Metabolic degradation of methylated glycans

Wider research context: Methylation of biomolecules plays an important role in many cellular regulation processes. For proteins and DNA, several methylating and de-methylating pathways are known. However, methyl groups are also found on glycan structures and for these, the modification mechanisms have not been investigated so far. Glycan structures play an important role in several recognition processes. Modifications on the sugar residues alter the specificity of attachment and binding events. Hypothesis/Objectives: In the current project we want to investigate the degradation of methylated glycan structures. Those organisms which are able to synthesise methylated glycans are expected to have also a mechanism to degrade these structures. Snail tissues have been shown to contain methyl groups on their protein linked glycans. Therefore they are candidates for enzymes which are able to degrade those structures metabolically. Approach: Demethylating enzyme activity of snail organelles will be determined using native and synthetic substrates. Proteins with the desired enzyme activity will be purified, sequenced, cloned and expressed. The native as well as the recombinant protein will be characterised for their biochemical and biophysical properties. Substrate specificities will be tested with native substrates as well as with other methylated molecules in order to get an overview on further potential applications. Sequence homology search will be performed in databanks of other organisms which are known to contain methylated sugar chains (other molluscs and parasites).

Glycosylation plays an important role in altering the properties of proteins and lipids, modifying their interactions by changing the biophysical characteristics of a target molecule significantly. Attached glycans contribute not only to physical properties, such as conformational stability, protease resistance, charge or hydrophilicity, but also modulate several types of recognition processes ranging from reproductive biology, self/non-self-recognition, cell-cell communication, cell trafficking, development, differentiation, host-pathogen or host-symbiont interaction, immune activation, cell death to even evolution. The biosynthesis of these glycans is a complex posttranslational event where a number of specific glycosyltransferases are involved. These modifications depend on the type of organism and its developmental and health state. Mollusca is a large and evolutionary very successful phylum which populates freshwater, marine and terrestrial habitats worldwide. Some species are recognized as important members of several ecosystems in terms of waste disposal and cleaning, others are used due to their nutritional value and shells. Especially snails and slugs, are ill-reputed and known as pests in agriculture or hosts of parasite life cycles. In general, their success in survival, their adaptability to changing environmental conditions and their immense potential in medical and pharmaceutical application, makes molluscs actually an interesting target for research. But, despite their significance, there is a general lack of knowledge regarding mollusc biochemistry or molecular biology. The glycan spectra of molluscs show a number of special features that do not occur in this form in vertebrates. The aim of the project was to identify enzymes that cause these modifications. In this project we were able to identify, clone, express and characterise several glycosidases and glycosyltransferases involved in the assembly and modification of mollusc N- and O-glycans: -galactosidases from Arion lusitanicus, Arion vulgaris and Crassostrea gigas, an UDP-N-acetylglucosamine:-1,3-D-mannoside -1,2-N-acetylglucosaminyltransferase I from Crassostrea gigas, UDP-Gal:glycoprotein-N-acetylgalactosamine -1,3-galactosyltransferases (T-synthases) from Biomphalaria glabrata, Pomacea canaliculata and Crassostrea gigas, and an -1,2-fucosyltransferase from Crassostrea gigas. Apart from the fucosyltransferase, all these glycoenzymes were described in molluscs for the very first time in this project. All of the enzymes where identified by homology search or purification from natural sources, cloned, expressed in an insect cell expression system, partly purified and characterised for their biochemical parameters, substrate specificities and three-dimensional structural features. Several of the methods had to be adapted to the needs of the molluscan enzymes. Even when the enzymes show some protein sequence homology with already known enzymes from other species, some of them display completely different features regarding their structural organisation, biochemical properties and substrate specificities. This confirms that molluscs have an enormous potential for the creation of newly designed glycans which could improve medical applications.