Expression And Immune Functional Analysis Of Transforming Growth Factor Beta In Amphioxus (Branchiostoma Japonicum)

Amphioxus or lancelet, a cephalochordate, has long been regarded as the livinginvertebrate most closely related to the proximate invertebrate ancestor of vertebrates.Amphioxus is becoming an emerging model organism for insights into the origin andevolution of vertebrates. Transforming growth factor beta (TGFβ) is a pleiotropiccytokine with important roles in mediating inflammatory response, includingproliferation, differentiation, migration, inflammation and immunosuppression. TGFβhas been shown to be widely present in vertebrates and invertebrates. MammalianTGFβ has been shown to exist in three distinct isoforms, TGFβ1, TGFβ2, and TGFβ3.Virtually every cell type in the body, including epithelial, endothelial, hematopoietic,neuronal, and connective-tissue cells, produces TGFβ and expresses receptor for it.Only one type of tgfβ was found in amphioxus genome and amphioxus tgfβ maybe the ancestral form of vertebrate tgfβ1, tgfβ2, tgfβ3genes. However, little is knownabout its functions in immune and inflammatory responses. Moreover, structural andfunctional insights into TGF molecules in invertebrates remain completely lacking.In this study, we report the immune functional analysis and structural and functionalinsights of tgfβ gene in amphioxus B. japonicum.In order to study the immune function of amphioxus TGFβ, the recombinantplasmid of amphioxus TGF mature peptide (mTGF) has been constructed,transferred to E.coli and induced to express. mTGFβ expressed in insoluble body withan abundant amount. We purified the insoluble body and renatured it. We found thehomodimerization of mTGFβ by cross-linking. The migration of non-activatedRAW264.7cells induced by recombinant mTGFβ was assayed using boyden chamber.The results showed amphioxus recombinant mTGFβ is able to induce the migration ofnon-activated RAW264.7cells. This suggests amphioxus TGFβ has similar function with vertebrate TGFβs, which could promote macrophage migration. We also testedthe effect of amphioxus TGFβ to activated RAW264.7cells, and found that it caninhibit the migration of activated RAW264.7cells. Its effect was similar to what havebeen found in the red seabream (Pagrus major) that the TGFβ can inhibit theactivated head kidney cells and peripheral blood cells. Both enhancing andsuppressing roles of mTGFβ on the RAW264.7cells migration indicated that theamphioxus TGFβ shared the similar bipolar nature with mammalian and fish TGFβ.The production of TNF-α and IL-1β production were detected by qRT-PCR andELISA. We demonstrate that recombinant mTGFβ is able to inhibit the expression ofTNF-α and IL-1β mRNA and the production of IL-1β in LPS-activated RAW264.7cells, suggesting that amphioxus TGFβ also plays an immune suppressive cytokinerole.In order to find the structural and functional insights of amphioxus TGFβ, themotif-deleted fragment with the C-terminal residues WSTD (53-56) of the TGFβmature peptide deleted was obtained by overlap extension PCR. We obtained therecombinant motif-deleted TGFβ (MD) and found that the motif WSTD is a site ofTGF critical for inducing or inhibiting the migration of macrophages such asRAW264.7.Altogether, these data suggest that amphioxus TGF is functionally similar tovertebrate TGF, suggesting an ancient origin of bipolar function of TGF proteins.TGFβ plays an immune suppressive cytokine role. It also highlights the importance ofthe motif WSTD of TGF for the induction and inhibition of macrophage migration.