flocculation is a result of the interaction of lectin-like proteins on the surfaces of yeast cells with carbohydrates from the cell walls of other yeast cells (28
). Flocculins are the cell wall proteins from S. cerevisiae
responsible for that interaction (4
). The FLO1
gene encodes a protein that leads to the Flo1-type flocculation phenotype, which is characterized by a strong inhibition capacity by mannose. Flo1p is believed to be highly mannose specific (12
). However, no direct proof for binding of the Flo1 protein itself to mannose has been reported, since previous results were obtained from experiments with whole cells, not with the purified protein.
In this study, the sugar binding domain of Flo1p was cloned and purified in S. cerevisiae
, and it was shown that N-Flo1p does indeed bind to carbohydrates. Using fluorescence spectroscopy, the binding of N-Flo1p to d
-mannose was demonstrated. Other carbohydrates, such as fucose, galactose, glucose, maltose, rhamnose, and sucrose, were also tested, but no binding was observed. Recently, inhibition experiments were performed using an S. cerevisiae
strain overexpressing Flo1p. The flocculation governed by the overexpressed Flo1p protein could be inhibited only by adding d
-mannose to the medium, and it was not inhibited by the other tested sugars, such as maltose, sucrose, glucose, and galactose (44
). Hence, our carbohydrate interaction analysis is in line with these results.
The binding of N-Flo1p was also determined by demonstrating its binding to mannose carbohydrates (mannan) present on the cell walls of intact yeast cells. Therefore, yeast cells were added to the purified protein solution. The proteins could be pulled down by the yeast cells, as the protein concentration was considerably (more than 40%) reduced, whereas mannose could inhibit binding to the cells, and no decrease in protein concentration could be observed (data not shown). Additionally, we attempted to demonstrate the binding of N-Flo1p to d-mannose by using other techniques, such as binding of the protein to d-mannose-coated agarose beads, the detection of N-Flo1p bound on yeast cells or on bovine serum albumin-mannose via anti-His antibodies (enzyme-linked immunosorbent assay [ELISA]), the binding of mannose-linked bovine serum albumin to N-Flo1p via surface plasmon resonance, and the binding of N-Flo1p to the 422 different mammalian glycans immobilized on a glycan array (Consortium for Functional Glycomics), where binding is also detected by anti-His antibodies. No single binding event was detected in any of these experiments (data not shown). Considering the fact that N-Flo1p is heavily glycosylated, we can explain the above results by steric hindrance, the poor accessibility of the His tag to anti-His antibodies, and the low binding affinity for mannose carbohydrates. In each experimental setup, mannose was immobilized on a matrix. It is possible that due to its extensive glycosylation, N-Flo1p was prevented from approaching the surface close enough to allow binding with the immobilized mannose. However, we succeeded in detecting an interaction between N-Flo1p and d-mannose carbohydrates by the fluorescence spectroscopy method because the carbohydrate could move freely in solution and find its best position toward the binding site.
The fluorescence titration data show a biphasic course which could best be fit by a two-binding-site model, indicating the presence of two distinct binding sites for mannose. The newly discovered binding site should contain at least one aromatic amino acid that interacts with mannose carbohydrates upon binding, which results in a decrease of fluorescence intensity. In previous reported research, only one binding site was considered. It was proposed that the carbohydrate binding activity of Flo1p is attributed to the VSWGT motif, a pentapeptide encompassing amino acids 226 to 230 (25
). By use of this model, carbohydrate recognition by the Flo1 protein was described, and the amino acids that are responsible for this recognition have been identified. It was suggested that the tryptophan residue on position 228 is involved in mannose recognition in the Flo1 flocculation phenotype (25
). This was confirmed in the present study by fluorescence spectroscopy, where tryptophan fluorescence was quenched upon binding of mannose.
Furthermore, the affinity of N-Flo1p for mannose carbohydrates was determined. The equilibrium dissociation constant is in the micromolar range for the binding site with high affinity, while the site with low affinity has a KD value in the millimolar range (). Similar values were found for the interaction of N-Flo1p with α-methyl-d-mannoside. Therefore, it was concluded that N-Flo1p is able to bind α-d-mannose, which is commonly found in nature when mannose chains are formed. For the interactions of N-deglycosylated N-Flo1p and N- and O-deglycosylated N-Flo1p with d-mannose, higher KD values were found (). This suggests that the glycans present on the protein increase the binding activity of the protein toward d-mannose.
The only quantitative measurement of the affinity between a Flo protein and a carbohydrate has been described for the interaction of a Flo1p homologue, named Lg-Flo1p, with mannose. Only one binding site was assumed, and the value for the dissociation constant was calculated to be 0.77 mM (13
). In the past, detailed research about the inhibition of Flo1 phenotype flocculation by sugars has been described on the cellular level. Mannose was added in different concentrations to flocculating yeast cells, inhibiting flocculation at a certain concentration. Very divergent results were obtained. Miki and coworkers quantified the MIC, and they found that up to 500 mM d
-mannose was required to completely inhibit flocculation (29
). Later, different sugars were ranked according to their inhibition capacity. It was shown that α-d
-mannose could disrupt the flocs at a concentration of only 25 mM (35
). More recently, this experiment was repeated using a yeast strain overexpressing Flo1p. It was observed that the flocculation mediated by Flo1p was fully inhibited by mannose only at a concentration of 1 M (44
In addition to the interaction between N-Flo1p and monomannose, the binding of dimannoses [α(1,2)-, α(1,3)-, and α(1,6)-dimannoses] was studied by fluorescence spectroscopy. It was found that dimannoses also bind to two different binding sites of N-Flo1p. The values for the dissociation constant at equilibrium are summarized in . The KD values for the binding site with high affinity are roughly three times higher than the values obtained for monomannose binding, which suggests that monomannose is a better ligand for the site with high affinity. In contrast, the KD values for the binding site with low affinity show that the affinities of the dimannoses are roughly three times higher than those for monomannose binding. This indicates that the binding site with low affinity has a preference for dimannoses over monomannose.
Moreover, we have shown on the protein level that N-Flo1p is able to interact with mannan (KD
= 16.53 ± 3.74 mg/ml), which is part of the cell walls of yeast cells (24
). This observation confirms the flocculation model, which assumes that flocculation occurs due to an interaction of cell wall proteins (flocculins) and the mannan layer of the cell walls of neighboring yeast cells (10
). Remarkably, only one binding site was detected for the interaction between N-Flo1p and mannan. Taking into account the results for dimannose binding, it was suggested that mannan binds only to the binding site of N-Flo1p with low affinity.
The model suggested by Kobayashi and coworkers (25
) is based on only one binding site, comprising the VSWGT binding motif. This pentapeptide allows one mannose residue to enter the binding pocket of Flo1p. We have shown that the binding site with high affinity has a preference for monomannose binding. Therefore, we assume that the Kobayashi model deals only with the binding site with high affinity.
The high-affinity binding site probably has a more enclosed binding pocket where a monomannose just fits in and can interact with the surrounding VSWGT amino acids. The binding affinity of mannose carbohydrates will decrease with their increasing molecular weight due to the reduced probability of fitting into the binding pocket, whereas the low-affinity binding site will have a more open structure with less specificity, since every size of mannose carbohydrates (up to mannan molecules) can bind to this site. It is hypothesized that the initial binding between two interacting yeast cells occurs predominantly by the binding of yeast cell wall high-molecular-weight mannose carbohydrates to the low-affinity binding site. Next, low-molecular-weight mannose carbohydrates (e.g., terminal mannose residues on mannose glycans) bind to the high-affinity binding site. Since the Flo protein is then already fixed in space by binding to the low-affinity binding space, the terminal mannose residues can bind more efficiently to the more enclosed high-affinity binding site. Therefore, the presence of the low-affinity binding site is necessary to increase the efficiency of binding to the high-affinity site. The result is a relatively strong interaction on the single-protein level. Cell-cell binding is further enforced by numerous multiprotein interactions, resulting in a strong cell-cell binding process (9
). Indeed, a high concentration of flocculins is found on the surface of a flocculating cell (3
Until now, no atomic structure of any Flo protein has been determined. However, the N-terminal part of a homologue of Flo1p (Lg-Flo1p) has been crystallized and diffracted to high resolution by X-ray radiation (13
), but the structure has not been solved yet. In this study, we have provided more information about the secondary structure of the N-terminal domain of Flo1p, based on CD measurements. We found that N-Flo1p consists mainly of β-sheets (37.7% β-sheets and 6.5% α-helices). This is in accordance with the recent literature about the PA14 domain. Recently, the N-terminal domain of Flo1p was assigned to a new domain family (PF07691) in Pfam (11
), called the PA14 domain. The flocculin N-terminal domain might therefore be considered one of the many PA14 domain variants. The PA14 domain is a conserved domain that has been discovered in a wide variety of bacterial and eukaryotic proteins, which include many glycosidases, glycosyltransferases, proteases, amidases, bacterial toxins such as anthrax protective antigen (PA), and also yeast adhesins. Most of the experimentally characterized PA14-containing proteins are involved in carbohydrate binding and/or metabolism (34
). The anthrax toxin PA also contains a PA14 domain. The crystal structure of this toxin indicates that the PA14 domain consists of a series of antiparallel β-strands (32
In conclusion, the flocculation mechanism was studied at the molecular level by purifying the lectin domain of the Flo1 protein. It was confirmed that the N-terminal domain of Flo1p was the binding part of the flocculin. We demonstrated the binding of N-Flo1p to d-mannose, α-methyl-d-mannoside, and different dimannoses. It was shown that N-Flo1p contains two binding sites, with high and low affinities, in the micromolar and the millimolar range, respectively. These results confirm the cellular flocculation model and extend it to the protein level.