[
European Worm Meeting,
2006]
Martin Gutternigg, Dorothea Lubich, Matthias Hackl, Katharina Paschinger, Ute Stemmer, Verena Jantsch1, Gnter Lochnit2, Ramona Ranftl, Petra Geier and Iain B. H. Wilson. Recent data indicates that in addition to the Golgi ?-mannosidases, the model nematode Caenorhabditis elegans also possesses, like insects, an N-acetylhexosaminidase activity putatively involved in N-glycan processing in the Golgi. The presence of such an activity is invoked, not just on the basis of the detected enzyme activity, but also to explain the absence of terminal N-acetylglucosamine residues on structures which require the prior action of N-acetylglucosaminyltransferase I during their biosynthesis. In order to understand the genetic basis for these activities, we have cloned cDNAs encoding members of both glycohydrolase families 20 and 38 from the worm. The encoded glycosidases were expressed in the yeast Pichia pastoris as soluble forms lacking putative cytoplasmic and transmembrane domains. Four glycohydrolase family 20 members were shown to cleave p-nitrophenyl-?-N-acetylglucosaminide and/or p-nitrophenyl-?-N-acetylgalactosaminide, but showed contrasting specificities with regard to N-glycan substrates. On the other hand, one glycohydrolase family 38 member was shown to be active using p-nitrophenyl-?-mannoside as a substrate and, in addition, had mannosidase II activity. Analysis of the glycans of the relevant mutant showed large-scale changes in the N-glycosylation spectrum. These, therefore, are the first data on the activity of Caenorhabditis glycosidases towards N-glycan substrates and should aid the further elucidation of N-glycan processing in this organism.
[
European Worm Meeting,
1998]
C. elegans has been a useful model organism for developmental studies because of its ease of culture, simple anatomy, and available genetics. These attractive features are now be augmented by a wealth of molecular data from a multitude of nematode research labs as well as data provided by the completion of the full genome sequencing project. Already, the genomic information is allowing accurate detection of C.elegans orthologues to various genes relevant in human diseases or other conserved pathways (Mushegian et al., 1997). The time is right to take a bioinformatics driven approach to functional studies. The combination of the complete genomic sequence, bioinformatics, and an experimentally facile organism makes C. elegans an excellent system in which to dissect complex signaling pathways. A large body of signal-transduction pathways involving seven-transmembrane G-protein coupled receptors mediate responses to different types of chemicals like odorants, neurotransmitters, hormones, and also to more !physical! stimuli, like osmotic pressure, temperature, pressure, etc. Many members of these types of pathways have been studied in some detail in C. elegans while other potential candidates have so far only been identified !in silico! (Sonnhammer & Durbin, 1997). Clearly, the nematode has been, and will continue to be, helpful in identifying potential roles for these factors in regulating behaviour and responses to environmental cues, or in crucial developmental processes. The emergence of RNA-mediated interference (RNAi) (Fire et al., 1998) provides a powerful technique that will facilitate the identification of phenotypes associated with the elimination of single or combinations of multiple signalling factors. To improve biochemical analysis in the worm, we aim to apply the new generation of protein 2D-gels analysis systems. We expect this to become a powerful tool (e.g. Bini et al., 1997), for example revealing mutation effects and more accurate proteomics. This is especially important for genes encoding transcription factors, where changes of the expression pattern might be detected most easily at the protein level. For such studies the main requirement is non-lethality, health and fertility of the mutation, so that enough material can be obtained for the analysis. REFERENCES Bini, L., Heid, H., Liberatori, S., Geier, G., Pallini, V. & Zwilling, R. (1997) Electrophoresis18, 557-562 Fire, A., Xu, SQ., Montgomery, M.K., Kostas, S., Driver, S.E. & Mello, C. (1998) Nature 391, 806-811 Mushegian, A.R., Bassett, D.E.Jr, Boguski, M.S., Bork, P. & Koonin, E.V. (1997) PNAS 94, 5831-5836 Sonnhammer, E. & Durbin, R. (1997) Genomics 46, 200-216