Abstract
Gastropods are a promising source for biotechnolgical useful resources, but also sometimes a host for pathogens or a pest in agriculture. Therefore it is extremely important to understand in detail their glycosylation abilities and analyse and characterize glycan structures as well as the enzymes involved in glycosylation processes.
Based on our previous projects dealing with the analysis of N-glycans, we know that gastropods combine in their glycans structural elements which are typical for mammals, plants, insects or nematodes. Especially the modification by fucoses and xylose show a high variability. In other organisms these sugar residues have been shown to play a major role in carbohydrate determined recognition processes. Their presence or absence is in some cases essential for function and binding. These glycan modifications depend on one hand on the availability of activated substrates and, on the other hand, on the presence of the corresponding fucosyltransferases and the xylosyltransferase. So far, from snail tissues some of these enzymes have been identified, but none of them has been cloned or compared in detail with those from other organisms.
The aim of this project is the purification and characterisation of the following enzymes which are responsible for the modification of N-glycans in snails: the core α1,6-fucosyltransferase, the core α1,3-fucosyltransferase, the α1,3-fucosyltransferase which fucosylates terminal sugars, the α1,2-fucosyltransferase which fucosylates terminal sugars and the β1,2-xylosyltransferase. The enzymes will be identified by specific enzyme assays, followed by the establishment of a cDNA library in an expression vector, which is then transfected into host cells. The transfected cells containing the functional cDNAs of the desired enzymes will be detected by antibodies and lectins and then isolated by the modified glycans expressed on their surface. These gastropod enzymes will be compared with the already known ones in terms of their substrate specificity and other biochemical parameters. To understand in detail the action of those enzymes which are important for binding and signalling will help to gain a deeper knowledge on invertebrate glycosylation. It will be the first time to obtain recombinant fucosyl- or xylosyltransferases from a gastropod source.
