Functional characterization of growth hormone releasing hormone and its receptor in amphioxus with implication for origin of hypothalamic-pituitary axis.

Growth hormone-releasing hormone (GHRH) has been widely shown to stimulate growth hormone (GH) production via binding to GHRH receptor GHRHR in various species of vertebrates, but information regarding the functional roles of GHRH and GHRHR in the protochordate amphioxus remains rather scarce. We showed here that two mature peptides, BjGHRH-1 and BjGHRH-2, encoded by BjGHRH precursor, and a single BjGHRHR protein were identified in the amphioxus Branchiostoma. japonicum. Like the distribution profiles of vertebrate GHRHs and GHRHRs, both the genes Bjghrh and Bjghrhr were widely expressed in the different tissues of amphioxus, including in the cerebral vesicle, Hatschek's pit, neural tube, gill, hepatic caecum, notochord, testis and ovary. Moreover, both BjGHRH-1 and BjGHRH-2 interacted with BjGHRHR, and triggered the cAMP/PKA signal pathway in a dose-dependent manner. Importantly, BjGHRH-1 and BjGHRH-2 were both able to activate the expression of GH-like gene in the cells of Hatschek's pit. These indicate that a functional vertebrate-like GHRH-GHRHR axis had already emerged in amphioxus, which is a seminal innovation making physiological divergence including reproduction, growth, metabolism, stress and osmoregulation possible during the early evolution of vertebrates.

Amphioxus adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U and A-to-I deamination of DNA.

Adenosine-to-inosine tRNA-editing enzyme has been identified for more than two decades, but the study on its DNA editing activity is rather scarce. We show that amphioxus (Branchiostoma japonicum) ADAT2 (BjADAT2) contains the active site 'HxE-PCxxC' and the key residues for target-base-binding, and amphioxus ADAT3 (BjADAT3) harbors both the N-terminal positively charged region and the C-terminal pseudo-catalytic domain important for recognition of substrates. The sequencing of BjADAT2-transformed Escherichia coli genome suggests that BjADAT2 has the potential to target E. coli DNA and can deaminate at TCG and GAA sites in the E. coli genome. Biochemical analyses further demonstrate that BjADAT2, in complex with BjADAT3, can perform A-to-I editing of tRNA and convert C-to-U and A-to-I deamination of DNA. We also show that BjADAT2 preferentially deaminates adenosines and cytidines in the loop of DNA hairpin structures of substrates, and BjADAT3 also affects the type of DNA substrate targeted by BjADAT2. Finally, we find that C89, N113, C148 and Y156 play critical roles in the DNA editing activity of BjADAT2. Collectively, our study indicates that BjADAT2/3 is the sole naturally occurring deaminase with both tRNA and DNA editing capacity identified so far in Metazoa.

Identification of ribosomal protein L30 as an uncharacterized antimicrobial protein.

Several ribosomal proteins have been shown to adopt for an antimicrobial function as antimicrobial proteins (AMPs). However, information as such is rather limited and their mode of action remains ill-defined. Here we demonstrated that amphioxus RPL30, BjRPL30, was a previously uncharacterized AMP, which was not only capable of binding Gram-negative and Gram-positive bacteria via interaction with LPS, LTA and PGN but also capable of killing the bacteria. We also showed that the residues positioned at 2-46 formed the core region for the antimicrobial activity of BjRPL30. Notably, both the hydrophobic ratio and net charge as well as 3D structures of the residues corresponding to BjRPL302-27 and BjRPL3023-46 from both eukaryotic and prokaryotic RPL30 proteins were closely similar to those of BjRPL302-27 and BjRPL3023-46, suggesting the antibacterial activity of RPL30 was highly conserved. This was further corroborated by the fact that the synthesized counterparts human RPL5-30 and RPL26-49 also had antibacterial activity. We show that the recombinant protein BjRPL30 executes antimicrobial function in vitro by a kind of membranolytic action including interaction with bacterial membrane through LPS, LTA and PGN as well as induction of membrane depolarization. Finally, we found that neither BjRPL30 nor its truncated form BjRPL302-27 and BjRPL3023-46 had hemolytic activity towards human red blood cells, making them promising lead molecules for the design of novel AMPs against bacteria. Altogether, these indicated that RPL30 is a member of AMP which has ancient origin and is highly conserve throughout evolution.