Characterization Of A Rel/NF-κB Homologue (Ab-Rel) In A Gastropod Abalone, Haliotis Diversicolor Supertexta And Development Of New Detection Strategies

Rel/NF-κB is a family of structurally related transcription activator proteins, which regulate gene expression in various physiological processes such as inflammation, immune response, apoptosis, embryonic morphogenesis, cell proliferation and differentiation. Rel\NF-κB signal transduction pathway is evolutionarily conserved, and has been intensely studied through insects to mammals. Recently, large amounts of evidence showed that aberrant regulation of NF-κB was involved in autoimmune diseases and different types of cancers, making this factor with other co-effectors involved in this signaling pathway potential candidates for drug discovery and development efforts.This paper report here for the first time that a homologue of Rel\NF-κB transcription factor, Ab-Rel, was identified and functionally characterized in the abalone, H. diversicolor supertexta, an economically important marine gastropod extensively cultured in the south coast of China. The full-length Ab-Rel cDNA has been cloned and sequenced, and the evolutionary relationships with those of other Rel proteins have been analyzed. The recombinant Ab-Rel protein was expressed in E. coli, and purified, as well as the biological activity of the recombinant protein was determined. Two different antibodies against Ab-Rel were raised to identify the molecular structure of the abalone NF-κB protein. The expression pattern of Ab-Rel has been analyzed, and NF-κB activity was functionally characterized in abalone. Ab-Rel has been successfully expressed and subcellularly located using EGFP fusion technique in insect cells. These results strongly suggest that Ab-Rel is a Rel homologue, which plays a conserved role in the immune response of the ancient invertebrate, abalone, allowing us to study the Rel\NF-κB signaling pathway in an evolutionary context. In addition, two new methods, the two-antibody indirect ELISA and the magnetocapture, were developed to isolate and detect this abalone transcriptional factor.Main results of this paper include the following parts: Molecular cloning and sequence analysis of Ab-Rel cDNAUsing degenerate primers, a 390 bp fragment was obtained by RT-PCR from haemocyte RNA of H. diversicolor supertexta. Subsequently, 3'-RACE and 5'-RACE were conducted on the basis of DNA sequence information from the above fragment to clone the Ab-Rel cDNA. The full-length cDNA has an ORF of 1752 bp encoding 584 amino acid residues. The 5' and 3' untranslated regions (UTR) contain 73 bp and 115 bp, respectively. A possible mRNA instability motif ATTTA and a polyadenylation signal AATAAA, 14 bp upstream the polyA tail, are found in the 3' UTR.Sequence analysis showed that the deduced amino acid sequence contains a Rel homology domain (RHD) in the N-terminal region, which includes a transcription factor immunoglobin-like folds (TIG) and nuclear localization signal (NLS). The consensus DNA binding motif RXXRXRXXC and a phosphorylation site RRPS are also observed within the RHD. Even though no conserved domains or recognizable protein motifs were observed in the C-terminal region, all results in the N-terminal region suggested that this cDNA encodes an abalone Rel protein, namely Ab-Rel (AY700781).Comparison of Ab-Rel in full-length amino acid sequence with other Rel proteins from invertebrates and vertebrates showed that an identity ranged from 22% to 46%, while on the highly conserved RHDs, the identity levels were from 43% to 72%. The highest identity, 46%, in an overall sequence and 72%, in RHD, were apparent in comparison with a C. gigas Rel homologue, Cg-Rel.The unrooted phylogenetic tree was built using the neighbor-joining method based on the available RHD amino acids sequences. Ab-Rel was branched with Cg-Rel and clustered with insect Rel transcription factors away from those of vertebrates. We can distinguish two classes of Rel proteins from this tree. Class I includes those containing the ankyrin-repeat domain in the C-terminal region such as p100, p105 and Relish. Class II appears to cover all the Rel proteins which have a TD in the C-terminal region. Ab-Rel was obviously located in the class II subfamily. The sequence and phylogenetic analysis suggest that a potential transactivation domain might exist in Ab-Rel, and that the NF-κB singnaling pathway might originate with the appearance of coelom. Expression of abalone NF-κB in Eschericia coli, determination of its biological activity and identification of its molecular structureThe plasmid containing the fragment corresponding to Rel homology domain (RHD) and transactivation domain (TD) of Ab-Rel was constructed respectively to express each recombinant protein in E. coli. Recombinant fusion proteins were purified by GST affinity columns, and purified proteins were used to immunize rabbits to produce two different antibodies.EMSA was carried out using DIG-labeled probe containing consensus NF-κB binding site to test the biological activity of the recombinant Ab-Rel protein. A specific binding activity was observed with recombinant Ab-Rel RHD, demonstrating that the recombinant Ab-Rel protein has biological activity to bind the consensus NF-κB binding site. This result indicates that the abalone transcriptional factor Ab-Rel has a functional similarity with those Rel family members.Western blotting was performed with the two different antibodies and abalone haemocyte extracts. Two proteins were detected by anti-,Ab-Rel RHD antibody, one of which is consistent with Ab-Rel, another one is smaller and suspected to be the small subunit of abalone NF-κB. This result was further confirmed by a Western blotting using anti-Ab-Rel TD antibody. In this experiment, only one protein, consistent with Ab-Rel was recognized by the Ab-Rel specific antibody. All results suggest that abalone NF-kB has a molecular structural similarity with its orthologue in insect and mammal.Expression analysis of Ab-Rel in H. diversicolor supertextaRT-PCR was conducted with total RNAs from foot muscle, mantle margin, gills, digestive gland and haemocytes of abalone. The results suggest that Ab-Rel, like most of the Rel genes, was ubiquitously expressed in all examined tissues. A relatively high amount of Ab-Rel transcripts were observed in haemocytes, suggesting that this transcription factor regulates immune-related genes in the abalone. The same result was also found in a northern blot with the specific probe to Ab-Rel. The size of the Ab-Rel transcript is about 1.9 kb, in close agreement with the length of the cDNA sequence.Western blot analysis of total extracts of abalone haemocytes using the anti-Ab-Rel antiserum revealed a protein with a molecular mass of about 65 kDa, which was in agreement with the predicted 64.2 kDa molecular weight of Ab-Rel, suggesting that no major post translational modification of the Ab-Rel protein occurred.Activation of Ab-Rel and its involvement in the immune response of abaloneTo investigate the effects of LPS stimulation on Ab-Rel expression, real-time PCR was conducted using RNA extracted from haemocytes of abalones after 0 h, 3 h, 6 h and 9 h of LPS injection. The control group, injected with equal volume of Saline, was made in parallel for the same time point. The same RNAs from LPS-injected animals were used in the northern blot to verify the result of real-time PCR. The result showed that Ab-Rel transcripts were not modified significantly by stimulation with LPS, suggesting that LPS cannot induce the expression of Ab-Rel at the mRNA level.In order to determine whether NF-κB activation in abalone is regulated at the protein level, and whether this activation is involved in the immune response, EMSA was carried out with total protein extracts from abalone haemocytes and DIG-labeled probes. A specific DNA-binding activity was determined, which appears to be increased in LPS-injected abalones. Meanwhile, a supershift was produced by the addition of an antibody against Ab-Rel. The specifically induced DNA-binding activity in LPS-challenged abalones was further confirmed in comparison with the normal group (no injection) and Saline-injected control group. The result demonstrates that DNA-binding activity of NF-κB was apparently induced in haemocytes of abalones after 1 h of LPS stimulation, while relative to the normal group (no injection), no obvious changes could be observed in Saline-injected control group. These results suggest that the NF-κB signaling pathway exists in the gastropod abalone and is involved in the immune response, which also agree with the previous conclusion that Ab-Rel expression was regulated at the protein level, rather at the mRNA level.Expression and subcellular location of Ab-Rel in insect cellsThe whole ORF of Ab-Rel was cloned into the transfer vector pFastBac^TMHT A, and the recombinant plasmid was transformed into DHIOBac competent cells to generate recombinant virus DNA, the bacmid. The host cells (Tn-5B1-4) were transfected with the bacmid, and whole cell extracts of the Tn-5B1-4 transfected with recombinant bacmid or blank bacmid were subjected to SDS-PAGE. A 70-kDa protein was specifically expressed in the host cells transfected with recombinant bacmid, which is consistent with the molecular mass of deduced protein. Western blotting was performed with anti-Ab-Rel antibody and whole cell extracts, an immunoreactive band corresponding to a 70-kDa protein was detected in the Tn-5B1-4 transfected with recombinant Ab-Rel bacmid. All these data showed Ab-Rel was successfully expressed in the insect cells.EGFP-,Ab-Rel fusion protein was expressed using the same system described as above. An expected 100-kDa recombinant EGFP-Ab-Rel protein was successfully expressed. The expressed products were located in the nuclei of the insect cells observed under a confocal fluorescent microscope.Detection of Ab-Rel with a two-antibody indirect ELISAA two-antibody indirect ELISA using anti-Ab-Rel RHD and TD antibodies was developed to detect Ab-Rel in different abalone tissues. This method is more sensitive than the one-antibody ELISA to detect the same sample from the abalone haemocytes. The two-antibody indirect ELISA was performed with protein extracts from foot muscle, mantle margin, gills, digestive gland and haemocytes of abalone. The results showed that Ab-Rel was ubiquitously expressed in all examined tissues. A relatively high amount of protein was observed in haemocytes. From the protein level, these data confirmed the results of expression analysis of Ab-Rel mRNA, strongly suggesting that Ab-Rel is pleiotropic and was involved in the immune response of abalone.Magnetocapture of abalone transcription factor NF-κBA specific double strand DNA probe was designed and produced by PCR, which has a biotinylated 5'-end with the 3'-end containing one consensus NF-κB binding site. The biotin endlabeled probe was linked to the magnetic beads coated with streptavidin to give them a binding capacity with the active NF-κB. After incubation with crude protein extracts from abalone hemocytes, the protein(s), DNA and magnetic beads complexes were subjected to magnetic separation, washing and elution. The enriched abalone NF-κB transcription factor were obtained and analyzed by SDS-PAGE. A protein with a molecular mass of about 65 kDa was isolated and enriched from crude extracts of abalone hemocytes. The protein specifically bound to the wild-type NF-κB probe with the consensus NF-κB binding site but did not bind to the mutant probe in which the consensus NF- KBbinding site was mutated. At least 3 proteins were eluted together with the 65-kDa protein, which was suspected to interact with Ab-Rel, reminding us that the magnetocapture method could become a better choice to study protein interaction due to its advantages of simplified manipulation and less mechanical stress. Compared with the Saline-injected or no-injected control groups, a higher amount of the 65-kDa protein was captured from the hemocytes of LPS-injected abalones, which confirmed the previous result that LPS can stimulate the NF-κB activity in abalone.Western blotting was performed with the eluted protein and the anti-Ab-Rel antiserum. A specific immunoreactive band, corresponding to the size of the enriched protein, was detected. All the results strongly indicated that the captured 65-kDa protein is Ab-Rel, and that this magnetocapture approach is highly specific for NF-κB.A 70-kDa His-tagged recombinant Ab-Rel protein was expressed in Trichopolusia ni cells using the Bac-to-Bac baculovirus expression system, and was also captured by the magnetocapture approach. Western blot was performed using His-tag monoclonal antibody and the proteins eluted from the magnetic separation assay. A single immunoreactive band, corresponding to a protein size of 70 kDa, was detected, suggesting that the captured 70-kDa protein was the expressed recombinant Ab-Rel protein.In conclusion, we have developed a novel strategy to isolate and detect NF-κB both in vivo and in vitro. Since only the activated NF-κB can be captured and separated from the whole cell extracts, this method was simultaneously used to detect the NF-κB activity. The magnetocapture method also would be useful for identifying interaction molecules due to its advantages of simplified manipulation and less mechanical stress for the analyte. These results indicate that this magnetocapture method is specific, rapid, reliable and versatile, providing a promising tool for studying Rel\NF-κB protein.