Research Projects

Molecular Glycobiology

 

Introduction

Since the pioneering work of Felix Hoppe-Seyler (1825-1895) on mucus glycoproteins in the "edible bird's nest", a Chinese delicacy representing the nest-cementing substance of the swiftlet, our knowledge on the third class of macromolecules, besides nucleic acids and proteins, the carbohydrates, has tremendously increased and research on this topic developed into what is nowadays referred to as "glycobiology". Owing to over one million publications during the last century listed in PubMed under the term "carbohydrates" the basic structural and to some extent also the functional aspects of glycans were unravelled as components of glycoconjugates, such as glycoproteins, glycolipids, proteoglycans, lipopolysaccharides or peptidoglycans. Glycomics, the structural profiling of complex glycans, has been pushed largely by the rapid developments in modern mass spectrometry which was initiated in the 1980ies by invention of FAB ionization and later of MALDI and ESI mass spectrometry. Concomitantly with the increasing knowledge on the structural aspects of glycans we got insight during the last decades into the multiple roles that glycans do play, far beyond a mere structural stabilization of proteins or their protection against proteolysis, in particular cell biological roles, which became already evident with the discovery of carbohydrate binding proteins or "lectins". Epitope masking of surface proteins by modulation of glycans, an important strategy in innate immunity to protect organisms against infection by pathogenic microbes, may have been one of the driving forces in evolution giving rise to the development of complex glycosylation machineries in cells and a correspondingly extreme heterogeneity of glycan structures. Adding to this, complex carbohydrates on cell surfaces play important roles in developmental processes, a finding which is in accordance with observations that specific glycan structures, like polysialic acid, appear in specific spatial and temporal patterns throughout normal development, but also in cancer. Modulation of cell surface glycans is associated with progression of cancer cells to an aggressive, metastatic phenotype and with immunological escape mechanisms. In particular the role of sialic acids and their precursors needs to be addressed as potential signaling molecules with impact on cell proliferation and differentiation, but also the pathophysiologcal implications mediated by the bifunctional key enzyme of sialic acid biosynthesis, UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). Another track of evidence supports a variety of intracellular functions of complex protein glycosylation. To these belong N- and O-glycosylation-dependent sorting and trafficking routes of glycoproteins,as for examplethe lysosomal targeting of hydrolases or the apical targeting of plasma membranousglycoproteins. Besides the classical protein modifications by N-linked and mucin-type O-linked chains a couple of rare O-glycosylation types (O-GlcNAc, O-Man, O-Fuc, O-Glc) came into the research focus, since these modifications seem to be involved in important regulatory mechanisms on the cellular and tissue levels.

 

Cancer-related Research

Our research efforts focus on mucin-type O-glycosylation, initially concentrating on tumor-associated carbohydrate antigens, later in a more general biological context. As model protein we investigated the structural aspects of MUC1 glycosylation, the first human mucin described and one of the most promising tumor markers with impact in diagnosis and in immunotherapy. Beyond a mere profiling of glycans expressed on the mucin from the normal (lactating) breast or from breast cancer cells, we tried to determine the changes in density and the sites of O-glycosylation associated with cancer development. In later studies we looked for the influence of the sites glycosylated on MUC1 tandem repeat peptides on their immunological properties (processing in the class I and class II pathways) to prepare the basis for a vaccine development.

 

Glycosylation and Cell Biology

Related to projects dealing with the regulation of O-glycosylation are studies on the type 1 transmembrane glycoprotein MUC1, which recycles via sorting endosomes through the secretory pathway back to the plasma membrane to gain in this way a maturation of its O- and N-glycosylation. We are interested to learn whether during recycling of MUC1 O-linked glycans are re-modelled in a way similar to N-linked glycans. Moreover, we are interested in the targeting of MUC1 for exosomal export and try to identify the structural requirements with respect to N-linked chains on the small subunit necessary for sorting to multy-vesicular bodies.

 

Rare O-Glycosylation-Types and their Control by Peptidic Elements

Other topics are related to rare modifications of proteins by O-mannosylation and by peripheral glycan modification with lacdiNAc. Both glycosylation events are under control of cis-regulating peptidic elements on the substrate protein. The aim is to unravel the structural requirements of this cis-control and to identify other protein substrates to finally get insight into the function of these rare glycans.

 

Methodological Developments

Other projects focus on the development of new techniques with regard to glycomics in SDS-gel-based platforms and to glycoproteomic approaches aiming at the improvement of O-glycoprotein identification.

Terminated Projects

• Pathomechanism of chronic cell damage in classical galactosemia (terminated)
• Protein specific O-glycosylation: Do cis-localized peptides control peripheral LacdiNAc modifications of complex O-glycans? (terminated)
• The role of recyclisation in the O-glycosylation of the transmembrane glycoprotein MUC1 (terminated)
• Control mechanisms in the initiation of protein O-mannosylation (terminated)
• Lectin functions of Trefoil Family Factor (TFF) peptides (terminated)

Current Projects

• Differential proteomics of statin-induced effects in differentiated muscle cells with particular emphasis on O-mannosylation-dependent pathways
• Fucoidans and fucosylated milk oligosaccharides as anti-infectiva against norovirus infections
• A novel class of human milk oligosaccharides based on 6’-galactosyllactose cores and with relationship to prebiotic oligogalactoses (GOS)