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Cell-cell adhesion by Flo1 and Flo11 proteins from Saccharomyces cerevisiae

woensdag, 29 juni, 2011 - 17:00
Campus: Brussels Humanities, Sciences & Engineering campus
Faculteit: Science and Bio-engineering Sciences
D
2.01
Katty Goossens
doctoraatsverdediging

Controlled cell adhesion is regulated by specific cell adhesion molecules. The adhesins from
Saccharomyces cerevisiae are called flocculins. Flocculins are responsible for the interaction
with the cell wall of adjacent cells leading to the formation of multicellular clumps (flocs),
which sediment out of solution. This is known as the process of flocculation. Flocculins are
also responsible for the adhesion to substrates, for flor and mat formation or for invasive
and pseudohyphal growth. In nature, these morphological transitions are physiological
responses to starvation and stressful conditions, offering cells protection or help them to
forage for nutrients. These phenomena have been studied for many years, although the
research was mainly focused on the cellular level.
In this study, we focus on the flocculins, which are responsible for S. cerevisiae cell
adhesion, to extend the knowledge to the protein level. In particular, the Flo1 and Flo11
proteins, belonging to the flocculin family, were investigated in detail. Members of this
family are highly mannosylated cell wall proteins. They have a modular configuration that
consists of three domains (N]terminal, central and C]terminal domain). The C]terminal
domain contains a glycosylphosphatidylinositol (GPI)]attachment site, with which the protein
is covalently bound to the cell wall. The central domain contains many tandem repeats and a
lot of serine and threonine residues. These amino acids are prone to extensive Oglycosylation
and therefore this domain enables the flocculins to obtain a long, semi]rigid
rod]like structure. The N]terminal domain is the active domain. All together, the general idea
is that the protein is attached to the cell wall and sticks out, reaching for other cells or
substrates to interact with.
The most interesting part of the flocculins is clearly the N]terminus, and therefore, our
research is directed to the purification, characterisation and investigation of the binding
properties of the N]terminal domains of the Flo1 (N]Flo1p) and Flo11 (N]Flo11p) flocculins in
order to understand essential aspects of the mechanism behind yeast adhesion. Most of the
work has been performed on N]Flo1p and is described in chapters III to VI. Chapter VII is
dedicated to N]Flo11p.
In chapter III, the N]terminal domain of Flo1p (N]Flo1p), a flocculin responsible for the
strong flocculation of the yeast cells, was isolated. Therefore, the N]terminal domain was
identified and cloned in different microorganisms: E. coli, S. cerevisiae and P. pastoris. The
expression and the purification of N]Flo1p were optimised in each expression system and
the binding to mannose using fluorescence spectroscopy, was evaluated. N]Flo1p purified
from S. cerevisiae and P. pastoris were able to bind mannose. Further experiments were
performed with N]Flo1p from S. cerevisiae.
The characteristics of N]Flo1p are described in chapter IV. Therefore the secondary
structure was analysed with circular dichroism and it was shown that the protein is both Oand
N]glycosylated. Then, the type of glycans decorating the protein was determined using
different techniques such as ESI]mass spectrometry and anion exchange chromatography.
The binding properties of N]Flo1p are described in chapter V. The binding of N]Flo1p to Dmannose,
ƒ¿]methyl]D]mannoside, various dimannoses and mannan, confirmed that the Nterminal
domain of Flo1p is indeed responsible for the sugar]binding activity of the protein.
Furthermore, it was demonstrated that calcium is needed for the binding event. Moreover,
fluorescence spectroscopy data suggest that N]Flo1p contains two mannose carbohydrate
binding sites with different affinities.
In chapter VI, the homotypic interaction of N]Flo1p and the role played by the protein
glycans in this interaction were studied. Firstly, it was demonstrated that N]Flo1p shows a
homotypic adhesion phenotype, since it interacts with glycans on other N]Flo1p in the
presence of calcium. Secondly, it was shown that N]Flo1p]derived glycans aggregate in the
presence of calcium. Combining those two major results, the flocculation mechanism was
refined and extended at the molecular level by suggesting that the mechanism of flocculation is
based on two types of interactions: lectin]carbohydrate interactions, extended with
carbohydrate]carbohydrate interactions.
In the last chapter, the purification of the N]terminal domain of Flo11p from S. cerevisiae
was described. The secondary structure was determined and the presence of O]glycans on
the protein was demonstrated. Furthermore, it was shown that N]Flo11p shows a homotypic
interaction, but does not bind mannose.