Immunol. 9:551–557.
17. Dunne, D. W., F. M. Jones, and M. J. Doenhoff. 1991. The purification,characterization, serological activity and hepatotoxic properties of two cat-ionic glycoproteins (alpha 1 and omega 1) from Schistosoma mansoni eggs.Parasitology 103:225–236.
18. Emanuelsson, O., H. Nielsen, S. Brunak, and G. von Heijne. 2000. Predictingsubcellular localization of proteins based on their N-terminal amino acidsequence. J. Mol. Biol. 300:1005–1016.
19. Fischer, R., K. Kohler, M. Fotin-Mleczek, and R. Brock. 2004. A stepwisedissection of the intracellular fate of cationic cell-penetrating peptides.J. Biol. Chem. 279:12625–12635.
20. Gibbs, B. F., A. Rathling, D. Zillikens, M. Huber, and H. Haas. 2006. InitialFc epsilon RI-mediated signal strength plays a key role in regulating basophilsignaling and deactivation. J. Allergy Clin. Immunol. 118:1060–1067.
21. Haisch, K., et al. 1999. Purification of morphologically and functionallyintact human basophils to near homogeneity. J. Immunol. Methods 226:129–137.
22. Haisch, K., et al. 2001. A glycoprotein from Schistosoma mansoni eggs bindsnon-antigen-specific immunoglobulin E and releases interleukin-4 from hu-man basophils. Parasite Immunol. 23:427–434.
23. Hatzi, E., Y. Bassaglia, and J. Badet. 2000. Internalization and processing ofhuman angiogenin by cultured aortic smooth muscle cells. Biochem. Bio-phys. Res. Commun. 267:719–725.
24. Hicks, G. R., and N. V. Raikhel. 1995. Protein import into the nucleus: anintegrated view. Annu. Rev. Cell Dev. Biol. 11:155–188.
25. Hodel, M. R., A. H. Corbett, and A. E. Hodel. 2001. Dissection of a nuclearlocalization signal. J. Biol. Chem. 276:1317–1325.
26. Hoglund, A., P. Donnes, T. Blum, H. W. Adolph, and O. Kohlbacher. 2006.MultiLoc: prediction of protein subcellular localization using N-terminaltargeting sequences, sequence motifs and amino acid composition. Bioinfor-matics 22:1158–1165.
27. Horton, P., et al. 2007. WoLF PSORT: protein localization predictor. Nu-cleic Acids Res. 35:W585–W587.
28. Hu, W., P. J. Brindley, D. P. McManus, Z. Feng, and Z. G. Han. 2004.Schistosome transcriptomes: new insights into the parasite and schistosomi-asis. Trends Mol. Med. 10:217–225.
29. Hua, S., and Z. Sun. 2001. Support vector machine approach for proteinsubcellular localization prediction. Bioinformatics 17:721–728.
30. Jones, A. T. 2007. Macropinocytosis: searching for an endocytic identity androle in the uptake of cell penetrating peptides. J. Cell. Mol. Med. 11:670–684.
31. Kalderon, D., W. D. Richardson, A. F. Markham, and A. E. Smith. 1984.Sequence requirements for nuclear location of simian virus 40 large-T anti-gen. Nature 311:33–38.
32. Kalderon, D., B. L. Roberts, W. D. Richardson, and A. E. Smith. 1984. Ashort amino acid sequence able to specify nuclear location. Cell 39:499–509.
33. Kasturi, S. P., and B. Pulendran. 2008. Cross-presentation: avoiding traf-ficking chaos? Nat. Immunol. 9:461–463.
34. Kiefer, P., P. Acland, D. Pappin, G. Peters, and C. Dickson. 1994. Compe-tition between nuclear localization and secretory signals determines thesubcellular fate of a single CUG-initiated form of FGF3. EMBO J. 13:4126–4136.
35. Kwon, D. S., G. Gregorio, N. Bitton, W. A. Hendrickson, and D. R. Littman.2002. DC-SIGN-mediated internalization of HIV is required for trans-en-hancement of T cell infection. Immunity 16:135–144.
36. la Cour, T., et al. 2003. NESbase version 1.0: a database of nuclear exportsignals. Nucleic Acids Res. 31:393–396.
37. Mathieson, W., and R. A. Wilson. 2010. A comparative proteomic study ofthe undeveloped and developed Schistosoma mansoni egg and its contents:the miracidium, hatch fluid and secretions. Int. J. Parasitol. 40:617–628.
38. Mayerhofer, H., et al. 2009. Cloning, expression, purification, crystallizationand preliminary X-ray crystallographic analysis of interleukin-4-inducingprinciple from Schistosoma mansoni eggs (IPSE/alpha-1). Acta Crystallogr.Sect. F Struct. Biol. Cryst. Commun. 65:594–596.
39. McGraw, T. E., L. Greenfield, and F. R. Maxfield. 1987. Functional expres-sion of the human transferrin receptor cDNA in Chinese hamster ovary cellsdeficient in endogenous transferrin receptor. J. Cell Biol. 105:207–214.
40. Nair, R., and B. Rost. 2005. Mimicking cellular sorting improves predictionof subcellular localization. J. Mol. Biol. 348:85–100.
41. Nakai, K., and P. Horton. 1999. PSORT: a program for detecting sortingsignals in proteins and predicting their subcellular localization. TrendsBiochem. Sci. 24:34–36.
42. Park, K. J., and M. Kanehisa. 2003. Prediction of protein subcellular loca-tions by support vector machines using compositions of amino acids andamino acid pairs. Bioinformatics 19:1656–1663.
43. Rapoport, T. A., B. Jungnickel, and U. Kutay. 1996. Protein transport acrossthe eukaryotic endoplasmic reticulum and bacterial inner membranes. Annu.Rev. Biochem. 65:271–303.
44. Regnault, A., et al. 1999. Fcgamma receptor-mediated induction of dendriticcell maturation and major histocompatibility complex class I-restricted an-tigen presentation after immune complex internalization. J. Exp. Med. 189:371–380.
45. Schramm, G., et al. 2003. Molecular characterization of an interleukin-4-inducing factor from Schistosoma mansoni eggs. J. Biol. Chem. 278:18384–18392.
46. Schramm, G., et al. 2006. IPSE/alpha-1: a major immunogenic componentsecreted from Schistosoma mansoni eggs. Mol. Biochem. Parasitol. 147:9–19.
47. Schramm, G., et al. 2007. Cutting edge: IPSE/alpha-1, a glycoprotein fromSchistosoma mansoni eggs, induces IgE-dependent, antigen-independentIL-4 production by murine basophils in vivo. J. Immunol. 178:6023–6027.
48. Shatkay, H., et al. 2007. SherLoc: high-accuracy prediction of protein sub-cellular localization by integrating text and protein sequence data. Bioinfor-matics 23:1410–1417.
49. Simpson, J. C., et al. 2004. A role for the small GTPase Rab21 in the earlyendocytic pathway. J. Cell Sci. 117:6297–6311.
50. Soeiro Mde, N., M. M. Paiva, H. S. Barbosa, N. Meirelles Mde, and T. C.Araujo-Jorge. 1999. A cardiomyocyte mannose receptor system is involved inTrypanosoma cruzi invasion and is down-modulated after infection. CellStruct. Funct. 24:139–149.
51. Szafron, D., et al. 2004. Proteome Analyst: custom predictions with expla-nations in a web-based tool for high-throughput proteome annotations. Nu-cleic Acids Res. 32:W365–W371.
52. van Liempt, E., et al. 2007. Schistosoma mansoni soluble egg antigens areinternalized by human dendritic cells through multiple C-type lectins andsuppress TLR-induced dendritic cell activation. Mol. Immunol. 44:2605–2615.
53. Vives, E., P. Brodin, and B. Lebleu. 1997. A truncated HIV-1 Tat proteinbasic domain rapidly translocates through the plasma membrane and accu-mulates in the cell nucleus. J. Biol. Chem. 272:16010–16017.
54. Wuhrer, M., et al. 2006. IPSE/alpha-1, a major secretory glycoprotein anti-gen from schistosome eggs, expresses the Lewis X motif on core-difucosy-lated N-glycans. FEBS J. 273:2276–2292.
55. Wynn, T. A., R. W. Thompson, A. W. Cheever, and M. M. Mentink-Kane.2004. Immunopathogenesis of schistosomiasis. Immunol. Rev. 201:156–167.
56. Xie, D., A. Li, M. Wang, Z. Fan, and H. Feng. 2005. LOCSVMPSI: a webserver for subcellular localization of eukaryotic proteins using SVM andprofile of PSI-BLAST. Nucleic Acids Res. 33:W105–W110.
57. Yu, C. S., C. J. Lin, and J. K. Hwang. 2004. Predicting subcellular localiza-tion of proteins for Gram-negative bacteria by support vector machinesbased on n-peptide compositions. Protein Sci. 13:1402–1406.
