Kluyveromyces bulgaricus Yeast Lectins ISOLATION OF N-ACETYLGLUCOSAMINE AND GALACTOSE-SPECIFIC LECTINS: THEIR RELATION WITH FLOCCULATION*

Kluyverornyces bulgaricus is a yeast which, upon culture in a calcium-enriched glucose-peptone medium, flocculates. Its flocculation can be reversed by the ad- dition of galactose. In this paper, it is shown that two lectins can be isolated either from the concentrated culture broth or from the supernatant of deflocculated cells suspended in galactose solution. The nor

Lectins (1) are a class of sugar-binding proteins which have been evidenced not only in plants and animals (2) but also in microorganisms, specially in bacteria (3). In most cases, lectins of microorganisms are associated with pili or cell walls; however, lectins may be excreted into the surrounding medium as in the case of bacteria (4) or of a yeast mutant (5). While bacteria lectins may be involved in adhesion to host cells (for a review, see Ref. 3), the function of yeast lectins is not yet well documented. Yeast lectins could recognize a saccharide structure at the surface of another cell and so be involved in the flocculation mechanism (6). Yeast lectins could also be involved in the mating process as suggested by the properties of the sexual agglutinin from Saccharomyces kluyveri 17 cells which binds glycopeptides of S. kluyveri 16 cells through their sugar moiety (7,8).
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Flocculation of yeast cells can be reverted by addition of specific sugars (9) showing that a sugar binding recognition mechanism takes place in addition to the classical calcium bridge mechanism.
Kluyveromyces bulgaricus yeast cells are known to spontaneously flocculate upon growth. Their aggregates are not dissociated by suspension in Helm's buffer at pH 4.5 (IO), but are easily deflocculated by suspension in the same buffer containing D-galactose (11). On the basis of this observation, we have been motivated to look for the presence of a galactosebinding protein produced by this yeast. The results presented here show (i) that two lectins are not only associated with the walls of K. bulguricus cells but are also excreted by them, (ii) that these two lectins, which have similar molecular weights and are able to specifically agglutinate red blood cells, act as agglutinins at pH 4.5, and (iii) that the galactose-specific lectin induces the flocculation of K. bulguricus cells at pH 4.5.

MATERIALS AND METHODS
Microorganism and Culture Conditions-The flocculent yeast K.
Lectin Isolation from the Culture Medium-The cells were harvested at 4 'C by centrifugation at 3,000 X g, for 10 min, and the supernatant was neutralized to pH 7.4 with 2 M NaOH. The precipitate obtained upon neutralization was removed by centrifugation at 10,000 X g for 15 min, and the supernatant was kept up to several weeks at -20 "C without loss of activity. The solution so obtained was applied onto an affinity chromatography column (1.5 X 5 cm; flow rate, 0.3 ml/min) of Ultrogel AcA 22 (IBF-RCactifs, Villeneuvela-Garenne, France) substituted with 1,6-diaminohexane, then succinylated, and finally activated with a water-soluble carbodiimide and reacted with p-aminobenzyl-thio-P-D-GlcNAc (12). After washing with 200 ml of Tris buffer (pH 7.4, 50 mM tris(hydroxymethy1)aminomethane, 0.1 M NaCI, 3.75 mM CaC12, 3 mM NaN3), elution was started with the same buffer supplemented with 1 M NaCl (100 ml), then with 0.1 M acetic acid (100 ml), and finally with 0.05 M hydrochloric acid (100 ml). The protein content of the eluate was monitored at 280 nm. Five-milliliter fractions were collected. Fractions eluted with the washing buffer, which are free of P-D-G~cNAc binding protein, were pooled. This solution was chromatographed on an affinity column (1.5 X 7 cm) of Ultrogel AcA 22 activated and substituted with p-aminophenyl-a-D-Gal, as described above, and sequentially eluted as described above.
The cells were then deflocculated in a 0.2 M D-Gal solution in the same buffer and centrifuged (11). The supernatant could be kept several weeks at -20 "C without any loss of activity. This deflocculated cell extract was chromatographed on P-D-GIcNAc affinity column under the previously described conditions. In order to eliminate D-Gal from the D-GlcNAc nonbinding fraction, proteins were precipitated with absolute ethanol at 4 "C overnight (ethanol/extract ratio = 4) and suspended again in Tris-HC1 buffer, pH 7.4, prior to chromatography on p-aminophenyl-a-D-Gal coupled to activated U1trogel AcA 22, as described above.
Hemagglutination and Hemagglutination Inhibition Tests-Twofold serial dilutions of lectin solution (50 pl) in wells of a microtiter plate were incubated for 1 h at room temperature with 5% erythrocyte suspension (50 pl) in phosphate-buffered saline (pH 7.4, 8 mM Na,HPO,, 1.5 mM KH,PO,, 137 mM NaCl, 2.7 mM KCl, 0.5 m M MgCl,, 1 mM CaCl,, 3 mM NaNJ and examined for agglutination. Sheep and rabbit erythrocytes were used for titration of D-GlcNAc specific and D-Gal specific lectins, respectively. Activity was expressed as titer, the reciprocal of the maximal dilution of lectin that gave visible agglutination. Hemagglutination was also tested at pH 4.5, in Helm's buffer with sheep and rabbit erythrocytes pretreated with 0.25% glutaraldehyde to resist to pH and osmotic shocks (15). Inhibition tests were performed in the same way, but serial dilutions of inhibitors solution (50 pl) were previously incubated for 15 min at room temperature with 50 p1 of a lectin solution giving an agglutination titer of 4.
Flocculation and Flocculation Inhibition Tests-Yeast flocculation was assayed in microtiter plates by mixing together 50 pl of 2-fold serial dilutions of lectin solution in the Helm's buffer, pH 4.5, and 50 p1 of yeast suspension (2 X 108cells/ml) in the same buffer. A positive reaction resulted in formation of aggregates (flocs), whereas nonflocculating cells settled to the bottom of the well in a diffuse layer (16), as determined either by visual reading or under a microscope.
Other Biochemical Methods-Sodium dodecyl sulfate/polyacrylamide gel electrophoresis was performed on a 8-cm-long slab gel containing 9% acrylamide (17). Molecular weight was calculated from calibration with standard markers kit (Sigma). Gel electrophoresis under nondenaturing conditions was done according to Ornstein (18) in 12.5% acrylamide gels.
Proteins were estimated by using the method of Bradford (19) or by their absorbance at 280 nm.

RESULTS
Isolation and Purification of the GlcNAc Specific Lectin from the Culture Medium-K. bulgaricus cells flocculate at the beginning of the exponential growth phase (after 24 h in culture) and the medium neutralized to pH 7.4 was able to agglutinate human erythrocytes to a similar extent irrespective of the A, B, or 0 blood group. This result prompted us to isolate the responsible factor for hemagglutination. We previously demonstrated that cell wall mannan of this flocculent yeast contained D-GlcNAc and observed that p-nitrophenyl-P-D-G~cNAc inhibited erythrocyte agglutination (20). On these bases, the neutralized culture medium was chromatographed on Ultrogel AcA 22 substituted with P-D-G~cNAc. The elution profile is presented in Isolation and Purification of the Gal Specific Lectin from the Cells-The unretarded fraction was found to agglutinate rabbit erythrocytes a t p H 7.4 and glutaraldehyde-fixed rabbit erythrocytes a t p H 4.5, and to flocculate yeasts a t p H 4.5. In order to eliminate D-Gal from the P-D-G~cNAc nonbinding fractions, proteins were precipitated with absolute ethanol and resuspended in Tris-HC1 buffer, pH 7.4, prior to chromatography on Ultrogel AcA 22 substituted with D-Gal. Calculation of activity before and after precipitation showed that less than 50% of the lectin activity was lost during this step. The elution pattern is quite similar to that of Fig. lb. The fraction eluted with 0.05 M HC1 had the same agglutination properties as the homologous fraction isolated from the culture medium (Fig. Ib). The D-Gal specific lectin was obtained with a yield of 0.2 mg from 1 g of wet yeasts.
Purity and Molecular Weight Estimation of the Lxctim-Purity of the lectins was substantiated by polyacrylamide (9% and 7% acrylamide) gel electrophoresis at pH 8.3 in the gel slab, in the presence or in the absence of 15 mM mercaptoeth-an01 and 0.1% sodium dodecyl sulfate. One major band and several minor bands were observed for the four lectin preparations, and a second step of affinity chromatography was required to achieve complete purification. Lectins were eluted from their respective affinity gels with 0.2 M D-GlcNAc or D-Gal. Under these conditions, only one band was observed in both cases for the two lectins, prepared either from the culture medium or from the deflocculated cells (Fig. 2b). Molecular weights were estimated under denaturing conditions to be 61,000 and 65,000 for the D-GlcNAc or D-Gal specific lectins, respectively (Fig. 2a).
Influence of the pH on Hemagglutination Properties-The results on the study of agglutination of erythrocytes at pH 7.4 in phosphate-buffered saline and at pH 4.5 in Helm's buffer are summarized in Table I. The D-GlcNAc specific lectin did not agglutinate glutaraldehyde-treated sheep erythrocytes at pH 7.4 but did at pH 4.5. In contrast, the D-Gal specific lectin did agglutinate glutaraldehyde-treated rabbit erythrocytes at both pH values. In addition, the D-Gal specific lectin, which did not agglutinate untreated or glutaraldehyde-treated sheep erythrocytes at pH 7.4, did agglutinate the glutaraldehydetreated sheep erythrocytes at pH 4.5.
Hemagglutination Inhibition Test of the D-G~NAc Specific Lectin-The sugar specificity of the lectin purified on agarose coupled with D-GlcNAc was examined by hemagglutination inhibition tests using sheep erythrocytes. p-Nitrophenyl glycosides and sugars giving the strongest inhibitory effect are presented in Table 11. The similarity of the results obtained with lectins isolated either from the culture medium or from cells supports the view that there is only one D-GlcNAc binding protein. a-and 8-p-Nitrophenyl derivatives of D-GlcNAc had the same efficiency as hemagglutination inhibitors. Conversely, the lectin was inhibited with a lower concentration of p-nitrophenyl-8-D-glucoside in comparison with pnitrophenyl-a-D-glucoside. The p-nitrophenyl-N-acetyl-Dglucosaminides are more efficient than free D-GlcNAc, showing the importance of the hydrophobic moiety of the ligand.     Table 111. Identical results were obtained with both excreted and cell-associated lectins. Among the monosaccharide derivatives tested, 0-andp-nitrophenyl galactosides exhibited the strongest inhibitory activity, which appears to be independent of their anomeric configuration. The presence of the hydrophobic aglycone also increases the inhibitory efficiency. The hydroxyl group in position 6 of galactose is not involved in the binding, since Dfucose is as efficient as D-galactose. Sugars listed below were found to be inactive even when concentrations were over 0.1 M: L-fucose, L-galactose, D-and L-glucose, D-and L-mannose, D-galactosamine, D-glucosamine and D-mannosamine, 6phosphate derivatives of D-galactose, D-glucose, and D-mannose,p-nitrophenyl-a-and o-D-glucosides, andp-nitrophenyl-8-D-mannoside.

Hemagglutination inhibition test of the purified *Gal specific lectin from the culture medium of K. bulgaricus or released upon incubation in the presence of D-Gal, from the yeast cell wall, at pH 7.4, using untreated rabbit red blood cells
Flocculation and Flocculation Inhibition Test-The common technique for measurement of flocculation consists of sedimentation speed evaluation combined with light extinction determination. In the present work, important variations in the viscosity of the Helm's buffer were introduced by the presence of sugars in the deflocculation test. Under such conditions, it was necessary to apply the method used by Burke et al. (16) in testing the inhibition of Hansenula wingei sexual agglutination factor. A typical assay of deflocculation is presented in Fig. 3 and the most efficient sugar derivatives for inhibition of flocculation are listed in Table IV. These inhibitors are similar to those used to inhibit the agglutination of rabbit erythrocytes at pH 7.4 and at pH 4.5, but the minimum amount completely inhibiting flocculation was slightly different: D-fucose, D-galactose, and Gal-P-l-4-Man were more efficient in flocculation inhibition than in agglutination inhibition, while the other sugars were more efficient in agglutination inhibition than in flocculation inhibition. DISCUSSION The flocculation of K. bulgaricus was reversed by addition of D-Gal ( l l ) , although this hexose was absent from isolated  cell wall polysaccharides of this yeast (21). The specificity of the interaction suggested a lectin-like mechanism, but isolation of the functional mediator was still lacking. The purification of two lectins from the calcium-enriched culture medium by affinity chromatography corroborated the previous hypothesis. The lectins are excreted in the medium, but a part of the activity remains associated with the outer layer of the cell wall, and upon deflocculation with D-Gal, a further quantity of lectins was released in the medium. According to the osidase activity investigations, the isolated lectins did not belong to the category of glycosidases that "act as lectin" at neutral pH, described by Shannon (22). The D-GlcNAc specific lectin agglutinated sheep erythrocytes at pH 7.4 and at pH 4.5, but had no effect on deflocculated yeasts at pH 4.5. This result is in agreement with previous observations indicating the important decrease of D-GlcNAc residues in cell wall mannans for flocculent cultures of this yeast (20), and so, the absence of flocculating effect could be the consequence of the absence of the relevant receptor on the wall. The physiological role of this D-GlcNAcbinding protein remains to be studied. Investigations of the specificity of this lectin showed that the presence of acetamido groups on C-2 probably promoted the recognition because Dglucosamine is not an inhibitor, and the corresponding Dglucosides are about 10 times less effective than the N-acetyl-D-glucosaminides. The substitution of C-1 by a hydrophobic aromatic group considerably increased the inhibitory effect of D-GlcNAc residues. In addition, the strong inhibitory effect of p-nitrophenyl-N,N'-diacetyl-@-D-chitobioside suggests the binding site of the lectin is extended.

Flocculation inhibition test at pH 4.5 of the purified D -G~ specific lectin from the culture medium of K. bulgaricus or released upon incubation in the presence
Evidence is also given for the excretion of a D-Gal specific lectin and for the fixation of this protein at pH 4.5 on rabbit erythrocytes and yeasts. Both lectins agglutinated glutaraldehyde-treated sheep erythrocytes at pH 4.5, but did not at pH 7.4. This result indicates that both lectins are more active in acidic than in neutral medium and may explain the observation that the extent of yeast flocculation which occurs at pH 4.5 decreased upon neutralization (22).
Polyacrylamide gel electrophoresis showed the monomeric nature of the two lectins, and this result implied that at least two binding sites should be present on each lectin, in order to create a bridge between adjacent red blood cells. The molecular weight (60,000) of the isolated lectins is close to that of S. kluyueri 17 cell sexual agglutinin which has been shown to be a proteolytic product of a larger macromolecule of the cell wall (8). Further works are required to determine whether the K. bulgaricus lectins have a larger molecular weight in situ and whether the 60,000 lectins are released by an endogenous protease, directly or indirectly activated by the incubation of the cells in the presence of D-Gal.
To our knowledge, this is the first report on the excretion of a lectin implicated in the flocculation mechanism of K. bulgaricus. However, excretion of lectins by other yeast species was recently shown by Basu et al. (5) for Saccharomyces mutants and by Critchley and Douglas (23, 24) for Candida albicans. In the first report, a D-Gal specific lectin was isolated, whereas in the second example a D-GlcNAc specific adhesion was evidenced. The nature of the receptors of the D-Gal specific lectin on K. bulgaricus cell wall is not yet known; we are currently investigating the inhibitory effect of oligosaccharides produced by acetolysis of cell wall polysaccharides.