Kluyveromyces bulgaricus Yeast Lectins ISOLATION OF TWO GALACTOSE-SPECIFIC LECTIN FORMS FROM THE YEAST CELL WALL*

Incubation of galactose treated Kluyveromyces bul- garicus yeast cells in EDTA/phosphate-buffered saline led to an extract possessing hemagglutinating and yeast flocculating properties. Purification of this extract by affinity chromatography and gel filtration gave two lectin forms, Kb-CWL I and Kb-CWL 11, with an apparent molecular mass of 38,000 and 150,000 Da, respectively. Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis revealed that Kb-CWL I and Kb-CWL I1 were dimeric and octameric of a subunit of 18,900 Da. At high concentration, purified Kb-CWL I associated to give Kb-CWL 11. This association seemed to be independent on pH. The two lectin forms were glycoproteins, the peptide counterpart was very rich in Lys, Glu, and Gly, and the carbohydrate part rep- resented 1% of the whole molecule and was composed of Glc, Man, and Ara. The two lectin forms (KB-CWL I and Kb-CWL 11) agglutinated human red blood cells and flocculated EDTA-treated K . bulgaricus yeast cells. The activity of both lectin forms required Ca2+ ions, while Sr2+ showed some competitive inhibition. Optimal activity was obtained within a pH range of 4- 6.5 for both forms. Temperatures of 80-90 OC for in the microorganism, at

Flocculation and sexual agglutination are the two types of specific cell-cell recognition phenomena reported in yeasts (1). The isolation of sexual agglutinins from many yeast species led to a better understanding of the mechanism of the sexual agglutination phenomenon (2-5). The sexual agglutinins were characterized by their covalent linkages to the cell wall, their monovalency, and their hapten (monosaccharides) nonsensitive activity (4-7). Such molecules failed to agglutinate cells when they were liberated from the cell wall, therefore their activity was always evidenced by an agglutination inhibition test in which cellular receptors of one mating type were saturated with the isolated sexual agglutinin of the other mating type before cellular contact of the two mating types (6, 7).
Although flocculation was shown to be a specific cell-cell recognition phenomenon, since it is hapten-specific, it's mechanism until now is poorly understood because of nonevidenced * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom requests for reprints should be addressed.
yeast cell flocculating factors. Hypothesis of lectin-carbohydrate interaction based on the deflocculating effect of sugars was described by many workers (8)(9)(10)(11). This lectin-carbohydrate interaction was presumed to occur between the hypothetic lectinic factor and it's presumed receptor, i.e. the cell wall peptidomannan. Although this wall heteropolymer has been the subject of many chemical analyses, showing different structural modifications between the flocculent and the nonflocculent molecule, it's role in the flocculation phenomenon has not yet been directly demonstrated.
In previous studies (12, 13) we reported the isolation of a galactose specific lectin from the culture broth of the yeast Kluyveromyces bulgaricus. This lectin was excreted by the cells when this yeast was cultivated aerobically in a calciumenriched medium. It's participation in the flocculation of this yeast was established (12, 13), although chemical analysis failed to demonstrate the presence of galactose residues in cell walls of this yeast (14). The aim of the present study was (i) to isolate and to determine the molecular form of a cell wall linked lectin, and (ii) to demonstrate the role of phosphopeptidomannans as the receptors of this lectin.

MATERIALS AND METHODS
Reagents-All chemicals were obtained from Sigma unless otherwise noted.
Microorganisms and Culture Conditions-The flocculent yeast K.
bulgaricus was grown in a bacto-peptone-glucose liquid medium as previously described (13). Saccharomyces cereuisiae X2180 was grown aerobically in a glucose, yeast extract, and ammonium sulfate medium.
Extraction of a Galactose-specific Lectin from the Cell-The cells were harvested at 4 "C by centrifugation at 3,000 X g, for 10 min, and treated with Gal, as previously reported (13). Cells obtained hereby were washed extensively with 0.01 M phosphate buffer (pH 7, 3 mM NaN3) and suspensed at a concentration of 4% (w/v) in the same buffer containing 5 mM EDTA (Prolabo). Cell suspension was incubated at 37 "C for 60 min under moderate agitation. Supernatant was collected by centrifugation, and cells were tested in Helm's buffer for residual flocculating activity (13). Supernatant was dialyzed at 4 "C for 48 h against distilled water (4 X 2.5 liters) and then lyophilized.
Preparation of Affinity Support-Unless otherwise noted, the cells of K. bulgaricus treated as described above were included in a nondenaturing 7.5% polyacrylamide gel preparation (15). Before the polymerization step, cells were introduced as 10% w/v suspension in distilled water in the place of water, then polymerization was realized by addition of N,N,N,N',-tetramethylethylendiamine. The gel containing the immobilized cells was cut into small pieces, extensively washed with Helm's buffer, pH 4.5, and stored at 4 "C in the same buffer containing 3 mM NaN3. A column of Sepharose 4B was also used for affinity chromatography.
Purification of the Gal -specific Lectin-20 mg of lyophilized extract were solubilized in 30 ml of Helm's buffer and applied onto an affinity chromatography column (20 X 1.5 cm; flow rate, 0.2 ml/min) of immobilized yeast cells equilibrated with Helm's buffer. After washing with 200 ml of Helm's buffer, elution was started with the same buffer supplemented with either 10 mM EDTA or 0. or galactose eluted fractions (4 ml/fraction) were dialyzed separately against Helm's buffer and titrated. The dialyzed fractions were lyophilized.
Extraction of Phosphpeptidornannan-The extraction of the phosphopeptidomannans from the flocculent and the nonflocculent yeast K. bulgaricus was performed by autoclaving in a neutral citrate buffer (18). Crude extracts were chromatographed onto a column (85 X 2.5 cm; flow rate, 0.16 ml/min) of Tris-acryl GF-2000 (IBF-Reactifs, Villeneuve la Garenne, France) equilibrated with a 3 mM NaN3 solution. Molecular mass estimations and chemical compositions of these molecules were the same as already reported (14). Phosphopeptidomannan of the yeast S. cerevisiue X2180 was also obtained by autoclaving the cells in a neutral citrate buffer (18). The crude extract was fractionated using cetyltrimethylammoniumbromide (IS), fraction I1 corresponding to the PPM' was used.
Preparation of RBC Bearing Yeast PPM-Human (A, B, or 0) or rabbit RBC were trypsinized and treated with glutaraldehyde (20). Before neutralization of the nonreacted aldehyde groups present on RBC surfaces, RBC were incubated at room temperature (22 "C) for 1 h as a suspension of 1% v/v in phosphate-buffered saline containing 0.1% PPM, RBC were then washed three times with PBS, and nonreacted aldehyde groups were neutralized prior to glycine treatment (20). lectins (Kb-CWL I and Kb-CWL 11) exhibit optimal binding to pH and Temperature Stability-The pH range over which the The abbreviations used are: PPM, phosphopeptidomannans; RBC, red blood cell; TBS, Tris-buffered saline.
The temperature stability of Kb-CWL I and Kb-CWL I1 was estimated by incubation of 300 pl of the lectins in Tris-buffered saline (TBS) (25 pg/ml) at different temperatures for 20 min, cooling them on ice, and titrating them with rabbit RBC and K. bulgaricus yeast cells. Divalent Cation Requirements-To examine the divalent cation requirements of Kb-CWL I and Kb-CWL 11, the agglutination tests were performed as described above, but using TBS without Ca2+ and in the presence of 20 mM EDTA. Flocculation tests were performed in 0.15 M sodium acetate/acetic acid without Ca2+ and in the presence of 20 mM EDTA. Another series of tests was performed by adding EDTA (20 mM in TBS or in Helm's buffer) to the agglutinated cells and monitoring the reversal of agglutination.
Hemagglutination and Flocculation Inhibition Tests-Kb-CWL I and Kb-CWL I1 were dissolved in TBS or in Helm's buffer at concentrations of 40 and 160 pg/ml, respectively. All carbohydrates to be tested for their inhibitory effect were dissolved in TBS or in Helm's buffer (at concentration up to 200 mM for mono-and oligosaccharides and 10 mg/ml for polysaccharides and glycoproteins as inhibitors). Serial 2-fold dilutions (50 pl) of the purified lectins were placed in the wells and equal volumes of inhibitor (50 pl) at a certain concentration were added. Mixtures were incubated for 60 min and 50 pl of the RBC suspension (lo7 RBC/ml) were added to each well.
Controls were the substitutions of the inhibitor solutions and of the purified lectins by TBS.
General Analytical Methods-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed under reducing condition (15 mM mercaptoethanol) on a 8-cm long slab gel containing 9% acrylamide as described by Laemmli (22). Gel filtration was performed on a column (97 X 1.5 cm; flow rate, 3.4 ml/min) of Sephadex G150 equilibrated with the Helm's buffer. Proteins were estimated by the method of Lowry (23), using crystallin bovine serum albumin as standard, or by their absorbance at 280 nm. Amino acids were determined after 5.6 M HCl hydrolysis under vacuum at 105 "C during  * Q, quantity of product in mg; s.a., specific activity expressed as K. bulgaricus yeast agglutinating unit/mg of product. 12 h, using a Technicon NC-2P autoanalyzer. Carbohydrates were determined by the phenol-sulfuric acid method (24) after 2 M HCI hydrolysis under vacuum at 105 "C during 2 h using a mixture of arabinose, glucose, and mannose (20,40, and 40%, respectively, w/v) as standard. Identification of monosaccharides was performed by descending paper chromatography (17) and as alditol acetate derivatives by gas-liquid chromatography at 210 "C with a 180-cm-long column of 33% Sp 2340 on chromosorb WAW-DMCS (100-120 mesh) as described elsewhere (25). A more rigorous identification of the carbohydrate derivatives was performed by gas-liquid chromatography coupled to mass spectrometry using alditol acetate derivatives, in a CPG-SM Carbo Erba 4160-Riber Mag R-lO-lOC, assisted by a Sidar l l l A informatic unit. Hexosamines were determined by the reagent of Elson Morgan following the procedure described by Ghuy-sen et al. (26), and by ion exchange chromatography using a Technicon NC-2P autoanalyzer.

RESULTS
Extraction and Purification of a Galactose-specific Lectin from the Cells-The flocculent yeast K. bulgaricus excreted Gal and GlcNAc-specific lectins in the culture medium (12, 13). These two lectins were also obtainable from whole cells by incubation with a 0.2 M Gal solution, in Helm's buffer, pH 4.5, in the presence of Ca2+. This treatment induced a dispersion of the flocs, but the cells, after washing in Helm's buffer showed a residual flocculation activity. Treatment of these yeast cells with 5 mM EDTA in 0.01 M phosphate buffer, pH 7, and centrifugation described under "Materials and Methods" led to a crude extract which after extensive dialysis showed hemagglutinating activity. This crude extract was chromatographed on the immobilized K. bulgaricus yeast cells in polyacrylamide gel column under the conditions described above. The elution profiles are presented in Fig. 1, A and B. Chromatography of the crude extract on a Sepharose 4B column gave the same results but with a better output (Fig.  2, A and B ) (Table I).
Purity and Molecular Mass Estimations-These were examined by both gel filtration and polyacrylamide (9% acrylamide) gel electrophoresis at pH 8.3. Gel filtration on Sephadex G-150 column of the purified fractions led to two products Kb-CWL I and Kb-CWL I1 with an apparent molecular mass of 38,000 Da and 150,000 Da, respectively (Fig. 3). Both products exhibited hemagglutinating and yeast flocculating activities. By polyacrylamide (9% acrylamide) gel electrophoresis, molecular masses of the subunits were estimated from semilog plots of molecular masses uersus mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions (Fig. 4). Results were the same for Kb-CWL I and Kb-CWL 11, the same band observed for the two fractions had a molecular mass estimated to 18,900 Da.
Chemical analysis of Kb-CWL I and Kb-CWL I1 showed that they were glycoproteins (Table 11). The peptide part of Kb-CWL I was very similar to that of Kb-CWL 11. The two lectins were very rich in Lys, Glu, and Gly ( Table 111). The carbohydrate counterpart of both products represented about 1% of the whole molecule. It was composed of Glc, Man, and a third compound showing, after transformation in alditol acetate, the same retention time.as arabinose. Mass spectrum fragmentation of this compound corresponded also to arabinitol penta acetate. These three monosaccharides Ara, Glc, and Man were present in a molar ratio of 1:2:2 (Table 11).
' Nonflocculent cells, obtained from culture of yeast in a Ca2+deficient medium as described (14). Main structure on the cell surface was low phosphorylated PPM (14).
Cells were obtained from culture in the Sabouraud medium. Main structure on the cell surface was high phosphorylated PPM. nyl-P-N,N"diacetyl chitobioside, p-D-N-acetyl glucosamine 1-phosphate, p-nitrophenyl-/3-lactoside. No osidase activity could be detected with either Kb-CWL I or Kb-CWL 11.
Agglutination Properties of Purified Kb-CWL I and Kb-CWL 11 and Demonstration of the Role of Phosphopeptidomannans-The results presented in Table IV, showed that agglutination properties of Kb-CWL I and Kb-CWL I1 were different. Indeed, 7-fold higher concentration of Kb-CWL I (220 pg/ml) was required in order to obtain titers for RBC   Table IV. c,d Cells used were the flocculent and the nonflocculent K. bulgaricus yeast cells treated with pronase. Treatment was realized as described by (14). comparable to those obtained with Kb-CWL I1 (35 pg/ml). Using trypsin-or pronase-treated RBC, only a slight difference between the two lectins RBC-agglutination capacity was observed. Both lectins agglutinated treated human 0-group RBC at higher titers than A and B ones. In the other hand, when trypsin-treated RBC were stabilized with glutaraldehyde and used as bearers of PPM extracted from flocculent or nonflocculent yeast K . bulguricus, such activated cells provided higher titers for both products, however, RBC-bearing phosphopeptidomannan from flocculent yeast were much more agglutinable by both lectins than RBC-bearing phosphopeptidomannans from nonflocculent yeast. RBC-bearing phosphopeptidomannan issued from S. cereuisiae X2180 showed no agglutination, and this was true for both charged and neutral fractions. Optimal binding activity was obtained within a pH range of 4-6 for both Kb-CWL I and Kb-CWL I1 ( Table V). Activities of Kb-CWL I and Kb-CWL I1 samples stored at room temperature (22 "C) for 2 weeks remained unchanged, while at 4 "C activities remained constant for several months. Incubation at 80-90 "C for 20 min reduced drastically the agglutinating activity of Kb-CWL I and Kb-CWL 11. Treatment of Kb-CWL I and Kb-CWL I1 with pronase or trypsin for 24 h discredit these molecules from their agglutinating activities. Among Ca2+, M$+, Mn2+, Zn2+, and Sr", only the presence of Ca2+ was required for the activity of Kb-CWL I and Kb-CWL I1 (Table VI). Addition of EDTA to a 10 mM final concentration reversed agglutination or flocculation of the lectin-RBC or lectin-yeast mixtures. EDTA-lectin preincubated solution showed neither RBC-agglutinating nor yeast-flocculating activities. EDTA-inhibited agglutinating or flocculating activity could be restored by addition of either Ca2+ (CaC12 30 mM) or, surprisingly, of Sr2+ (SrC12 30 mM).
Inhibition of Hemagglutination and Yeast Flocculating Properties of Kb-C WL Z and Kb-C WL ZZ-The sugar specificity of the purified lectins was examined by hemagglutination inhibition tests at pH 4.5 and 7, using rabbit erythrocytes, pnitrophenylglycopyranosides, and sugars giving the strongest inhibitory effect were reported in Table VII. Among the monosaccharide derivatives tested, p-nitrophenylgalactopyranosides exhibited the strongest inhibitory activity, and the inhibitory potency of their a-anomeric configuration was better than their @ one. The presence of a hydrophobic aglycon also increased the inhibitory efficiency. The hydroxyl group in position 6 of galactose could be replaced by a H atom without influencing the inhibitory efficiency, since D-fucose was as potent as D-galactose. Sugars listed below were found to be inactive even at concentrations over 100 mM; L-fucose, L-galactose, D-and L-glucose, D-and L-mannose, D-glUCOSamine, D-mannosamine, p-nitrophenyl (a and 8)-glucopyranosides, and p-nitrophenyl, ( a and 8)-D-mannopyranoside.
Inhibition studies on yeast flocculation were realized at pH 4.5 and 7. The most efficient sugar derivatives for inhibition of flocculation were presented in Table VII. These inhibitors were similar to those used to inhibit the agglutination of rabbit erythrocytes at pH 4.5 and 7. However, the minimum amount completely inhibiting the agglutination of rabbit erythrocytes was 4-fold higher than that in flocculation inhibition tests. Similary, p-nitrophenyl-a-D-galactopyranoside was a more potent inhibitor than the 8-anomeric configuration. DISCUSSION We have shown in previous papers (12, 13) that the yeast K. bulgaricus excreted GlcNAc-and Gal-specific lectins which had molecular masses of 61,000 and 65,000 Da, respectively. These lectins could be eluted from the yeast cell surface with 0.2 M galactose solution in Helm's buffer, which led to temporarily deflocculated yeast cells. Since upon washing with Helm's buffer such cells flocculated again, we postulated the presence of other lectins strongly linked to the cell wall. This study shows that yeast K. bulgaricus possesses beside the 65,000 Da Gal-specific lectin, two other Gal-binding lectins, Kb-CWL I and Kb-CWL 11, which are not found in the culture medium. The localization of these two last lectins is essentially on the cell surface. The fact that these two lectins are neither detectable in culture medium nor eluable from cell surface by treatment of yeast cells with a galactose solution, attests that these molecules are associated to the cell surface by any mechanism but carbohydrate-lectin interaction. However, these molecules under their cell surface-associated form recognize the carbohydrate moieties of other cells since, before their extraction, yeast cells flocculate upon elimination of galactose by washing with Helm's buffer.
Usually, lectins are composed of subunits (27). The molecular basis of the two lectins of K. bulgaricus seems to be identical, since only one type of subunit is detected for both forms. These results are supported by the results of the chemical analysis of the two lectins. Indeed, the similarity in amino acid composition as well as the identity of the osidic counterpart assign the same subunit structure to both lectins. Moreover, the association of Kb-CWL I at higher concentration can evolve Kb-CWL 11.
It is of great interest to note that these molecules contain a glycosidic counterpart different from the major polymers building up the cell wall, since arabinose is absent from the cell wall structure (14). This implies at least that these molecules do not undergo the same glycosylation pathway as glycoproteins building up the cell wall.
These two lectins Kb-CWL I and Kb-CWL I1 show the same agglutination profiles toward A, B, and 0 blood groups and rabbit erythrocytes. Higher titers are obtainable with RBC-bearing K. bulgaricus yeast PPM. These results corroborate those already reported (12, 14) and demonstrate that PPM represent a potential physiological receptor on the yeast cell wall surfaces for these lectins. The fact that these lectins cannot agglutinate RBC-bearing PPM (fraction 11) of S. cereuisiae, shows at least, that these later are not recognized by the two lectins. Nevertheless, RBC-bearing PPM from flocculent yeast K. bulgaricus provide always higher titers than RBC-bearing PPM from nonflocculent yeast. To our knowledge these results are the first direct demonstration of the role of yeast phosphopeptidomannan in flocculation, at the same time they show that this phenomenon is also governed by the structure of these wall polymers. Indeed, structural differences between PPM from flocculent and nonflocculent yeasts have been already described (14,10,29), but the