Fucosyl- and Xylosyltransferases in Gastropods

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 a1,6-fucosyltransferase, the core a1,3-fucosyltransferase, the a1,3-fucosyltransferase which fucosylates terminal sugars, the a1,2-fucosyltransferase which fucosylates terminal sugars and the ß1,2-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.

Molluscs are a large and evolutionary very successful phylum of animals. They are important members of several ecosystems in terms of waste disposal and cleaning but are mainly noticed negatively as a pest in agriculture or as part of the life cycle of parasites. Due to their success in survival and their adaption abilities to changing environments they are an interesting target for research in order to learn more about biosynthetic pathways in general. O-Glycosylation is a frequent post-translational modification of proteins. It contributes to physical properties, such as conformational stability, protease resistance, charge or hydrophilicity. It serves also as a recognition determinant in host-pathogen relationships, protein targeting and cell-cell interactions. As we know that glycosylation is the main factor in several biological recognition processes the detailed investigation of the snail glycosyltransferases is a first step to understand difficult recognition events, such as parasite-intermediate host interaction. So far, no data on the O-glycosylation machinery of gastropods have been available. In the current study glycosyltransferases from snail species should be cloned and expressed and characterised regarding their enzymatic activity. In the course of the project the first glycosyltransferases from mollusc origin (Biomphalaria glabrata) was identified, cloned and expressed. This enzyme, an UDP-GalNAc:polypeptide GalNAc transferase (ppGalNAcT; EC 2.4.1.41), initiates the first step in mucin-type O-glycosylation. The entire gene codes for a protein consisting of 600 amino acids which displays all functional parts which are characteristic for enzymes of this enzyme family. Sequence comparison with ppGalNAcTs from humans, flies and worms revealed high similarities in terms of structural architecture. Enzyme specificity studies and the analysis of biochemical parameters confirmed the close relationship to the family of yet known ppGalNAcTs. Furthermore, the second enzyme acting in the construction of O-glycans, a beta-galactosyltransferase, and also an alpha1,6-fucosyltransferase were identified. The results of the project confirm that the basic steps of O-glycosylation apply also for molluscs.