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One of the best-studied examples of cell to cell adhesion is the flocculation of baker's yeast. This interaction is defined as the nonsexual and calcium-dependent aggregation of cells, mediated by the binding of cell surface lectins (flocculins) to surface carbohydrates (mannans) of neighbouring cells [1-7]. The lectin–carbohydrate interaction is often characterized by rather low affinities, which are assumed to be compensated for by an avidity effect and/or a high local concentration of carbohydrates at the cell surface . The genetics of flocculation in the laboratory yeast Saccharomyces cerevisiae are well characterized and involve the expression of cell wall flocculins from at least one of four structural genes: FLO1, FLO5, FLO9, or FLO10 .
The flocculation phenotype of yeast is commonly described with respect to whether flocculation can be inhibited by mannose or by other sugars as well. This disruption of flocculation occurs as the sugar competitively displaces the cell wall mannans from the flocculin binding site. The phenotype governing flocculation is usually categorized as being Flo1-type or NewFlo-type and depends on whether the yeast flocculation can be inhibited by mannose, as in the first case, or by a broad range of sugars including mannose and glucose, as in the latter case . Lg-FLO1, a flocculin gene homologous to FLO1 first discovered in Saccharomyces pastorianus but, more recently, also reported in S. cerevisiae , has been shown to be responsible for the NewFlo phenotype .
Flocculation of yeast is very relevant in biotechnology applications, specifically as a separation tool to remove yeast cells from fermented products, such as beer, wine and ferments used in the distilling industry . NewFlo phenotype yeast is of particular importance in brewing because its sensitivity to wort sugars (glucose, maltose, maltotriose) ensures that flocculation is delayed until the fermentable sugars are depleted .
Flocculation is a complex process dependent on many factors, including the regulation of flocculation genes [14-17], cell surface factors (charge and hydrophobicity)  and the growth environment, such as pH, temperature, ethanol, oxygen and calcium availability [1, 2, 9, 19]. To fully understand this adhesion process, it is essential to understand all of the aspects involved, including the structural and functional properties of the flocculins. Until recently, little was known about the detailed biochemical properties of the flocculin–carbohydrate interactions because few studies had been conducted outside the complex cellular environment. Recent biochemical and structural studies involving the Flo1-type flocculins flocculin 1 from S. cerevisiae (Flo1p) and flocculin 5 from S. cerevisiae (Flo5p) have attempted to characterize the binding specificity of the flocculins towards terminal yeast mannans [20, 21] and to explain the structural basis for calcium dependency and mannose recognition .
The structure of yeast cell wall mannan consists of a long linear α-(1,6) linked mannose backbone to which α-(1,2) and α-(1,3) linked mannose side chains are attached. From structural studies of Flo5p, the flocculin recognizes the terminal α-(1,2) linked mannose side chains . However, in some yeasts, such as S. cerevisiae and the pathogenic yeast Candida albicans, the outer chains of the yeast mannans are further modified by mannosylphosphate, resulting in a net negative charge on the yeast cell surface . In S. cerevisae, these mannosylphosphate residues are added on to α-(1,2) side chains of cell wall mannans by a Golgi mannosyltransferase encoded by the MNN6 gene . The role of mannosylphosphorylation is not well understood. However, it has been proposed that it may play a role as a virulence determinant in C. albicans . In the present study, we examined the role of mannose phosphorylation on flocculation.
In the present study, we focused on the biochemical characterization of a NewFlo-type flocculin from the brewer's yeast Saccharomyces carlsbergensis (syn. S. pastorianus), which was previously cloned, expressed and purified. We present the first crystal structure of the lectin domain of flocculin 1 from brewer's yeast (Lg-Flo1p) resolved to 2.5 Å and compared it with the sugar-complexed structure of Flo5p and related epithelial adhesion 1 (Epa1p) domain from Candida glabrata. We also investigated the sugar-binding specificity of Lg-Flo1p towards a number of mono-, di- and polysaccharides derivatives, including phosphorylated sugars.