Research

Metabolic degradation of methylated glycans

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).

Start of the work: March 2020

Co-workers so far dealing with this project:

Julia Thoma
​​​​​​​doctoral thesis

Marilica Zemkollari
​​​​​​​doctoral thesis

Annalena Gamperl - master thesis 2022

If you are interested in a diploma work in this area, please contact me. 

Financed by:

FWF P33239-B (03/2020 - 02/2024)

Co-operation partner

Univ. Prof. Dipl. Ing. Dr. Reingard Grabherr
Department of Biotechnlogy, BOKU

Cloning and characterisation of glycosyltransferases from snails

Glycosylation is a posttranslational modification of proteins which is involved in a number of recognition, adhesion and transportation events. Snails are in some cases intermediate hosts of parasites and therefore the investigation of these processes are of medical relevance. 

Enzymes which are involved in the biosynthesis of these glycans are an interesting tool for the modification of artificial oligosacchardes. There are only few informations on snail enzymes. We want to change that now ! In the course of this project different fucosyltransferases and one xylosyltransferase shall be purified, cloned and characterised. 

The biochemical part of the work will be carried out at the Department of Chemistry under my supervision, the molecular biology part of the work will be supervised by Prof. Reingard Grabherr at the Department of Biotechnology.

Methods: biochemical methods (protein purification, enzym activity tests, characterisation) and molecular biology methods (cDNA-library, cell culture, cloning, expression ).

Co-workers dealing with this project:

Chantal Lucini
doctoral thesis 2014

Christopher Taus
doctoral thesis 2013

Teresa Riegler
diploma thesis 2010

Marita Preims
diploma thesis 2013

Financed by

FWF P22118-B20 (04/2010 - 03/2015)

Co-operation partner:

Univ. Prof. Dipl.-Ing. Dr. Reingard Grabherr
Department of Biotechnology, BOKU

Univ. Prof. Dr. Kiyoshi Furukawa
Nagaoka University of Technology, Nagaoka, Japan

O-Glycan structures in gastropods

O-Glycosylation is a posttranslational modification of proteins, which is involved in the transport and localization of proteins, their function and several recognition processes (e.g. Parasite - (intermediate) host). Only a few well investigated examples of non-mammals are available. 

Target of the project:
Analysis of the O-glycan spectrum of several gastropod species - and as far as possible - the localization and function of the specific structures within the body of the snail.

Methods: Established methods have to be optimised to gain the here necessary sensitivity.

Co-worker dealing with this project:

Herwig Stepan
​​​​​​​doctoral thesis 2012

Financed by:

FWF P20393-B11 (03/2008 - 12/2011)

Co-operation partner:

Univ. Prof. Dr. Rudolf Geyer
Justus Liebig Universität Giessen

N-Glycosylation of molluscs

Gastropods are not only food, the damage some vegetables and are sometimes intermediate hosts for parasites. Nevertheless not much is known about this group of organisms. 

We investigate the glycosylation potential of several species of snails (land- and water snails, with or without shell) by anaylzing their glycan profiles and by analyzing the enzymes (glycosyltransferases and glycosidases) which are involved in the biosythetic pathway of snail glycans.

Major questions are: if gastropod cells may be putative expression systems, if  already known carbohydrate-related allergens are present, and if there is a possibility for a specific manipulation of the glycosylation pathway in order to reduce damages in agriculture. 

Co-workers dealing with this project: 

Heidi Grabher-Meier
diploma thesis 1998

Sabine Bürgmayr
diploma thesis 1999

Karin Ahrer
diploma thesis 2001

Martin Gutternigg
diploma thesis 2003

Josef Voglmeir
diploma thesis 2006

Judith Rudolf
diploma thesis 2007

Sabine Bürgmayr
doctoral thesis 2003

Financed by:
FWF 13928 - BIO (4/2000 - 10/2003)

Co-operation partner:

Ass. Prof. Dr. Manfred Pintar
Institut für Zoologie, Department für Integrative Biologie und Biodiversität, BOKU

Purification of a xylosidase from potatoes

Some plants contain xylosidases which cleave specifically the beta-1,2-linkage of xylose in N-glycans. This enzyme was purified from potatoes by biochemical purification methods and furthermore characterized by its substrate specificity and biochemical parameters. 

This specific xylosidase can be used as a tool for N-glycan analysis, if there are some hints that a beta-1,2-linked xylose may be present.

Co-worker dealing with this project: 

Christian Peyer
diploma thesis 2000

Co-operation partners:

Prof. Dr. Arnold Stütz
​​​​​​​Technical University Graz

Prof. Dr. Pedro Bonay
Universidad Autonoma de Madrid

Comparison of alpha1,6-fucosyltransferases

Alpha-1,6-fucosylation linked to the asparagine-bound N-acetylglucosamine residue of N-glycans is a frequent structural determinant in mammalian cells.
Not much was known on the corresponding enzymes which generate this linkage in 1997. At that time only one was cloned (from porcine brain [E.C. 2.4.1.68]) and a few others characterized by their biochemical parameters and their substrate specificity. 

In a comparison study alpha-1,6-fucosyltransferases from different tissues (heart, lung, brain, liver) of different animals (cattle, pork, sheep, rabbit, chicken) were analyzed for their biochemical paramaters and compared with available data from literature. 

The enzymes were different in their patterns, which leads to the suggestions that there may be an enzyme family and not only one single enzyme responsible for the alpha-1,6-fucosylation of N-glycans. For alpha-1,3-fucosyltransferases such a enzyme family is already well characterized. 

The knowledge about the glycosylation potential of cells is very important if these cells are going to be used for the production of pharmaceutical relevant glycoproteins. 

Co-worker dealing with this project:

Sissi Struppe
diploma thesis 1997

Co-operation partner:

Prof. Dr. Wolfgang Wetscherek
Institut für Tierische Lebensmittel, Tierernährung, Ernährungsphysiologie; Department für Lebensmittelwissenschaften und -technologie; BOKU

Financed by:

FWF 12552-MOB (01/1998 - 12/2000)