Coates D et al. (1995) International C. elegans Meeting "CHARACTERISATION OF ZK643.3/4: A PUTATIVE CGRP-7TM NEUROPEPTIDE RECEPTOR"

Lynch AS, Briggs DA, Hope IA, Kolakowski LF, McGregor D, Coates D, Isaac RE

[International C. elegans Meeting, 1995]

Neuropeptides are essential for the coordination of growth, development, homeostasis, reproduction and behaviour in invertebrates. Although only a small number of peptides have been isolated from nematodes, it is clear from immunocytochemical studies that the nervous system of nematodes are endowed with a variety of peptidergic neurons. A putative receptor gene (ZK643.3/4) has been sequenced as part of the C.elegans genome sequencing project. It is predicted to have seven TM domains, and a G- protein binding site, has sequence identity within TM 3, TM6 and TM7 with members of the secretin/calcitonin/PTH sub-class of receptors and can be classed as a new member of this receptor family. Expression pattern studies using a fusion gene comprised of regulatory sequences of ZK643.3/4 and lac Z in C.elegans indicate that the receptor is localised to the muscle cells that control the opening and closing of the vulva and to muscle cells in the head region of adult worms. This pattern of gene expression is consistent with a role for ZK643.3/4 and a CGRP -like peptide in the control of head movement and of egg-laying. Full length cDNA was obtained by RT-PCR amplification. The 5' region of the transcript was identified using RT-PCR with an SL1 primer and an exon 1 primer, and confirmed using RNAse protection. Sequencing of the full length cDNA has highlighted the need to confirm gene structures predicted in genome sequencing projects by analysis of cDNAs. Comparison with the sequences of other members of the same sub-class of receptors has identified residues which are conserved, both within the transmembrane domains, and in several of the cytoplasmic loops. There is particularly high homology between the insect DH receptor and ZK643.3/4 in the third cytoplasmic loop.

Coates D et al. (2000) European Worm Meeting "The ADAMs family of C. elegans: what will the new members do?"

Coates D, Daulby J, Hooper NM, Hope IA

[European Worm Meeting, 2000]

ADAMs are a family of integral membrane glycoproteins containing a disintegrin and a metalloprotease domain. The physiological roles of the majority of ADAMs have yet to be elucidated, however some mammalian ADAMs are known to be involved in many diverse processes including sperm migration, sperm-egg binding and fusion, myoblast fusion, the processing of adhesion molecules, cytokines, cytokine receptors and extracellular protein domains, and in neural development. In C. elegans ADAMs are thought to be involved in cell fusion events in sperm and in epithelial cells (ADM-1), in early embryonic development (ADM-2) and in vulval development (SUP-17). In addition to these membrane anchored proteins C. elegans also has soluble ADAM-like proteases, for example GON-1, which plays an essential role in gonadal morphogenesis. We have identified four novel ADAM-like sequences in C. eleganswhich encode a TNFa converting enzyme (TACE) homologue and three soluble ADAM-like proteinases, and our aim is to determine the precise functions of these proteins. Initially we will examine their cellular location with reporter gene constructs and assess the importance of these proteinases by generating gene knockout mutants. We are also interested in finding out what the substrates of these ADAMs are and will biochemically characterise the recombinant proteins. The data we will attain will be related to the ADAM homologues found in humans.

Coates D et al. (2000) Biochem Soc Trans "Exploring the Caenorhabditis elegans and Drosophila melanogaster genomes to understand neuropeptide and peptidase function."

Isaac RE, Coates D, Siviter R

[Biochem Soc Trans, 2000]

Comparison of peptidase gene families in the newly released Drosophila melanogaster and Caenorhabditis elegans genomes highlights important differences in peptidase distributions with relevance to the evolution of both form and function in these two organisms and can help to identify the most appropriate model when using comparative studies relevant to the human condition.

Daulby J et al. (1998) European Worm Meeting "Characterisation of the sigma class glutathione S-transferases of C. elegans"

Isaac E, Daulby J, Coates D

[European Worm Meeting, 1998]

The Glutathione S-Transferases (GST) are a superfamily of enzymes that catalyse the nucleophilic addition of the tripeptide glutathione (GSH) to endogenous and xenobiotic electrophilic substrates. GSTs are principally detoxification enzymes, but have been implicated in the development of resistance to a range of pesticides, herbicides and a number of drugs. These enzymes also serve as non-catalytic carrier proteins (ligandins). Recently, a sigma class related GST isolated from Ascaridia galli, displayed a high level of specific activity in the GSH dependent isomerisation of prostaglandin H to prostaglandin E (1). This suggests that certain sigma GSTs may be involved in eicosanoid metabolism. We are investigating the possible functions of members of the sigma class GST of C.elegans. To date, seven sigma class GST-like genes have been identified in C.elegans. We have amplified the corresponding cDNA for each gene by PCR from a cDNA library, and successfully expressed six of the seven cDNA sequences in Escherichia Coli. The recombinant protein product of the R03D7.6 gene has GSH binding properties, allowing purification by affinity chromatography. This protein also shows high levels of activity towards the universal GST substrate 1-chloro-2,4-dinitrobenzene (CDNB). We intend to utilise each recombinant protein firstly to raise monoclonal antibodies to localise the native enzymes in vivo. Secondly, we will conduct a series of assays to analyse the substrate specificities of each recombinant protein. In addition we would like to inject combinations of dsRNAs prepared from the GST cDNAs, to study the effects of interfering with the expression of this GST family. 1 Meyer D.J., Muimo R., Thomas M., Coates D. and Isaac R.E. (1996) Biochem. J. 313, 223-227

Sakoguchi H et al. (2016) Biosci Biotechnol Biochem "Screening of biologically active monosaccharides: growth inhibitory effects of d-allose, d-talose, and l-idose against the nematode Caenorhabditis elegans."

Izumori K, Yoshihara A, Sakoguchi H, Sato M

[Biosci Biotechnol Biochem, 2016]

We compared the growth inhibitory effects of all aldohexose stereoisomers against the model animal Caenorhabditis elegans. Among the tested compounds, the rare sugars d-allose (d-All), d-talose (d-Tal), and l-idose (l-Ido) showed considerable growth inhibition under both monoxenic and axenic culture conditions. 6-Deoxy-d-All had no effect on growth, which suggests that C6-phosphorylation by hexokinase is essential for inhibition by d-All.

Sakoguchi H et al. (2016) Bioorg Med Chem Lett "Growth inhibitory effect of d-arabinose against the nematode Caenorhabditis elegans: Discovery of a novel bioactive monosaccharide."

Shintani T, Izumori K, Sato M, Sakoguchi H, Okuma K, Yoshihara A

[Bioorg Med Chem Lett, 2016]

Biological activities of unusual monosaccharides (rare sugars) have largely remained unstudied until recently. We compared the growth inhibitory effects of aldohexose stereoisomers against the animal model Caenorhabditis elegans cultured in monoxenic conditions with Escherichia coli as food. Among these stereoisomers, the rare sugar d-arabinose (d-Ara) showed particularly strong growth inhibition. The IC50 value for d-Ara was estimated to be 7.5mM, which surpassed that of the potent glycolytic inhibitor 2-deoxy-d-glucose (19.5mM) used as a positive control. The inhibitory effect of d-Ara was also observed in animals cultured in axenic conditions using a chemically defined medium; this excluded the possible influence of E. coli. To our knowledge, this is the first report of biological activity of d-Ara. The d-Ara-induced inhibition was recovered by adding either d-ribose or d-fructose, but not d-glucose. These findings suggest that the inhibition could be induced by multiple mechanisms, for example, disturbance of d-ribose and d-fructose metabolism.

Yoshihara A et al. (2019) Bioorg Med Chem Lett "Growth inhibition by 1-deoxy-d-allulose, a novel bioactive deoxy sugar, screened using Caenorhabditis elegans assay."

Sakoguchi H, Fleet GWJ, Izumori K, Shintani T, Sato M, Yoshihara A

[Bioorg Med Chem Lett, 2019]

The biological activities of deoxy sugars (deoxy monosaccharides) have remained largely unstudied until recently. We compared the growth inhibition by all 1-deoxyketohexoses using the animal model Caenorhabditis elegans. Among the eight stereoisomers, 1-deoxy-d-allulose (1d-d-Alu) showed particularly strong growth inhibition. The 50% inhibition of growth (GI₅₀) concentration by 1d-d-Alu was estimated to be 5.4mM, which is approximately 10 times lower than that of d-allulose (52.7mM), and even lower than that of the potent glycolytic inhibitor, 2-deoxy-d-glucose (19.5mM), implying that 1d-d-Alu has a strong growth inhibition. In contrast, 5-deoxy- and 6-deoxy-d-allulose showed no growth inhibition of C. elegans. The inhibition by 1d-d-Alu was alleviated by the addition of d-ribose or d-fructose. Our findings suggest that 1d-d-Alu-mediated growth inhibition could be induced by the imbalance in d-ribose metabolism. To our knowledge, this is the first report of biological activity of 1d-d-Alu which may be considered as an antimetabolite drug candidate.

Katane M et al. (2020) Biochim Biophys Acta Proteins Proteom "Biochemical characterization of d-aspartate oxidase from Caenorhabditis elegans: Its potential use in the determination of free D-glutamate in biological samples."

Katane M, Sekine M, Nakayama K, Homma H, Kuwabara H, Saitoh Y, Miyamoto T

[Biochim Biophys Acta Proteins Proteom, 2020]

d-Aspartate oxidase (DDO) is a flavin adenine dinucleotide (FAD)-containing flavoprotein that stereospecifically acts on acidic D-amino acids (i.e., free d-aspartate and D-glutamate). Mammalian DDO, which exhibits higher activity toward d-aspartate than D-glutamate, is presumed to regulate levels of d-aspartate in the body and is not thought to degrade D-glutamate in vivo. By contrast, three DDO isoforms are present in the nematode Caenorhabditis elegans, DDO-1, DDO-2, and DDO-3, all of which exhibit substantial activity toward D-glutamate as well as d-aspartate. In this study, we optimized the Escherichia coli culture conditions for production of recombinant C. elegans DDO-1, purified the protein, and showed that it is a flavoprotein with a noncovalently but tightly attached FAD. Furthermore, C. elegans DDO-1, but not mammalian (rat) DDO, efficiently and selectively degraded D-glutamate in addition to d-aspartate, even in the presence of various other amino acids. Thus, C. elegans DDO-1 might be a useful tool for determining these acidic D-amino acids in biological samples.

James CL et al. (1998) European Worm Meeting "Characterisation of a Neurotransmitter Transporter Gene (T25B6.7) in C.elegans"

James CL, Coates D, Isaac E

[European Worm Meeting, 1998]

Termination of neurotransmitter-mediated signalling at the synapse is mainly carried out by members of the sodium- and chloride- coupled neurotransmitter transporter protein (NTP) family, which take neurotransmitter into presynaptic and glial cells by linking it to a sodium concentration gradient. They have a putative structure of 12 transmembrane domains linked by hydrophilic loops. To date over 40 members of the NTP family have been cloned and charaterised mainly from verterbrates. T25B6.7 was predicted to encode a member of the NTP family by the genome sequencing consortium. The promoter driven LacZ expression pattern of T25B6.7 is observed in embryonic, larval and adult stages. In the adult hermaphrodite staining is seen in neuronal processes in the phryngeal region, in lateral neuronal processes, in the vulval D cells, in the anal sphincter, and nuclei posterior to the anus. The adult male has similar pattern, staining is also visible in the copulatory apparatus. In the L1 stage, expression occurs in nuclei along the lateral aspect of the worm and in the ~400 min. embryonic stage there are nuclei staining in the dorso-lateral aspect in the posterior of the embryo. The larval and embryonic patterns of expression suggest the V and T lineages some of which go on to form neurones, socket cells and sheath cells. Deletions were carried out in the T25B6.7::LacZ construct to investigate promoter/enhancer elements. A deletion leaving 796bp of 5! flanking sequence reduced the level of expression of the T25B6::LacZ construct. Sequence upstream of this site is homologuous to 5! flanking sequence of other genes in the cosmids F21G4 and R03G8. A full length cDNA for this gene has been isolated and sequence analysis reveals that this is an atypical member of the family. Secondary structure prediction programs suggest an 11 transmembrane domain protein flanked by long carboxy and amino termini. Double stranded RNA was transcribed from the cDNA and injected into C.elegans, this produced late embryonic arrest. Vesicles were visible in the arrested embryo. The expression pattern and RNA experiments suggest that T25B6.7 is important in developmental stages of C.elegans and may be involved in blast cell fate inductions. It is difficult to speculate on the role of the gene in adult stages as its DNA sequence and expression pattern offers no obvious clues.

Shintani T et al. (2019) J Appl Glycosci (1999) "D-Allose, a Stereoisomer of D-Glucose, Extends the Lifespan of Caenorhabditis elegans via Sirtuin and Insulin Signaling."

Izumori K, Sakoguchi H, Yoshihara A, Shintani T, Sato M

[J Appl Glycosci (1999), 2019]

D-Allose (D-All), C-3 epimer of D-glucose, is a rare sugar known to suppress reactive oxygen species generation and prevent hypertension. We previously reported that D-allulose, a structural isomer of D-All, prolongs the lifespan of the nematode Caenorhabditis elegans. Thus, D-All was predicted to affect longevity. In this study, we provide the first empirical evidence that D-All extends the lifespan of C. elegans. Lifespan assays revealed that a lifespan extension was induced by 28 mM D-All. In particular, a lifespan extension of 23.8 % was achieved (p< 0.0001). We further revealed that the effects of D-All on lifespan were dependent on the insulin gene daf-16 and the longevity gene sir-2.1, indicating a distinct mechanism from those of other hexoses, such as D-allulose, with previously reported antiaging effects.