ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 19’7, No. 2, October 15, pp. 367-378, 1979

Introduction of 0-Glycosidically Linked Mannose into Proteins Mannosyl Phosphoryl Dolichol by Microsomes from Fusarium solani f. pisi’ CHARLES Department

of Agricultural

L. SOLIDAY Chemistry

via

AND P. E. KOLATTUKUDY2 and Program

in

Washington State University, Pullman,

Biochemistry and Biophysics, Washington 99164

Received April 4, 1979 Cutinase, a glycoprotein containing 0-glycosidically linked carbohydrates, is induced in glucose-grown Fusarium solani f. pisi by cutin hydrolysate. Microsomal preparations from the induced cells catalyzed mannosyl transfer from GDP-mannose to glycolipid and glycoprotein fractions but not into oligosaccharide lipids. Maximal rates of mannosyl transfer into glycolipids and glycoproteins were obtained with 5 mM Mg2+ and 10 mM Mn2+, respectively. Mannosyl transfer into glycolipids and glycoproteins showed pH optima of 8.0 and 7.0, respectively, and both transfers showed an apparent K, of about 2 PM for GDPmannose. The mannosyl lipid was identified as p-D-mannoSy phosphoryl dolichol by thinlayer and ion-exchange chromatography, as well as by analyses of the products derived from it by acid and base treatments. The fungal microsomal preparation also catalyzed mannosyl transfer from GDP-mannose to exogenous dolichol phosphate. This transfer was stimulated maximally by 0.09% Triton X-100 and showed a pH optimum at pH 8.0. The apparent K, values for dolichol phosphate and GDP-mannose were 120 and 2.3 pM, respectively. The product derived from exogenous dolichol phosphate was identified as P-Dmannosyl phosphoryl dolichol as indicated above. The endogenous mannosyl acceptor lipid from this fungus was isolated by DEAE-cellulose chromatography. Analysis of the pnitrobenzoyl derivatives of the base hydrolysis products of this acceptor lipid by highperformance liquid chromatography showed that the major components of this dolichol were G5 and Cloo. The microsomal preparation also catalyzed the transfer of mannose from exogenous mannosyl phosphoryl dolichol to glycoproteins with a pH optimum of 7.5 and an apparent K, of 1.7 PM. Analyses of the p-elimination products of the glycoproteins generated from both GDP-mannose and dolichol phosphoryl mannose showed that single mannosyl residues were transferred to hydroxyl groups of the endogenous proteins. Exogenous cutinase was not glycosylated even after denaturation, sulfitolysis, or removal of carbohydrates by HF hydrolysis. Sodium dodecyl sulfate electrophoresis indicated that cutinase and its possible precursors were among the in vitro glycosylation products. Bacitracin and amphomycin but not tunicamycin inhibited the mannosyl transfer reactions.

Cutinase, an extracellular enzyme which catalyzes the hydrolysis of the cuticular hydroxy fatty acid polymer, cutin, has been recently isolated from several pathogenic fungi (l-4). With the recent finding that ’ Scientific Paper No. SP 5316, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, Wash. 99164. This work was supported in part by NSF Grants PCM-74-09351 and PCM-77-00927. ’ Author to whom inquiries should be made. 367

this enzyme is necessary for the fungal penetration of the cuticle during infection by Fusarium solani f.pisi (5, S), it has become clear that the production of this enzyme is an important phase in pathogenesis. These fungal enzymes are glycoproteins containing monosaccharides Oglycosidically linked to serine, threonine, and two novel amino acid residues, phydroxyphenylalanine and P-hydroxytyrosine (7). Particulate preparations from a variety of organisms have shown that glycoOOO3-9861/79/120367-12$02.00/O Copyright 0 19’79by Academic press, Inc. All rights of reproduction in any form reserved.

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sylation of proteins proceed by the transfer of a monosaccharide from a sugar nucleotide to glycolipid intermediates followed by the transfer of the carbohydrates from the lipid to the protein (8, 9). Whereas the function of dolichol phosphoryl sugar as the glycolipid intermediate in the formation of the N-glycosidic bonds of glycoproteins appears to be universal in eucaryotic organisms (8), the participation of the glycolipid in the formation of 0-glycosidic bonds in glycoproteins has been reported only in a few cases (10-14). In this paper we demonstrate that a particulate fraction from F. solani pisi catalyzes the transfer of mannose from GDPmannose to dolichol phosphate and from the resulting glycolipid to endogenous proteins. Evidence is presented that this process introduces the mannose residue Oglycosidically attached to the proteins. The major endogenous acceptor lipid in the organism is shown to be C,, and C,,, dolichol phosphates. MATERIALS

AND METHODS

Dolichol (pig liver), dolichol phosphate, GMP, GDP, GDP-mannose, and bacitracin were purchased from Sigma Chemical Company. p-Nitrobenzoyl chloride was from Aldrich Chemical Company. GDP[U-Wlmannose (170 mCi/mmol) was purchased from AmershamSearle. [W]Mannosyl phosphoryl dolichol from pig aorta was a gift from Dr. A. D. Elbein. Amphomycin and tunicamycin were gifts from Bristol Laboratories and Eli Lilly, respectively. F. solanipisi, obtained from Dr. Lee Hadwiger, was maintained on potato dextrose agar. Aliquots of a suspension of the spores were added to 100 ml of 0.2% glucose in a mineral medium (pH 7.2) contained in Roux culture bottles. After 5 days of fungal growth at 23°C glucose was depleted and a dispersion of cutin hydrolysate (8 mg in 0.2 ml of water) was added to induce cutinase production (15). The fungal mycelia were recovered by filtration after about 16 h of induction. The mycelia from 6-10 bottles were homogenized with a Ten Broeck glass homogenizer in 12-20 ml of 0.05 M Tris-HCl, pH 7.5, containing 2 mM mercaptoethanol. The homogenate was centrifuged at 10,OOOg for 10 min, and the supernatant was centrifuged at 105,OOOg for 90 min. The pellet was resuspended in buffer and centrifuged at 105,OOOg for 60 min. This washed pellet was suspended in 1 to 1.5 ml of the above buffer and O.l-ml aliquots containing 0.3 to 0.7

mg of microsomal protein were used,as the enzyme source. Incubation conditions and product isolation. Initial incubations were conducted in 0.05 M Tris-HCl buffer, pH 7.5, containing 2 mM mercaptoethanol, 5 mM MgCl,, 5 mM MnCl,, and 1 pM GDP-[UJ’C]mannose (80,000 cpm) in a total volume of 0.3 ml. After incubation at 30°C in a gyrating water bath the reaction was stopped by the addition of 0.7 ml of water and 2 ml of CHCl,:CH,OH (21). The glycolipid, oligosaccharidelipid, and the glycoprotein fractions were isolated using 2:l (v/v) mixture of CHCl, and CH,OH and l&10:3 (v/v) mixture of CHCI,, CH,OH, and H,O as described before (16). The protein pellet resulting from this procedure was solubilized by heating it in a boiling water bath for 15 min with 250 ~1 of 4% sodium dodecyl sulfate (SDS)3 containing 2 mM P-mercaptoethanol. An aliquot was assayed for “C in ScintiVerse (Fisher Scientific Co.) as a scintillation fluid. Aliquots of the glycolipid and oligosaccharide extracts were evaporated to dryness and assayed for ‘C in a counting fluid consisting of 30% ethanol and 70% toluene containing 4 g Omnifluor (New England Nuclear) per liter. Unless otherwise specified the above procedure was used for measuring mannosyl transferase activity with GDP-mannose as the substrate. Characterization of glycolipid. DEAE-cellulose in acetate form (17) was equilibrated with 99% CH,OH. After the glycolipid fraction was applied the column was washed with 99% methanol, and the labeled glycolipid was eluted with a O-O.3 M linear gradient of ammonium acetate in 99% methanol. The desired fractions were evaporated to dryness, the residue was dissolved in CHC&, and ammonium acetate was removed by extraction with water. The glycolipid fraction was also subjected to thin-layer chromatography on O&mm layers of silica gel G with the following solvents: A, CHCl,:CH,OH:H,O (60:35:6), B, CHCl,: CH,OH:acetic acid:H,O (25:X&2), C, CHCI,:CH,OH: coned ammonium hydroxide (75:25:4). The glycolipid fraction was subjected to mild acid hydrolysis (16) with 0.01 N HCl in 50% l-propanol at 100°C for 10 min. The samples were neutralized with NaOH and extracted with CHCl,:CH,OH (2:l). Both aqueous and organic phases were assayed for radioactivity and the products in the aqueous phase were examined by paper chromatography. The glycolipid fraction was also treated with 0.1 N NaOH with 90% l-propanol at 65°C for 20 min (18). After neutralizing the samples with acetic acid, they were extracted with 3 Abbreviations used: SDS, sodium dodecyl sulfate; hplc, high-performance liquid chromatography; solvent A, CHC&:CH,OH:H,O (60:35:6); solvent B, CHCl,:CH,OH:acetic a&H*0 (25:15:5:2); solvent C, CHCl,:CH,OH:concd ammonium hydroxide (72254); tic, thin-layer chromatography.

0-GLYCOSIDICALLY CHCl,:CH,OH as above.

LINKED

MANNOSE

(21) and the two phases were analyzed

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369

reaction mixture was acidified and the polyprenols were recovered by extraction with CHC&. p-Nitrobenzoyl derivatives of the polyprenols were prepared as described by Keenan et al. (23). High-performance liquid chromatography (hplc) of the p-nitrobenzoyl derivatives of polyprenols were performed with a Waters Associates, Inc., ALC/GPC 244 instrument equipped with a Waters PBondapak C,, reversephase column (30 cm x 4 mm) using 90% (v/v) lpropanol:H,O at 0.5 ml/min flow rate. The p-nitrobenzoyl derivatives were detected by their absorbance at 256 nm with a Waters Model 450 variable wavelength detector. Electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed according to the method described by Maize1 (24). The 14Clabeled glycoprotein fractions, dissolved by heating them at 100°C for 3 min in 2% SDS containing 2 mM P-mercaptoethanol, were subjected to electrophoresis on 17 x BO-mm 10% polyacrylamide-SDS gels with 15 mA/gel. The gels were frozen and sliced into 3-mm sections; each section was treated with 0.8 ml of 30% H,Oz at 60°C until the gel dissolved, and l“C was assayed with ScintiVerse. Proteins of known molecular weight were subjected to SDS electrophoresis as indicated above; the gels were stained overnight in Coomassie blue and destained electrophoretically in 7% acetic acid and 5% methanol.

Characterization of glycoprotein fraction. The protein pellet obtained from the reaction mixture was dissolved in 4% SDS containing 2 mM P-mercaptoethanol. This solution was subjected to gel filtration with Bio-Gel P-2 (1.3 x 45 cm) and Sephadex G-25 (1.3 x 45 cm) both in the presence of 0.1% SDS in 10 mM ammonium acetate buffer, pH 7.5. The fractions corresponding to the exclusion volume were pooled, lyophilized, and passed through a Dowex 2 X B-400 anion-exchange column (0.7 x 7 cm) to remove the SDS (19). The glycoprotein fractions were pooled, lyophilized, and the residue was dissolved in 1.5 ml of 0.2 M KOH prepared in 50% dimethylsulfoxide, 40% water, and 10% ethanol. To this solution 80 mg of NaBH, was added and the mixture was incubated at 40°C for 4 h. After acidification with acetic acid, the reaction mixture was lyophilized. The residue was dissolved in a minimal volume of 10 mM ammonium acetate buffer, pH 7.5 and an aliquot was subjected to paper chromatography with isobutyric acid:concd. ammonium hydroxide:H,O (57: 4:39) and 95% ethanol:1 M ammonium acetate, pH 7.5 (5:2). Another aliquot was subjected to gel filtration with Bio-Gel P-Z column (1.3 x 45 cm, >400 mesh). Carbohydrates in the column efluent fractions (1.1 ml) were assayed by the phenol-sulfuric acid method (20) and 0.5-ml aliquots were assayed for 14C. The radioactive monomer fraction was lyophilized and the RESULTS AND DISCUSSION residue was subjected to paper chromatography with the solvent system indicated above. Mannosyl Transfer from GDP-[U-14C] The protein pellet isolated from the incubation Mannose to Glycolipid and Glycoprotein mixture was dispersed by sonication in 2 ml of 0.05 M Fractions Tris-HCI, pH 7.7, containing 10 mM CaCl, and incubated at 30°C with 2 mg Pronase for 24 h folCutinase is known to be induced by cutin lowed by a second 24 h incubation with another 2 mg hydrolysate in glucose-grown F. solani pisi of Pronase. The reaction mixture was placed in a upon depletion of glucose from the medium boiling water bath for 3 min, centrifuged, and the (15). This induced cutinase production consupernatant passed through a Bio-Gel P-2 column tinues for 24-48 h. To study the glycosylawith 0.05 M Tris-HCl, pH 7, buffer. tion reactions involved in the synthesis of Isolation and identification of the endogenous accutinase, crude microsomal fractions were ceptor lipid. About 500 g (wet wt) of F. solani pisi micelia was homogenized in CHCI,:CH,OH (2:l) and obtained from mycelia harvested 16 h into the total lipids were recovered by the method of Folch the induction period. Upon incubation of the microsomal fraction with GDP-[U-14C]manet al. (21). This lipid fraction was applied to a DEAEcellulose (acetate) column (1.3 x 30 cm) and the column nose the glycolipid fraction and the lipid was washed with 2.5 bed volumes of CHCl,:CH,GH depleted residue became labeled; however (21) followed by a O-O.4 M linear gradient of ammonium the oligosaccharide-lipid fraction was not acetate in CHCl,:CH,OH (2:l). Aliquots (0.4 ml) of labeled. These results suggested that the eluant fractions (5 ml) were extracted with water mannosyl residues were being transferred and the lipid material was assayed for mannosyl acfrom GDP-mannose to polyprenols and proceptor activity with the particulate enzyme preparateins (25-27). tion using the standard assay conditions indicated Time course studies revealed that there elsewhere. The fractions containing the acceptor of radioactivity activity were pooled and extracted with acidified was a rapid accumulation fraction followed by a water. The lipid recovered was treated with 4% KOH in in the glycolipid 94% ethanol under N, at 70°C for 30 min (22). The slower accumulation of radioactivity in the

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120

TIME (mid FIG. 1. Time course of the transfer of mannose from GDP-mannose to the glycolipid and glycoprotein fractions by the microsomal preparation from F. soloni pisi. The incubation mixtures contained 0.80 mg of protein and the other experimental conditions were as described in the text.

glycoprotein fraction (Fig. 1). This time course is consistent with the notion that mannose is transferred from GDP-mannose via glycolipids (8, 9). Although decrease in the glycolipid fraction was not significant within the 2-h incubation used in these experiments, significant decrease in glycolipid was observed in 10 min when similar incubations were carried out with microsomes from rapidly growing F. solani pisi (data not shown). In general agreement with results obtained with other systems (8, 9) Mg2+ was the preferred cation for [14C]mannose transfer from GDP-[UJ4C]mannose to the glycolipid fraction, whereas Mn2+ was the most effective cation to enhance incorporation of [14C]mannose into glycoproteins (Table I). The effect of increasing concentrations of the preferred cations on the rate of incorporation of [14C] mannose from GDP[U-14C]mannose showed saturation at 5 lllM Mg2+ for transfer of radioactivity to the glycolipid, and at 10 mM Mn2+ for the transfer of [14C]mannose to the glycoprotein fraction. Transfer of mannose into glycolipid occurred optimally at pH 8.0 with 0.05 M TrisHCl, whereas the optimum pH for mannose transfer into the glycoproteins was about 7.0 with 0.05 M Tris-maleate. In contrast, the pH optimum for the glycolipid formation in Aspergillus niger was pH 9.0 (28), and mannosyl transfer into proteins showed pH

optimum of 6.8 in A. niger (29) and 6.0 in Aspergillus owzae (30). In general, pH optima for glycosyl transferases in other tissues vary from 6.0 to 9.0. The transfer of [14C]mannose from GDP[U-14C]mannose to glycolipid was linear to 1.1 mg/ml of protein and mannosyl transfer to glycoproteins increased linearly with increasing microsomal protein up to 0.6 mg/ml. Increasing the GDP-mannose concentration showed a typical substrate saturation pattern and from linear doublereciprocal plots apparent K, values of 2 p and 1.6 j..kM were obtained for the transfer of mannose from GDP-mannose into glycolipids and glycoproteins, respectively. These values are within the range of values previously reported for particulate glycosyl transferases from other sources (8). Characterization

of the Labeled Glycolipid

Since dolichol phosphate is known to be a carrier lipid involved in glycoprotein synthesis in eucaryotic organisms (8, 9), the mannosyl lipid isolated from the present microsomal preparation was subjected to chromatographic analyses similar to those used for the identification of mannosyl TABLE EFFECT

I

OF CATIONS ON MANNOSYL TRANSFER GDP-[U-WIMANNOSE INTO GLYCOLIPID AND GLYCOPR~TEIN FRACTIONS

FROM

Relative rate (%) of mannosyl transfer into Cation”

Glycolipid

0 Mn*+ Wt+ Nil+ co*+ Ca2+ Fez+

20 44 100 25 49 23 3

Glycoprotein 16 100 74 7 22 35 404b

a Each reaction mixture containing 10 mM cation and 0.5 mg protein was incubated at 30°C for 10 min and the other conditions were as described in the text. b The incorporation of label into the glycoprotein fraction in the presence of Fe*+ was an artifact as gel filtration in the presence of SDS showed that W was not associated with macromolecules.

0-GLYCOSIDICALLY

LINKED

MANNOSE

phosphoryl dolichol. DEAE-cellulose chromatography showed that the present fungal glycolipid cochromatographed with porcine mannosyl phosphoryl dolichol (Fig. 2). Thinlayer chromatography on silica gel G with neutral (A), acidic (B), and basic (C) solvent systems revealed a single labeled component with R, values quite similar to those previously observed (31) for mannosyl phosphoryl dolichol (R, values: 0.35, 0.88, and 0.18 with solvents A, B, and C, respectively). In agreement with this conclusion was the finding that the glycolipid was stable to mild treatment with alkali (0.1 N KOH, 37°C 30 min), but was labile to mild acid treatment (0.01 N HCl, lOO”C, 10 min). The product of this acid treatment was identified as mannose by paper chromatography suggesting that mannose was glycosidically attached to dolichol phosphate. Upon treatment of the glycolipid with 0.1 N NaOH (in 90% propanol) at 60°C for 20 min, 85% of the radioactivity was rendered water soluble, and this product was identified to be mannose l-phosphate by paper chromatography. These results suggest that the mannosyl phosphoryl lipid had P-D configuration just as previously found in other tissues (8, l&32). Addition of 1 mM GDP but not GMP nearly completely inhibited the incorporation of mannose from GDP-mannose into both the glycolipid and glycoprotein fraction. Incubation of the purified [‘*C]mannosyl lipid and the particulate preparation with 1 mM GDP resulted in the production of water-soluble labeled products (presumably GDP-mannose) whereas 1 mM GMP did not produce such

FIG. 2. DEAE-cellulose (left) and thin-layer (right) chromatography of the mannosyl-lipid generated from GDP-[UJ4C]mannose by the microsomal preparation. A Berthold radioactivity scanner was used to detect 14C on tic.

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l/S (PM), 0

200 [ DOLIGHOL- PO41 (p Ml 100

FIG. 3. Effect of increasing the concentration of dolichol phosphate on the rate of mannosyl transfer from GDP-mannose to mannosyl phosphoryl dolichol. The reaction mixtures containing 0.05~1 Tris-HCI, pH 8, 0.09% Triton X-100, 2 mM pmercaptoethanol, 5 mM MgCl,, 1 PM GDP-[U-W]mannose, 0.5 mg microsomal protein, and varying amounts of dolichol phosphate in a total volume of 0.3 ml were incubated at 30°C for 10 min.

labeled products. These results are consistent with the conclusion that the mannosyl lipid intermediate is mannosyl phosphoryl dolichol. Mannosylation Phosphate

of Exogenous

Dolichol

Since the results discussed above suggested that the glycolipid intermediate generated by the microsomal preparation from F. solani pisi was mannosyl phosphoryl dolichol, we tested whether exogenous dolichol phosphate could be mannosylated by the same particulate preparation. The addition of porcine dolichol phosphate to the microsomal preparation with GDP-[U-‘*C]mannose resulted in a 5- to 20-fold increase in [ ‘*C]mannose incorporation into the glycolipid fraction. Triton X-100 further stimulated the transfer of mannose from its sugar nucleotide to the glycolipid fraction. As the concentration of the detergent was increased the rate of mannose transfer into the lipid fraction increased up to a detergent content of 0.09% (-55% stimulation) and further increases severely inhibited the mannosyl transfer. The pH dependence of the glycosylation of the exogenous dolichol phosphate was identical to that observed for the

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mannose acceptor lipid eluting at 0.13 M ammonium acetate (Fig. 4). A similar pro-zfile of mannose acceptor activity was obe.3 * tained when porcine dolichol phosphate 0 was chromatographed and the fractions =2 were analyzed in the same manner. The 5 effect of mild acid (0.01 N HCl, lOO”C, 10 2 1 min) and alkaline (0.1 N NaOH, 37”C, 1 h) & L treatments of the mannosyl acceptor lipid a Oo 100 fraction on its ability to be mannosylated ELUTION VOLUME (ml) was determined. The fungal acceptor lipid FIG. 4. Isolation of the endogenous mannosyl was found to be stable to mild alkali and acceptor lipid from F. solani pisi by anion-exchange to the mild acid treatments. These results chromatography. The solid line shows the acceptor are consistent with the conclusion that the activity of aliquots of each fraction. endogenous mannosyl acceptor is dolichol phosphate. To determine the chain length of the mannosyl transfer into the endogenous endogenous dolichol phosphate the acceptor glycolipid. Increasing the concentration of lipid fraction was subjected to alkaline dolichol phosphate resulted in a typical hydrolysis (4 N KOH, 7O”C, 30 min) and the substrate saturation pattern (Fig. 3), and resulting free polyprenols were converted from linear double-reciprocal plots the apderivatives. High-presparent K, was calculated to be 1.2 x 1O-4 M. into p-nitrobenzoyl sure reverse phase liquid chromatography With saturating concentration of dolichol showed that the major dolichols were C,, phosphate the apparent K, for GDP-manwith smaller amounts of others and Go0 nose was found to be 2.3 PM, which is al(Fig. 5). This chain length distribution is most identical to that obtained without similar to that reported for dolichol acexogenous dolichol phosphate. The labeled glycolipid generated in the ceptor lipids found in algae (33), plants (34), and mammalian systems (8, 19). In yeast presence of exogenous dolichol phosphate (Saccharomyces cereuisiae), on the other had chromatographic properties (DEAEhand, the accceptor lipid fraction contained cellulose, tic with solvents A, B, and C) shorter dolichols, C,,-C,, (35). Aspergillus identical to those of porcine mannosyl phosphoryl dolichol. Furthermore, acid niger was reported to contain dolichols C,,and base treatments of this glycolipid fraction gave results identical to those obtained with P-D-mannosyl phosphoryl dolichol as discussed above. FUNGAL CM 2:l

ACCEPTOR

Identification Lipid

of Endogenous

LIPID

Acceptor

The endogenous mannosyl acceptor lipid was isolated from a chloroform-methanol extract of F. solani pisi mycelia by anionexchange chromatography on DEAE-cellulose. After the total lipid extract was applied to the column, the neutral lipids were eluted with chloroform-methanol (2: 1, v/v) and the acidic lipids were eluted by a O0.4 M ammonium acetate gradient. The addition of aliquots of the column fractions to microsomal preparations with GDP[U-14C]mannose showed a single peak of

I

0

20

TIME FIG.

porcine alkaline Fig. 4. number

40

60

(mid

5. The hplc of p-nitrobenzoyl derivatives dolichol and the polyprenols generated hydrolysis of the fungal fraction shown The numbers on the peaks indicate of isoprene units in each molecule.

of by in the

0-GLYCOSIDICALLY

LINKED

MANNOSE

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C,,, in chain length with C,,, and C,,, as the most abundant components (36). However, in this case the chain length analysis was not done on the endogenous glycosyl acceptor phospholipid fraction, but on the more abundant neutral derivatives (e.g., fatty acid esters) which were capable of acting as glycosyl acceptor lipids after saponification and chemical phosphorylation (37). In the present case, only the naturally occurring acidic mannosyl acceptor fraction was examined for the chain length distribution. Therefore, in this ease it is quite cerFIG. 6. Effect of the concentration of [U-W]mantain that the C&C,,,, dolichols serve as the nosy1 dolichol phosphate on the rate of mannosyl glycosyl carrier lipid. It is possible that transfer to the glycoprotein fraction by the microfilamentous fungi utilize longer dolichols somal preparation of F. solani pisi. Incubation mixtures containing 0.7 mg microsomal protein in 0.05 M than those used by yeast. Transfer of Mannose from Mannosyl Phosphoryl Dolichol to the Glycoprotein Fraction

Tris-maleate, pH 7.5, were incubated for 30 min at 30°C; specific activity of the substrate was 1’79 Ciimol.

divalent cation, whereas no such require[UJ4C]Mannosyl phosphoryl dolichol was ment could be shown for the transfer of produced from GDP-[UJ4C]mannose and mannose from the glycolipid to the glycoexogenous dolichol phosphate with the protein. In fact, the addition of EDTA, microsomal preparation as described above, had a slight stimulatory effect on the and the glycolipid was purified by thin-layer mannosyl transfer to the glycoproteins chromatography. This labeled glycolipid, (-50% at 0.5 InM). In contrast to the present when incubated with the fungal microsomal finding, Mg2+ was reported to be required preparation, gave rise to a labeled glyco- for introduction of the first 0-glycosidically protein fraction. The pH optimum for this attached mannose residue in the glycopromannosyl transfer was found to be 7.5 in teins of yeast (10, 11) and N. crassa (13). 0.05 M Tris-maleate buffer, and the rate of However, lack of divalent cation dependency transfer was linear with time of incuba- and lack of inhibition by EDTA for the tion for 30 min at 30°C. The rate of in- transfer of mannose from the glycolipid to corporation of mannose from the glycolipid glycoproteins has been previously observed into the protein fraction increased linearly in bacterial (38) and mammalian (22, 39) up to a microsomal protein concentration systems. of 1.15 mg/ml. A typical Michaelis-Menten substrate saturation pattern was obtained Characterization of the Glycoprotein for mannosyl phosphoryl dolichol and from Fraction linear double-reciprocal plots the apparent K, was calculated to be 1.7 PM (Fig. 6). When SDS-solubilized labeled glycoproThe addition of GDP did not stimulate tein fractions were chromatographed on the incorporation of radioactivity from Bio-Gel P-2 or Sephadex G-25 columns in labeled glycolipid into the glycoproteins, the presence of 0.1% SDS, 90-95% of the but in fact, showed a slight inhibition label was excluded by the gels and eluted suggesting that a direct transfer of labeled at the void volume indicating a covalent mannose from the glycolipid to the protein attachment of labeled mannosyl residues to occurred without involving an indirect route macromolecular components. To further via GDP-mannose. The transfer of mannose test whether the label incorporated into from GDP-mannose to the glycolipid or the lipid-free residue was in glycoproteins, glycoproteins showed a requirement for a the labeled residue generated from GDP-

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in the incorporation of mannose from ,GDPmannose into the glycoproteins, the labeled proteins generated from the exogenous labeled glycolipids should be similar to those produced from GDP-[U-14C]mannose. In fact examination of the labeled proteins derived from labeled glycolipid by gel filtration in the presence of SDS and analysis of p-elimination products gave results identical to those obtained with the labeled protein generated from GDP-mannose. Thus the particulate preparation from F. FIG. ‘7. Gel filtration of the alkaline NaBH,solani pisi catalyzed transfer of mannosyl treated glycoprotein fraction generated from GDP- residues from mannosyl phosphor-y1 dolichol [U-%]mannose in the presence of 5 mM MnCl,. The to the hydroxyl groups in proteins. dashed lines represent markers for molecular weight Under the conditions of the fungal growth in decreasing size, dextran blue, stachyose, maltoused in this study, cutinase and a nontriose, and mannose. specific esterase are the major extracellular proteins produced by this fungus (15), and [U-14C]mannose was subjected to Pronase both of these proteins contain O-glyattached mannose. To test digestion. Over 90% of the radioactivity was cosidically rendered soluble by this treatment, and whether the proteins mannosylated in vitro upon gel filtration on Bio-Gel P-2 the bulk contained these two enzymes, the labeled of this radioactivity was not excluded in- proteins derived from GDP-[U-14C]mannose gel dicating the glycoprotein nature of the were subjected to SDS-polyacrylamide electrophoresis and the distribution of product. in the various regions of the To determine the size and linkage of the radioactivity gel was examined (Fig. 8). There were carbohydrates introduced into the protein under the experimental conditions, the glycoprotein fraction recovered from the IO3 M. Wt. 68 26 12 above SDS chromatography step was sub1p ‘7 1 jected to alkaline-NaBH, treatment after removal of SDS by anion-exchange chromatography. Upon gel filtration of the resulting products on a Bio-Gel P-2 column, about 90% of the label coeluted with an authentic mannose sample (Fig. 7). The radioactivity of the p-elimination products migrated with authentic mannitol when subjected to paper chromatography. Very little radioactivity was associated with oligosaccharides; the small amount (- 10%) of label eluted at the exclusion volume could represent incomplete p-elimination, poly20 40 60 saccharides, or proteins containing N-glyMbBlLlTY (mm) cosidically attached carbohydrates. In any FIG. 8. SDS-polyacrylamide gel electrophoresis of case it is quite clear that the bulk of the labeled glycoprotein fractions generated from GDPmannose residues incorporated into the [U-Wlmannose by microsomal preparations from glycoprotein fraction constituted single glucose-grown F. solani pisi. I, Microsomes from mannosyl residues 0-glycosidically attached cells which were 16 h into cutinase induction; NI, to proteins. control noninduced; I - NI, difference; N, nonspecific If the glycolipids are in fact intermediates esterase; C, cutinase.

0-GLYCOSIDICALLY

LINKED

MANNOSE

considerable amounts of radioactivity in proteins which did not migrate significantly into the gel and a substantial amount of 14C was also associated with the dye front. The other major labeled components were in the 25,000 to 50,000 molecular weight range. It is possible that the radioactive components in this range represent cutinase (C in Fig. S), its precursor (N in Fig. 8), and intermediate forms varying in the degree of glycosylation. The results of similar analysis of the labeled proteins, generated from GDP-[U-14C]mannose by the particulate preparations from glucose-grown F. solani pisi cultures, which were not induced to synthesize cutinase, were compared with the above results obtained with induced cultures. The labeling of proteins in the molecular weight range of cutinase and its suspected precursor was less pronounced in the case of the noninduced cultures. To determine the most significant differences between the proteins glycosylated by the membranes from the induced and noninduced cultures, the values obtained with the latter were subtracted from the former (I - NI, Fig. 8). It is apparent that the nonspecific esterase and cutinase regions, although not exclusively, were preferentially labeled by the membranes from the induced cultures. Since these two proteins are known to have multiple sites of attachment of carbohydrates (7, 40), it is possible that the preferential labeling of intermediate size proteins represents partially glycosylated moieties. Although cutinase and the nonspecific esterase account for the major portion of the extracellular protein (15), they account for only a much smaller proportion of the labeled proteins generated in the in vivo glycosylation system. These results are similar to those reported with myeloma cells and oviduct tissues which synthesize relatively large amounts of K-type immunoglobulin light chains and ovalbumin, respectively. SDSpolyacrylamide electrophoresis of labeled proteins generated from GDP-[U-14C]mannose by microsomal preparations from oviduct showed that ~10% of the label was present in the major secretory protein (41). Similary, K chains constituted only l&20%

INTRODUCTION

INTO

PROTEINS

375

of the proteins in vitro glycosylated by particulate preparations from myeloma (42). Attempts to Glyeosylate Exogenous Cutinase In view of the fact that some exogenous proteins have been reported to be glycosylated in vitro (32,43,44) we attempted to glycosylate cutinase using the particulate preparation from F. solani pisi. This preparation did not catalyze mannosyl transfer from either GDP-mannose or mannosyl phosphoryl dolichol to exogenous purified cutinase. Neither acid treatment (1 M HCl, lOO”C, 30, 60, and 120 min) nor sulfitolysis, which have been used on proteins to render them suitable as exogenous glycosyl acceptors (32, 43), was successful in the present case. It is possible that cutinase does not contain suitable free sites for glycosylation. However, carbohydrate-depleted cutinase, prepared by HF hydrolysis (45), also failed to be glycosylated by the fungal particulate preparation. Since HF treatment is known to remove the 0-glycosidically attached carbohydrates, releasing the hydroxyl groups of the amino acids involved (45), it is most probable that sites suitable for glycosylation were present in this cutinase preparation. Therefore, the lack of glycosylation was probably because this protein was unable to traverse the membrane and reach the luminal region where glycosylation most probably occurs. However, attempts to make this substrate available to the glycosylating enzyme by ultrasonic treatment of the enzyme-substrate mixture failed. Recent studies with hen oviduct membranes also showed that some proteins could not serve as exogenous glycosyl acceptors, whereas other proteins did, suggesting that secondary and/or tertiary structures could restrict glycosylation of exogenous proteins. In the present case an additional restriction could have been imposed by the carbohydrate moiety attached by an amide linkage to the Nterminus of cutinase (46). It is noteworthy that exogenous carbohydrate-depleted and denatured carboxypeptidase y was also not glycosylated by yeast microsomal preparations (47).

376

SOLIDAY

The Effect of Antibiotics on Glycolipid Glycoprotein Synthesis

AND

KOLATTUKUDY

and

Antibiotics such as bacitracin, amphomycin, and tunicamycin have been used to study glycosylation because of their inhibitory effects on selective phases of the glycosylation pathway. Tunicamycin is known to inhibit specifically the formation of dolichol pyrophosphoryl N-acetylglucosamine but not the synthesis of mannolipid (48,49). In agreement with these findings this antibiotic at concentrations up to 30 pM did not affect the mannosyl transfer from GDP-mannose to either the glycolipid or glycoprotein fractions in the present fungal microsomal preparation. Ampho-

mycin, a known inhibitor of the synthesis of bacterial peptidoglycans (50) and mammalian lipid-linked saccharides (51), also inhibited mannosyl transfer from GDPmannose into both mannosyl phosphoryl dolichol and glycoproteins catalyzed by the present enzyme system (Table II). With exogenous dolichol phosphate even more severe inhibition of mannosyl transfer to both the lipid and protein fraction was observed. The addition of bacitracin to the fungal particulate preparations inhibited the incorporation of mannose from GDPmannose into both the glycolipid and glycoprotein fractions (Table II). These results are contrary to those obtained with

TABLE

II

EFFECT OF ANTIBIOTICS ON MANNOSYL TRANSFER TO GLYCOLIPID AND GLYCOPROTEIN FRACTIONS

Experiment

Ia

With 20 nmol exogenous dolichol phosphate Experiment

II”

With 20 nmol exogenous dolichol phosphate

Experiment

III0

[‘QMannosyl phosphor-y1 dolichol substrate

Antibiotic (concn)

Glycolipid (cpm)

Amphomycin (PM) 0 50 loo 200 0 40 100

2020 1130 340 80 18400 5600 840

Bacitracin (m@ 0 5 10 0 1 5 10 Bacitracin ma 0 5 10

4790 2720 1680 50300 43500 14400

Percentage inhibition

44 83 96 70 95

43 65 13 71 82

Glycoprotein (cpm)

650 310 140 65 3970 440 220

1920 1200 600 5840 5390 2180 1170

840 300 200

Percentage inhibition

53 79 90 89 95

37 68 8 63 80

65 76

0 The reaction mixtures containing, in addition to the designated concentration of antibiotic, 0.05 M TrisHCl, pH 8, 2 mM /+mercaptoethanol, 5 mM MgCl%, 0.09% Triton X-100, 1 PM GDP-[U-Y?]mannose, and 0.4-0.6 mg of microsomal protein in a total volume of 0.3 ml, were incubated 10 min. b Reaction mixtures, containing 0.05 M Tris-maleate, pH 7.5,2 mM /3-mercaptoethanol, 0.09% Triton X-100, 0.5 PM [Ylmannosyl phosphoryl dolichol, and 0.5 mg of mircosomal protein in a total volume of 0.3 ml, were incubated 60 min.

0-GLYCOSIDICALLY

LINKED

MANNOSE

yeast where the addition of this antibiotic enhanced mannosyl phosphoryl dolichol synthesis (52) but inhibited glycosylation involving N-acetylglucosamine (53). Our results are, however, in agreement with the report that bacitracin inhibited mannosy1 transfer to the glycolipid in Phaseolus aureus and Daucus carota (54) and in pig aorta (55). If the inhibitory action of bacitracin in this fungal glycosylation system were due to complex formation between the divalent cation and endogenous dolichol pyrophosphate (therefore reducing the available dolichol phosphate as mannose acceptor), as suggested in the case of bacterial systems (56), the addition of exogenous dolichol phosphate should overcome the inhibitory effects. However, the addition of excess exogenous dolichol phosphate failed to overcome the inhibitory effect of bacitracin on [14C]mannose transfer from GDP-[U-14C]mannose to either glycolipid or glycprotein (Table II). Furthermore, bacitracin also inhibited the transfer of mannose from [14C]mannosyl phosphoryl dolichol to the glycoprotein fraction (Table II), a process which, in this system, does not involve divalent cation. These results suggest that bacitracin inhibits mannosyl transfer by its effect on the enzymes involved in mannose transfer rather than by depletion of the lipid acceptor as proposed for bacterial cell-wall biosynthesis (56). The results obtained with enzyme preparations from pig aorta could also not be explained on the basis of the complex formation involving dolichol pyrophosphate and divalent metal ion (56, 57). ACKNOWLEDGMENTS We thank Dr. A. D. Elbein mannosyl phosphoryl dolichol.

for a sample

of labeled

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