Molecular characterization, tissue distribution, subcellular localization and actin-sequestering function of a thymosin protein from silkworm

Abstract

We identified a novel gene encoding a Bombyx mori thymosin (BmTHY) protein from a cDNA library of silkworm pupae, which has an open reading frame (ORF) of 399 bp encoding 132 amino acids. It was found by bioinformatics that BmTHY gene consisted of three exons and two introns and BmTHY was highly homologous to thymosin betas (Tβ). BmTHY has a conserved motif LKHTET with only one amino acid difference from LKKTET, which is involved in Tβ binding to actin. A His-tagged BmTHY fusion protein (rBmTHY) with a molecular weight of approximately 18.4 kDa was expressed and purified to homogeneity. The purified fusion protein was used to produce anti-rBmTHY polyclonal antibodies in a New Zealand rabbit. Subcellular localization revealed that BmTHY can be found in both Bm5 cell (a silkworm ovary cell line) nucleus and cytoplasm but is primarily located in the nucleus. Western blotting and real-time RT-PCR showed that during silkworm developmental stages, BmTHY expression levels are highest in moth, followed by instar larvae, and are lowest in pupa and egg. BmTHY mRNA was universally distributed in most of fifth-instar larvae tissues (except testis). However, BmTHY was expressed in the head, ovary and epidermis during the larvae stage. BmTHY formed complexes with actin monomer, inhibited actin polymerization and cross-linked to actin. All the results indicated BmTHY might be an actin-sequestering protein and participate in silkworm development.

Figure 1. Profile of silkworm BmTHY gene.

(a) Schematic representation of BmTHY gene. (b) ORF sequence and predicted amino acid sequence of BmTHY gene. (c) BmTHY contains two intact THY domains.

Figure 2. Multiple amino acid sequence alignment of BmTHY with homologous proteins.

The identical residues were shaded in black, while the similar residues were shaded in gray, Ruler shows the number and position of residue. BmTHY, TcTHY, DrTHY and ApTHY represent thymosin superfamily protein of Bombyx Mori, Tribolium castaneum, Drosophila melanogaster, Apis mellifera respectly; HuTB4X:Tβ4(X-Chromosome,Homosapiens);HuTB4Y:Tβ4(Y-chromosome,Homosapiens);BtTB10:Tβ10(Bos Taurus);SsTB12:Tβ12(Salmo salar);SsTB11:Tβ11(Salmo salar).

Figure 3. The tertiary structure of BmTHY.

(a)The tertiary structure of BmTHY(strands); (b)The tertiary structure of BmTHY(Molecular Surface).

Figure 4. Expression and purification of the His-tag-BmTHY fusion protein.

Samples were resolved by 12% SDS-polyacrylamide gel electrophoresis under reducing conditions. A: Expression of fusion protein in Rosetta (DE3); M: protein molecular weight marker (low); 1: Rossetta (pET-28a-BmTHY) without induction; 2: Rossetta (pET-28a-BmTHY) after induction; B: Purification of the His-tag fusion protein in Rosetta (DE3); M: protein molecular weight marker (low); 1: supernatant of E.coli Rosetta/pET-28a-BmTHY induced by IPTG after supersonic treatment; 2: purified fusion protein expressed in E.coli Rosetta.

Figure 5. Analysis of the His-tag-BmTHY fusion protein by MS.

Figure 6. Determine of polyclonal antibody titer by ELISA.

ELISA was used to determine the following ratio for antibodies against rBmTHY: positive serum extinction value/negative serum extinction value (P/N)≥2.1 was positive; 1.5≤P/N<2.1 was suspicious expression; P/N<1.5 was negative. From this, the titer of the antibodies was greater than 1∶25600 at a concentration of 10 µg/mL.

Figure 7. Western blotting analysis of the His-tag-BmTHY fusion protein expression.

Samples were resolved by 12% SDS PAGE under reducing conditions (M,1,2,3 SDS-PAGE; 1′, 2′,3′ Western blotting). M: protein molecular weight marker (low); 1: purified fusion protein expressed in E.coli Rosetta; 2: supernatant of E.coli Rosetta/pET-28a-BmTHY induced by IPTG after supersonic treatment; 3: E.coli Rosetta/pET-28a. Arrow indicates the fragment of the His-tag fusion BmTHY.

Figure 8. Transcription and expression level of BmTHY in different development stages of Bombyx mori.

(a) Analysis of BmTHY expression was performed by RT-PCR. Relative BmTHY expression was determined in relation to the corresponding BmTHY expression level in the silkworm moth: ΔΔC_T (stage) = ΔC_T (stage)−ΔC_T (egg); (b) Western blotting analysis of the expression levels of of BmTHY in different development stages. 1,egg;2,pupa; 3,larva;4,moth.

Figure 9. Transcription and expression level of BmTHY in different tissues of fifth-instar larva of Bombyx mori.

(a) Transcription level of BmTHY in different tissues of fifth-instar larva of Bombyx mori. Total RNA from the silk gland, spiracle,midgut, Malpighian tubule, head, ovary, testis,fatty body, and epidermis of fifth-instar larva was used as a template for RT-PCR. Relative BmTHY expression was determined in relation to the corresponding BmTHY expression level in testis: ΔΔC_T (tissue) = ΔC_T (tissue)−ΔC_T (testis); (b) Western blotting analysis of the expression levels of of BmTHY in different development stages. 1:Silk gland;2:Spiracle;3:Midgut; 4: Malpighian tubule;5:Head;6:Ovary; 7: Testis;8:Fatty body;9: Epidermises.

Figure 10. Subcellular localization of BmTHY in Bm5 cells by immunofluorescence.

A1–A4,B1–B4: Experimental group, using anti-BmTHY polyclonal antibody;C1-C4: negative control group, using negative rabbit serum;A1,B1,C1: Cells under transmitted light. A2,B2,C2: DAPI staining;A3,B3,C3: BmTHY subcellular localization as indicated by Cy3-labeled secondary antibody;A4,B4,C4: merged image.

Figure 11. Electrophoretic analysis of the interaction of actin with BmTHY.

a,Effect of BmTHY and Tβ4 on the mobility of actin in nondenaturing polyacrylamide gel. NPAGE of muscle G-actin plus BmTHY or Tβ4 shows that both BmTHY and Tβ4 shift the mobility of actin in a stoichiometric fashion. Lane 1,2 µg of G-actin ; Lane2 and 5,blank; Lane 3, 0.6 µg of Tβ4 and 2 µg of G-actin ; Lane 4, 1.2 µg of Tβ4 and 2 µg of G-actin ; Lane 6, 0.6 µg of BmTHY and 2 µg of G-actin ; Lane 7, 1.2 µg of BmTHY and 2 µg of G-actin. b,sedimentation analysis of the effect of BmTHY and Tβ4 on salt-induced actin polymerization. 100 µL of G-actin was mixed with:100 µL of G-buffer (lane 1); 100 µL of KCl-buffer (lane 2); 66 µL of Tβ4 (lane 3); 66 µL of BmTHY (lane 4);33 µL of Tβ4 (lane 5); 33 µL of BmTHY (lane 6);The molar ratio of BmTHY (Tβ4) to actin was 0.57∶1 in lanes 3 and 4 and 0.28∶1 in lanes 5 and 6. The final actin concentration was 5.8 µM in each assay. Each pellet was solubilized in 50 µL of SDS sample buffer. c,cross-linking of BmTHY and Tβ4 to G-actin. G-actin was incubated with either BmTHY (lane 2, 3 and 4) or Tβ4 (lane 5 and 6) plus 1-ethyl-3-(3-dimethylaminopropyl) carbodimide for 2 h at 25°C. The molar ratio of BmTHY (Tβ4) to actin was 1∶5 in lanes 2, 3 and 5 and 1∶9 in lanes 4 and 6. Lane 1 was G-actin. Lane 7 was protein molecular weight marker. Each aliquots were taken up into SDS sample buffer and analyzed by SDS-PAGE on a 10% gel under reducing conditions.