Endothelial monocyte activating polypeptide II (EMA-PII) is a cytokine that is specifically induced by apoptosis.Its precursor (pro-EMAPII) has been suggested to be identical to p43, which is associated with the multi-tRNA synthetase complex. Herein, we have demonstrated that the N-terminal domain of pro-EMAPII interacts with the Nterminal extension of human cytoplasmic arginyl-tRNA synthetase (RRS) using genetic and immunoprecipitation analyses. Aminoacylation activity of RRS was enhanced about 2.5-fold by the interaction with pro-EMAPII but not with its N-or C-terminal domains alone. The N-terminal extension of RRS was not required for enzyme activity but did mediate activity stimulation by pro-EMAPII. Pro-EMAPII reduced the apparent K m of RRS to tRNA, whereas the k cat value remained unchanged. Therefore, the precursor of EMAPII is a multi-functional protein that assists aminoacylation in normal cells and releases the functional cytokine upon apoptosis.Aminoacyl-tRNA synthetases (ARSs) 1 catalyze ligation of their cognate amino acids to specific tRNAs. Although basic architecture of the core domain is well conserved among ARSs, unique peptide extensions have been found in the N-or Cterminal ends of metazoan enzymes (1-3). Although these extensions have been thought to be involved in heterologous molecular interactions, their functional significance is not yet understood.A macromolecular protein complex consisting of at least nine different ARSs has been found in higher eukaryotes (1-3). This multi-ARS complex also contains three nonsynthetase components, p18, p38, and p43 whose functions are not clear (4 -7). Among these nonsynthetase components, p43 has been proposed to be a precursor of a tumor-specific cytokine, endothelial monocyte-activating polypeptide II (EMAPII) based on over 80% sequence identity between the two proteins (6). EMAPII was originally identified in the culture medium of murine fibrosarcoma cells induced by methylcholanthrene A (8). It triggers an acute inflammatory response (9, 10) and is involved in development-related apoptosis (11).The precursor for EMAPII (pro-EMAPII) is processed at the Asp residue of ASTD/S sequence to release the C-terminal cytokine domain of 23 kDa (11). Its C-terminal domain shares homology with the C-terminal parts of methionyl-tRNA synthetases of prokaryotes, archaea and nematode, and also a yeast protein, Arc1p/G4p, which interacts with methionyl-and glutamyl-tRNA synthetases. The N-terminal domain of pro-EMAPII does not show homology to any known proteins, and its function has not been understood.EMAPII is expressed in a wide range of cell lines and normal tissues (12) and its mRNA level is unchanged during apoptosis (11) although its production is induced by apoptosis. The present work was designed to address whether pro-EMAPII is identical to p43 and to understand its function in the normal cell. The results showed that pro-EMAPII is associated with the N-terminal extension of human arginyl-tRNA synthetase (RRS), facilitating aminoacylation reaction. EXPE...

Aminoacyl-tRNA synthetases (tRNA synthetases) of higher eukaryotes form a multiprotein complex. Sequence elements that are responsible for the protein assembly were searched by using a yeast two-hybrid system.Human cytoplasmic isoleucyl-tRNA synthetase is a component of the multi-tRNA synthetase complex and it contains a unique C-terminal appendix. This part of the protein was used as bait to identify an interacting protein from a HeLa cDNA library. The selected sequence represented the internal 317 amino acids of human bifunctional (glutamyl-and prolyl-) tRNA synthetase, which is also known to be a component of the complex. Both the C-terminal appendix of the isoleucyl-tRNA synthetase and the internal region of bifunctional tRNA synthetase comprise repeating sequence units, two repeats of about 90 amino acids, and three repeats of 57 amino acids, respectively. Each repeated motif of the two proteins was responsible for the interaction, but the stronger interaction was shown by the native structures containing multiple motifs.Interestingly, the N-terminal extension of human glycyl-tRNA synthetase containing a single motif homologous to those in the bifunctional tRNA synthetase also interacted with the C-terminal motif of the isoleucyl-tRNA synthetase although the enzyme is not a component of the complex. The data indicate that the multiplicity of the binding motif in the tRNA synthetases is necessary for enhancing the interaction strength and may be one of the determining factors for the tRNA synthetases to be involved in the formation of the multi-tRNA synthetase complex.Aminoacyl-tRNA synthetases play an essential role in cellular protein synthesis by catalyzing attachment of their cognate amino acids to tRNAs. Many eukaryotic aminoacyl-tRNA synthetases are distinguished from their prokaryotic counterpart in their abilities to form supracomplexes through self assembly or association with protein synthesis machinery (1-5) and cellular structures (6, 7). Most intriguing among them is a multi-tRNA synthetase complex generated by the assembly of many aminoacyl-tRNA synthetases and a few other protein factors of unknown function (8-10). The exact structure of the complex is still controversial because different forms of the complex have been isolated depending on the purification methods and organisms. The eukaryotic tRNA synthetases have been grouped depending on their abilities to form the multi-tRNA synthetase complex (8). The class I enzymes includes isoleucyl-, leucyl-, methionyl-, aspartyl-, bifunctional (glutamyl-and prolyl-), glutaminyl-, lysyl-, and arginyl-tRNA synthetases that have been consistently identified as the components of the multi-tRNA synthetase complex (11, 12). It is not clear whether other tRNA synthetases are loosely associated with or completely independent of such a complex.Understanding the structure of the multi-tRNA synthetase complex is important to find the functional linkage between the catalytic activities of this complex and other cellular processes. Many different approa...

No abstract

No abstract

The foot and mouth disease virus (FMDV) is an RNA virus composed of single stranded positive sense RNA. FMDV has been known to infect cloven-hoofed animals, including pigs, cattle, and sheep. FMDV is rapidly spreading outward to neighboring regions, often leading to a high mortality rate. Thus, early diagnosis of FMDV is critical to suppress propagation of FMDV and minimize economic losses. In this study, we report the generation and characterization of polyclonal and six monoclonal antibodies against VP1 through immunoblotting and immunofluorescence microscopy analyses. These VP1 antibodies will be useful as tools to detect serotypes A and O of FMDVs for diagnostic usage.