Masaomi Minaba et al. (2007) International Worm Meeting "Development of heterotrimeric G protein signalling manipulation by exogenous G-protein coupled receptors originated from evolutionarily distant animals using C. elegans model."

Masaomi Minaba, Yusuke Kato

[International Worm Meeting, 2007]

The GPCR-G protein signalling regulates various physiological functions in a wide variety of organisms including plants and animals. Therefore, such physiological functions can be affected by manipulation of the GPCR-G protein signal transduction. Our interest is in the use of GPCRs derived from evolutionarily distant organisms for the manipulation of G protein signalling. GPCRs recognise a wide variety of ligands. Although some ligands are conserved in many organisms (e.g. acetylcholine, dopamine, etc.), others are recognised only in few organisms (e.g. peculiar peptide hormones, biogenic amines, etc). The GPCRs recognising such unique ligands are often found in evolutionarily distant organisms. If such GPCRs can couple with the target G<font face=symbol>a</font>, we can manipulate the GPCR-G protein signalling of transgenic individuals by using specific ligands that do not activate any receptors in wild-type individuals. We are interested in developing such manipulation system using C. elegans model. To test the functional coupling of a GPCR from an evolutionarily distant organism and a C. elegans G protein, we selected the mammalian G<font face=symbol>a</font>_i/o coupled receptor, human muscarinic acetylcholine receptor M₂ subtype (M₂), and the nematode G<font face=symbol>a</font>_i/o orthologue, GOA-1, as a model. A fusion protein of M₂ and GOA-1 was expressed using a baculovirus expression system. The ligand binding properties of M₂ in the GOA-1 fusion protein agree with that of the G<font face=symbol>a</font>_i1 fusion protein, indicating that the M₂ is functional. The binding of GTP<font face=symbol>g</font>S was increased by stimulation with the agonist, carbamylcholine, in a dose-dependent manner. The increase of GTP<font face=symbol>g</font>S binding was completely inhibited by the antagonist, atropine, suggesting that carbamylcholine induced the substitution for GTP<font face=symbol>g</font>S in GOA-1. In addition, carbamylcholine caused the decrease of GDP affinity in GOA-1. These results indicate that M₂ functionally couples with GOA-1. The affinity of M₂ for agonists, but not for antagonists, decreases on interaction with G<font face=symbol>a</font>_i/o in the presence of guanine nucleotides. The affinity for carbamylcholine of the fusion protein decreased in the presence of GTP, whereas the affinity for atropine was not affected by GTP, suggesting that M₂ in the fusion protein interacts with GOA-1 as well as with G<font face=symbol>a</font>_i/o in a GTP-sensitive manner. In conclusion, the human M₂ can activate the nematode GOA-1. We are presently trying other mammalian GPCRs for coupling with various nematode G proteins.

Kohtaro Kusaka et al. (2005) International Worm Meeting "Screening of novel immunodeficient mutants using opportunistic infection model"

Kohtaro Kusaka, Yusuke Kato, Masaomi Minaba

[International Worm Meeting, 2005]

Human imunodeficiency is caused by AIDS, aging, and so on. In such immunodeficient individuals, opportunistic infections are often caused by pathogens which are incapable of causing infection in immunocompetent individuals. We are attempting to screen immunodeficinet mutants using C. elegans as a model for opportunistic infection. Escherichia coli BL21(DE3) expressing EGFP is almost nonpathogenic for C. elegans on NGM agar plates. Wild-type worms grow and proliferate without any obvious disorders under the existence of BL21(DE3). However, this bacterium was detected in the intestine of app. 2% of adult worms 4 days after hatch, and the ratio of bacteria-detected worms increased with aging [1]. Dying worms were often filled with bacteria. In rare cases, a small number of bacteria was detected also in younger worms. These observations are identical to the symptom of opportunistic infection, i.e., BL21(DE3) is nonpathogenic to immunocompetent worms but parasitic/pathogenic to immunodeficient worms caused by aging or accidental debilitation. Thus, disorders which are not seen in wild type animals are expected to arise if immunodeficient mutants are cultured with this bacterium. In some EMS-induced mutant, EGFP-expressing BL21(DE3) was accumulated in the uterus. Such phenotype was never observed in wild-type worms. Those mutants can grow up to be adult since no significant disorder affects development before vulva formation, but are sterile. Further characterization of those mutants is presently progressing. [1] Garigan et al., (2002) Genetics 1616, 1101.

Satoshi Ueno et al. (2004) East Asia Worm Meeting "Immune defense of worms: Another novel immune-inducible antimicrobial peptide, ASABF-6Cys-alpha, in Ascaris suum."

Yusuke Kato, Satoshi Ueno, Masaomi Minaba, Ajitha Pillai, Hong Zhang

[East Asia Worm Meeting, 2004]

ASABFs are antimicrobial peptides isolated from nematodes including Caenorhabditis elegans. ASABF-type antimicrobial peptides contain an alpha-helix and a pair of anti-parallel beta-sheets stabilized by 4 intramolecular disulfide bridges. Previously, we reported that mRNAs encoding ASABF-alpha, beta, gamma, and delta increased in the pig roundworm, Ascaris suum, by injection of heat-killed Escherichia coli OP50. On the other hand, we have also reported ASABF-6Cys-alpha containing only 3 disulfide bridges. We tested the immune-inducibility of ASABF-6Cys-alpha. The transcripts encoding ASABF-6Cys-alpha was dramatically induced in the body wall, intestine, and reproductive organs. Especially, over 100 times increment was observed in the body wall and intestine. We are presently trying to prepare a recombinant ASABF-6Cys-alpha and characterize its biological activities.