The self-binding of Saccharomyces cerevisiae flocculation glycoproteins: protein-glycan and glycan-glycan interactions

S. cerevisiae cells possess a remarkable capacity to adhere to other cells, which is called flocculation. Flocculation is defined as the phenomenon wherein yeast cells adhere in clumps and sediment rapidly from the medium in which they are suspended. These cell-cell-interactions are mediated by a class of special cell wall proteins, called flocculins that stick out of the cell walls of flocculent cells and selectively bind mannose residues present in the cell walls of adjacent yeast cells. Flocculins consist of three domains. The first one is the binding domain, which acts as a Ca2+-dependent lectin and is both O- and N-glycosylated. The middle domain consists of a variable number of repeats and is heavily O-glycosylated. The protein is anchored to the cell wall by a GPI-anchor in the C-terminal domain.
In this work, we have studied the N-terminal domain of the Flo1-protein (N-Flo1p), which is the most important flocculin responsible for flocculation of yeast cells. Firstly, we determined if glycosylated Flo1 proteins are able to bind themselves, given the affinity between the flocculin binding site and mannose-containing glycans. Secondly, we assessed whether and to which extent carbohydrate-carbohydrate interaction contribute to Flo1 self-binding. N-Flo1p was expressed and purified from S. cerevisiae as a glycosylated protein. Both the protein and the enzymatically released glycans were analysed using Atomic Force Microscopy (AFM). Morphologies of protein and glycan samples after addition of calcium ions or EDTA were observed using "tapping mode" in air. AFM images show calcium-mediated aggregative phenomena and revealed a new aspect of flocculin adhesive behaviour.
The protein-protein-interaction was confirmed with Surface Plasmon Resonance (SPR). It was shown that N-Flo1p binds to N-Flo1p, and a lower binding capacity was observed when the protein was N-deglycosylated. Therefore, the glycans play an important role in this binding event.
All together, these results refine and extend the flocculation model by suggesting that the mechanism of flocculation is based on two types of interactions: Flo1p can bind to mannose-containing glycans present on other Flo1p proteins and the high mannose glycans present on the flocculins can bind to each other.