a mannose-binding protein from the cell surface of flocculentsaccharomyces cerevisiae (ncim 3528):...

A mannose-binding protein from the cell surface of¯occulent Saccharomyces cerevisiae (NCIM 3528):its role in ¯occulation

V. S. JAVADEKAR1, H. SIVARAMAN1, S. R. SAINKAR2 AND M. I. KHAN1*

1 Division of Biochemical Sciences, National Chemical Laboratory, Pune-411 008, India2 Special Instrumentation Laboratory, National Chemical Laboratory, Pune-411 008, India.

A cell surface lectin was isolated and puri®ed to homogeneity from the cell walls of a highly ¯occulent strain ofSaccharomyces cerevisiae (NCIM 3528) by chromatography on DEAE-cellulose, phenyl Sepharose and SephacrylS-300. It showed a molecular mass of 40 kDa on SDS±PAGE. It is an acidic protein with a pI of 4.0 and contains 44%hydrophobic amino acids. The N-terminal sequence up to 10 amino acid residues showed at least 70% homologywith the predicted N-terminal sequence of the putative FLO1 as well as FLO5 gene products. The mannose-bindingnature of the lectin was indicated by its high af®nity and speci®city towards the branched trisaccharide of mannose,a ligand which also inhibits the ¯occulation of yeast cells. Immuno¯uorescence studies con®rmed the presence oflectin on the yeast cell surface and lectin-speci®c IgGs prevented ¯occulation of the cells. This cell surface mannose-speci®c lectin probably plays an important role in ¯occulation, with the branched trimannoside on the cell wallbeing the apparent carbohydrate receptor. The N-terminal sequence data gives a primary indication that the lectincould be a product of one of the FLO genes. Copyright # 2000 John Wiley & Sons, Ltd.

KEY WORDS Ð cell wall protein; ¯occulence; microbial lectin; puri®cation; Saccharomyces cerevisiae

INTRODUCTION

Lectins, sugar binding proteins of non-immuneorigin with no catalytic activity, are ubiquitous innature and are known to play an important role incell recognition (Goldstein et al., 1980). They havebeen associated with the non-sexual ¯occulationof yeast cells, a widespread phenomenon exhibitedby many yeast genera. Flocculation is believed tobe due to calcium-dependent bonding between thecell surface proteins (lectins) on ¯occulent cellsand speci®c sugar residues intrinsic to the mannanpresent in the yeast cell wall (Miki et al., 1982).Straver et al. (1994) partially puri®ed a heat-stable, protease-insensitive agglutinin from the cellwall of both non-¯occulent and non-constitutively¯occulent mannose-sensitive brewer's yeast strain,S. cerevisiae. Shankar and Umesh-Kumar (1994)puri®ed a 13 kDa cell surface lectin from a¯occulent strain of S. cerevisiae that binds

speci®cally to mannose and mannans isolatedfrom yeast cell walls as well as to intact cells, inthe presence of calcium. Although these lectinswere proposed to be associated with ¯occulation,the precise nature of the lectin and the ligandinvolved in this interaction is still not very clear.

A number of chromosomal genes involved in¯occulation have been reported. These include thedominant genes FLO1 (FLO4), FLO2, FLO5 andFLO8±11; and the recessive or semidominantgenes ¯o3, ¯o6, ¯o7, fsu1, fsu2 and fsu3 (Holm-berg, 1978; Holmberg and Kielland-Brandt, 1978;Stewart and Russell, 1977; Russell and Stewart,1979; Russell et al., 1980; Johnston and Reader,1983; Yamashita and Fukui, 1983; Siero et al.,1993; Teunissen and Steensma, 1995; Wan-Shengand Dranginis, 1996; Siero et al., 1997). The FLO1gene has been studied in some detail with respectto its gene product and is predicted to code for alarge 1537 amino acid residue structural protein,rich in serine and threonine (Bidard et al., 1994;Watari et al., 1994; Bony et al., 1995). Thestructure of the putative FLO gene product has

*Correspondence to: M. I. Khan, Division of BiochemicalSciences, National Chemical Laboratory, Pune-411 008, India.E-mail: [email protected]

YEAST

Yeast 2000; 16: 99±110.

Received 10 April 1999Accepted 22 August 1999

CCC 0749-503X/2000/020099±12$17.50Copyright # 2000 John Wiley & Sons, Ltd.

been predicted in some detail on the basis of thegene sequence. Although a direct role of theputative Flo1p in the ¯occulation has beensuggested (Teunissen et al., 1995, Bony et al.,1995) there is no experimental evidence for it.Also, it is far from clear whether FLO genes codefor the cell surface lectin, which apparently isinvolved in ¯occulation.

The exact nature of the receptor involved in¯occulation has not yet been elucidated. Stratford(1992) made an effort to characterize the lectinligand using known mnn mutants which vary inwall mannan structure and suggested that lectinreceptors were the outer chain mannan sidebranches, two or three mannose residues in length.

In the present communication, we report thepuri®cation and characterization of a cell surfacelectin, sharing 70% N-terminal sequence homol-ogy with the FLO1 and FLO5 genes, from aconstitutively and highly ¯occulent strain of S.cerevisiae, and present evidence for its involve-ment in ¯occulation. The role of branchedtrimannoside as a putative lectin ligand has alsobeen discussed.

MATERIALS AND METHODS

Materials

Diethyl amino ethyl (DEAE)-cellulose (DE-52)was purchased from Whatman (UK); phenylSepharose CL-4B, Sephacryl S-300 and Sepharose4B were from Pharmacia (Sweden). Ampholines,sodium dodecyl sulphate (SDS)±polyacrylamidegel electrophoresis (PAGE) low molecular weightmarkers (SDS 70L), Freund's complete adjuvant,¯uorescein isothiocyanate (FITC), glycoproteins,and various monosaccharides and disaccharideswere obtained from Sigma (USA). Mannoseoligosaccharides were purchased from Dextralaboratories (UK). All other chemicals used wereof analytical grade.

Yeast strains and culture conditions

The yeast strain used in the present study was ahighly ¯occulent Saccharomyces cerevisiae (NCIM3528), obtained from the National Collection ofIndustrial Microorganisms (NCIM), NationalChemical Laboratory, Pune, India. It was main-tained at 4uC on MGYP agar slants containing(g/l): malt extract, 3.0; glucose, 20.0; yeast extract,3.0; peptone, 5.0; and agar, 20.0. The yeast cellswere grown in Erlenmeyer ¯asks (1 litre capacity)

containing 300 ml liquid MGYP medium, at 30uCwith shaking (200 rpm).

Isolation of yeast cell wall

Cells of S. cerevisiae (NCIM 3528) grownaerobically in MGYP medium for 24 h at 30uC,were harvested by centrifugation (6500rg,20 min) and washed twice with distilled water.The cell walls were isolated according to themethod of Vega and Dominguez (1986) and storedat x20uC.

Extraction of cell wall

10 g cell wall, obtained from 30 g of cells, weresuspended in 30 ml extraction buffer consisting of10 mM Tris±HCl (pH 7.2) and 0.5% non-ionicdetergent, Nonidet P-40 and homogenized for2.5 min (10 cycles of 15 s each) in a cell homo-genizer (B. Braun, Melsungen, West Germany).The supernatant was collected by centrifugation(9000rg, 30 min, 4uC) and stored. The extractionwas repeated four times, the cell wall extracts werepooled and used as the crude lectin preparation.

Puri®cation of the lectin

The crude extract was loaded onto a DEAE-cellulose column (2r40 cm) pre-equilibrated with10 mM Tris±HCl buffer, pH 7.2. The column waswashed with the same buffer till the A280 was<0.05. The bound protein was then eluted with alinear gradient of NaCl (0±1 M) in Tris±HCl buffer(10 mM, pH 7.2). The active fractions were pooled,adjusted to 4 M NaCl and loaded onto a phenylSepharose column (1.2r15 cm) pre-equilibratedwith 10 mM Tris±HCl, pH 7.2, containing 4 M

NaCl. The column was washed with a decreasinggradient of NaCl (4±0 M) and the bound lectin waseluted with a gradient of ethylene glycol (0±50%)in Tris±HCl buffer (10 mM, pH 7.2). The activefractions were pooled, dialysed against Trisbuffered saline (TBS) consisting of 10 mM

Tris±HCl buffer and 145 mM NaCl, pH 7.2,concentrated and loaded onto a Sephacryl S-300column (1.4r110 cm) pre-equilibrated with TBS.The active fractions were pooled, dialysed exten-sively against TBS, lyophilized and stored at 4uC.

Protein estimation

Protein was determined by the method ofMarkwell et al. (1978) using crystalline bovineserum albumin as standard.

100 V. S. JAVADEKAR ET AL.

Copyright # 2000 John Wiley & Sons, Ltd. Yeast 2000; 16: 99±110.

Carbohydrate content

Total neutral sugar content was estimated bythe phenol sulphuric acid method of Dubois et al.(1956), using mannose as a standard: 200 mg ofS. cerevisiae (NCIM 3528) cell wall lectin in400 ml distilled water was mixed with 400 ml of5% (w/v) phenol and incubated for 10 min atroom temperature; 2 ml concentrated sulphuricacid was then added to the mixture, allowed tocool for 20 min and the absorbance was mea-sured at 490 nm. Glycoprotein staining of thelectin was carried out by subjecting the puri®edlectin to SDS±PAGE, followed by staining withthymol-sulphuric acid (Glossmann and Neville,1971).

Molecular mass and pI determination

The relative molecular mass of the lectin wasestimated by SDS±PAGE (10% gel) according toLaemmli (1970) using SDS low molecular weightmarkers (Mr 14 000±66 000) for calibration. Thegels were stained with Coomassie Brilliant BlueR-250. Isoelectric focusing was carried out accord-ing to Chinnathambi et al. (1995), using ampho-lines in the pH range 3±10.

Haemagglutination assay

Haemagglutination and haemagglutination inhi-bition assays were carried out according toJoshi et al. (1997). For haemagglutination activitydetermination, two-fold serial dilutions of a lectinsolution (50 ml) in a microtitre plate were incu-bated at room temperature for 60 min with equalvolume of 3% suspension of rabbit erythrocytes inTBS and examined. The activity was expressed astitre, i.e. the reciprocal of the highest dilutionof the lectin that gave complete agglutination.Haemagglutination inhibition was performed insimilar manner, but serial dilutions of the inhibitorto be tested (25 ml) were preincubated at 25uC for30 min with 25 ml of protein of titre 4. Aliquots(50 ml) of rabbit erythrocyte suspension wereadded and the plates were read after 60 min ofincubation at room temperature.

Measurement of ¯occulation

The cells were grown in MGYP medium,harvested after 24 h and washed twice withdouble-distilled water. After de¯occulation using100 mM ethylenediamine tetra-acetic acid (EDTA)(pH 7.4), the cells were washed twice with 2 mM

EDTA (pH 7.4) and were suspended in the latter.The cell density was adjusted spectrophotometri-cally (A600=1.5, maintained in all experiments)and the EDTA was removed by pelleting the cells.The pellet was washed with double-distilled waterfollowed by suspension in the ¯occulation buffer(50 mM sodium acetate buffer, pH 4.5, containing5 mM CaCl2). 5 ml suspension, in 25 ml conical¯asks, was incubated on a rotary shaker (150 rpm)for 1 h at 25uC and then allowed to stand for2 min. The samples (0.1 ml) were removed fromjust below the meniscus, dispersed completely in100 mM EDTA and the free cell concentrationswere determined spectrophotometrically. Theeffect of the sugars on ¯occulation was checkedaccording to Masy et al. (1992). To check theeffect of lectin-speci®c IgGs on ¯occulation, thecells (A600=1.5) were incubated with the lectin-speci®c IgGs (100 mg) for 60 min at room tem-perature on a rotary shaker and the ¯occulationwas then initiated by the addition of 5 mM CaCl2.After 60 min incubation, the extent of de¯occula-tion was determined spectrophotometrically aswell as microscopically.

Amino acid composition

The amino acid composition of the puri®edlectin was determined on an automated aminoacid analyser (Hewlett-Packard Ti series 1050,with HP ¯uorescence detector). The sample washydrolysed in 200 ml of 6 M HCl at 110uC for 20 hand then subjected to analysis. Total tryptophanwas determined according to Spande and Witkop(1967). Total cysteine was determined accordingto Cavallini et al. (1966).

Electroblotting and N-terminal sequence analysis

Electroblotting of the lectin was carried outaccording to LeGendre et al. (1993). The N-terminal amino acid sequence of the ®rst 10residues of the lectin was determined by subjectingthe blot to Edman degradation on an automatedprotein sequencer (Shimadzu model PSQ-1).

Immunological methods

Antibodies against puri®ed S. cerevisiae (NCIM3528) cell wall lectin were raised in New Zealandwhite rabbits by immunizing them with approxi-mately 2 mg of the protein. Subcutaneous injec-tions of the lectin in phosphate buffered saline(PBS) mixed with equal amount of Freund's

MANNOSE-SPECIFIC CELL SURFACE LECTIN FROM FLOCCULENT YEAST 101


complete adjuvant were administered over aperiod of 3 months at 15 day intervals. Theantiserum was separated from the blood afterthe last injection and immunoglobulin Gs (IgGs)were puri®ed according to Dunbar and Schwoe-bel (1990). The lectin-speci®c IgGs were puri®edby af®nity chromatography on lectin±Sepharoseaccording to the method of Harlow and Lane(1988a). The puri®ed lectin (2 mg) was coupledto Sepharose 4 B using divinylsulphone, thematrix was equilibrated in PBS and the puri®edIgGs were passed through the column. Afterwashing the column with PBS, the bound IgGswere eluted sequentially by 0.1 M glycine(pH 2.75) and 0.1 M triethylamine (pH 11.75).The eluted lectin-speci®c IgGs were dialysedagainst PBS and concentrated by ultra®ltration.Labelling of the IgGs with FITC was carried outaccording to Harlow and Lane (1988b) andimmuno¯uorescence tests on intact S. cerevisiaecells (A600=1.5) were performed using FITC-labelled lectin-speci®c IgGs (100 mg) by themethod of Harlow and Lane (1988c).

RESULTS

Extraction and puri®cation of the lectin

The lectin was extracted repeatedly (at leastfour times) from the freshly prepared cell walls(10 g wet wt) of ¯occulent S. cerevisiae (NCIM3528) by homogenization in the presence of 0.5%Nonidet P-40. The activity of the lectin wasmeasured during puri®cation and characteriza-tion by haemagglutination assay. Extractionyielded approximately 180 mg of crude protein,with a haemagglutination titre of 256. The crudeextract contained the detergent, as well as aconsiderable amount of carbohydrate, whichcould be removed by chromatography on DEAE-cellulose and phenyl Sepharose, respectively.Subsequent gel ®ltration on Sephacryl S-300yielded a homogeneous protein. The puri®edlectin obtained after the above steps contained6±10% carbohydrate. Glycoprotein staining of thepuri®ed lectin, however, did not reveal thepresence of covalently linked carbohydrate (datanot shown). The requirement of ethylene glycol toelute the bound lectin suggests the hydrophobicnature of the protein. The total yield of thepuri®ed lectin from 10 g (wet wt) cell wall was300 mg.

Molecular mass and pI determination

The molecular mass of the lectin as determinedby SDS±PAGE was 40 kDa (Figure 1). The pI ofthe lectin was 4.0.

pH and temperature stability of the lectin and¯occulation

Comparison of pH and temperature stability ofthe puri®ed S. cerevisiae (NCIM 3528) cell walllectin, and ¯occulation of the cells, was carriedout. The lectin was incubated in universal bufferwith a pH range of 3±10 for 60 min at 25uC andthe haemagglutination activity was determined.The ¯occulation level of the cells at different pHswas checked by suspending cells in buffers (50 mM,pH range 3±10), containing 5 mM CaCl2. Afterincubation for 60 min on a rotary shaker(150 rpm), the extent of ¯occulation was deter-mined. The lectin was stable at acidic and neutralpH values but gradually lost its activity at alkalinepH values, whereas S. cerevisiae (NCIM 3528)cells showed more or less similar ability to¯occulate at all pH values (3±10) with slightlyhigher ¯occulation at acidic pH.

For temperature stability, the lectin was incu-bated in TBS, whereas the cells were incubated inacetate buffer (50 mM, pH 4.5) containing 5 mM

CaCl2 at various temperatures (8±70uC) for60 min. The lectin was found to be unstable athigh temperatures, with complete loss of activityoccurring at 60uC. The cells also lost their ability

Figure 1. SDS±PAGE of S. cerevisiae (NCIM 3528) cell walllectin. Lane 1, Molecular weight markers: bovine serumalbumin (66 000), ovalbumin (45 000), glyceraldehyde-3-phos-phate dehydrogenase (36 000), carbonic anhydrase (29 000),trypsinogen (24 000) Lane 2, puri®ed cell wall lectin.



to ¯occulate with increase in temperature, withalmost complete de¯occulation occurring at 70uC.

Effect of metal ions on lectin activity and¯occulation

The effect of metal ions on ¯occulation as wellas lectin activity was checked. The lectin wasincubated for 60 min in the presence of variousmetal ions (5 mM) in TBS at 25uC and thehaemagglutination activity was determined. Thecells were incubated in Tris±HCl (pH 7.2) contain-ing metal ions (5 mM) on a rotary shaker(150 rpm) for 60 min at 25uC and the ¯occulationlevel was checked. The puri®ed lectin did not showan obligatory requirement for added metal ionsfor haemagglutination activity. The activityremained unaffected in the presence of Ca2+,Na+ and Zn2+, whereas all other metal ions wereinhibitory. In case of S. cerevisiae (NCIM 3528)cells, at pH 7.2, although almost 80% of the cellscould ¯occulate in the absence of the metal ions,addition of Ca2+ and Zn2+ increased the extent of¯occulation. Na+ and Mg2+ ions did not affectthe ¯occulation levels, whereas Ba2+, Mn2+ andAl3+ caused almost complete de¯occulation.Treatment with EDTA resulted in completeinhibition of the haemagglutination activity ofthe lectin, and also abolished ¯occulation of thecells (see Figure 3a), indicating that trace amountsof metal ions are required for the lectin activity aswell as ¯occulation.

Amino acid composition and N-terminalsequencing of the lectin

Amino acid analysis of the lectin indicatedits hydrophobic nature, with approximately 44%hydrophobic amino acids (Table 1). The aminoacid residues at positions 5 and 6 could not bedetermined unambiguously during the N-terminalsequencing of the lectin due to high backgroundnoise. The ®rst 24 amino acids at the N-terminusof the FLO1 gene product represent a signalsequence, whereas the sequence of the nativeprotein probably starts at position 25 (Teunissenet al., 1993). The homology search carried outusing Saccharomyces Genome Databases showedthat the N-terminal sequence of our lectin shared70% homology with the putative FLO1 and FLO5gene product (Figure 2). Other FLO1 homologues,such as FLO10 and FLO9, shared 60% and 40%N-terminal sequence identity, respectively, withthe lectin. The amino acid in the ®fth position of

all FLO genes is cysteine (Figure 2). The inabilityof Edman's degradation procedure to identifyunderivatized cysteine residues probably explainsits failure in detecting the ®fth residue duringsequencing. The homology search indicated thatthe probability of other Saccharomyces proteinshaving homology with this sequence is slightlyhigh for 40% homology and very low for 70%homology.

Sugar speci®city of the lectin and ¯occulation

Sugar speci®city of S. cerevisiae (NCIM 3528)lectin was determined by haemagglutination inhi-bition assay. The effect of sugars on ¯occulationwas investigated by incubating the cells withvarious sugars (1 M) for 60 min at 25uC on arotary shaker and the free cell concentration wasdetermined spectrophotometrically. When testedwith monosaccharides, haemagglutination by thepuri®ed lectin was inhibited by aminated sugars aswell as methyl a-mannoside at a concentration of62 mM (Table 2), whereas mannose could inhibitthe lectin only at 125 mM. None of the othersugars tested could inhibit haemagglutination at aconcentration as high as 250 mM. The ¯occulationof S. cerevisiae (NCIM 3528) cells was almostcompletely inhibited by mannose and methyla-mannoside at a concentration of 1 M. All other

Table 1. Amino acid composition of the cell walllectin.

Amino acid Mol/Mol 40 kDa

As (x) 35Thr 20Ser 26Gl (x) 40Gly 47Ala 46Cys* 4Val 23Met 6Ile 16Leu 29Tyr 10Phe 13His 6Lys 19Arg 18Pro 31Trp** 4

*Cys was determined according to Cavallini et al. (1966).**Trp was determined according to Spande and Witkop (1967).



sugars tested failed to de¯occulate the cells.Amongst the oligosaccharides, mannose disac-charides did not inhibit the lectin activity at thehighest concentration tested (260 mM), but thelectin showed high af®nity towards the branchedtrimannoside, with the lowest inhibitory concen-tration being 2 mM (Table 3). The pentasaccharideof mannose also could not inhibit the lectinactivity, even at a concentration of 100 mM.

Glycoproteins having asparagine-linked carbo-hydrate chains have branched trimannoside as apart of the core structure of their glycosylmoieties. Haemagglutination inhibition carriedout using glycoproteins showed the lectin tobe highly speci®c towards Man-9 glycopeptide(obtained from soyabean lectin) and was inhibitedat low concentration (0.2 mg/ml). It also showedsome af®nity towards fetuin (Type III from fetal

calf serum) and ®brinogen (Fraction I fromhuman plasma). Chitobiose and Man b 1±4GlcNAc, which also occur in the pentasaccharidecore structure of the glycosyl moiety of theseglycoproteins, could not inhibit lectin activity,con®rming the speci®city of the lectin towardsthe branched trimannoside. However, the lectinshowed an odd pattern of haemagglutinationinhibition in presence of high mannose-typeglycoproteins such as ribonuclease B, yeastinvertase and ovalbumin. Although partial inhibi-tion was observed in all wells, complete inhibitioncould not be obtained, even at a concentration of2.5 mg/ml (data not shown).

Trimannoside, speci®c for S. cerevisiae (NCIM3528) cell wall lectin, also completely inhibitedthe ¯occulation of cells (Figure 3b). The concen-tration required to inhibit the ¯occulation was

Figure 2. Comparison of the N-terminal sequence of S. cerevisiae (NCIM 3528) cell wall lectin with that deduced from FLOgenes. The arrow indicates the predicted signal cleavage site of the FLOp. Asterisks represent amino acids which could not beidenti®ed.

Table 2. Inhibition of haemagglutination activity of the cell wall lectin and ¯occulation of S. cerevisiae (NCIM3528) cells in presence of monosaccharides.

Haemagglutination inhibitory Free cells**Monosaccharide concentration (mM) (%)

Glucose ±* 23D-Glucosamine 62 NDGalactose ± 15D-Galactosamine 62 NDMannose 125 86D-Mannosamine 62 NDMethyl a-mannoside 62 87

ND=Not determined.*No inhibition at the highest concentration tested (250 mM). Other sugars, such as N-acetyl glucosamine, methyl a-glucoside,N-acetyl galactosamine, methyl a-galactoside, methyl b-galactoside, xylose, lyxose, ribose, arabinose, fucose, rhamnose were non-inhibitory to the haemagglutination activity at the highest concentration tested (250 mM).**Percentage of free cells were determined spectrophotometrically after incubation of the cells in presence of various sugars (1 M)for 60 min at 25uC on a rotary shaker, as described in Materials and Methods. Free cell concentration in the absence ofmonosaccharides was 12%. Sugars such as xylose, lyxose, ribose, arabinose, fucose and rhamnose were non-inhibitory to the¯occulation of S. cerevisiae cells at 1 M concentration.



high (10 mM) compared to that of haemaggluti-nation, which can be correlated to the clustereffect of the natural ligand present on the yeastcell wall.

Localization of the lectin and de¯occulation ofcells with lectin-speci®c IgGs

The puri®ed lectin was immobilized on Sephar-ose 4-B and the lectin-speci®c IgGs were puri®edusing this as an af®nity matrix. Of the total IgGs,15% were found to be lectin-speci®c. These werelabelled with FITC and were used to localize thelectin on the intact yeast cells. Figure 4 shows S.cerevisiae (NCIM 3528) cells uniformly stainedwith FITC-labelled lectin-speci®c IgGs, indicatingthe presence of the lectin on the cell surface. In thecase of budding cells, however, intense staining of

the buds and at the neck of the mother±daughterjunction can be observed, suggesting the preferredlocalization of the lectin at these places.

The ¯occulation ability of S. cerevisiae (NCIM3528) cells was checked in the presence of theunlabelled lectin-speci®c IgGs. Figure 3c showscomplete failure of the cells to ¯occulate in thepresence of lectin-speci®c IgGs.

DISCUSSION

The phenomenon of asexual ¯occulation of yeastcells, leading to reversible cell aggregation andrapid sedimentation of the aggregates from thesuspending medium, is in¯uenced by variousenvironmental, physiological and genetic factors(Stewart and Russell, 1981). Although molecularmechanisms of ¯occulation are still not very clear,

Table 3. Inhibition of haemagglutination activity of the cell wall lectin in presence of oligosaccharides andglycoproteins.

Oligosaccharide Inhibitoryor glycoprotein Oligosaccharide structure concentration

Mannobiose Man a 1±3 Man ±*Mannobiose Man a 1±2 Man ±Mannobiose Man a 1±6 Man ±Chitobiose GlcNAc b 1±4 GlcNAc ±

Man b 1±4 GlcNAc ±

Mannotriose 2.5 mM

Mannopentaose ±

Man-9 glycopeptide 0.2 mg/ml

Fibrinogen1 10.0 mg/ml

Fetuin2 20.0 mg/ml

Abbreviations: G=D-galactose; GN=N-acetyl glucosamine; M=D-mannose; N=N-acetyl- neuraminic acid.*No inhibition at the highest concentration tested.1,2Townsend et al. (1982), Green et al. (1988).



the involvement of cell surface lectin-like mole-cules in ¯occulation is now generally accepted.

The ¯occulent strains are known to exhibit two

different ¯occulation phenotypes, viz. Flo1 andNewFlo, which differ in their sugar speci®city aswell as salt, protease and pH sensitivity of the¯occulation (Stratford and Assinder, 1991). Flo1strains are generally laboratory strains constitu-tively expressing ¯occulence throughout growth,and contain the genes FLO1, FLO5 or FLO8,whereas strains with the NewFlo phenotype aregenerally brewing strains, the genotype of which isnot known.

The ¯occulent strain of S. cerevisiae used in thepresent investigation has been isolated from abrewery and the genotype of the strain is notknown. It is likely to be a Flo1 strain, since it is aconstitutively ¯occulent strain exhibiting typicalFlo1 ¯occulation characteristics, such as theability to ¯occulate in a broad range of pHvalues and speci®city for mannose and its deriva-tives. The puri®ed cell wall lectin shows pH andtemperature stability, EDTA sensitivity and sugarspeci®city similar to that of ¯occulating cells. Theslightly higher stability of ¯occulation of the cells

(a) (b)

(c) (d)

Figure 3. Inhibition of ¯occulation of cells of S. cerevisiae (NCIM 3528) in presence of: (a) EDTA (100 mM); (b) branchedtrimannoside (10 mM); (c) lectin-speci®c IgGs (100 mg); (d) cells in ¯occulation buffer without any de¯occulation agent.

Figure 4. Localization of S. cerevisiae (NCIM 3528) cell walllectin by FITC-labelled lectin-speci®c IgGs. The S. cerevisiaecells (A600=1.5) were stained with 100 mg of labelled lectin-speci®c IgGs and examined under a ¯uorescence microscope.



at high temperatures and alkaline pH values overthe isolated lectin could be due to the higherstability of the lectin±carbohydrate complexformed during ¯occulation, as well as the stabilityprovided by the cell wall. Metal ions are known toaffect ¯occulation in a pH-dependent manner. Atneutral or high pH values, low concentrations ofmetal ions such as Ca2+, Mg2+, Zn2+ and Cd2+

are shown to be equally effective in inducing¯occulation (Calleja, 1987) and our ®ndings areconsistent with that. Metal ions other than Ca2+

promote ¯occulation indirectly by causing Ca2+

leakage from cells, which in turn initiates ¯occula-tion (Stratford, 1989). This may be the reason whyMg2+ ions were found to favour ¯occulation inspite of being inhibitory to lectin activity.

S. cerevisiae (NCIM 3528) cell wall lectin seemsto be different from those reported from other¯occulent S. cerevisiae strains with respect to itsmolecular mass and temperature stability. In caseof sugar speci®city, however, it showed speci®citytowards mannose similar to the lectins from other¯occulent strains (Shankar and Umesh-Kumar,1994; Straver et al., 1994). Its higher af®nitytowards the oligosaccharides was expected, sincethese are known to represent the natural ligand ofthe lectin. The lectin exhibited high af®nity forMan-9 glycopeptide. The inability of high man-nose-type glycoproteins to inhibit the lectin, inspite of having many Man-9 oligosaccharide units,could be due to the stearic hindrance offered bythe protein moiety of the glycoproteins in lectin±carbohydrate interaction.

The lectin-speci®c IgGs were found to inhibitthe ¯occulation completely, suggesting the roleof lectin in ¯occulation. However, it is possiblethat the IgG molecule inhibits ¯occulation non-speci®cally due to stearic hindrance, thus affectingthe lectin±carbohydrate interaction. To rule outthis possibility, we generated monovalent Fabfragments of the lectin-speci®c IgGs, which werealso found to be equally effective in inhibiting the¯occulation (data not shown).

The monovalent nature of the protein involvedin ¯occulation has been suggested previously(Stewart et al., 1995). Isolated S. cerevisiae(NCIM 3528) lectin, however, has the ability toagglutinate rabbit erythrocytes, indicating itsmultivalency. Being hydrophobic in nature, thelectin has a tendency to aggregate. Aggregation ofother lectin molecules isolated from other ¯occu-lent strains also has been reported (Shankarand Umesh-Kumar, 1994; Straver et al., 1994).

Since the lectin molecules tend to aggregate, thepossibility of generation of apparent multivalencycannot be denied, which in turn results in theagglutination of rabbit erythrocytes.

The N-terminal domain of the putative FLO1gene product shares common features with legumelectins (Bidard et al., 1995; Teunissen et al., 1995).Inhibition of ¯occulation by Concanavalin A(Con A) has been reported, which can be reversedby the addition of methyl a-mannoside (Miki et al.,1982; Stratford, 1993) and it was also con®rmed inour laboratory. These observations indicate thatthe lectin involved in ¯occulation shares acommon carbohydrate receptor with Con A; thebranched trimannoside (Baenziger and Fiete,1979). The cell wall lectin from S. cerevisiae(NCIM 3528) also showed high speci®city andaf®nity towards the branched mannose trisacchar-ide. The inhibition of ¯occulation by the branchedtrisaccharide of mannose provides direct evidencefor the involvement of the lectin in ¯occulationand strongly suggests that the trimannoside couldbe the putative carbohydrate receptor of the lectinduring ¯occulation.

Repeated extraction of the cell walls by homo-genization in presence of the detergent was neededto extract the majority of the lectin, whichsuggested ®rm anchoring of the lectin to the cellwall. The ability of the lectin to become solubi-lized with the detergent indicated its non-covalentlinkage to the cell wall ®brils. Recently, Bony et al.(1997) suggested a similar non-covalent but ®rmassociation of the putative Flo1p with the cell walland proposed that it is a true cell wall proteinwhich is only transiently anchored in the plasmamembrane by the GPI anchor. Immuno¯uor-escence studies using FITC-labelled antibodieswere carried out on intact yeast cells withuntreated cell walls, thus preventing the accessionof IgGs to the inside of the cells. These studiessuggest the presence of the lectin on the surface ofS. cerevisiae (NCIM 3528) cells. A more pro-nounced ¯uorescence observed at the neck of themother±daughter junction in budding cells impliedlocalization of the lectin at these junctions. This isconsistent with the studies of Bony et al. (1998),describing the distribution of Flo1p on the cellsurface of the FLO1 and FLO5 strains of S.cerevisiae, which revealed polarized distribution ofFlo1p on the cell surface at the bud tips and at themother±daughter neck junction.

N-terminal sequence analysis of the puri®edlectin yielded a sequence of up to 10 residues,



which, although not really ideal to seek forhomology, was suf®cient to give an indicationthat the puri®ed lectin is encoded by the ¯occula-tion gene homologous to the FLO1 gene. Based onthe sequence homology, it has been proposed thatthe products of FLO5 and FLO9 genes are 96% and94% similar to Flo1p, respectively, whereas Flo10pis 58% similar (Teunissen et al., 1995). The overallproperties assigned to these gene products fromnucleotide sequence data are: a molecular mass ofabout 170 kDa; high Ser-Thr contents; heavyglycosylation; and the presence of hydrophobic Nand C terminal domains. The S. cerevisiae (NCIM3528) lectin, however, differs from the putativegene product of these FLO genes in aspects such asmolecular mass and the percentage of Ser-Thrresidues. It has a much lower molecular mass thanthat proposed for a FLO gene product and thepercentage of Ser-Thr residues in lectin is less (12%)than the predicted value.

On the basis of gene sequence, the putativeFlo1p has been predicted to consist of a hydro-phobic N-terminus (248 amino acids) exposedtowards the cell surface and containing thesaccharide binding site; the middle rigid, stem-like cell wall-spanning region (1162 amino acids)consisting of heavily glycosylated Ser-Thr-richrepeats; and the hydrophobic C-terminus (103amino acids) (Teunissen et al., 1995). It has alsobeen predicted that the Flo1p could be attached tothe plasma membrane and span the whole cellwall, with middle extended Ser-Thr-rich repeats toreach out to the outer surface of the cell andexpose the N-terminal region (Bidard et al., 1995).Strain polymorphism at the level of Ser-Thr-richrepeated sequences has already been reported(Watari et al., 1994). The most deleted forms ofthe Flo1p, lacking Ser-Thr-rich repeats, were alsoshown to induce ¯occulation, although withreduced intensity (Bidard et al., 1995). It ispossible that the strain used in the presentinvestigation contains a gene homologous to theFLO1 gene encoding for a low molecular weightprotein which may be a highly deleted version ofthe Flo1p. The high intensity of ¯occulationobserved in this strain would require the N-terminal regions of even a highly deleted form ofthe protein to span the cell wall and to extend tothe outer cell surface, contrary to the earlierobservation of Bidard et al. (1995). The possibilityof the puri®ed lectin being a proteolytic cleavageproduct formed during the extraction also cannotbe ruled out. In a recent report, however, it has

been suggested that Flo1p does not necessarilyspan the whole cell wall, as suggested earlier, butthat it is more likely that the protein is onlytransiently anchored to the plasma membranebefore being incorporated in the cell wall, and islocated in the external mannoprotein layer of thecell wall (Bony et al., 1997). This raises thepossibility of post-translational modi®cation ofthe Flo1p, resulting in a low molecular weight®nal product, which is incorporated in the outerlayer of the cell wall.

In conclusion, the cell surface lectin, isolatedand puri®ed from the constitutively and highly¯occulent brewer's strain, S. cerevisiae (NCIM3528), in the present investigation, is a putativeactive molecule in ¯occulation behaviour. Theinvolvement of the lectin in ¯occulence exhibitedby this strain is supported by the following®ndings: (a) lectin-speci®c IgGs could preventthe ¯occulation; (b) the strongest ligand of thelectin, a branched trimannoside, inhibited the¯occulation; and (c) there was a positive correla-tion between lectin stability and the ¯occulation ofcells under different environmental conditions.The N-terminal sequence and the localization ofthe lectin showed similarity with that of theputative Flo1p indicating that this ¯occulation-speci®c lectin could be possibly encoded by one ofthe FLO genes.

ACKNOWLEDGEMENTS

We thank Professor A. Kolaskar, Department ofBioinformatics, University of Pune, for allowingus to carry out the Saccharomyces GenomeDatabase search. This work was supported bythe Department of Science and Technology,Government of India. The award of a SeniorResearch Fellowship to V. S. Javadekar by theCouncil of Scienti®c and Industrial Research isgratefully acknowledged.

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a mannose-binding protein from the cell surface of flocculentsaccharomyces cerevisiae (ncim 3528):...

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