Understanding how extra-cellular polysaccharides (EPS) are (1) synthesized inside the cell and (2) translocated across biological membranes is the primary objective of our research. EPS are biopolymers of enormous biotechnological and biomedical value and we hope that our research will help us engineer polysaccharides with defined properties, e.g. by altering the carbohydrate composition and/or the polymer length.
Cellulose, a linear polymer of glucose molecules, is the most abundant biopolymer on earth. It forms the major component of the plant cell wall where it confers stability to the cell allowing a growth based on pressure (turgor). Bacteria also form cellulose, mainly Gram-negative species when transitioning from growth in solution to a sessile form (formation of biofilms). The bacterial cellulose synthase complex is an inner and outer membrane spanning system that consists of at least 3 subunits (BcsA, BcsB, BcsC and possibly BcsZ). BcsA is an integral membrane protein with 8-10 predicted transmembrane helices and belongs to the family of processive glycosyltransferases. BcsB carries an N-terminal signal sequence for posttranslational translocation into the periplasm and is anchored to the inner membrane via a C-terminal transmembrane helix. The BcsC subunit likely forms a beta-barrel structure in the outer membrane, preceded by a large, periplasmic domain. A complex of BcsA, B and C might span the cell envelope, thereby forming a conduit for the synthesized polysaccharide to outside of the cell. We are interested in studying the individual components of the cellulose synthase complex both from a structural as well as a functional point of view. To this end, we express and purify the individual subunits as well as defined sub-complexes for structural analysis and are performing in vitro cellulose synthesis reactions with the purified components. In addition, we plan to use single particle electron microscopy to study the entire cellulose synthase complex.
Hyaluronan (HA) is a major component of the extracellular matrix in vertebrates and affects numerous physiological processes, such as cell adhesion and migration, and cell differentiation and proliferation. Because of its broad physiological significance, altered expression levels of HA correlate with various pathological conditions, examples include autoimmune diseases and cancer. HA is a linear polysaccharide consisting of alternating glucuronic acid and N-acetyl glucosamine residues and can reach several microns in length. In eukaryotes as well as in some Gram-positive bacteria, the polymer is synthesized by the membrane embedded hyaluronan synthase (HAS) and is transported to the outside of the cell either by the very same HAS or by an additional factor. We are addressing this ambiguity by reconstituting the HA polymerization and translocation reaction in vitro. In addition, we seek to crystallize the enzyme in different, biologically relevant, conformations either in a detergent solubilized state or in artificial lipid bilayers, such as cubic phases and bicelles.
