| Karyopherins also called Importins, including alpha- and beta-type, are important transport proteins in eukaryotic cells that carry cargoes across nuclear pore complexes into or out of nucleus. Karyopherin alphas(KPNAs) cannot carry cargoes across nuclear pore complexes(NPCs) by themselves but they can act as receptors attaching cargoes to Karyopherin beta 1(KPNB1) and forming trimers which can across NPCs and finally release cargoes in nucleoplasm. Karyopherin betas(KPNBs) include Importin betas, Exportins and Transportins. Importin betas transport cargoes into nucleus and only Karyopherin beta 1(KPNB1) can recognize KPNAs to form complexes. Exportins transport cargoes out nucleus while transportins transport cargoes either into or out nucleus.After BLAST-searched the domestic silkworm genome database(Silk DB) we found three predicted KPNAs and one predicted KPNB. We had designed primers and successfully cloned the c DNAs of the four genes in Bombyx mori and Antheraea pernyi. The open reading frames(ORFs) of KPNAs is 1563, 1515 and 1551 bp coding 520, 504 and 516 AAs respectively. The similarity of these KPNAs ORF or proteins between Bombyx mori and Antheraea pernyi are respectively 99.55%/100% 、 99.93%/100% and 99.68%/99.50%. The KPNAs of Bombyx mori and Antheraea pernyi are respective classed into α1,α2 or α3 subfamily by constructing phylogenic tree. All insects have three kinds of KPNAs at most except drosophila who has four and each KPNA is classed into a different subfamily. Thus we named the three KPNAs of Bombyx mori and Antheraea pernyi as KPNA1, KPNA2 and KPNA3 according the subfamily they belonging to. The KPNB of Bombyx mori and Antheraea pernyi ORFs are 2661 and 2658 bp coding 886 and 885 AAs respectively and the similarity of ORFs or AAs are respectively 80.16% or 91.12%. After aligning protein sequences and constructing phylogenic tree we found the KPNB of Bombyx mori and Antheraea pernyi most similar to KPNB1 from other organisms so we named the KPNB as KPNB1.We had cloned ORFs of KPNA1, KPNA2, KPNA3 and KPNB1 of Bombyx mori and Antheraea pernyi into plasmid p ET-28(a+) for prokaryote expression and found these recombinant proteins all expressed successfully and correctly. After analyzing the sequence of these proteins we found KPNA1, KPNA2 and KPNA3 all have one importin beta binding domain(IBB) and 8, 4 or 7 typical armadillo/beta-catenin-like repeats(ARM). KPNB1 have 19 typical Huntingtin, Elongation factor 3, protein phosphatase 2A and TOR1 repeats(HEAT repeats). They construct IBB_N and IBB binding domain. Hydrophobicity analysis display there are hydrophilic regions about 100 AAs long at the N terminal of KPNAs and the remain regions of KPNAs and KPNB1 are mixed with hydrophobic and hydrophilic AAs and homologous modeling results indicate they are all nestle structure constructed by tens of alpha helix. In vitro GST pull-down experiments also show the interaction between KPNB1 and IBB of KPNAs but not between KPNB1 and KPNAs without IBB.We detected the m RNA expression levels of KPNAs or KPNB1 in tissues of Bombyx mori and Antheraea pernyi and found KPNAs and KPNB1 widely express in all tissues. In Bombyx mori KNPA1 m RNA level is highest in integument, second in malpighian tube, hemocyte and midgut, and low in fat body and silk gland; KPNA2 m RNA level is highest in hemocyte and low in other five tissues; KPNA3 and KPNB1 m RNA levels are higher in hemocyte, malpighian tube and midgut and low in other three tissues. In Antheraea pernyi KPNA1 m RNA levels are higher in malpighian tube, hemocyte and midgut and low in integument, fat body and silk gland; KPNA2 are highest in fat body and low in other five tissues; KPNA3 are higher in hemocyte, fat body and silk gland while KPNB1 are higher in hemocyte, malpighian tube and silk gland.To make clear KPNAs and KPNB1 m RNA expression variation to innate immune response in fat body and hemocyte, we injected 5th 3 day larval with Gram positive bacterium(Micrococcus luteus or Bacillus subtilis), Gram negative bacterium(Escherichia coli), virus(Nuclear polyhedrosis virus) or fungus(Beauveria bassiana).In the fat body of Bombyx mori, KPNAs m RNA levels were generally low at 1 and 8 h, but increased at 4 and 12 h. KPNA1 was most sensitive to E. coli, B. bassiana, and Nuclear polyhedrosis virus(NPV), KPNA3 to M. luteus, E. coli, and B. bassiana, and KPNA2 was not as sensitive as the other two KPNAs to the four microbes. KPNB1 m RNA levels in response to E.coli and M. luteus exhibited no significant variation at 1, 4 or 8 h, but at 12 h they increased by 2.97-fold and 4.93-fold, respectively. When responding to B. bassiana and NPV, KPNB1 m RNA levels slightly fluctuated.In the hemocytes of Bombyx mori, KPNA and KPNB1 m RNA levels were generally first downregulated and then upregulated. At 4 h they decreased to basal levels. They were most sensitive to B. bassiana and upregulated to 15.54, 19.32, 8.09, and 3.93-folds than that of PBS treated larvae, respectively, at 8 h.In the fat body of Antheraea pernyi, KPNA1 and KPNB1 m RNAs expression were all downregulated. KPNA2 increased to 10.4 and 7.2 times at 12 and 48 h when responsing to B. subtilis; increased to 9.8 and 84.2 times at 0.5 and 48 h when responsing to E. coli; increased to 6.1 and 38.4 times at 3 and 6 h when responsing to NPV and increased to 23.3, 11.2 and 22.2 times at 0.5, 3 and 6 h when responsing to B. bassiana. KPNA3 increased to 9.8, 6.4, 5.7 and 13.8 times at 0.5, 3,12 and 48 h when responsing to B. subtilis; increased to 45.7 and 96.1 times at 0.5 and 48 h when responsing to E. coli; increased to 69.23 times at 6 h when responsing to NPV and increasd to10.4 and 80.3 times at 3 and 6 h when responsing to B. bassiana.In the hemocytes of Antheraea pernyi, KPNAs and KPNB1 at 24 h increased to 15.9, 39.9, 2.17 and 2.77 times respectively while decreased at other times when responsing to B. subtilis. When responsing to E. coli the four protein m RNA levels increased to 3.8, 10.1, 5.5 and 20.3 times at 0.5 h and except KPNA3 the other three proteins increased to 21.9, 23.1 and 3.7 times at 24 h and 9.7, 10.8 and 5.7 times at 48 h. When responsing to NPV, KPNA3 increased to 12.4 times at 6 h and KPNA2 increased to 2.0 and 2.3 times at 3 and 24 h respectively. When responsing to B. bassiana KPNB1 m RNA levels were low constantly; KPNA1 increased to 13.5 and 13.7 times at 3 and 24 h; KPNA2 increased to 132.5 and 5.6 times at 3 and 24 h and KPNA3 increased to 41.1 times at 3 h.Through KPNA pull-down assay with native proteins of tissues, we found the protein, heat shock transcription factor(HSF), is transported into nucleus by KPNA3 in Bombyx mori. At 4 and 24 h after heat shock under 45°C for 30 minutes male pupae of Bombyx mori appeared m RNA expression peaks of HSP19.9, HSP20.4 and HSP25.4. The facts that knocking-down KPNA3 or HSF using RNA interference method both eliminate the peak at 24 h indicated it is KPNA3 that transport HSF into nucleus to increase the three HSPs transcription. And GST pull-down assay also testified the interaction between GST-KPNA3 and His-HSF in vitro. |
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