Sección de Microbiología, Facultad de Farmacia, Universidad de Valencia, Avda V. Andrés Estelles s/n, 46100 Burjassot (Valencia), Spain1
Author for correspondence: Salvador Mormeneo. Tel: +34 96 3864682. Fax: +34 96 3864682. e-mail: salvador.mormeneo{at}uv.es
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ABSTRACT |
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Keywords: cross-linking, activity inhibition, cell wall, yeast
Abbreviations: TGase, transglutaminase
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INTRODUCTION |
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Previously, we described the presence of transglutaminase (TGase) activity in the walls of Candida albicans, producing covalent cross-links between proteins. Inhibition of this enzyme decreased the covalent incorporation of several proteins, which, in turn, disrupted both the regeneration of protoplasts and the yeastmycelium transition (Ruiz-Herrera et al., 1995 ). Moreover, the cell wall acceptors of TGase activity have been shown to be virulence determinants of C. albicans (Staab et al., 1999
; Sundstrom, 1999
).
TGases (EC 2.3.2.13) catalyse covalent cross-linking between proteins by forming an amide bond between the -carboxyamide group of appropriate peptide-bound glutamine moieties and the
-amino group of specific peptide-bound lysine residues (Folk, 1980
). TGases with different physiological roles have been described in mammals, plants and other organisms (Ichinose et al., 1990
; Serafini-Fracassini et al., 1995
; Chandrashekar & Mehta, 2000
), many related to the formation of resistant structures having protective functions. A TGase immunologically related to mammalian TGase catalyses cross-linking of the cell wall in the green alga Chlamydomonas reinhardtii (Waffenschmidt et al., 1999
) and recently, using computer analysis, proteins with homology to eukaryotic TGases have been described in S. cerevisiae and Schizosaccharomyces pombe (Makarova et al., 1999
).
Here, we present evidence of a TGase activity in the wall of S. cerevisiae; the effects of its inhibition by cystamine suggest that it plays important roles in the organization of the cell wall.
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METHODS |
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Preparation of cell-free extracts.
Cell-free extracts were obtained by breaking cells with glass beads in a Braun cell homogenizer, in 50 mM Tris/HCl (pH 7·4), containing 1 mM phenylmethylsulfonyl fluoride and 1 µg pepstatin A ml-1. The extracts were centrifuged at 2000 g to pellet the cell walls, which were washed twice in 50 mM phosphate buffer, twice with 2 M NaCl and twice with distilled water. The supernatant was centrifuged at 40000 g in a Beckman JA 20 rotor to separate the membrane fraction (pellet) from the cytosol (supernatant). Alternatively, to obtain small amounts of purified walls, the cells, mixed with glass beads, were broken by agitating on a Vortex mixer for 30 s periods, with cooling periods of 1 min in an ice bath until all of the cells were disrupted, and treated as above. In each case, breakage was assessed by phase-contrast microscopy.
TG assay.
TG activity of the different fractions was quantitated by incorporation of a radioactively labelled amine substrate, as previously described (Barsigian et al., 1988 ; Simon & Green, 1985
), except that [14C]lysine was used instead of [3H]putrescine. TGase reaction mixture was in a final volume of 1 ml 50 mM Tris/HCl (pH 7·4) containing 2·5 µCi (92·5 kBq) [14C]lysine (9·2 mCi mmol-1, 340·4 MBq mmol-1) and enzymic fraction. Following 5 h incubation at 30 °C with shaking, the reaction was stopped by addition of 2 ml 10% TCA.
After at least 2 h on ice, samples were centrifuged; the pellet was resuspended in 2·5 ml 10% TCA, heated in a boiling water bath for 10 min, and recentrifuged. Sediment was washed twice with 10% TCA, twice with 5% TCA and twice with ethanol prior to measurement of radioactivity. To determine TGase activity using the endogenous wall acceptors, the same protocol was used without the addition of N,N'-dimethylcasein as exogenous acceptor.
Solubilization of wall proteins, gel electrophoresis and fluorography.
Purified walls labelled with [14C]lysine were sequentially solubilized with SDS, Zymolyase (Miles Laboratories) and chitinase (Sigma), as previously described (Ruiz-Herrera et al., 1995 ). Released proteins were separated from the insoluble residue by centrifugation (3000 g, 10 min) and the radioactivity measured. Samples containing 10000 c.p.m. were subjected to electrophoresis and subsequent fluorography, as has been described (Ruiz-Herrera et al., 1995
). The molecular masses were determined by reference to molecular mass markers (Bio-Rad).
Analysis of sensitivity to cystamine.
The method for testing the sensitivity of S. cerevisiae strains exposed to different concentrations of cystamine was similar to that described by van der Vaart et al. (1995) . Briefly, the different strains were grown to early exponential phase and diluted to an OD600 of 0·4 (3x106 cells ml-1); 3 µl aliquots containing approximately 104 cells or 1/10 serial dilutions of each culture were spotted onto a series of YPD plates containing varying amounts (0 to 100 mM) cystamine. Differences in the growth of the strains were recorded after incubation of the plates at 28 °C for 48 h.
Analysis of sensitivity to Zymolyase.
S. cerevisiae X2180-1A cells from early exponential culture, incubated in the presence or absence of cystamine, were adjusted to an OD600 of 0·5 in 10 mM Tris/HCl (pH 7·5) containing 25 µg Zymolyase 20T ml-1. The decreases in optical density were monitored over 10 min periods, according to the method of van der Vaart et al. (1995) .
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RESULTS |
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DISCUSSION |
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Using [14C]lysine as precursor, most of the radioactivity cross-linked in the cell wall (Table 1), whereas only residual binding was detected in the cytosol. A similar radioactive distribution was demonstrated for C. albicans (Ruiz-Herrera et al., 1995
), and in both fungal species the cross-linking was inhibited by cystamine, indicating a TGase-mediated process. Almost 30% of S. cerevisiae TGase was in a membrane preparation and this enzyme might be a precursor of the wall TGase. However, TGase activity functioning intracellularly has been described in other systems (Thacher & Rice, 1985
; Chowdhury et al., 1997
).
Whereas TGases in higher animals always require Ca2+ for activity, Ca2+ is not essential for TGase activity in plants and micro-organisms (Aeschlimann & Paulsson, 1994 ; Chandrashekar & Mehta, 2000
). Although the different cations had differing effects on wall TGase activity, as already described for hepatocyte TGase (Barsigian et al., 1988
), this experiment was not definitive because the wall can have a natural background of cations. The results suggest that other cations were, in general, less effective than Ca2+ in supporting TGase activity, and the fact that EDTA inhibited the enzymic activity (Fig. 1
) supports the hypothesis that wall TGase activity is dependent on divalent cations.
To investigate the distribution of endogenous acceptors of TGase in cell walls, the enzyme reaction was carried out in the presence of purified walls that later were sequentially solubilized by SDS, to release non-covalently linked proteins, and with Zymolyase and chitinase, in order to solubilize the proteins cross-linked covalently to polysaccharides. SDS solubilized approximately 50% of the radioactivity apparently cross-linked by the wall TGase; Zymolyase and chitinase subsequently released only small amounts (3%), in contrast to the higher proportions of covalently linked wall proteins that others report being solubilized by ß-glucanases and chitinase (Klis, 1994 ; Kapteyn et al., 1999
). The fact that high levels of the proteins apparently labelled radioactively by the wall TGase were resistant to the hydrolytic enzymes (45%) suggests the existence of unknown proteinaceous material forming part of the core of the wall structure.
The apparent TGase acceptors released by SDS had low molecular masses (50 kDa) in contrast to the predominantly high molecular masses of the materials released by Zymolyase or chitinase (
180 kDa) (Fig. 2
), suggesting a possible precursorproduct relationship. That is, the measurable molecular mass of SDS-soluble TGase acceptors may have increased as these acceptors were cross-linked to the wall by TGase activity. This type of high-molecular-mass material, which was described by others as a product of TGase activity (Barsigian et al., 1988
; Martinez et al., 1989
), had a similar electrophoretic mobility to the wall supramolecular complexes termed building blocks (Kollar et al., 1997
; Kapteyn et al., 1999
). Thus, S. cerevisiae TGase may be involved in the formation of these supramolecular complexes.
To determine whether protein cross-linking through TGase is important for the growth of S. cerevisiae, we made use of the TGase inhibitor cystamine (Simon & Green, 1985 ; Martinez et al., 1989
). Adding cystamine to the liquid growth medium reduced the level of growth of S. cerevisiae X2180-1A proportionally to the concentration of the inhibitor (Fig. 3
). Cystamine also altered the cell morphology (Fig. 4
), and increased the sensitivity of cells to Zymolyase (Fig. 6
). These results suggest that inhibition of growth by cystamine is due, at least in part, to the effect of cystamine on cell wall TGase activity.
Recently, using computer analysis, proteins with homology to eukaryotic TGases were detected, for the first time, in S. cerevisiae and Schizosaccharomyces pombe (Makarova et al., 1999 ). S. cerevisiae disruptants of the genes YPL096 and YDL117W, detected as candidates for TGase genes by computer analysis, were purchased from Euroscarf, and no significant differences between TGase activities in these strains and in wild-type organisms were detected (data not shown). Moreover, the YPL096W/PNG1/LPG12 gene encodes a soluble protein that, when expressed in Escherichia coli, exhibits peptide N-glycanase activity (Suzuki et al., 2000
) while the YDL117W/CYK3/D2275 gene encodes a protein possibly involved in cytokinesis (Zhu et al., 2000
). Thus there is no obvious relationship between these gene products and the TGase activity characterized during the present study. However, from the results presented here we conclude that TGase activity is indeed present in S. cerevisiae, where it plays important roles in cell wall organization.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 2 July 2001;
revised 4 January 2002;
accepted 15 January 2002.
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