| High-mobility group box family (HMG) genes are ubiquitous in vertebrates,including mammals, birds, amphibians and fishes. HMGB, a highly conserved DNA-bindingprotein, plays an important role in maintaining nucleosome structures, transcription,inflammation and cell proliferation. Lampreys are considered to be the most scientificallyaccessible model of the remaining jawless vertebrates. Researches in the evolution anddevelopment of immunological structures in lamprey may contribute to better understandhow vertebrates could have evolved. So far researches have focused mainly on HMGB inmammalian, little is known about the existence of HMGB in lamprey. To elucidate themolecular phylogeny of the HMGB genes in the primitive vertebrate, we have cloned threehomologues of HMG-box genes, called Lj-HMGB1, Lj-HMGB2and Lj-HMGBX, from acDNA library generated from lymphocyte-like cells of the Japanese lamprey (Lampetrajaponica), an Agnathan that occupies a critical phylogenetic position between invertebratesand vertebrates. The open reading frames (ORFs) of Lj-HMGB1, Lj-HMGB2andLj-HMGBX were627bp,585bp and678bp long and encoded208,194and225aminoacids, respectively. The calculated molecular masses of Lj-HMGB1, Lj-HMGB2, andLj-HMGBX were24,086Da,22,177Da, and25,271Da, respectively. The analysis of thededuced amino acid sequences indicated that these three putative Lj-HMGB proteins containfour domains: HMG-box A, HMG-box B, an acidic carboxyl-terminal tail and a linker. Aphylogenetic analysis revealed that the Lj-HMGB proteins fall outside the vertebrate clade;Lj-HMGBX is descended from a gene ancestral to the mammalian HMGB1/2/3. Thisdiscovery implies that there was a gene duplication event in the HMGB1/2/3gene family thatoccurred after the divergence of the vertebrates (Cyclostomata) from the Cephalochordataand Urochordata at least450million years ago (MYA). The Lj-HMGB1, Lj-HMGB2andLj-HMGBX genes were detected in most tissues of the lamprey by RT-PCR. The expressionof Lj-HMGB1, Lj-HMGB2and Lj-HMGBX was detected in the erythrocyte, leukocyte,kidney, heart, liver, gill, gut, muscle, and buccal gland of the lamprey, except that theexpression level of Lj-HMGB2was weak in the buccal gland. In addition, the level ofLj-HMGB1expression was higher than that of Lj-HMGB2and Lj-HMGBX in most of thetissues. Our findings provide insight into the phylogeny of the HMGB genes in vertebrates.We examined Lj-HMGB1expression in lymphocyte-like cells, and the kidneys, heart, gills, and intestines of lampreys before and after the animals were challenged with LPS andConA. Lj-HMGB1was initially expressed at a higher level in the heart, but after treatmentwith LPS and ConA only the gills demonstrated a significant up-regulation of expression.Recombinant lamprey HMGB1protein (rLj-HMGB1) was expressed as a histidine-taggedfusion protein in Rosetta blue cells. The purified rLj-HMGB1migrated as a single band on a12%SDS-PAGE gel with a molecular mass of approximately27,000Da. We next generated arabbit anti-Lj-HMGB1polyclonal antibody. Western blots showed that the anti-Lj-HMGB1antibody recognized native Lj-HMGB1in lamprey tissues, including the heart, kidneys, gills,intestines and lymphocyte-like cells. To investigate the DNA-binding ability of lampreyHMGB1, purified rLj-HMGB1was incubated with lamprey GAPDH polynucleotides (101bpand12bp in length), and the DNA-rLj-HMGB1complexes were analyzed usingelectrophoretic mobility shift assays (EMSAs). We used human HMGB1(Hu-HMGB1) as apositive control and observed the expected reduction in mobility with both the101-bp and the12-bp DNA fragments. In addition, the rLj-HMGB1protein also reduced the mobility of the101-bp and the12-bp DNA bands in a dose-dependent manner, suggesting that the amount ofrLj-HMGB1limits the reaction. To confirm that Lj-HMGB1binds directly to DNA,pEGFP-N1DNA was incubated with rLj-HMGB1, BSA, or Hu-HMGB1in the presence orabsence of DNase I. Both rLj-HMGB1and Hu-HMGB1protected the DNA from degradation,whereas BSA provided no protection, indicating that rLj-HMGB1competitively binds to thepEGFP-N1DNA and protects it from degradation. We measured the concentration of TNF-αreleased by THP-1monocytic cells in the presence of rLj-HMGB1. rLj-HMGB1significantlystimulated the secretion of TNF-α from the THP-1cells in a time-dependent manner. Thehighest TNF-α level was detected after24h of incubation. The TNF-α level decreasedthereafter and reached50%of the maximum at48h. We compared the amount of TNF-αreleased in the presence of Lj-HMGB1to that released when the cells were exposed toHu-HMGB1or LPS and found that similar amounts of TNF-α were released under allconditions. To ensure that contaminants from the purification process were not responsiblefor the release of TNF-α, we tested a related, but irrelevant, His-tagged protein (Lj-HBP1).Treatment with Lj-HBP1did not cause a release of TNF-α. Our results suggest thatrLj-HMGB1stimulates THP-1monocytic cells to release proinflammatory cytokines. Todetermine whether rLj-HMGB1can induce the proliferation of tumor cells, human breastadenocarcinoma cells (MCF-7) were cultured in the presence of rLj-HMGB1for24h.Compared with the negative control group (Lj-HBP1), rLj-HMGB1and Hu-HMGB1exhibited similar, significant increases in MCF-7cell proliferation in a dose-dependent manner despite their evolutionary divergence.In conclusion, we have cloned three homologue of HMGB from lymphocyte-like cells ofL. japonica. The recombinant Lj-HMGB1protein bound double-stranded DNA and inducedthe proliferation of human adenocarcinoma cells to a similar extent as human HMGB1. Wefurther revealed that Lj-HMGB1was able to induce the production of tumor necrosis factor-α(TNF-α), a pro-inflammatory mediator, in activated human acute monocytic leukemia cells.These results suggest that lampreys use HMGB1to activate their innate immunity for thepurpose of pathogen defense. |
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