YEAST

v o ~7:. 7 17-726 ( I99 1 )

Cell Wall Glucomannoproteins of Saccharomyces cerevisiae mnn9 JOHANNA VAN RINSUM*, FRANS M. KLlS A N D HERMAN VAN DEN ENDE Department of Moleculur Cell Biology. Biotechnology Center, University of Amsterdam, Kruislaan 318, 1098 S M Amsterdam. The Netherlands Received 31 January 1991; accepted 20 May 1991

Mannoproteins were isolated from Saccharomyces cerevisiue mnn9 mutant cell walls by laminarinase digestion and purified by affinity and anion-exchange chromatography. The purified mannoprotein fraction contained three predominant proteins with molecular masses of 300 kDa, 220 kDa and 160 kDa. These compounds were absent in an SDS extract of cell walls or in a hot-citrate extract of mnn9 cells. The carbohydrate part of the purified mannoproteins consisted of (N-acetyl)glucosamine, mannose and glucose in a molar ratio of 1:53:4. 0-Glycosidically linked chains, containing 70% of the mannosc, were released by mild 0elimination. N-Glycosidically linked chains, representing 80% of the (N-accty1)glucosamine and 20% of the mannose, were released by peptide N-glycosidase F (PNGase F) digestion. Complete degradation of protein by alkaline hydrolysis released besides the N- and 0-glycosidically linked chains, another type of carbohydrate chain containing the residual (N-acetyl)glucosamine, mannose and most of the glucose in a molar ratio of l:l7:l8. Glucose was P-glycosidically linked. Theresultsindicatethat P-glucoseislinked toPNGase F-resistant N-linkedchainsprcsentoncell wallmannoproteins. We propose that these chains are responsible for the linkage between mannoproteins and glucan in the cell wall. KEY WORDS - Mannoprotein; glucan; mannan;

N-glycosylation.

INTRODUCTION The cell wall of Succhuromyces cerevisiae is composed of equal parts of P-1,3-#-1,6-glucan and mannan and a small amount of chitin (Manners et al., 1973a.b; Fleet and Phaff, 1981; Ballou, 1982; Cabib et al., 1982). In Cundidu ulbicans, whose cell wall structure resembles that of S. cerevisiue (Shepherd rt ul., 1985), P-1,6-glucan is covalently linked to chitin, a P-1,Clinked homopolymer of N-acetylglucosamine (Surarit et ul., 1988). The mannan fraction consists of complex glycoproteins carrying large mannose polysaccharides, N-glycosidically linked to asparagine residues via a diacetylchitobiosyl unit, as well as short mannosyl chains. 0-glycosidically linked to serine or threonine residues (Ballou, 1982). Two types of mannoprotein exist. One type can be extracted from cell walls by SDS and comprises 80% of the cell wall protein (Valentin et al., 1984; Elorza ei al., 1985). The other type can be isolated from cell walls by p1.3-glucanase digestion (Pastor et al., 1984; Elorza et ul., 1985). 0749-503x19 I !070717- 10 S05.(w) D 1991 by John Wiley &Sons Ltd

The SDS-extractable mannoproteins are only 0mannosylated, except for a 29 kDa mannoprotein, and have relatively low molecular masses ( < 100 kDa) (Pastor et ul., 1984; Elorza et al., 1985; Sanz et al., 1989), whereas the P-glucanaseextractable mannoproteins contain both N- and 0linked carbohydrate chains (Pastor et al., 1984; Frevert and Ballou, 1985; Elorza er al., 1985).Since 90% of the cell wall mannose in wild-type cells is N-glycosidically linked (Cohen and Ballou, 198I ; Zuecoeral., 1986),the P-glucanase-extractablemannoproteins account for the majority of mannose present in wild-type cell walls. The ability of P-1,3-glucanase to liberate mannoproteins from cell walls suggests that they are covalently linked to glucan (Pastor ef ul., 1984).Another indication for a covalent linkage between glucan and mannoproteins is the presence of glucose in P-glucanase-extracted mannoproteins (Kitamurd, 1982; Shibata et ul., 1983; Elorza et ul., 1988). Furthermore, isolated glucan fractions contain mannose residues (Fleet and Manners, 1976, 1977), protein (Andaluz et ul., 1988) or N-glycosidically

718

J. VAN

RINSUM, F. M. KLIS AND H. VAN DEN ENDE

linked chains (Tkacz, 1984). Regeneration of protoplasts in the presence of papulacandin B, which inhibits glucan synthesis, results in the secretion of mannoproteins into the medium (Murgui et al., 1985, 1986). This suggests that the formation of an intact cell wall glucan network is necessary for the incorporation of mannoproteins. It has been suggested that the mannoproteins are linked to p1,6-glucan (Fleet and Manners, 1977; Tkacz, 1984; Shepherd et a [ . , 1985). The nature of this binding is still unknown. We have studied the attachment of glucan to mannoproteins in cell walls of the mnn9 mutant. This mutant has truncated N-glycosidically linked manno-oligosaccharide chains (Tsai et al., 1984). Our results show that cell wall mannoproteins contain P-glucose present on chains, which can be distinguished from 0-or N-glycosidicallylinked chains by their resistance towards mild p-elimination or peptide N-glycosidase F (PNGase F) respectively.

pH 5.5, containing 1 mM-PMSF and laminarinase (Sigma, U.S.A.; 0.5 U P-1,3-glucanase/g ww cell wall). The mixture was incubated at 35°C for 4 h. The undigested cell wall material was pelleted at 10 000 x g for 10 min. The supernatant was desalted on a Bio-Gel P-6 column (Econo-Pac lODG, BioRad, Richmond, CA, U.S.A.) equilibrated in water, and lyophilized. To extract non-covalently linked mannoproteins, cell walls were extracted in 2% SDS at 100°C for 5 min (Elorza et al., 1985). Total cell mannoproteins were extracted by autoclaving mnn9 cells in 0.02 Mcitrate buffer, pH 7.0 for 90 min (Nakajima and Ballou, 1974).

Extraction of mannoproteins To extract covalently linked mannoproteins, cell walls were suspended in 100 mM-sodium acetate,

8-Elimination

Concanavalin A-afinity chromatography The desalted, lyophilized laminarinase digest from cell walls was dissolved in 50mM-Tris-HC1, pH 7.4, containing 0.25M-NaC1, and applied on a Concanavalin A (Con AkSepharose (Pharmacia AB, Uppsala, Sweden) column (3 ml bed volume/ MATERIALS AND METHODS mg protein), equilibrated with the same buffer. The column was washed with 3 bed volumes of 5 0 ~ Yeast strains and growth Tris-HC1, pH 7.4, containing 0.5 M-NaC1, and the Saccharomyces cerevisiae X2180-1B ( M A T a ; mannoproteins were eluted with 2 bed volumes of referred to as wild type) was obtained from the 50 mM-Tris-HC1, pH 7.4, containing 0.5 M-NaCl Yeast Genetic Stock Center (Berkeley, CA, U.S.A.) and 0.5 M-methyl-a-D-mannopyranoside (Sigma, S. cerevisiae LB347-1C (mnn9, MATa) was kindly U.S.A.).The eluate was lyophilized, desalted on a donated by Dr L. Ballou (Dept of Biochemistry, Bio-Gel P-6 column and lyophilized again. University of California, Berkeley, CA, U.S.A). Cells were grown at 28°C in YPD medium (1% (w/ DEAE-Trisacryl anion-exchange chromatography v) yeast extract (Gibco), 1YO (w/v) Bactopeptone The desalted, lyophilized mannoprotein fraction (Difco), 3% (w/v) glucose). was dissolved in 20mM-Tris-HC1, pH 7.4, and fractionated on a DEAE-Trisacryl (Pharmacia AB, Isolation of cell walls Uppsala, Sweden) column (0.5 x 4-0 cm/mg proWild-type cells and mnn9-a cells were collected in tein) equilibrated with 20 mM-Tris-HC1, pH 7.4. the exponential phase with a continuous flow centri- The mannoproteins were eluted with 300 mM-NaCl fuge at 1000 x g . The cells were washed 3 times in 20 mM-Tris-HC1, pH 7.4. with 10 mM-Tris-HC1, pH 7.4, containing 1 mMphenylmethylsulfonyl fluoride (PSMF). A cell a-Glucosidase and p-glucosidase digestion suspension (0.1 g wet weight (ww)/ml) in 10mMMannoproteins were incubated with a- or pTris-HC1, pH 7-4, 1 mM-PMSF, was shaken in a glucosidase (Boehringer Mannheim, Germany; EC Bead-Beater with glass beads (diameter 0.5 mm, 3.2.1.20 and EC 3.2.1.21, respectively; 1 U enzyme/ 10 g beads/g ww cells) at 0°C 5 times for 1 min. Glass mg mannoprotein in a total volume of lOOp1) in beads were separated from the cell walls by filtration 100 mM-sodium acetate, pH 6-8 or 4.5, respectively, on a nylon filter. Cell walls were pelleted and washed at 37°C for 24 h. The products were separated by gel 3 times with 1 M-NaCI containing 1 mM-PMSF and filtration on a Bio-Gel P-6 column equilibrated in 3 times with 1 mM-PMSF. water.

Mannoproteins were subjected to p-elimination under reductive conditions by incubation in 0.1 M-

CELL WALL GLUCOMANNOPROTEINSOF SACCHAROMYCES CEREVISIAE M ” 9

NaOH, containing 1 M-NaBH,, at 20°C for 24 h. The excess of BH; was eliminated by the addition of 4 M-acetic acid at 0°C until a pH of 5 was reached. The released carbohydrate chains were separated from the protein by gel filtration chromatography on a Bio-Gel P-2 (Bio-Rad, Richmond, CA, U.S.A.) column (1 cm x 100 cm) equilibrated in water. Peptide N-glycosidase F (PNGase F) digestion Mannoproteins, dissolved (6 mg/ml) in 100 mMNa,HPO,, pH 8.0, containing 0.5% (w/v) SDS and 100 mM-P-mercaptoethanol, were boiled for 5 min. De-N-glycosylation was performed in an incubation mixture containing 6 mg mannoprotein, 0.4% (w/v) SDS, 100mM-Na,HP04, pH 8.0, 3% (v/v) Triton X-100, 50 mM-EDTA, 0.8 mM-PMSF and 20mU PNGase F (Boehringer Mannheim, Germany; EC 3.5.1.52) in a total volume of 1.8 ml. PNGase F was introduced by three sequential additions at 24-h intervals. The mixture was incubated at 37°C for 72 h. Triton X- 100 was removed by passing the incubation mixture over Amberlite XAD-2 (Sigma, U.S.A.; Cheetham, 1979). The de-N-glycosylated mannoproteins were separated from the N-glycosidic carbohydrate chains by gel filtration chromatography on a Bio-Gel P-10 (Bio-Rad, Richmond, CA, U.S.A.) column (1 cm x 100 cm) equilibrated in water. Isolation of glucose-containingcarbohydrate chains Incubation of mannoproteins in 0.1 M-NaOH containing 1 M-NaBH, at 37°C for 72 h resulted in complete protein degradation. The excess of BH, was eliminated by the addition of 4 M-acetic acid at 0°C until a pH of 5 was reached. Cations were removed by passing the incubation mixture over Dowex AG50 (X2, 10&200 mesh, H+-form; BioRad, Richmond, CA, U.S.A.). The eluate and two bed volumes of 10mM-formic acid wash were pooled and lyophilized. Boric acid was evaporated as methylborate by repeated additions of methanol containing 5% (v/v) of acetic acid. SDS-polyacrylamide electrophoresis (SDS-PAGE) Electrophoresis was performed on linear gradient (2.2-20%) polyacrylamide gels according to Laemmli (1970). Gels were stained by the silver staining method as described by De Nobel et al. ( I 989).

719

High-pH anion-exchange chromatography with pulsed amperometric detection To determine the carbohydrate composition, the various desalted fractions were hydrolysed in 2 Mtrifluoroacetic acid (TFA) at 100°C for 4 h. Since this results in deacetylation of N-acetylglucosamine (GlcNAc), no discrimination between GlcNAc and glucosamine (GlcNH,) could be made. The GlcNH, detected was referred to as GN, indicating that either GlcNAc or GlcNH, is present. The TFA was evaporated, and the monosaccharides were separated on a CarboPac PA1 anion-exchange column (4 x 250 mm; Dionex, Sunnyvale, CA, U.S.A.), equipped with a CarboPac PA guard column (3 x 25 mm, Dionex). Elution was performed with 15 mM-NaOH and monosaccharides were detected with the pulsed amperometric detector PAD I1 with a gold electrode (Dionex; Hardy et al., 1988). Reference monosaccharide solutions were used to determine retention times and concentration-dependent peak areas. Analytical methods Protein was assayed with the BCA-protein assay reagent (Pierce, Rockford, IL, U.S.A.) with bovine serum albumin as reference protein. Carbohydrate content was measured with the phenol-sulphuric acid method (Dubois et al., 1956) with mannose as reference. Reducing sugars were detected by the Nelson-Somogyi copper reduction method (Spiro, 1966). Other materials Laminarin (p- 1,3-glucan) was purchased from Fluka AG, Buchs, SG, Switzerland. Pustulan (p1,6-glucan) was obtained from Calbiochem, La Jolla, CA, U.S.A. Mannan, various p-nitrophenyl substrates, mannitol, glucitol, Nonidet P-40 and fluorescamine came from Sigma, U.S.A. BioGel P-60, acrylamide, bisacrylamide, N,N,N,Ntetramethylethylene-diamine and Triton X-100 were obtained from Bio-Rad, Richmond, CA, U.S.A. A standard protein electrophoresis calibration kit came from LKB, Bromma, Sweden. Endo-P-N-acetylglucosaminidase H (Endo H, EC 3.2.1.96) was purchased from Boehringer Mannheim, Germany. Glucosaminitol was prepared by incubating GlcNAc in 0-1 M-NaOH, containing 1 M-NaBH,, at 20°C for 4 h; the resulting

720

J. VAN RINSUM, F.

lane

a

b

C

d

e

f

M. KLIS AND H. VAN DEN ENDE

g

Figure 1. SDS-PAGE patterns of various mannoprotein fractions isolated from mnn9 cells. Lane a: SDS extract from cell walls; lane b: whole cell mannoproteins isolated by hot-citrate extraction; lane c: purified mannoproteins from the hot-citrate extract; lane d: laminarinase extract from cell walls; lane e: Con A-Sepharose-retarded fraction of laminarinase; lane f: purified cell wall mannoproteins; lane g: purified cell wall mannoproteins after Endo H digestion. The positions of several standard proteins as well as those of major compounds present in the mannoprotein fraction are indicated.

the digest contained various proteins with molecular masses in the range of 35-740 kDa. From the laminarinase preparation no compounds were retarded on Con A-Sepharose (Figure 1, lane e). When mannoproteins present in the laminarinase RESULTS digest were purified by Con A-Sepharose affinity Isolation andpurEfication of cell wall mannoproteins chromatography and anion-exchange chromatography on DEAE-Trisacryl, ten different proteins Mannoproteins, covalently linked to glucan, can were found with molecular masses in the range be liberated from the cell walls by P-1,3-glucanase of 3 5 4 5 0 kDa; bands at 160 kDa, 220 kDa and digestion (Kitamura, 1982; Shibata et al., 1983; 300 kDa represented the major compounds (Figure Pastor et al., 1984). We used laminarinase, which I , lane f). SDS, which extracts the non-covalently contained besides P- 1,3-glucanase activity also 7 % linked mannoproteins from cell walls (Valentin et and 6% of P-1,6-glucanase and a-mannanase al., 1984; Elorza et al., 1985),liberated a different set activity respectively, when tested with pustulan and of mannoproteins with masses in the low molecular mannan as substrates. Laminarinase was free of range (Figure 1, lane a). Mannoproteins were also protease activity towards casein when tested isolated by hot-citrate extraction, precipitated with according to Evans and Ridella (1984), and methanol (Nakajima and Ballou, 1974) and purified showed no exo-a-mannanase, exo-P-glucanase, by Con A-Sepharose affinity chromatography and exo-chitinase, or acid phosphatase activity towards DEAE anion-exchange chromatography. Analysis by SDS-PAGE of this mannoprotein fraction the corresponding p-nitrophenyl substrates. Analysis of the laminarinase digest of mnn9 cell (Figure 1, lanes b and c), showed the presence of walls by SDS-PAGE (Figure I , lane d) revealed that one major compound with a molecular mass of N-acetylglucosaminitol was deacetylated by hydrolysis in 2 M-TFA at 100°C for 4 h. All other chemicals were of analytical grade.

72 1

CELL WALL GLUCOMANNOPROTEMS OF SACCHAROMYCES CEREVISIAE M " 9

Table 1. Purification of mannoproteinsfrom 10 g ww mn9 cell walls

YORecovery

Step

(mg)

(%)

(mg)

(%)

GN

Man

Glc

Molar ratio GN:Man:Glc

Cell walls Laminarinasedigest Con A-Sepharose DEAE-Trisacryl

210 62* 8 6

100

376 256 13 11

100 68 3 2

100 55 1.9 1.1

100 49 16 14

100 74 0.5 0.3

1: 4:18 1: 4124 1:36: 5 1:53: 4

Protein

Carbohydrate

30 4 3

Abbreviations: GN = (N-acety1)glucosamine; Man = mannose; Glc= glucose; ww = wet weight. *Corrected for the amount of protein present in laminarinase.

150 kDa. This compound was not present in the laminarinase digest or in the SDS extract of cell walls. According to Frevert and Ballou (1985), this protein is a cell wall mannoprotein. The carbohydrate part of the isolated cell walls of mnn9 consisted of 77% glucose, 19% mannose and 4% G N (N-acetylglucosamine or glucosamine, see Materials and Methods section). Cell walls isolated from wild-type cells consisted of 54% glucose, 43% mannose and 3% GN. In both wild-type and mnn9 cell walls the absolute amount of glucose was approximately the same (30 mg/g ww cell wall); only the amount of mannose was decreased in mnn9 cell walls, due to the absence of the outer chain in the N-glycosidically linked chains (Tsai et al., 1984). Table 1 summarizes the composition of fractions obtained by laminarinase digestion of mnn9 cell walls and purification of the mannoproteins from the digest. Laminarinase extracted 30% of the proteins, 49% of the mannose and 74% of the glucose present in the cell wall. The laminarinase preparation itself contained only a small amount of glucose, representing less than 0.5%of the total glucose present in the digest. Of the G N present in the cell walls, 55% was extracted by laminarinase (Table l), of which one fifth part derived from the laminarinase preparation. The laminarinase preparation contained no mannose. When the laminarinase digest was passed over Con A-Sepharose, only 13% of the protein and 32% of the mannose was retained. In this retained fraction 1.9% of the G N was recovered and the fraction still contained glucose (Table 1). This result suggested that glucose was covalently linked to the mannoproteins, since control experiments had shown that glucose, laminarin and pustulan were not retarded on Con A-Sepharose. Nevertheless, to remove any possible contaminating glucan, the mannoprotein fraction was subjected to anion-

exchange chromatography on DEAE-Trisacryl, on which laminarin and pustulan are not retained. As shown in Table 1, the resulting mannoprotein fraction still contained a small, but significant amount of glucose. This result indicates that we had isolated a set of mannoproteins containing covalently linked glucose. Mannoproteins isolated by hot-citrate extraction and purified by affinity and anion-exchange chromatography contained less glucose than those purified from the laminarinase digest. The molar ratio GN:mannose:glucose of the mannoproteins purified from the hot-citrate extract was 1 :38:1. When laminarinase digests of wild-type cell walls were subjected to affinity chromatography on Con A-Sepharose, the retained mannoproteins also contained glucose (0.3 mg/mg protein). However, the analysis of small amounts of glucose in the presence of a large excess of mannose was inaccurate. Moreover, wild-type mannoproteins gave diffuse bands on SDS-PAGE (results not shown), probably due to the variation in length of the outer chain. For these reasons further experiments were performed on mnn9 cell wall mannoproteins. The anomeric conjiguration of glucose N-Glycosylation orginates in the lumen of the endoplasmic reticulum, where the unit GlcNAc,Man,Glc, is transferred from a dolicholpyrophosphate carrier to asparagine residues of proteins. In later processing events, the three aglucose residues and one mannose residue are removed (Kukuruzinska et al., 1987). It is unlikely that the glucose present in our mannoprotein fraction is due to the presence of unprocessed N-linked carbohydrate chains, since we extracted them from isolated cell walls. However, to rule out this possibility, we incubated the mannoproteins with a- and P-glucosidase. The products were separated by gel

722

J. VAN RINSUM, F. M. KLIS AND H. VAN DEN ENDE

Table 2. Sensitivity of mannoproteins towards a- and (3-glucosidase Step

%Recovery GN Man Glc

i p

I0

c

-

d

e

f

Y

0.5

a-Glucosidase

Residual substrate Product

I

100 0

100 0

99.3 0.7

8 0

0.0

8-Glucosidase

Residual substrate Product

FRACTION NUMBER

100 0

97.2 2.8

54.6 45.4 Composition of t h e pooled f r a c t i o n s .

Abbreviations: G N = (N-acety1)glucosamine; Man = mannose; Glc = glucose. Mannoproteins were incubated with a- or @-glucosidase;products were separated on Bio-Gel P-6 and the compositions of both residual mannoprotein and product were determined by highpH anion-exchangechromatography with pulsed amperometric detection.

filtration and the monosaccharide compositions were determined (Table 2). It appeared that only 0glucosidase could liberate glucose from the mannoproteins, indicating that they contained p-glucose. However, no conditions were found in which more than 45% of the glucose was released, suggesting that part of the glucose residues is resistant to pglucosidase. To our knowledge the specificity of pglucosidase for various p-glycosidic linkages has never been determined, so no indications can be given for the type of linkage between mutual glucose residues or between glucose and mannoproteins. /I Elimination Purified mannoproteins were subjected to pelimination at 20°C for 24h; the products were separated by gel filtration and pooled fractions were analysed for monosaccharide composition (Figure 2). The released 0-linked chains, identified by the presence of mannitol, are present in pool c-g. Protein, most of the GN, glucose and 29% of the mannose eluted with the void volume. Longer incubation periods or higher temperatures did not result in the release of more 0-linked chains, but only resulted in protein degradation. It appeared that approximately 70% of the mannose residues is 0-glycosidically linked to protein. The 0-linked chains were not further analysed. The heavy 0-glycosylation of the mannoproteins is consistent with the high content of serine and threonine in cell wall proteins (Shibata et al., 1983; Frevert and Ballou, 1985; Lipke et a/., 1989).

pool :

GN

a b c d e f g

1 1 0 0 0 0 0

molar r a t i o Man : Manol : Glc

: 19.5 : 17.3 : : : :

5 . 3 . 2 . 1 . : 17.9

:

0

: 4.6

:

0

: 0.7 0 0 0 0 : 7.5

8 8 3 6

: : : :

:

1 1 1 1 1

: : : :

% recovery

GN

Man

Glc

87 10

29 3 19 19

81 2

18

0 0

0 0 0

0 0

16